DNA Alkylaiion and Cress-linking Agents As Compennds and Payloads for Targeted

Therapies

Related Applications and Grant Support

This application claims the benefit of priority of United States provisional

applications nos. 11862/288,572, filed January 29, 2017 and 0862/417,650, filed November 4, 2016, entitled "DN A Alkylaiion and Cross-linking Agents as PaySods for Targeted

Therapies", each of said applications being incorporated by reference in its entirety herein.

This invention was made with government support under grant nos. IDP2-C A 186575, R01 GM 110506 and 5T32G 06754 awarded by National Institutes of Health. The government has certain rights in the invention

Field of the invention

The present invention is directed to novel small molecules- that alkylate and cross-link DNA. These molecules are easily-prepared and modified to adjust DMA binding and alkylaiion properties. The molecules may contain a cleavable protecting group (prodrug) to allow for specific activation in selected settings arid often comprise a targeting element attached to the molecules by way of a cleavable linker which may be cleaved to facilitate therapy. Methods of synthesizing these compounds are also disclosed as are important intermediates in the process of synthesis.

Background and Overview of the Invention

Precolibactins arid col.ibacti.us are natural products produced by select commensal, extraintestinal, and probiotic E. coli. The metabolites are encoded by a hybrid potyketide synihase- nonribosomal peptide synthetase (PKS--N PS) gene cluster termed clb or pks) clh~ E.. coli strains induce DN A damage in eukaryotic cells and are thought to promote colorectal cancer formation, but the gene cluster is also found in the probiotic strain Nissle 1 17, which is used in Europe for the treatment of ulcerative colitis, diarrhea, and other

gastrointestinal disorders. ' Mature precolibactins are substrates for the 12-transmembrane multidrug and toxic compound extrusion transporter ClbM, which mediates their transfer to the bacterial periplasm." ¹ There, the colibacilli peptidase C!bP converts precolibactins to genotoxic co!ibactins via removal of an A'-acyl-D-asparagrae side chain. ⁵ Mutation of ClbP abolishes cellular DNA damaging-acti v t ' ⁰¹ ' and A-myiistoyl-D-asparagijie and closely related analogs have been identified in wild type clb ^': £. coil cultures.*^ Whether the differential production of biosynthetically-related but distinct metabolites, or other factors (such as the requirement for celS-to-cell contact to observe cytopathic effects ²⁸ "*') underlie the seemingly con radictory phenotypes associated with the clb gene cluster, remains unresol ved.

Despite extensive efforts, fully mature {pre)eoiibactins prior to the present invention, had not yet been isolated in homogenous form. This has been attributed to the low levels of natural production of the metabolites, their instability under fermentation conditions, and the ini½mmaiion-dependant expression of the h gene cluster. The metabolites i, ^5c 2 3. ^',h and 4 ^b were obtained in vanishingly small quantities (2.5-55 pg/L for 2-4) from the fermentation broth of wild-type or genetically-engineered clb ^" E, coii and implicated as intermediates or shunt metabolites in the colibacilli biosynthetic pathway. Based on the isolation of 2 (Figure 1), as well as HRMS analysis, retrospective bioinformatics, and isotopic labelling, the structure of precolibactin A was proposed as S. ^¾t Key elements within 5 include a

hydrophobic .^-terminal fragment, a spirocye!ic ammocyclopropane, and a miazoline--- thiazole tail. The presence of the thiazoSine- thiazoSe fragment was inferred by MS/MS analysis; ⁹ * the alternate isomer, containing a thiazole-thiazoHne sequence, could not be excluded. In addition, the absolute stereochemistry of the tbiazoiine ring was not determined. A compound with an exact mass corresponding to 5 was observed in impurified extracts, but all efforts to isolate this structure were hampered by decomposition. ⁹ ' ¹ The pyridone structure 6 was also recentl proposed based on biosynthetic considerations, isolation of 4, and HRMS analysis, but like 5, was obtained in quantities too minute ⁵⁰ to permit full characterization. ¹¹ 4 was shown to weakly cross-link DNA in viiro ^{' 'A} suggesting that the colibactins may damage DNA by induction of replication-dependant SBs. ¹² Although detailed structure™ functi on analyses of the colibactins had not been conducted prior to the present invention, the aminocyclopropane fragment of 4 is reminiscent of yatakemycin, CC-1065, and the duocaimycins, which have been shown to alkylate DMA via nucl.eoph.ilic ring-opening, ¹³ and the biheterocycHc fragment may serve as a DNA intercalation motif. ³⁴

Pursuant to the present invention, the inventors have focused on understanding the molecular basis of coSibactin-induced DNA damage. Advanced preco!ibactins arise from linear precursors of the generalized structure shown as 1 (Scheme 6 ₅ Figure 15 A). The linear precursors were suggested to transform to unsaturated lactams 2 tha i are processed by Clb J? to generate unsaturated (minium ions 3 (colibactins), which alkylate DMA by cyclopropane ring-opening (grey pathway). ^' '' ⁸ However, this mechanistic hypothesis is ostensibly incompatible with subsequent isolation ⁹ and synthesis ³⁰ efforts that lead to the identification and unequivocal structural assignment of precolibactias A (7), ^{! 5} B (8), and C (9). which contain a pyridone residue (Figures 1 and ! SB), ^' Precolibactias A-C (7-9, Figure 15B) were obtained from cibP mutaat strains; these deletion strains were employed to promote accumulation of the precolibactin metabolites, if 7-9 are the genotoxic precursors, the data outlined above ⁵ suggests that amines such as 5 resulting from ClbP-mediated processing in the wild type strains are responsible for the cytopathicity of the clb cluster, as these cannot readily -convert to unsaturated hniniura ions such as 3. Precolibactin€ (9, Figure 1 SB) was demonstrated to be a substrate for ClbP. ^Ui in earlier synthetic work, the inventors showed that the double dehydrative cycltzafion of the relati vely stable N-acylated linear precursors (1) to pyridones such as 7-9 (Figure 1 and 15B) was facile under mildly acidic or basic conditions (c.f., l-»2~ 4, Scheme 6, Figure 15 A). ¹⁰ The unsaturated lactam intermediates (2) could be detected by LC/ S analysis, bui they were not isolable, arguing against their interception b ClbP in the biosynthesis. The inventors reasoned that the colibactins may instead form by ClbP processing of isolable linear precursors 1 directly. Sequential cyclodehydration reactions proceeding through the

vinylogous ureas 6 would then provide 3 (red pathway), it follows from this analysis that precoiibaetms A-C (7-9) are non-natural cycHzation products deriving from the absence of ClbP in the producing organisms, and are unlikely to be genotoxic. To test this hypothesis, the inventors modified their synthetic strategy ¹⁰ to allow access to the deacylated pyridone derivatives 5 and the analogous unsaturated iminiuni ions 3. The inventors show that the imiiiium ions are potent DNA alkylation agents while the corresponding pyridone structures are not. In addition, the mentors rigorously define the stracti»-e-functio» relationships of 3 that are required for or enhance DNA alkylation activity. Finally, the synthetic studies support the alternative biosynthetic pathway involving the interaiediacy of the vinylogous amide 6 en route to 3. Collectively, our data lend further support to the hypothesis that unsaturated (minium ions 3 are responsible for the genotoxic effects of the clb gene cluster and support the conclusion that precolibactins A- C (7-9) (and other pyridone-containing isolates) are off-pathway fenuentatkm products derived from the absence of a functional clbP gene. This work constitutes the first structure - function studies of colibacilli metabolites and provides a foundation to begin to conned. ^' the disparate phenotypic effects of the clb cluster with metabolite structure.

Brief Description of the Figures

Figure 1 shows the structures of isolated, predicted and synthesized eolifoae&n metabolites. Fermentation yields (micrograms of product per liter of fermentation broth) are shown in parentheses.

Figures..!--? ^' shows numerous synthetic chemical steps to afford compounds accordin to the present invention (which includes intermediates).

Figure 8, Scheme 1, shows the chemical syntliesis of carboxyiic acid 10, Reagents and conditions: a. (.S)~hex-5-en-2-amin.e hydrogen chloride (8), l-ethyl-3~(3- diinetjiylaininopropy!)carbodiimide (EDOHC1), hydroxybenzotriazole (HOST). NjSf- diisopropyleihyk rne (DIPEA), DMF, 23 °C, 84%; b. HCL CHjC - 1 ,4-dioxane (7:1), 23 °C, >99%; e. My istoyl chloride, triethySamine (£t ₃ N), DMF, 23 °C, 82%; d. RuC , NaI0 ₄ , H ₂ CMEtOAc-C¾CN (3:2:2), 50 °C _S 95%.

Figure 9, Scheme 2, A. Shows the Synthesis of the ihiazolrae-thiazole 17. B. Shows the Synthesis of the thiazole - thiazoiine 23. C. Shows the Synthesis of the bithiazoSe 27. Reagents and conditions: a. L-( -^-cysteine ethyl ester hydrochloride, EtjN, CH ₃ OH, 23 °C, 85%; b. NH ₃ , CH ₃ OH-H ₂ 0 (2: 1), 23 °C, >99%; c. Lawesson's reagent, CH ₂ Cl _2i 23 °C, >99%; d. bromopyravic acid, Et ₃ N, CH ₃ OH, reflux, 71 %; e. HCL C¾C1 ₂ ~1 ,4-dioxane (4: 1 ), 23 °C, >99%; f silver trifluoroacetate (AgOTFA), EtjN, DMF, 0 °C, 63%; g. HCl, C¾C! ₂ -- 1 ,4-dioxane (4: 1), 23 °C, >99%; h. ethyl bromopyruvate, CaC0 ₃ , EtO , 23 °C, 74%: i. N¾, C¾OH- ¾0 (2:1), 23 °C >99%; j. trifluoroacetic anhydride (TFAA), Et ₃ N, CH ₂ C! ₂ , 0-->23 °C, 84%; k. L-(+)-eysieine _; Et ₃ N _? CH3OH, reflux, 97%; 1. HCl Ce ₂ Ci ₂ - l ,4~dioxane (8:1), 23 °C, >99%; m. AgOTFA, Et ₃ N, DMF, 0 °C, 69%; n. HCL CH Cly-J .,4-dioxane (4: i), 23 °C, >99%; o. N¾ C¾OB--H ₂ 0 (2: 1), 23 °C, >99%; p. Lawesson's reagent, CH ₂ C¾, 23 °C, >99%; q. bromopyruvic acid, CaCO _¾ EtOH, 23 °C, 58%; r. HCl, CH ₂ a -l,4-dioxane (4:1), 23 °C, >99%; s. AgOTFA, Et ₃ N, DMF, 0 °C, 72%; t. HCl, CH2CI2-I .,4-dioxane (4: 1), 23 °C, Figure .10, Scheme 3, Shows the Synthesis of the acyclic advanced precursors 29a-c, Reagents and condittonsr a. Carbonyl diin dazole (CDI), 4 A molecular sieves, DMF, then malonic acid haif-thioester, magnesium ethoxide (Mg(OEt)j), 23 °C, 95%; b, 17, 23, or 27, AgOTFA, E , DMF, 0 °C 90% (29a); 87% (29b); 86% (29c).

Figure 1 .1 , Scheme 4, A. Cy odehydratton of the linear precursors 29a~c, Reagents aad conditions: a, ¾CO _?; OfeOH.0 °C, 79% (30a); 80% (30b); 83% (30c) or & ¾C0 _3s dimethyl sulfoxide, 24 °C (for 29b). B. UV trace (254 ni») of the cyclization of 29b using potassium carbonate in dimethyl sulfoxide at 24 °C.

Figure 12 A. Mass-selective (mlz ^ 816.3788) LC/HRMS-QTOF analysis of the ethyl acetate extracts of elb ^* E. coil Ac bP (top), synthetic 5a (middle), and co-injection {bottom). B. Mass-selective (mk ^» 816.3788) LC/OR S-QTOF analysts of the ethyl acetate extracts of elb ^* E. coli AcIhP (top), synthetic 5b (middle), and co-injection (bottom). Y axis

corresponds to relative ton intensity (x!O ⁴ ).

Figure 13, Scheme 5. A. The originally predicted (5a) and revised (7) structures of precoHbactin A, B, Synthesis -of the -revised, structure of preeoti actin A (7). Reagents and conditions: a. bromopyruvic acid, CaC0 ₃ , EtOH, 23 °C, 74%; b. HQ, CHsC -dtoxane (3: 1 ), 23 °C, >99%; c. AgOTFA, Et ₃ N, DMF, 0 °C ₅ 55%; d, HCI, CHaCfe-l ,4-dioxane (3:1), 23 °C, >99%; e. AgOTFA, Et ₃ N, DMF, 0 °C; f. ₂ C0 ₃ , CH ₃ OH, 0 °C, 67% (two steps); g. L~ cysteine, A-hydroxysucciiiiiBide (NHS), EDC*EfCl, E N, DMF, 0->23 °C, 89%.

Figure 14 shows the Mass-selective (mfz ^■ · ^■ 816.378S) LC/HRMS-QTOF analysis- of the ethyl acetate extracts of cfi E. coli AclhP (top), synthetic 7 (middle), and co-injection (bottom).

Figure 1 A, Scheme 6 shows proposed mechanisms of action and divergent reactivity of precoHbactin precursors. The gray bail in each of compounds 1 -6 in the scheme denotes a variable region of the compound.

Figure 15B shows the chemical Structures of preeo!ibactins A (7), B (8), and C (9).

Figure 16, Scheme 7 shows the synthesis of the unsaturated inline 15a and the pyridone 17a. Structures of the rV»methy!.amides 15b and 17b. Reagents and conditions: (a) silver trifliioroacetate (AgOTFA), Et,,N, DMF, 0 °C; (b) concentrate f om 0.5% HC<¼H-5% ¾OH~€¾CN, 23 °C, 87% (two steps); (c) propyl pliosphonie anhydride solution (T3P), jV-methylmorpholine, A^N-dimemylethylenediamine, THF, 23 °C, 93%; (d) trifluoroacetic acid (TFA), C¾Cl _2i 0 °C; aqueous NaHC0 ₃ , 23 °C, 62%; (f) K ₂ O _h CH ₅ OH, 0~»23 °C, 78% (two steps); (g) T3P, A-memyhnorpho!hie, A'-diiiiethyk'thylenediamine, THF, 23 °C, 46%; (ft) TFA, CH _S C1 _2> 0 *C, 86%,

Figure 17 A, DNA aikylation assay employing linearized pBR32 DNA and the pyridone derivatives 17a or 1 b or the unsaturated imines 15a or 15b. Conditions; Linearized pB 322 DNA (20 μΜ in base pairs (bps)), 15a (100, 10, t, 0.5, or 0.1 μΜ% 15b (500, 100, 10, or 1 μΜ), 17a (500 or 100 μ ) or 17b (500 or 1 0 μΜ), 37 °C, 15 h. Cispiatin (CP: 100 μΜ) and methyl medianesulfonate (MMS; 500, 300, or 10 μ.Μ) were used as positive controls for DNA cross-linking and aikylation, respectively. DNA was visualized using SybrGoid, B. DNA aikylation assay employing linearized pBR322 DNA and the unsaturated imine 15a. Conditions: Linearized pBR322 DNA (20 μΜ in base pairs), 15a (100, 10, 1, 0.1 , 0.05, 0.01, or 0.005 μ ), 37 °C, 15 h.

Figure 18 A. increase in the melting temperature of calf thymus DNA treated with increasing amounts of the pyridone 17a. Conditions: 2.09 mM aH ₂ P0 ₄ , 7.13 mM Na ₂ BP0 ₄ , 928 μΜ Na^EDTA, 1.01 mM DMSO, pH 7.18. The pyridone 17a was incubated with ctDNA for 3 h prior to UV thermal denaturation experiments (260 nm, heating rate: 0,5 °C/tnin). [DNA] ~ 32.0 mM bps. B. rime-dependent modulation of the melting temperature of calf thymus DN A treated with 1 or 2 bp equiv of the imine 15a. Conditions: 2,09 mM NaFbPQ*, 7.13 mM a ₂ HP0 ₄ , 928 μΜ Na ₂ EDTA, 1.01 mM DMSO, pH 7.18. The imine 15a was incubated with ctDNA for 5 mm, 1 h, 3 h, 6 h, or ! 5 h prior to UV thermal denaturation experiments (260 mil, heating rate: 0.5 °C/min . [DNA] ~ 32.0 mM bps. Time-dependent DNA aikylation assay employing linearized pB 322 DNA and the unsaturated imine 15a, Conditions Linearized pBR322 DN (20 μΜ in. base pairs), 15a (t pM), 37 ^S C, 0.1-15 h.

Figure 1 A. Structures of the dimer 1 c and the # /w ^« dimethyl derivati ve 15d. B. DNA aikylation assay employing linearized pBR322 DNA and the dimer 15c or the #e/»-dimethy! derivative 5d. Conditions: Linearized pBR322 DN A (20 μΜ in base pairs). 15c (10 μΜ) or 154 (500 or 100 pM), 37 °C, 0.1-15 h. Figure 20 snows the Rrag-opersing of the unsaturated irmne 15b by propanediol. Conditions: j ^p -toluenesulfonic acid monohydrate, CHjCN-propaaethioi-DMF (6:2:1), 23 °C, 34%,

Figure 21 stiows A, Simctures of the analogs 15 i, 19, and 15j. B. DNA alkylation assay employing linearized pBR322 DNA and the unsaturated lactam 19 or the unsaturate imine iSi Conditions: Linearized pBR322 DNA (20 uM in base pairs), 19 (500, 100, 10, or 1 μ ) or 15j (500, 100, 10, or 1 μΜ)„ 37 °C, 15 h.

Figure 22 shows the various chemical moieties on a representative generic chemical structure of compounds according to the present invention.

Figure 23, Scheme Si, shows the synthesis of the dimeric unsaturated imine lSc> Reagents and conditions: (a) propylphosphonic anhydride solution (T3P), A-methylmorphpiine,, N _t N- bis(3-aminopropy1)ffletbyianwne, THF, 23 °C, 58%; (b) tritluoroaeetk acid (TFA), 0¾ί¾, 0 °C, then aqueous NaHC<¾, 23 °G, 73%.

Figure 24, Scheme S2 shows the synthesis of the unsaturated imine ISd. Reagents and conditions: (a) silver triflaoroacetate (AgOTFA), Et ₃ N, DMF, 0 ⁰ C, 40%; (h) HO, C¾Cb- l,4~di«xane (1:1 Q→23 °C, >99% _. ; (c) 10, AgOTFA, E N, DMF, 0 °C, 34%; (d) propylphosphonic anhydride solution (T3P), A-niethyhHorpholine, N,N- dimethy!eth l nedtamine, THF, 23 °C, 83%; (e) tritluoroacetic acid (TFA), CH2CI2, 0 °C, then aqueous NaHCOs, 23 °C, 67%.

Figure 25 , Scheme S shows the synthesis of the unsaturated imines 15b, ISe-i. Reagents and conditions: (a) methylamme, A^A ¾methyI~ 1. ,3-diamin.opropane _s ;¥-(ter/- butoxycarbonyI)-t,2-diaminoeth-ane, A^/er/^uto eari^^ A^A-bis- (&T/-buto ycarbonyi)~A ^? ' ~(2~aminoetl^ (Sll), orA V-bis-(i-?ri-butoxycarbony1)-

N' -(4-airiinobiityi)-giianidine (Sll), propylphosphonic anhydride solution (T3P), Λ - methylmorphoime, THF, 23 °C; 79% (14b), 95% (14e), 81% (14f), 48% (14g), 67% (Mb), 72% (141); (b) tritluoroacetic acid (TFA), C¾C¾, 0 *C, then aqueous aHCO _¾ 23 °C; 39% (15b), 76% (15e), 49% (IStl, 45% (lag), 34% (15b), 48% (ISi).

Figure 26, Scheme S4 show the synthesis of the unsaturated inline 1 . Reagents and conditions: (a) ethyl bromopyruvate, iso-prop ol, 83 °C, then d--/eri-butyl diearbonate. aqueous KHCOj, 1,4-dio ane, 0 °C, 66%; (b) Natl, iodomethane, DMF, ~5->15 °C, then LiOH, H ₂ 0, 15 °C, 98%; (c) HCl, CH ₂ Cl ₂ -l,4-dioxiMe (3:1), 23 X, >99%; (d) AgOTf A, Et ₃ N, DMF, 0 °C, 95%; (e) HCl, CH ₂ Clr-l,4-dioxane (3:1), 23 °C _S >99%; (f) AgOTFA, Et ₃ N, DMF, 0 °C, then ₂ C0 _3> CH ₃ OH ₅ Q™»23 X\ 63% (g) propylphosphonic anhydride THF, 23 ^¾ C, 58%; (b) trifluoroacetic acid (TFA), CEsCIs, 0 *C, 81%.

Figure 27 shows the Time-dependent modulation of the melting- temperature of calf thymus DNA treated! with 1 or 2 bp equiv of the imine 15a. Conditions: 2,09 m.M NaFbPGt, 7.13 niM NaaHPO 9 8 uM Na-?EDTA, 1.01 niM DMSO, pH 7.18. The imine -15» was incubated with ctDNA for 5 mitt, 1 h, 3 h, 6 li, or 15 !h prior to U V thermal denaturati!on experiments (260 ran, heating rate: 0.5 °€/min). [DNA] ~ 32.0 mM bps. The T _m was defined as the temperature at. which half of the duplex DNA was unwound, and was determined by the maximum of the first derivative of the thermal denaturation profile.

Figure 28 shows the increase in the melting temperature of calf thymus DNA after treatment with increasing amounts of the pyridone 17a. Conditions: 2.09 mM

7.13 mM a ₂ HP0 ₄ , 928 μΜ Na ₂ £DTA, 1.01 mM DMSO, pH 7.18. The pyridone 17a was incubated with ctDNA for 3 h prior to UV thermal denaruration experiments (260 nm, heating rate; 0.5 C/min). [ DN A] ~ 32.0 mM bps. The T _m was defined as the temperature at which half of the duplex DNA was unwound, and was determined by die maximum of the first derivative of the thermal denaturation profile.

Figure 29 shows no significant modulation, of the melting temperature of calf thymus DNA was observed on treatment with 2 bp equiv of me- yridone 17t> or 1 bp eqaiv -of the imine 15b. Conditions: 2.09 mM N ¾P04, 7.13 mM Na ₂ HP0 ₄ , 28 μΜ Na ₂ EDTA, 1.01 mM! DMSO, pH 7.18. The pyridone 17b and the imine 15b were incubated with ctDNA for 5 min, 3 h, or 15 h prior to UV thermal denaturation experiments (260 nm, heating rate: 0.5 °C/min). [DN A] - 32.0 mM hps.

Figure 30 shows no significant modulation of Ae melting temperature of calf thymus DMA was observed on treatment with 2 bp equiv of the unsaturated imine 15b. Conditions:

2.09 mM! Na¾P0 , 7.13 mM a ₂ HP0 ₄ , 28 μΜ Na ₂ BDT _s 1.01 mM DMSO, pH 7.18. The imine 15b was incubated with ctDNA for 5 min, 1 h, 3 h, 6 b, or 15 h prior to UV thermal ^■ denatutation experitneirts (260 nm _} heating rate: 0.5 °C min). [DNA] ^::: 32.0 mM bps. The- T _m was defined as the temperature at which half of the duplex DNA was unwound, and was determined by the maximum of the first derivative of the thermal denaturation profile.

Figure 31 shows no significant modulation of the melting temperature of calf thymus DNA was observed on treatment with 2 bp equiv of the pyridone J:7b. Conditions: 2,09 oiM Nai¾P04, ?.1.3 mM a ₂ HP0 ₄ , 28 μΜ Na ₂ EDTA, 1.01 mM DMSO, pH 7.1.8. The pyridone 17b was incubated with ctDNA for 5 rain, 1 h, 3 h, 6 h, or 15 fa prior to UV thermal

denaturation experiments (260 nni, heating rate: 0.5 °C/min). [DNA] - 32.0 mM hps. The T _w was defined as the temperature at which half of the duplex DNA was unwound, and was deien-Rffied by the maximum, of me first derivative of the thermal denaturation profile.

Figure 32 shows a comparison of the ^l H NMR of the -unsaturated iirdne 15b (top) and the propaiiethiol adduct product IS (bottom). ^! H speciroscopic data were recorded in DMSO- i¾ (600 MHz (15b), 500 MHz (1.8), 23 °C).

Figure 33 shows the results of a DNA alkylation assay DNA alkylation assay

employing linearized pBR322 DNA and the derivatives 15a and 15e— i to probe the influence of the cationic residue on DNA alkylation activity. Conditions: Linearized pBR322 DNA (20 μΜ in base pairs), ISh (I , 0.1, or 0.01 u ), 15i (1 , 0.1 , or 0.01 μΜ), 15f (1, 0.1, or 0.01 uM), 15 (1, 0.1, or 0.01 uM) _s 15a (1 , 0.1 , or 0,01 μΜ), 15e (i , 0.1, or 0.01 μΜ), 37 °C, 15 h. Methyl methanes lfonate ( MS; 500 or 100 μΜ) was used as a positive control for DNA alkylation. DN A was visualized using SybrGold.

Figure 34, Table S I shows a comparison of selected ^! H and ^{5 >} C NMR Data of 15b and 18.

Brief Description of the I nven tion

In embodiments, the present invention is directed to compounds according to the chemical structure 1:

Wliere X is N o C-R;

W is M, N-R^, C-R ^' or CR.fR) (preferably the variable bond between W and the ^' adjacent carbon atom is a double bond and W is or C-R);

Each Z is independently S, O, N-R ^N or C-R(R);

Each R is independently H, a C Ce (preferably C C; alky group optionally substituted with one or two hydroxy I groups or up to three halogen (F, CI , Br, I, preferabl F or CI, most often F) groups, or a 0-(Ci-C:¾) alkoxy group;

Each R* is independently H or a CrCe (preferably O-C3) alkyl grou optionally substituted with one or two hydroxy! groups or up to three halogen groups, preferably H or methyl;

Q is 0, S, N(R ^J ) or C(R¾ ^Q ;

R R* and R ³ are each independently H or a Cj-C« (preferably C ₁ -C3) alkyl group which is optionally substituted with one or two hydroxy! groups;

When D is , or R is (the double bond is the same in both moieties);

Where R ^A is H or an optionally substituted C{-C¼ alkyl or alkene group, preferably H or a Cj- C;5 alkyl, most often methyl;

R ⁱ and R ² are each independently E, a Ct-Ce (preferabl C1-C3) alky! group which is optionally substituted with, one or two hydroxy! groups or up to three halogen groups, a protecting group (Pa), preferably a BOC group) or a targeting element TE which is linked to the nitrogen by linker group he which is optionally cleavable;

' " is absent, H, a Ci-C<j (preferably C1-C3) alkyl group which is optionally substituted with one or two hydroxyi groups, a protecting {¾), preferably a BOC group) or a targeting element TE which is linked to the nitrogen by a linker group Lc which is optionally

cleavable;

¾ and R ^x are eac independently H or a a Ci-Cy, (preferably Cj~C¾) alky! group which Is optionally substituted with one or two hydroxyi groups or up to three halogen groups;

i is 1-4, preferably 2-4;

j is 1-3;

each n is independently l _t 2 or 3 (preferably 1);

R ^B! and R are each independently E, a Ct-CV. (preferably Cj-C¾) group which is optionally substituted with one or two hydroxyi groups o up to three halogen groups or together ^bi and R ^iU form a cyclopropyl or cyclobutyl group (preferably, R ^B1 and R ^B" are each

independently H, methyl or together form a cyclopropyl group);

R ^c is H, a C _f Cn optionally substituted alkyl or alkene group (preferably substituted with one or two hydroxy! groups, up to five halo groups) or a and R ₂ are each independently H or a Ci-Q, optionally substituted alkyl group and n l is 1-8

(preferably I , 2, 3, 4 or 5), a protecting group {¾) (preferably a BOC group) or a targeting element TE which is linked to X' by a linker group L which is optionally cleavable, or R ^c forms a dimer compound through a cova!etH linker group L which is optionally cleavable, said dimer compound having the. general chemical structure;

Where X, W, Z, Q, X ¹ , D, R% R ⁴ , n, R ^B1 and R ⁸² are the same as above; and

L is a linker group which is optionally cleavable and covatently links the dimeric portions of the molecule to each other, or

a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof.

In preferred aspects of compound I, each n is 1 , W is C~R, R is H or methyl, X is N, Z is S or N~R ⁴ \ R is H or methy Q is C(R ^Q )R ^Q , each R ^Q' is independently Η· or methyl, preferably both are H, X ^! is ΝΉ or N-meth l, ^A is if or methyl (preferabl H), R ^A in R ⁴ is methyl, R ^B! aml R ⁸² are each independently H or metliyi or together form a cyciopropyl group and L is a linker as otherwise described herein, preferably L is a a polyethylene glycol linker having between 2 and 12 ethylene glycol units or a -(CH2)mN( Q¾) _m - group where R is H or a C Cs alkyl group (preferably if or methyl) and each m is independently f om 1-1,2 (preferably, 1-10, more preferably ! , 2, 3, , 5, or 6).

In another embodiment, the invention is directed to compounds according to the chemical structure II:

Where X is ^' N or C-R;

W is N, N-R ^N , C-R or CR(R) (preferably the variabl bond between W and the adjacent carbon atom is a double bond);

Each Z is independently S, O, N-R or C-R(R);

Each R is independently H, a€rQ> (preferably C j -€ _¾ ) afky! group optionally substituted with one or two hydroxy! groups or up to three halogen groups, or a 0-(Ci-Q) aikoxy group; Each R ^N is independently H or a Ci-C§ (preferably C1-C3) alky! grou optionally substituted with one or two hydroxy! groups or up to three halogen groups, preferably H or methyl; Q is O, S, (R ⁵ ) or€( ^Κ°- X ⁵ is O, S, N(R ³ ) or C(R¾ ^X ;

R ^! , ^{' 2} and. R ^" are each independently H or a C Q (preferabl Ci-Q) alkyl group which is optionally substituted with one or two hydroxy! groups or up to three halogens groups;

Where R ^A is H or an optionally substituted Cj-Cs alkyl of aikene group, preferably H or a Cj- Cs alkyl most often methyl;

R ^Ni and R ^N2 are each independently H, a Ci-Ce (preferably C _\ ~CW} alkyl group which is optionally substituted with one or two hydroxy! groups or up to three halogen groups, a protecting (PG) (preferably a BOC group) or a targeting element ¾ which is linked to the nitrogen by a linker Lc which is optionally oleavable;

R ^Q and R ^x are each independently H or a a Cj-C* (preferably C1-C3) alkyl group which is optionally substituted with one or two hydroxy! groups or up to three haiogen groups:

i is 1 -4, preferably 2-4;

j is 1-3;

R. ^t and R ^m are each independently E, a C C< _> (preferably C ₁ -C3) group which is optionally substituted with one or two hydroxy! groups or up to three halogen groups, or together R ^Bi and R ^bi form a cyclopropyl or cyclobutyl group (preferably, R ⁸¹ and R ^B * are each

independently H, methyl or together form a cyclopropyl group);

R is H, a C Ci ₂ optionall substituted- alkyl or aikene group (preferably substituted with one or two hydroxy! groups, up to five halo groups) or a -(€¾)« R1R2 group where Rj and R2 are each independently B or a C _. rQ; optionally substituted alkyl group and n is 1 -8

(preferably t, 2, 3, or 5), a protecting (P ₀ ) (preferably a BOC group) or a targeting element ¾ which is linked to X ⁵ (preferably through nitrogen) by a linker L which is optionally cleavable, or R ^c forms a dimer compound, through a covalent linker group L which is optionally cleavable, said dimer compound having the general chemical structure:

Where X, W, 2, Q, X , R% R ^' , R ^' and R. ^" " are the same as above; and L is a. linker group which is optionally cleavable and which covaiently links the dimeric portions of the molecule to each other, or

In preferred aspects of compound II, W is C-R, R is H or methyl, X is N, Z is S or N~R ^N , R ^N is H or methyl, Q is N-H or C(R ^Q )R ^Q where each R° is independently H or methyl, preferably both are H, X ¹ is NH or -methyl , R ² is H or methyl (preferably H), R ^A in R ⁴ is methyl.

R ^Bi nd R ^B2 are each independently H or methyl or together form a cyclopropyl group and L is a linker as otherwise described herein, preferably L is a polyethylene glycol linker having between 2 and 12 ethylene glycol units or a -((¾) _m N(R)(CH; } _ff i- group where R is H or a O- Cj a!kyi group (preferably H or methyl) and each m is independently from 1-12 (preferabiy, 1~I0, more preferably I , 2, 3, 4, 5, or 6).

In another embodiment, the present invention is directed to certain- referred compounds according to the general chemical structure III:

Where X is N or C-R;

W is N, N-R , C-R or CR( ) (preferably the bond between W and the adjacent carbon atom is a double bond);

Each Z is independently S, O, N-R or C-R(R); Each R is independently II, a CrC? alky! group . ^■ optionally substituted with one or two hydroxy! groups or up to three halogen groups, or a O-(Ci-Cs) alfcox group;

Each R ^N is independentl H or a C ₁ -C3 a!kyl group optionally substituted with one or two hydroxy! groups or up to three haloge groups, preferably H or methyl;

R.\ ^{' 2} and ^" are each independently H or a C Cs a!kyi group which is optionally substituted with one or two hydroxy! groups or up to three halogen groups;

R ^A is H or an optionally substituted C Cg alky! or alkene group, preferably H or a C ₁ -C3 alkyl, most often methyl;

R ^Bi and R ^B2 are each independently E, a Cr ¾ alkyl group which is optionally substituted with one or two hydroxy! groups or up to three halo groups (F, CS, Br or I, preferably O or F _s . most often F) or together R ⁰ⁱ and R ^hI form a cyclopropyl or cyelobutyl group (preferably, R ^{rf l} and R ^B2 are each independently H, methyl or together form a cyclopropyl group);

R ^c is H, a Ci-Cn optionally substituted alkyl or alkene group (preferably substituted with one or two hydroxys groups, up to fi ve halo groups) or a -((¾ , ¾ Ι¾ group where Rj and R ₂ are each independently H or a Ci-Ca optionally substituted alkyl group and n is 1-8

(preferably 1, 2, 3, 4 or 5), a protecting (P ₀ ) (preferably a BOC group) or a targeting element

Tg which is linked to X ¹ (preferably a nitrogen) by a linker L _(: which is optionally eleavabie or R forms a dkuer compound through a covalent linker group L which is optionally eleavabie, said dimer compound having the general chemical structure:

Where X, W, Z _s R, R ^N , R R ² , R ³ , R ^A , R ^Bi aud R ^m are the same as above; and L is a linker group which covalently l inks the dimeric portions of the molecule to each other, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof.

in preferred aspects of compound HI, W is C-R, R is H or methyl, X is , Z is S or N-R , R ^N is H or methyl, Q is C{ R ^Q )R ^Q , each R ^Q is independently E or methyl, preferabl both are H,

X* is H or N-methyl, R ² is or methyl (preferably H), R ^A in R ⁴ is methyl, R ^H) and R ^B2 are each independently H or methyl or together form a cydopropyl group and L is a linker as otherwise described herein, preferably L is a polyethylene glycol group having ^' between 2 and 12 ethylene glycol units or a ~(C¾) _m N(R)(CH ₂ ) _m - grou where R is H or a Q-Cj aiky! group (preferably H or methyl) and each ra is independently from 1 -12 (preferably. 1 -10, more preferably 1 , 2, 3, 4, 5, or 6).

Preferably, the compound is according to the chemical structure IV;

Where X, Z, R ¹ , R ^* , .R ³ , R ^A , R ^B! , ^¾ and R are the same as for compound III above (the bond between the two carbons is preferably a double bond),

or a pharmaceutically acceptable salt, stereoisomer thereof. In preferred embodiments, the variable bond between carbons is a double bond. In preferred embodiments, X is preferably N; Z is preferably S, O, N-H or N-C% (more preferably S); R is preferably H, meihyi or O e; R. ^J is preferably or methyl; R. , R ^* and R: are each independently preferably H or methyl; R ^A is preferably H or a Ct-Cs alkyl, preferably methyl; R ^Bi and R ^B2 are each independently H, methyl or together form a cyclopropyl group and is methyl, a -{(¾)«- ^' (C¾) ₂ group where a is 1 , 2, 3 or 4 (preferably 2), forms a guanidme group with the nitrogen to which it is attached or R ^c forms a dime* compound through linker L where L is preferably a -{CH2) _m H(R)(G¾) _m - group where R is H or a Cj-C alkyl group (preferably H or methyl) and each m is independently f om 1- 12 (preferably, 1 -10, more preferably 1, 2, 3, 4, 5, or 6). in one embodiment, preferred compounds according to the present invention include compounds of the chemical structure:

or

a pharmaceutical salt, stereoisomer, solvate or polymorph thereof. in alteraative embodiments, the coiBpound is according to the chemical stractiire V:

Where Q is C¾. N-H or N-Me;

X ^s is O, S, N(R ³ ) or C(R )R ^X ;

R ² and R ^J are each independeiitly H or a Cj-Ce (preferably CJ-C- _Λ ) alky! group whic is optionally substituted with one or two hydroxy! groups or up to three halogen groups: R ^A is H or an optionally substituted Cj-C* a!ky] or alkene group, preferably H or a O-C3 aifcyl, most often methyl;

R ^N{ and R ^m are each independently H, a C C< _> (preferably C ₁ -C3) alky] group which is optionally substituted with one or two hydroxyl groups or u to three halogen groups, protecting (Po) _> preferably a BOC group, or a targeting element g which is linked to the nitrogen by a linker L _f.: which is optionally cleavable;

Each R ^x is independently H or a Ci-Q (preferably C ₁ -C3) alkyt group which is optionally substituted with one or two hydroxyl groups or up to three halogen groups (preferably F or CI, more often F);

i is 1-4, preferably 2-4;

R ^&1 arid R ⁸² are each independently H, a Ci-Q> _. {preferably C.VQ) group which is optionally substituted with one or two hydroxyl groups or tip to three halogen groups (preferably F, CI, Br or Ϊ, preferably CI or F, most often F) or together R ^B1 and R ^{'bi '} form a cyclopropyi or cyclobutyl group (preferably. R ^m and R ^B2 are each independently H, methyl or together form a cyclopropyi group);

R' ^" is H, a Cr-Cn optionally substituted alkyl or alkene group (preferably substituted with one or two hydroxy! groups or up to five halo groups), a

are each independently H or j-Gs optionally substituted alkyl group and l is 1-8

(preferably 1 , 2, 3, 4 or 5), a protecting Po„ preferably a BOC group, or a targeting element g which is linked to X ^! (preferably a nitrogen) by linker L< _: which is optionally cleavalble, or R forms a dkner compound through a covalent linker group L which is optionally cleavable, said diraer compound having the general chemical structure:

Where Q, R ² , R ^A , i, R ^H \ R ^f,2 ₅ R ^N1 , R \ X* and L are the same as for compound V above, and L is a linker grou which is optionally cleavable and which covalently links the dimeric portions of the molecule to each other, or

a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof In preferred embodiments, the variable bond between carbons is a double bond, R ² is H or methyl, R ^A is methyl, i is L R ^B, and R ^a~ are each H, methyl or together form a cyclopropyi group, NR ^N, and NR ^{! *} are each independently H, methyl, a protecting group (preferably a BOC) or a a targeting element ¾ which is linked to the nitrogen by an optionally cleavable linker L _c , X is N-H or N-raethy and L is a linker group -{CH _{2 m} Ni (CB:? _m - where R is H or a Cj -C3 alkyl group (preferably H or methyl) and each m is independently from 1 -12 (preferably, 1- 10, more preferably 1 , 2, 3, 4. 5, or 6). in another embodiment, the present invention is directed to compounds according to the chemical structure:

Where Q is C¾ or -H;

X ¹ is O, S, N(R ³ ) or C(R ^X )R ^X ;

R ² and R ^J are each independently H or a CrCs (preferably Cj-Cs) alkyl group which is optionally substituted with one or two hydroxy! groups;

Each R ^x is independently H or a Ci-Cc (preferabiv C ₁ -C3) alky! group which is optionally substituted with one or two bydroxyl groups;

R ^A is H or an optionally substituted C|-C¼ alkyl or alkene group, preferabl H or a Cj-C? alkyl, most often methyl;

R' " is H, a Ci-Q (preferably C ₁ -C3) alkyl group which is optionally substituted with one or two hydroxy! groups, a protecting {!¾), preferabl a BOC group, or a targeting element ¾ which is linked to t!ie nitrogen by an optionally cleavable linker L^;

R ^{rf l} and R ^B2 are each independently H, a Ci-Cc (preferably Ci~C ₃ ) group which is optionally substituted with one or two hydroxyl groups or up to three halo groups (F, CI, Br or 1, preferably CI or F, most often F) or together R ^Bi and R ^S2 form a cyclopropyl or cyciobirtyl group (preferably, R ^m and R ^b2 are each independentl H, methyl or together form a cyclopropyl group); and

R is if, a C Cj ₂ optionally substituted alkyl or alkene group (preferably substituted with one or two hydroxyl groups, up to five halo groups) or a -(€¾) _ui NR ₃ R;! group where j and R2 are each independently H or a Ci-Ca optionally substituted alkyl group and nl is 1-8

(preferably 1 , 2, 3, 4 or 5), a protecting (?«), preferably a BOC group) or a targeting element ¾ which is linked to X (preieiabiy a nitrogen) by a Sinker Lc which is optionally c!eavable, or R ¹ forms a dimer compound through a covalent linker group L which is optionally cSeavabie, said dimer compound having the genera! chemical structure:

Where Q, ² , R ^A , R ^{rf l} , R ^B2 _S R ^N, and X ^f are the same as above for compound VI, and L is a linker group which covalentiy Jinks the dimeric portions of the molecule to each other, or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. In preferred embodiments, the variable bond between the two carbons is a double bond. In preferred embodiments, SR. ^" is H or methyl, R ^' is H or methyl, R and R " are each independently H, methyl or together form a cyclopropyl group, R ^? is H, methyl, a protecting group (Ρ«), preferably a BOC group, or a targeting element ¾ which is linked to the nitrogen by an optionally cleavable linker Lc, ¹ is N-H or N~merhyl and L is a linker group

~(CH ₂ ) _m N( )(Cfi ₂ ) _m - where R is H or a Ci-Qj alkyl group (preferably H or methyl ) and each m is independently from 1-12 (preferably, 1-10, more preferably 1 , 2, 3, 4, 5, or 6).

In other embodiments, the present invention is directed to pharmaceutical

compositions which are principally used for treating cancer comprising an effective amount of a compound having anticancer activity as otherwise described herei in combination with a pharmaceutically acceptable carrier, additive or exeipient, optionally in combination with at least one additional bioactive agent, in most instances at least one additional anticancer agent.

In further embodiments, the present invention is directed to methods of treating cancer comprising administering to a patient in need an effective amount of compound or pharmaceutical composition as described herein.

In stil l other embedments, the present invention is directed to methods of chemical synthesis as set forth in the schemes which are presented herein, including the attached figures and the compounds which are also described herein. I» still other embodiments, compounds according to the present invention include one or more of the folio mu:

Or a non-salt or alternative salt form, including a pharmaceutically acceptable salt form or Stereoisomer thereof,

Or a salt form, including a pharmaceutically acceptable salt form or stereoisomer thereof.

Or a salt form, including a pharmaceutically acceptable salt form or stereoisomer thereof.

Or a salt form, including a phartsraceuticaty acceptable salt form or stereoisomer thereof.

The present invention also is direct to anticancer compounds of according to the general chemical structures:

Where R is a targeting element Τβ such as a non-c leasable or cleavabie moiety which optionally has anticancer activity or a moiety such as an acid labile moiety including an acid

an antibody or antibody fragment,

cysteine-cathepsin labile moiety; and

" is an amine group, preferably diamine, triamine, tetracaine, even more preferably a

-NH(CH ₂ ) _m N R~ group where m is an integer from 1-6 ( 1 , 2, 3, 4, 5 or 6) and R ^' and R are each independently H, C Q alkyl which is optionally substituted with one or two

hydroxy! groups or is a groups which forms a guan dine grou with the nitrogen to which it is attached

or a pharmaceutically acceptable salt or stereoisomer thereof

In embodiments, the present in vention is directed to compounds according to the chemical structure:

CH,

Where R is is an amine group, preferably a diamine, triamine, tetramine, even more

preferably a s-NH(CH ₂ ) _m H ^tD R ^2Ls group where m is a integer from 1-6 (1, 2, 3, 4 _# 5 or 6) ί Y . . . . . . .

and R ^' and R"" are each indepen Q-Q alkyl which is optionally substituted with

one or two hydroxy!, groups or is a groups which forms a guanidine group with the nitrogen, to which it is attached. or a pliarroaceuticaOy acceptable salt or stereoisomer thereof

The present invention is also directed to methods of chemical synthesis as described in the schemes presented, herein and in the figures attached hereto.

In certain embodiments the present invention is directed to a chemical synthesis as set forth below:

Compound 22 is converted to compound 23 by reacting compound 1.6 of figure 9 with compound 22 in a silver catalyst such as AgCFsCOs in a weak base (e.g., trietliyl amine) and sol vent (e.g. DMF) at reduced temperature (e.g. 0 "€) to produce the compound 23 G i 63% yield.

High YieW 50%

22 63%

23

Compound 23. 23G or a related compound, produced above, is converted to compound 24 24G or a related compound in acid (e.g. HO) in solvent (methylene chlorine- dioxane mixture) at about room temperature (e.g. 23 X) under conditions to remove the Boc amine protecting group. The reaction proceeds in quantitative or close to quantitative yield.

23© 9S% 24G

23 ^> 99% 24

17

27

Compounds 9 aad.240, 9 aad 17, G, 9 and 24 aad 9 aad 27(abo ve) are reacted together in the presence of a silver calayst (e.g., AgCJ¾C<¼) in a weak base (e.g., trietliylamme) and solvent

(e.g. DMF) at reduced temperature (e.g. 0 "C) to produce compound 25.

2S

28

2SD

In an alternative embodiment, compound i3(BL is an amine blocking group, preferably a Bqc group). is exposed to propylphqsphonic anhydride (T3P) in the presence of an amine to provide a peptide on the earboxyiie acid position. The amine may be any amine group containing a primary amine, but preferably is an amine which contains at least one cationic group after formation of the amide bond, and can include a diamine, triamine, tetramme, preferably a H ₂ (€H ₂ )„ _i R ^{UJ iD} where m is an integer from 1 -6 (1 , 2, 3, 4, 5 or 6) and R ^m and ^' R ^2D are each independently H, Cj -Cg alkyl which is optionally substituted with

one or two hydroxy! groups or is a groups which forms a guanidine group with the nitrogen to which it is attached. The protecting group is subsequently removed in

trifiuoroaeetic acid and aqueous bicarbonate to produce the final product 15a.

The above embodiments, and additional embodiments of the present invention ares readily gleaned from a review of the detailed description of the invention which follows.

Detailed Description of the Invention

The following terms shall be used throughout tire ^■ ■specification: to describe the present invention.. Where a term is not specifically defined herein., that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless, the context clearly dictates otherwise, between the upper and l ower limit of that range and any other stated, or intervening value in that stated range is encompassed within the invention. The upper and lower limits, of these smaller ranges that may independently be included in the smaller ranges are also

encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention, in instances where a siibstifuent is a possibility in one or more Markush groups, it is understood that only those substituents which form stable bonds are to be used.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly ^' understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those

described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

Furthermore, the following terras shall have the definitions set out below.

The term "patient" or "subject" is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, hi most instances, the patient or subject of the present invention is a human patient of either or both genders.

The term "effective" is used herein, unless otherwise indicated, to describe an amount of a compound or component which, when used within the context of its use, produces or effects an intended result, whether that result relates to the prophylaxis and/or therapy of an infection and/or disease state within the context of its use or as otherwise described herein. The terra effective subsumes all other effective amount or effective concentration terras (including the term "therapeutically effective"} which are otherwise described or used in the present application.

The term "compound" is used herein to describe any specific compound or bioaciive agent disclosed herein, including any and all stereoisomers (including diasiereorners, individual optical isomers/enantiomers or racemic mixtures and geometric isomers), phaonaceuticaiiy acceptable salts and prodrug forms. The term compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein, it is understood that the choice of substituents or bonds within a Markush or other group of substituents or bonds is provided to form a stable compound from those choices withi that Markush or other group. The symbol signifies that a bond is either a single bond or a double bond. In all compounds, where a variable bond is presented, the variable bond between two atoms is preferably a double bond. The term '' liarmaceaiically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

"Hydrocarbon" or "hydmcarbyl" refers to any monovalent (or divalent in. the case of alkyJene groups) radical containing carbon and hydrogen, which ma be straight branch- chained or cyclic in nature. Hydrocarbons include linear, branched and cyclic hydrocarbons, including alkyl groups, alkylene groups, saturated and ^' unsaturated hydrocarbon groups including aromatic groups both substituted and unsubstiiuted, aikene groups (containing double bonds between two carbon atoms) and alkyne groups (containing triple bonds betwee two carbon atoms). In certain instances, the terms substituted alkyl and alkylene are

sometimes used synonymously.

"Alkyl" refers to a fully saturated monovalent radical containing carbon and

hydrogen, and which may be cyclic, branched or a straight chain. Examples of alkyl groups are methyl, ethyl, n-butyl, n-hexyl, n-heptyl, n-octyl, n~nonyl, n-deeyt isopropyl, 2~methyl- propyl. cyclopropyL cyclopropylmettryi cyclobutyl, cyclopentyl. cyclopentylethyi,

cyclohexylethyl and cyclohexyl. Preferred alkyl groups are Q-Q alkyl groups, "Alkylene" refers to a fully saturated hydrocarbon which is divalent (may be linear, branched or cyclic) and which is optionally substituted. Preferred aikylene groups are Ct-Q alkylene groups. Other terms used to indicate subsiitutuent groups in compounds according to the present invention are as conventionally nsed in the art

The term "ary ^* or "aromatic", in context, refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., benzene or phenyl). Other examples of aryl groups, in context, may include heterocyclic aromatic ring systems "heteroaryl" groups having one or more nitrogen, oxygen, or sulfur atoms in the ring (5- or 6-membe.reci heterocyclic rings) such as imidazole, furyl. pyrrole, pyridyl, furanyl, thiene, thiazole.

pyridine, pyrimidrae, pyrazine, triazoie, oxazole, among others, which may be substituted or unsubstiiuted as otherwise described herein.

The term "substituted ⁵ ' shall mean substituted at a carbon or nitrogen position within a molecule or moiety within context, a hydroxyl, carboxyl, cyano (O ^s N), nitre (NOs), halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl, especially a methyl group such as a trifluoron ethyl), alkyl group (preferably, C Ci?, more preferably, Cs-Cs), alkoxy group

(preferably, Ci-Ce, alkyl or aryl, including phenyl, and substituted phenyl), a C _. Q tbioether, ester (both oxycarbonyl esters and caiboxy ester, preferably, Ci-Q alkyl or aryl esters) Including alkylene ester (such thai attachment is on the alkylene group, rather tha at the ester function which is preferabl substituted with a Ci~C< _> alkyl or aryl group), thioester

(preferably, Cj-Ce alkyl or aryl), halogen (preferably, F or CI), nitto or amine (including a five- or six-membered cyclic alkylene amine, former including a Ct-C¾ alky! amine or Ci-C(> dialkyi amine which alkyl groups may be substituted with one or two hydroxy! groups), amido, which is preferably substituted with one or two Ci-Q _: alkyl groups .(including a carboxamide which is substituted with one or two Ci-G, alkyl groups), alkanol (preferably, Cj-Ci, alky! or aryl), or alkanoic acid (preferably, C Q alkyl or aryl) or a thiol (preferably, Ci-C _f i alkyi or aryl), or thioalkanoie acid (preferably, Cj-CV, alkyl or aryl). Preferably, the term "substituted" shall mean within its context of use alkyl, alkoxy, halogen, ester, keto, nitro, cyano and amine (especially including mono- or di~ Ci~C _& alkyl substituted amines which may be optionally substituted with one or two hydroxy! groups). Any substitutable position in a compound according to the present invention may be substituted in the present invention, but often no more than 3, more preferably no more than 2 substituents (in some instances only 1 or no substituents) is present on a ring. Preferably, the term "unsubstituted" or within context a bond which is imsubsituted shall mean substituted with one or more H atoms.

The term "tumor" is used to describe a malignant or benign growth or tumefacent.

The term "neoplasia" refers to the uncontrolled and progressive mu plicatkm of tumor cells, under conditions that would not elicit, or would cause cessation of. Multiplication of normal cells. Neoplasia results in a "neoplasm", which is defined herein t mean any new and abnormal growth, particularly a new growth of tissue, in which the growth of cel ls is uncontrolled and progressi ve. Thus, neoplasia, includes "cancer", which herein refers to a proliferat ion of tumor cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis. The cancer may be "nai ve", metastatic or recurrent and includes drug resistant and multiple drug resistant cancers, all of which may be treated using compounds according to the present invention. As used herein, neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject or host, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplastics include benign tumors and malignant tumors (e.g., colon tumors) that are either invasi v or noninvasive. Malignant neoplasms are distinguished from benign neoplasms in that the former show a greater degree of anapiasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. Examples of neoplasms or neoplasias from which the target cell of the present invention may be derived include, without limitation, carcinomas (e.g., squamous-ceil carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal ceil carcinomas), particularly

those of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly E wing's sarcoma,

tiemangiosarcoma, Kaposi's sarcoma, liposarconia, myosarcomas, peripheral

neuroepithelioma, and synovia! sarcoma; tumors of the central nervous system (e.g. , gliomas, astrocytomas, oligodendrogliomas, ependymomas, gSiohastomas. neuroblastomas,

ganglioneuromas, gangliogliomas, medulJobiastomas, pineal ceil tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ- line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine endometrial cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixed types of neoplasias, particularly carcinosarcoma and HodgkkVs disease; and tumors of mixed origin, such as WACMs' tumor and teratocarcinomas, which may be treated by one or more

compounds according to the present invention. See, (Beers and Berkow (eds,). The Merck Manual of Diagnosis and Therapy. 17.sup.th e l (Whhehouse Station, N.J.: Merck Research Laboratories, 1999) 973-74, 76, 86, 88, 991.

In certain particular aspects of the present invention, the cancer which is treated is metastatic cancer. Metastatic cancer may be found in virtually all tissues of cancer patient in late stages of the disease, including the lymph system/nodes (lymphoma), in bones, in bladder tissue, in kidney tissue, liver tissue and in virtually any tissue, including brain (brain cancer/tumor). Thus, the present invention is generally applicable and may be -used to treat any cancer in any tissue, regardless -of etiology. In other instances, the cancer which is treated, including prophylactical y treated, is a recurrent cancer, which often recurs after an ini tial remission. The present compounds also may be used to reduce the likelihood of a cancer recurring and for treating a cancer whic has recurred. In further instances the present compounds may be used to treat cancer stem cells, which often occur in metastatic and recurrent cancers.

The term "targeting element", "cancer cell targeting element", "Tg CTE" or "cell targeting element" is used to. describe that portio of a chimeric compound according to the present invention which comprises at least one moiety which is capable of selectively binding to a cancer cell. Targeting groups for including in chimeric compounds according to the present invention include small molecules which bind to folate receptors (folate receptor binding moiety), antibody-type CCT _E s such as monoclonal antibodies (especially a

humanized monoclonal antibody) such as herceptm or antibody fragments (FAB), including single chain variable fragment (scFv) antibodies which bind to cancer cells, a PSMA binding moiety or a YSA peptide (which binds to Ephrin A2 (EphA2), as otherwise described herein. The targeting element ΤΈ may also include a peptide (e.g. a low pH insertion peptide), an antibody or antibody fragment.

roup according to the chemical structure , or a cysteine- cathepsin moiety.

The term "folate receptor binding moiety" (FR.BM) or (F ) is used to describe a folate moiety which binds to cancer cells selectively and is used in the present invention to target folate receptors on cancer cells which are often overexpressed or hyperexpressed on cancer cells compared to normal cells. The folate receptor, given its selective heightened expression on cancer cells compared to normal cells represents an excellent selective target to bind compounds according to the present invention to cancer cells for uptake into ceils where the intercalating moiety may exhibit its antiproliferative activity, resulting in cancer cell death. Folate receptor I is often overexpressed in numerous numerous cancer cells including ovarian, breast, uterine, cervical, renal, lung, colorectal and brai cancer cells, thus making it an important targeting site for compounds according to the present invention.

Folate receptor binding moieties for use in the present invention include the following chemical structures:

where X _F is 0(0), S(O), S(0) ₂ , CR _F R _F , O, S or N-R _P ,

where i½ is H or a C1-C3 alkyl (preferably H).

The term "prostate specific membrane antigen" or "PSMA" according to the chemical structure is directed to a cancer cell targeting moiety that binds to prostate specific membrane antigen (PSMA) which is frequently overexpressed or hyperexpressed in cancer cells.

PSMA, although found on prostate cancer cells, including metastatic prostate cancer cells, are also found on virtually all other cancer cells and may be used to selectively target compounds according to the present invention to cancer cells. A number of metastatic and recurrent cancers also hyperexpress PSMA compared to naive cancers and PSMA may represent a particularly useful binding site for metastatic and/or recurrent cancers.

PSMA binding moieties include moieties according to the chemical structure:

Where X* and ? are each independently C¾, 0 _; KB or S;

X ₃ is 0 _? C¾ NR.*, S(O), S(C¾ -S(0) ₂ 0 _; -OS(0) ₂ , or OS(0) ₂ 0; R is E, Cs-Cj alky! group, or a -QO ^' Ct-C ₆ ) group;

k is an integer from 0 to 20, 8 to 12, 1 to 15, 1 to 10, 1 to 8, 1 to 6. 1, 2, 3, 4, 5 or 6;

or a salt or enantiomer thereof.

A preferred FSMA binding group (CCTE) for rise in the present invention is the

Where k is 2. 3 or 4, preferabl 3 or 4. This CCT _¾ group, as well as the others, optionall has an amine group or other fimctioimi group at the distill end of the alkyiene group (k) such that k is formed from, for example, a lysine amino acid, such that the amine group or other functional group may participate in farther reactions to form a linker, a connector group [CON], a multifunctional group [MULTICON] or may be linked directly to an (ACM) as otherwise described herein.

The term "blocking group" refers to a group which is introduced into a molecule by chemical modification of a function group to obtain chemoselectivtty in a subsequent chemical reaction. It plays an important role in providing precursors to chemical components which provide compounds according to the present invention. Blocking groups may be used to protect functional groups on ACM groups, CCTe groups, connector molecules and/or linker molecules in order to assemble compounds according to the present invention. Typicai blocking groups are used on alcohol groups, amine groups, carboriyl groups, earboxylk acid groups, phosphate groups and alkyne groups among others.

Exemplar aleohol/hydroxyl protecting groups include acetyl (removed by acid or base), benzoyl (removed by acid or base), benzyl (removed by hydrogenolysis. β- methoxyethoxyraedjyl ether (MEM, removed by acid), dimemoxytrityi [bts-(4- methoxyphenyl)phenyhnethyl j (DMT, removed by weak acid), methoxymeihyi ether (MOM, removed by acid), methoxytrityl [(4~methoxyphenyl)diphenylmethyl], (MMT, Removed by acid and hydrogenolysis), p-meAoxylbenzyl ether (PMB, removed by acid, hydrogenolysis, or oxidation), methyithiomethyl ether (removed by acid), pivaloyl (Piv, removed by acid, base or reductant agents. More stable than other ac l protecting groups, ietraliydropwaoyl (THP, removed by acid), tetrdiydrofnran (THF, removed by acid), trityl (triphenyl methyl, (Tr, removed by acid), si!y! ether (e.g. trimetliyl.sil.yl or TMS, ferf-butyldimethy!si!yl or TBDMS, trww-propylsilyloxymethyl or TOM, arid tnisopropylsilyl or TIPS, all removed by acid or fluoride ion such as such as NaF, TBAF (teira-i¾-btt ylaramom ^' um fluoride, I-!F-Py, or HF~NEt¾); methyl ethers, (removed by TMSI in DCM, MeCN or chloroform or by BBt¾ in DCM) or ethoxyethly! ethers (removed by strong acid).

Exemplary amine-protecting groups include carhobenzylox (Cbz group, removed by hydrogenolysis), p- ethoxylbenzyl carbon : ( oz or MeOZ group, removed by

hydrogenolysis), teit-butyioxycarhonyl (BOC group, removed b concentrated strong acid or by heating at elevated temperatures), 9-Fluorenylmethyjoxycarbon l (FMOC group, removed by weak base, such as piperidine or pyridine), acyl group (acetyl, benzoyl, pivaloyl, by treatment with base), benzyl (Bn groups, removed by hydrogenolysis), carbamate, removed by acid and mild ^' heating, p-methoxybenzyl (PMB, removed by hydrogenolysis), 3,4- dimethoxybenzyl (DMPM, removed by hydrogenolysis), p-methoxyphenyi (P P group, removed by ammoni um cerium IV nitrate or CAN); tosyl (Ts group removed by concentrated acid and reducing agents, other sulfonamides, Mesyl, Nosy! & Nps groups, removed by samarium iodide, tributyl tin hydride.

Exemplary carbonyl protecting groups Include acyct ical and cyclical acetals and ketals (removed by acid), aeykls (removed by Lewis acids) and dithianes (removed by metal salts or oxidizing agents).

Exemplary carboxyiic acid protecting groups include methyl esters (removed by acid or base), benzyl esters (removed by hydrogenolysis), teri-butyl esters (removed, by acid, base and reductants), , esters of 2,6-disubstituted phenols (e.g. 2,6-dimethylphenol, 2,6- diisopropylphenol, 2,6-di-tert-butylphenol, removed at room temperature by DBU-cata!yzed methanolysis under high-pressure conditions, silyl esters (removed by acid, base and organometaiSic reagents), orthoesters (removed by mild aqueous acid), oxazoline (removed by strong hot acid (pH < 1 , T > 100 °C) or strong hot alkali (pH > 12. T > 100 °C)).

Exemplar phosphate group protecting groups including eyanoethy! (removed by weak base) and methyl (removed by strong nucleophUes, e.g. thiophenol/TEA). Exemplary terminal alkyrie protecting groups include propargyl alcohols and siiyl groups.

The term "antibody", also referred to art immunoglobulin (Ig), is a protein, which is Y-shaped and produced by B-ee!Js that the immune system uses to identity and neutralize foreign objects in the body, such as pathogens, including viruses, bacteria and cancer cells, which the immune system recognizes as objects to the immune system. As used herein, antibody includes, but is not limited to, monoclonal antibodies. The following disclosure from U .S . Patent Application Document No. 20100284921 , the entire contents of which are hereby incorporated by reference, exemplifies techniques that are useful in making antibodies which may be modified and employed in chimeric compounds of the instant invention.

Pursuant to its use in the present invention, the antibody is preferably a chimeric antibody. For human use, the antibody is preferably a humanized chimeric antibody.

[A]n anti-target-structure antibody ... may be monovalent, di alent or polyvalent in order to achieve target structure binding. Monovalent immunoglobuli ns are dinners (HL) formed of a hybrid heavy chain associated through disulfide bridges with a hybrid light chain. Divalent immunoglobulins are tetramers (H2 L2) formed of two dimers associ ated through at leas t one disulfide bridge.

As discussed above, the term antibody for use in the present invention includes compounds which exhibit binding characteristics comparable to those of the antibodies, and include, for example, hybridized and single chain antibodies, as well as fragments thereof. Methods of producing such compounds are disclosed in FC Application os. WO

1993 21319 and WO 1 89/09622. These compounds include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hyperv ariable regions of the antibodies raised against targets on cancer ceils pursuant to the practice of the present invention. These may be readily modified to link these CCTMs to the (ACM), thus forming chimeric compounds hereunder.

Compounds according to the present invention which serve to bind to target cancer cells include fragments of antibodies (FAB) that have the same, or substantially the same, binding characteristics to those of the whole antibody . Such fragments may contain one or both Fab fragments or the F(ab¾ fragment Preferably the antibody fragments contain all six complement determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or fi ve complement determining regions, are also functional. The functional equivalents are members of the IgG immunoglobulin class and subclasses thereof, but ma be or may combine any one of the following immunoglobulin classes : IgM, IgA, IgD, or IgE, and subclasses thereof. Heavy chains of var ous subclasses, such as the IgG subclasses, are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, hybrid antibodies with desired effector function are produced. Preferred constant regions are gamma 1 (IgGl ), gamma 2 (lgG2 and IgG), _. gamma .3 (igG3) and gamma 4 il:gG4}. The light chain constant region can be of the kappa or lambda type. in another approach, the monoclonal antibodies may be advantageously cleaved by proteolytic enzymes to generate fragments retaining the target structure binding site. For example, proteolytic treatment of IgG anti bodies with papain a t neutral pH generates two identical so-called "Fab" fragments, each containing one intact light chain disulfide- bonded to a fragment of the heavy chain (Fc). Each Fab fragment contains one antigen-combining site. The remaining portio of the IgG molecule is a dimer known as "Fc". Similarly, pepsin cleavage at pH 4 results in the so-called F(ab')2 fragment.

Single chain antibodies or Fv fragments are polypeptides that consist of the variable region of the heavy chain of the antibody linked to the variable region of the light chain, with or without an interconnecting linker. Thus, die Fv comprises an antibody combining site. Hybrid antibodies also may be employed as CMTs in the chimeric compounds according to the present invention. Hybrid antibodies have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region of a monoclonal antibody from each stable hybridoma.

Methods for preparation of fragments of antibodies (e.g. for preparing an antibody or an antige binding fragment thereof having specific binding affinit for a cancer cell target are readily known to those skilled in the art. See, for example, Coding, "Monoclonal

Antibodies Principles and Practice", Academic Press (1983), p. 1 19-123. Fragments of the monoclonal antibodies containing the antige binding site, such as Fab and F(ab')2 fragments. may be preferred m therapeutic applications, owing to their reduced .«mn««ogat»icity. Such fragments are less immunogenic than the intact antibody, which contains the immunogenic Fc portion. Hence, as used herein, the term "antibody" includes intact antibody molecules and fragments thereof that retain antigen binding ability.

When the antibody used in the methods used in the practice of the in vention is a monoclonal antibody, the antibody is generated using an known monoclonal antibody preparation procedures such as those described, for example, in Harlow et al. (supra) and in Tuszynski et al. (Blood 1988, 72: 109-1 15), Generally, monoclonal antibodies directed against a desired antigen are generated from mice immunized with the antigen using standard procedures as referenced herein. Monoclonal antibodies directed against full length or fragments of target structure may be prepared using the techniques described in iiarlow et al. (supra).

Chimeric animal-human monoclonal antibodies may be prepared by conventional recombinant DNA and gene transfection techniques well known in the art. The variable region genes of a mouse antibody-producing myeloma cell line of known antigen-binding specificity are joined with human immunoglobulin constant region genes. When such gene constructs are transfected into mouse myeloma cells, the antibodies produced are largely human but contain antigen-binding specificities generated in mice. As demonstrated by

'Morrison et al., 984, ^' Proc. Natl. Acad Sei. USA 81 :685.1 -6855, both chimeric heavy chain V region exon (VH) tuman heavy chain C region genes and chimeric mouse light chain V region exon (V )-human light chain gene constructs may be expressed when txansfected into mouse myeloma cell lines. When both chimeric heavy and light chain genes are transfected into the same myeloma cell, an. intact H2L2 chimeric antibod is produced. The methodology for producing such chimeric antibodies by combining genomic clones of V and C region genes is described in the above-mentioned paper of Morrison et al., and by

BouSianne et al. (Nature 1984, 312:642-646}. Also see Tan et al. (J. Immunol. 1985,

.1 5:3564-3567) for a description of high level expression from a human heav chain

promotor of a human- mouse chimeric chain after transfection of mouse myeloma cells. As an alternative to combining genomic DNA, cD A clones of the relevant V and C regions may be combined for productio of chimeric antibodies, as described by Whitte et al. (Protei Eng. 1987, 1 :499-505) and Liu et al. (Proc. Natl. Acad. Scl USA 1987, 84:3439-3443). For examples of the preparation of chimeric antibodies, see the following U.S. Pat. Hos 5,292,867; 5,0 1 ,313; 5,204,244; 5,202,238; an 5,169,939. The entire disclosures of these patents, and the publications mentioned in the preceding paragraph, are incorporated herein by reference. Any of these recombinant techniques are available for production of

rodent/human chimeric monoclonal antibodies against target structures.

When antibodies other than human antibodies are modified for incorporation into chimeric compounds pursuant to the present in vention, it may be necessary to reduce the imraunogenicity of the murine antibody. To further reduce the immiraogenicity of murine antibodies, "humanized" antibodies have been constructed in which only the minimum necessary parts of the mouse antibody, the coirmtementarity-detenmning regions (CDRs), are combined with human V region frameworks and human C regions (Jones et a!., 1986, Mature 321 :522-525; Verhoeyen et al, 1 88, Science 239: 1534-1536; Hale et aL 1 88, Lancet 2: 1394-1399; Queen et a!., 1989, Proc. Natl. Acad. Sci. USA 86: i 002940033). The entire disciosui ^' es of the aforementioned papers are incorporated herein by reference. This technique results in the reduction of the xenogenei c elements in the humanized antibody to a minimum. Rodent antigen binding sites are built directly into human antibodies by transplanting only the antigen binding site, rather than the entire variable domain, from a rodent antibody. This technique is available for production of chimeric rodent/human anti-target structure antibodies of reduced human immunogenicity."

The term antibody fragment or "FAB" is used to describe a fragment -of an antibody which substantially .maintains the same binding characteristics of the whole antibody, but el iminates other chemical features of the ant ibody which may complicate administration and produce untoward immunogenic responses in a patient.

The term "single-chain antibody variable fragment" or "scFv* is used to describe an artificial construct that links the sequences encoding the V« and V . domains of an antibody into single polypeptide chain and lacks the rest of the antibod molecule. Because the antigen-binding site of an antibody is formed in a cavity at the interface betweein \% and V _L domains, the scFv preserves the antigen binding activity of the intact antibody molecule. Normally the \½ and V; domains are parts of different polypeptide chains (the heavy and light chains, respectively), but in the scFv they are joined into a single polypeptide that can be fused genetically to other proteins, for example, proteins on cancer cells to be targeted.

These scFvs may form the basis of effective CCTMs on chimeric compounds according to the present invention. The term "linker" (designated as "L", "(L)" "Lc" or (Lcf in compounds according to the present invention) is used to describe a chemical moiety which, when present in chimeric molecules according to the present invention, eovalently binds a (ACM) group to a (CC¾) group, optionally through one or more [CON] groups and/or one or more alternative linker groups. The linker group may be cleavable or nondeavable depending on the function of the CCTR group or the compo und itself (in the case of dimeric compounds according to the present invention). In general, antibody or antibody related (CCT ^' E) groups described above are generally, but not exclusively linked to a (ACM) group through a cleavable linker group. Other CCTES often are linked to (ACM) groups through a non-cleavable linker group.

Typical cleavable linker groups (L), which may be represented as (la), for use in the present invention are represented by any chemical structure which is compatible with the chemistry of the chimeric compounds and their administration to a patient and readily cleave in or on a cell in which the chimeric molecule is introduced, in general, the cleavable linker for use in compounds according to the present invention is at least one chemical moiety, more often at least two chemical moieties in length to upwards of 100 or more moieties in length. These linkers are presented in detail hereinbelow. Often, one or more linkers, especially cleavable linker groups may be linked to one or more non-cleavable (non-labile) linker groups either directly or through a connector group (CON) or multicomiector group

(MULTKX)N) as otherwise described herein. These form a more complex linker group.

Cleavable or labile linkers (Lex) allow the [ACM] moiety to be cleaved from the (CCTM) in compounds according to the present invention order to provide a maximal effect in the cell, by allowing the ACM to be cleaved from, the CC TM after the compound targets the cancer cell, facilitating entr of the ACM into the cell which causes cleavage breakage and/or intercalation of the cell 's DMA, causing cytotoxicity and cell death. These labile linkers include hydrolytically labile (acid labile) linkers, reductively labile linkers

(principally disulfide linkers which are reductively cleaved by intracellular glutathione or other disulfide reducing agent) and enzyraaficaliy labile linkers (protease substrates).

In certain embodiments according to the present invention, the cleavable linker Le _t . is a disulfide wherei one of the sulfurs in the disulfide group is provided by a cysterayi residue alone or as an oligopeptide ranging from about I to about .10 amino acid units in length, often L 2 or 3 amino acid units in length.. I» certain embodiments the oligopeptide is represented by a glutamyl eysteinyl dipeptide (with the amide formed between the sidechain carboxyiic acid of the glutamic acid and the amine of the eysteinyl residue), a glycinyl cysteiny! dipeptide. an alatimyl eysteinyl dipeptide or a lysinyl eystioyl dipeptide. The dipeptide may ^¬ be linked (mated) with another dipeptide of similar or- different structure each having a eysteinyl residue linked to the eysteinyl residue of the other dipeptide, or the dipeptid may be linked with a mercaptide such as an alkyl mercaptide (which is further substituted with a group which can further link the cleavable iinker to another group, such as a non-cieavable (non-labile) linker an (ACM) group or a (CCTE) group or a connector group, etc.

In other embodiments the cleavable linker group (Lei.) is an oligopeptide (containing a disulfide group as described above) or other linker which contains an ester group which may readily clea ved . For example, a iinker may consist of a dipeptide such as a glutamyl eysteinyl group which provides a disulfide link to a linker (such as a alkylene group or polyethylene glycol group) which can form an integral connector molecule (such as a difunctional triazole CON group or a MULTICON group) as otherwise described herein, or alternatively bind directly to an ACM group or a CCTM group.

Cleavable or labile linkers (Lc) may comprise a group represented by the chemical structures:

where R is an -ethylene glycol group, a methylene grou or an amino acid, preferably an ethylene glycol group or an animo acid and n in this labile linker is from 0 to 10, often from I to 6, or 1 to 3 and where points of attachment (as indicated) are to other portions of the cieavable or labile linker (Le), a difunetional connector moiety (CON), a iion-cleavable (non- labile) linker (LN) _> or a multifunctional connector molecule [MULTICONJ, through which an [ACM] functional group and a [CC ^' IM functional group are linked as otherwise described herein;

X is O, N-R ^AL or S;

R ^AL is H or a C3 -C3 alkyl group (often H or Me, most often H);

Y is 0 or S and

Z— Me, Et„ IPr, tBu, Ph, each of which may be optionally substituted with one or more halogen groups (especiall from three up to five Fs, preferably no more than three Fs) and where said Fh group may be further optionally substituted with a C · ··<- ^' ; alkyl group (which itself may be substituted with up to three halogens, preferably F) or OMe.

Exemplary reduciive!y cleaved moieties (by glutathione, other reductive species within the eel!) include chemical formula:

Where R is independently an ethylene glycol group, a methylene group or an amino acid where at least one amino acid (that which provides one of the sulfurs in the disulfide group) is a cysteinyi group (often, (R)n i a glutamyl cysteinyl or tysinyl cysieinyl dlpeptkie) and n in this labile linker is from 0 to 10, often from 1 to 6, or 1, 2 or 3 and where points of attachment (as indicated) are to other portions of the cleavable/labile linker [LciJ, a difunetional connector molecule or group (CON ), a non-labile linker (NLL) or a

multifunctional connector group molecule [MULTiCON] as otherwise described herein.

Another reductively cleaved linker (Lei.) which is often used in compounds according to the present invention is represented by the following structure:

Where Xu is -(Olak iC K ~iC¾} _mL -<, N n, NR _{L (CO), S, SO or S(C% , or a

nucieophilic or electrophilic functional group (which can be further reacted to form a eovalent Hnk);

a nucleophilie or electrophilic functional group (which can be further reacted to form a eovalent link);

t _t . is H or a Ct-Cj alkyl group;

Each « _¾. is independently 1, 2, 3, or 4 (often, each n¾ _. is 2);

mU is 0, 1 , 2, 3, 4, or 5 (preferably 0);

mi: ^* is 1 _s 2, 3, 4 or 5; and

ntis 0-20, 1-15, 2-10. 1-6, 1, 2, 3, , 5, 6 _f 7, or 8.

In certain embodi ments of the above compounds as described above, Xu and X are optionally functional groups on the linker moiety (pre-linker molecules), for example, nucieophilic and/or electrophilic groups which are reacti ve with a corresponding electrophilic and/or nucieophilic group on the ACM, CCT _E , [CON] group or another linker molecule so that the ACM group. CC¾. [CON] group or another linker molecule can be eovalent linked or coupled to the linker group. In certain embodiments, X _u and/or groups are

nucieophilic groups such as amine groups, hydroxyl groups, sulfhydryl groups or

nucieophilic carbon groups (e.g., carbanions) which couple and form covalent bonds with a corresponding electrophilic group such as an ester groups (which may be activated), aeyl groups (activated), or other electrophilic groups such as nichloromethylmethyiiniinoester ( 3-0 ^::: -i(C€¾)) _s among others, on the ACM, CC¾ [CON] moiety or alternative linker molecule. Alternatively, Xu and/or X _u ma be nucieophilic groups such as an amine group, a hydroxyl group or a sulfhydryl group which are reactive with a correspondeing electrophlic groups as described above, in these pre-linker molecules, each of Xu and X _Tj2 may be a nueleoptulic and an electrop lic group. This approach applies to a!l Sinkers provided herein which may be presented as pf elinker compoimds capable of coupling with fuiictionai groups on the ACM, CCT _E; [CON] or alternative linker components of the present compoimds.

In certain embodiments, the reductively cleaved linker (La,) is a moiety according to the chemical strac n

Where ¾is 0-20, .1 -1.5, 2-10, 1-6, 1 , 2, 3, 4, 5, 6, 7, or 8.

In certain alternative embodiments, a partial cleavage linker containing an alkynyl containing functional group (which ultimately forms a connector group) is according to the chemical structure:

This linker may be reacted with other components (which may include CCTK groups, ACM groups, [CON] groups or alternative linkers containing a hydroxy! group to react with the tric oromethyimethyliminoester (-0-C==NH(O¾) functional group at one end of the molecule and an azide group at the alkynyl functionality according to the present invention to form exemplary compounds according to the present invention.

In alternative versions of this approach, the linker molecule is according to the chemical structure:

Where the iochlorom thylmetliyliminoester (-0-C~NH(CCl ₃ ) functional groop may be used, to covalently link a hydroxyl groo to form an ether and the disulfide groop is used to bind to cysteinyl group of an antibody or other oligo- or polypeptide.

Exemplary eazymatical ^' ly cleaved labile linkers include those according to the chemical structure:

Where the protease (cathepsin) substrate is a a. peptide containing from 2 to 50 amino acid units or more, often 2 to 25 amino acid units, 2 to 15 amino acid units, 2 to 10 amino acid units, 2 to 6 amino acids, 2 to 4 amino acids, 2, 3 or 4. Often, the protease substrate, above contains, comprises, consists essentially of or consists of the following peptides the point of attachment being at the distal ends of the peptide:

-GIy~Phe~Leu~Giy- ;

-Ala-Leu-Ala-Lett ;

~Phe-Arg~ ;

-Phe-Lys- ;

-Val-Cit~ (valine-citriliune)

-Val-Lys-

-Val-Ala- and

where R (above) is an ethylene glycol group, or a methylene group and n is from 0 to 10, often from 1 to 6, or 1 to 3 and where points of attachment (as indicated) are joined to other portions of the labile linker, a difunctional connector group or molecule (CON), a non-labile linker (NLL) or other moiety as described herein. Other enzyme labile linkers are the beta-gloeosida.se labi le tinkers according to the chemical structure:

Where the points of attachment are joined to other portions of the labile linker, a di functional connector moiety (CON), a non-labile linker (NLL) or a multifunctional connector group or molecule [ ULTiCON] as otherwise described herein.

In each of the above labile linkers, at the point of attachment in each group, the labile linker may be further linked to a non-labile linker as otherwise described herein, preferably a (poly)ethyiene glycol group of from 1 to 12 glycol units (often 2 to 8 glycol units or 4 to 6 units) or an alkylene chain from 1 to 20 methylene units, often 1 to 10 methylene units, often 1 to S methylene units, more often 1 to 6 methylene unit, often 2 to 4 methylene units.

Preferred non-labile linkers include, for example, (polyethylene glycol linkers ranging in length from 2 to about 100 ethylene glycol units, preferably about 2 to 10 ethylene glycol units, about 2 to about 25, about 2 to about S 5, about 2 to about 14, about 4 to about 30 units, hi other preferred embodiments, the non-cleavable linker (L ) is a polyethyleiie-co- polypropylene (PEG/PPG block copolymer) linker ranging from 2 to about 100, about 2 to about 25, about 2 to about 15, about 2 to about 14, about 2 to about 10, about 4 to about 10. combined ethylene glycol and propylene glycol units.

(Poiy)alkylene chains as otherwise described herein are also preferred L _N for use in the present invention. When present, these have 1 to about 100 units, often about 2 to 10 units, about 2 to about 25, about 2 to about 15, about 2 to about 14, about 4 to about 10 units. Ljv; for use in the present invention may also contain one or more connector CON moieties as otherwise described herein which chemical ly connect separate (two or more) LN portions, the entire portion being labeled LN- I addition, a non-c!eavable linker L may be linked through at least one connector moiety CON (as described in greater detail herein) to a cleavable linker Lc in order to provide a linker moiety. In certain preferred embodiments, the non-labile linker (NLL) is represented by the following exemplary structures (note that the NLL may contain one ore more CON moieties as discussed above):

, COLLAGEN LINKER . , , and , among numerous others, as described herein. where n and n ^s are each independently 0 to 100, preferably 1 to 1 0, more preferably about 2 to aboirt 20, about 2 to about 10, about 4 to about 10. about 4 to about 8.

The non-labile linker group N LL may also be a linker according to the chemical formula:

where _a is H or a Cj- a!ky!, preferably CJh, most often H;

m is an integer from 1 to 12, often 1 , 2, 3, , 5, or 6;

m" is an integer 1 , 2, 3, 4, 5, or 6, often 6;

t is- 0, 1 , 2, 3, 4, 5, or 6; aid

iL is 0 or 1 , often 1 ; or

a Sinker according to the structure:

Where q Is aa integer Scorn 0- 2, preferably 1 , 2, 3, 4, S or 6;

q' is 1 to 12, often 1 , 2, 3, 4, 5 or 6 and

it is 0 or 1 _s preferably 1.

The two above linkers may be linked together to provide farther linkers which- are often used in compounds according to the present invention:

Where q is an integer from 0-12, preferably 0, 1 , 2, 3, 4, 5 or 6;

q' is 1 to 12, often 1 , 2, 3, 4, 5 or 6;

iL is 0 or I .; and

Ri. is an amino acid or an oligopeptide (which term includes a dipeptide) as othenvise described herein, especiai!y including lysine, dilysine, or glycinelysine.

Another linker according to the present invention includes a linker based upon succminiide according to the chemical formula:

K is H or Ci-j alkyl, preferably H; So is CI½ Ci¾0; or C¾Ci¾0;

i is 0 or 1 ; and

in is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 1-5).

In certain additional embodiments, the linker group is an amino acid, a dipeptide or an oligopeptide containing from 1 to 12, preferably 1 to 6 amino acid monomers or more. In certain embodiments, the oligopeptide is a dipeptide and the dipeptide is a dilysine or a glyeinelysine dipeptide. When lysine is used as an amino acid in an oligopeptide linker, the sidechain alkylene amine may be used to link other linker groups or other components in the ^• molecule. The dipeptide or oligopeptide may he considered a cleavable linker or non- eieavable depending upon the nature of the peptide, in certain additional embodiments, as discussed above, the linker group NLL is a group

polyproline linker

a group

or a polypropylene glycol or polypropylene-co-polyethylene glycol linker having between 1 and 100 glycol raits (1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, i to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 , 2, 3, 4 or 52 and 50, 3 and 45);

Where R _s is H, Cj-O a!kyl or aikanol or forms a eye Sic ring with R ¹ (prolme) and ^" ' is a side chain derived of an amino acid preferably selected from th group consisting of alanine (methyl), arginine (propylenegaariidine), asparaghie (inethylenecarboxyairride), aspartic acid (ethatioic acid), cysteine (thiol, reduced or oxidized di-thiot), glutamiiie (ethylcarboxyamide), glutamic acid (propanoic acid), glycine (H), histidirre (memyleneimidazole), isoleocme ( 1 - niethylpropane), leucine (2-methylpropane), lysine (buryleneamine), methionine

(ethylmethylthioether), phenylalanine (benzyl), proline (R ³ forms a cyclic ring with R* and the adjacent nitrogen group to fomi a pyrrolidine group), serine (methanol), threonine (ethanol, 1- Irydroxyethane), tryptophan (rnethyleneindo!e), tyrosine (methylene phenol) or valine

(isopropyl);

XE is a bond, O, N-R _NA , or S;

¾ _A is H or C ₁ -C3 aikyl, preferably H;

i is an integer from 0 to 6 (0, ! , 2, 3, 4, 5, or .6);

m ^w is an integer from 0 to 25, preferably 1 to 10, 1 to 8, 1, 2, 3, 4, 5, or 6;

m is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30,

1 to 15, 1 to 10, 1 to 8, 1 to 6, L 2, 3, 4 or 5; and

n is an integer from 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, I to 45, 1 to 40, 2 to 35, 3 to 30, I to 15, 1 to 10, 1 to S, 1 to 6, 1, 2, 3, 4 or 5; or L may also be a linker according to the chemical formula;

Where Z and Z ^* are eac independently a bond, -(C3¾)i»Q, * 0¾-$ ₅ -(C%)rN-R ,

wherein said -(CI¾ group, if present in Z or Zyis bonded to [ ACM], [CCTE], or an optional difunctional connector group [CON], if present;

Each R is independently H„ or a C3-C3 alky! or alkanoi group;

Each R ^* is independently B or a Ci-Cj alky! group;

Each Y is independently a bond, O, S or -R;

Each i is independently 0 to 100, 1 to 100, 1 to 75, 1 t 60, 1 to 55, 1 to 50, .1 to 45, 1 io 40, 2 to 35, 3 to 30, I to 15, i to 10, 1 to 8, i to 6, I , 2, 3, 4 or 5;

D is

{C¾ a bond, or D may be

or a polypropylene glycol or polypropylene-co-po yethy ene glycol linker having between 1 and 100 glycol units (1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 52 and 50, 3 and 45);

with the proviso that Z, Z' and D are not each simultaneously bonds;

j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45 , 1 to 40, 2 to 35, 3 to 30, I to 1 , Ϊ to 10,

I to 8, I to 6, 1 , 2, 3, 4 or 5;

m (within this context) is an integer trom 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 , 2, 3, 4 or 5; and

II (within this context) is an integer from about 1 to 100, about 1 to 75, about 1 to 60, about 1 to 50, about I to 45, about I to 35, about I to 25, about 1 to 20, about 1. to 15, 2 to 1 , about 4 to

12, about 5 to 10, about 4 to 6, about 1 to 8, about i to 6 , about 1 to 5, about 1 to 4, about 1 to 3, etc.).

of is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, I to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 30, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;

m" is an integer between 0 to 25, preferably 1 to 10, 3 to 8, 0, 1, 2, 3, 4, 5, or 6;

Ώ' is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, I to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to

10, 1 to 8, I to 6, 1 , 2, 3, 4 or 5;

X ¹ is O, S or N-R;

R is as described above;

Ra is H, C Cj alky! or alkanol or forms a cyclic ring with R" ^* (proline) and R ³ is side chain derived of an amino acid preferably selected from the group consisting of alanine (methyl), arginine (propylene uanidme), asparagine (raethylenecarboxyamide), aspaitic acid (ethanoic acid), cysteine (thiol, reduced or oxidized di-thiol), glutamine (ethylcarboxyamide), glutamic acid (propanoic acid), glycine (H), hisiidioe ( etiiyleneimidazole), isoleucine ( 1- rnethy I propane), leucine (2-methy!propane), lysine (buryleneatnine), methionine (ethyimethylthioether), phenylalanine (benzyl), proline (R ³ forms a cyclic ring with R« and he adjacent nitrogen group to form a pyrrolidine group)., serine (methanol), threonine (ethanol, 1 - hydroxyethan ), tryptophan (methyleneindole), tyrosine (methylene phenol) or valine (isopropyl).

It is noted that each of the linkers (both cleavable and non-cleavable linkers) identified in the present application may be further linked with connector molecules/moieties

[CON] molecules/moieties, [ACM] groups and [CCTR] groups through amide groups (which include alkylene groups on either or both sides of the amide group containing one to fi ve methylene units), keto groups (which include alkylene keto groups containing one to five methylene units on either or both sides of the keto group), amine groups ( which include alkylene amine groups containing one to five methylene units on either or both sides of the amine group), urethane groups ( which include alkylene groups containing one to fi ve methylene units on either or both sides of the urethane moiety)., alkylene groups (containing from 1 to 5 methylene units), urea groups (which include alkylene groups containing one to five methylene units on either or both sides of the urethane moiety) amino acids, succinimide groups or other moieties compatible with the linker chemistry in order to link components of the molecules. It is noted that in the case of polyethylene gl ycol and polypeptide linkers, the use of an additional group (eg, alkylene amine or other group as described above) or a second linker group ma be useful for joining the linker to another component of the molecule, including a [COM] group.

Additionally, more than one linker group identified herein may be linked together to form a linker group as otherwise used in the present compounds, consistent with the stability of the linker chemistries . These extended linkers are often, though not exclusively, linked through [CO ] connecting groups as otherwise described herein.

In certain embodiments according to the present invention, linker molecules are provided which contain at least one and preferably at least two functional groups through which ACM, CCTB, connector [CON] groups or even additional linker groups may be covalently linked to provide compounds according to the present invention. The functional groups are generally at or are proximal!y located at the distil ends of a linker molecule and may be electrophilic and/or nucleophiiic groups or may be readily functional ized to functional groups (electrophiiic and'or nueleophilic groups) which may be used to covaleittJy link other molecules ACM moieties, CC ^' I E moieties, [CON] molecules or even additional Sinker molecules) to the linker molecule.

In certain embodiments, functional groups on the linker moiety, include, for example, nueleophilic and or electrophiiic groaps which are reactive with a corresponding electrophiiic and/or nueleophilic group on the ACM, CCTE group so that the ACM group or the CCT¾ group can be eovalent linked or coupled to the linker group. In certain embodiments, Xu and/or X groups are nueleophilic groups such as amine groups, hydroxyl groups, sulmydryl groups, azide groups (for reaction with an aikyol group to form tria ole connector molecules) or nueleophilic carbon groups (e.g., earbanions) which couple and form eovalent bonds with a corresponding electrophiiic groups such as ester groups (activated), acyl groups (activated), or other electrophiiic groups such as trichloromethylmethyliminoester (-O^^ HCCCls ), or aifcyrryi groups (reactive with azide groups) among others, on the ACM or CCTE.

Alternatively, these functional groups may he used to link additional linker molecules, ACM and/or CC¾ groups through connector [CON] molecules.

Another difunctional linker molecule for use in the present invention is

Where the tHchioromethyimeihyiimhioestei (~0-C=NH(CCi;;) functional grou is reactive with a free hydroxy! group and the alkynyl group is reacti ve with an azklo group to form a triazole connector [CON] moiety.

The term ''difunctional eonnneetor group" or [CON] is used to describe a difunctional group which connects two (or more) portions of a linker group to extend the length of the linker group, in certain embodiments, a linker group is reacted with or forms a [CON] group with, another linker group to form m extended linker group or with another moiety such as a ACM moiety or CCTE moiety to link the linker to that moiety. The reaction product of these groups results in an identifiable connector group [CON] which may be distinguishable from the linker group as otherwise described herein, but is integral to same and essentially forms a portion of the linker group. It i farther noted that there is often overla between the description of the Afunctional connector group and the linker group, especially with respect to more common connector groups such as amide groups, oxygen (ether), sulfur (ihioether), carbonyl or amine linkages, urea or carbonate -OC(0)0- groups, etc. as otherwise described herein. It is noted that a difunctional connector molecule [CON] used hereunder is often connected to one or two parts of a linker group which binds [ACM] to [CCT _E ]- Alternatively, a [CON] group may be directly linked to a [ACM] group or more often, a [CCTE] group, as well as a [MULTICONJ group as described herein. L CCTE, CCTE and/or ACM groups optionally include [CON] groups to facilitate the binding of a linker group to the CCTE group and'or the ACM group. .

Common difunctional connector groups [CON] which are used in the present invention, principally to link one end of a linker to another end of a linker to provide a longer linker or to connect a linker (and essentially become integral to the linker) to a ACM or CCTE group and include

Where X ² is C¾, O, S, NR. ⁴ , S(Q), S(0) ₂ , ~S(0) ₂ 0, ~OS(0) ₂ , or OS(0) ₂ 0;

Χ ^' · is absent, C¾, O, S, NR. ⁴ ; and

R ⁴ is H, a Ci-C.¾ aikyl or alkanol group, or a -C(0){C Q;) group. an amide, keto group, urethane or urea ;

m in CL is an integer from 0 to 12, often 0, 1 ,2,3,4,5 or 6;

and iL is 0 or I , often ! ; in other embodiments [CON] is a

group.

In certain embodiments, tlie [CON] group is often linked through the amine of the triazo ^' le or the succinimide moiety to a c!eavable or non-cleavab e linker or to an ACM group or CC¾ group.

The term ^4t multifunctional connector", symbolized by [MULTICON], is used, to describe a chemical group or molecule which is optionally included in chimeric compounds according to the present invention which link at least one or more linker groups (which may be cSeavabie or iion-deavable), difenciiona! connector groups (CON)., (ACM) groups or (CCTE) groups as otherwise described herein. The connector grou is the resulting moiety which forms from the facile condensation of at least three separate chemical fragments which contain reactive groups which can provide connector groups as otherwise described to produce chimeric compounds according to the present invention, it is noted thai a multifunctional connector moiety or molecule (MULTICON] is readily distinguishable from a linker in thai the multifunctional connector is the result of a specific chemistry which is used to provide chimeric compounds according to the present invention.

Connecting moieties in the preseni invention include at least one multifunctional moiety or molecule [MULTICQ j which contains three or more functional groups which may be used to covalently bind (preferably, through a linker) to at least one [ACM] group (preferably more than one) and at least one [CCTE] group (preferably more than one), thus linking each of these functional groups into a single compound. Multifunctional connector groups for use in the present invention include monies which have at least three or more functional groups which can bind to linkers to which are bound [ACM] and/or [CCTrO groups in order to provide compounds which contain at least one [ACM] and [CCTE] groups, but preferably more than one of each of these groups pursuant to the present invention. These multifunctional connector moieties may also bind to other multifunctional connector molecules in order to create compounds containing a number of [ACM] and [CCTE] groups as defined herein. ultifunctional connector molecules [MULTICON] comprise any molecule or moiety which contains at least three groups which may be linked to [ACM], [CCTE] and/or linkers (non-labile linkers or labile linkers) and/or other connector groups (including difunctional and multifunctional connector groups) and often comprise five or six-membered aryl or heteroaryi groups (especially six-membered ring groups) exemplified by mul tifunctional, especially afunctional or teiraftmetiona! aryl or heteroaryi groups, including phenyl, pyridyl, pyrimidinyl, 1 ,3,5-triazinyl, 1,2.3-triazinyl, 1 ,2,4-triazmyl groups, each of which is substituted with at least 3 and up to 6 functional groups. These functional groups ma be derived from nucieophilie or electropMtc groups on the multifunctional connector molecule precursor (the .multifunctional connector molecule which forms the [MULTICON] moiety in final compounds according to the present invention) which are condensed onto linker group (each of which contains, for example an [ACM] group or a [CCT _K ] group) which contains a group which can be linked to the [MULTICON] moiety, [MULTICON] groups which are used in the present mvention preferably include substituted phenyl, pyridyl pyrimidinyl and 1,3.5-triazinyl, 1,2,3-triazinyl. 1 ,2.4-triazmyl groups, and other groups of multifbnctionality especially including groups accordin to the chemical structure:

where Y is OH or N; and

Each X ^5* is independently derived from an electrophilic or nucleophilic group, preferably

(€¾)„ ^■ · or (C¾) _!f «00;

the substitutent R ^{i tjN} is H or a C ₁ -C3 alkyl, preferably H or CH ₅ ,

n" is G, 1, 2 or 3 and

r is an integer from 1 -12, often 1, 2, 3, 4, 5 or 6.

The term "pharmaceutically acceptable salt" or "salt" is used throughout the specification to desc ribe a salt form of one or more of the compositions herein which are presented to increase the solubility of the compound in saline for parenteral delivery or in the gastric juices of the patient 's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids well known in the pharmaceutical art. Sodium and potassium salts may be preferred as neutralisation salts of carboxylic acids and free acid phosphate containing compositions according to the present in venti on. The terra "salt" shall mean any salt consistent with the use of the compounds according to the present invention. In the case where the compounds are used in

pharmaceutical indications, including the treatment of prostate cancer, including metastatic prostate cancer, the term "salt" shall mean a pharmaceutically acceptable salt, consistent with the use of the compounds as pharm aceutical agents.

The term "coadmi istr tion ^' '' shall mean that at least two compounds or compositions are administered to th patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time. Although compounds according to the present invention may be co-administered to patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, provided that effective concentrations of all coadministered compounds or compositions are found in the subject at a given time. Chimeric antibody- recruiting compounds according to the present invention may be administered with one or more additional anti-cancer agents or other agents which are used to treat or ameliorate the symptoms of cancer, especially prostate cancer, including .metastatic prostate cancer. The term "anticancer agent" or "additional anticancer agent" refers to a compound other than the chimeric compounds according to the present invention which may be used in combination with a compound according to the present invention for the treatment of cancer. Exemplary anticancer agen ts which may be coadministered in combination wit one or .more chimeric compounds according to the present invention include, for example, antimetabolites, inhibitors of topoisoroerase 1 and II, alkylating agents and microtuboJe inhibitors (e.g., taxol), among others. Exemplary anticancer compounds for use n the present invention may include everoli os, trabec edin, abraxane, TLK 286, AV-299, DN-101 , pazopanib, GSK690693, R.TA 744, ON 09IQ.Na, AID 6244 (ARRY- 142886), AM -107, ^' 00-258, GSK461364, AZD 1 152, enzastaurin, vandetaaib, ARQ-1 7, M .-0457, MLN8054, PHA-739358, R-763, T- 263, a FLT-3 inhibitor, a VEGPR inhibitor, an. EGFR TK inhibitor, an aurora, kinase inhibitor, a ΡίΚ-i modulator, a Bcl-2 inhibitor, an HDAC iahbiior, a c-MET inhibitor, a PARE inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an. IGFR-T inhibitor, an aati~HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a lAK/STAT inhibitor, a checkpoint- 1. or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase ( ek) inhibitor, a VEGF trap antibody, pemettexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicm. oregovomab, Lep-eto, noiatre ed, azd2171, hatabulin. ofatamamab (Arzerra), zanolimumab, edotecark, tetrandrine, rubitecan, tesmilifene, oblimersea, ticili.mu.mab., ipilimumab, goss pol, Bio 1 1 1 , 13M-TM-601 , ALT- 1 10, BIO 140, CC 8490, cilengitide, gmiateean, IL -PE38QQR, [NO 1001 , IPdRj RX-0402, iueanthone, LY 3176.15, neuradiab, v ^' itespan. Eta 744, Sdx 102, takmpanel, atrasentan, Xr 31 1 , romidepsia, ADS- 100380, sunitinlb, 5 >uorouracil, vorlnostat, etoposide, gerneitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5 ^< -deoxy--5 1isorom1dine, vincristine, temozo!omide, ZK. -304709, seliciclib; PD0325901 , AZD-6244, eapecitabine, L-Gktamic acid, N ~ 4-[2.(2~aa¾ino-4 ₅ .7. dihydro-4-oxo~I H - pyrroio[2,3- d ]pyrimidsn-5~yl)ethyi]benzoyll-, dtsodtum salt, heptahydrate, campiothecm, PEG~labeled irinotecan, tamoxifen, toremifeue citrate, anastrazole, exemesiane, letrozole, DES(diethylstilbesirol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC- 1 CI I. , CHIR-258.); 3-[S-(me hyteulfonyIpiperadinemethyl}- indolyy-quinoione, vatalanib, AG-0I3736, AVE-0005, the acetate salt of [D- Ser(Bo t ) ,Azgly 10 ] (pyro-Glu-His-Trp-Ser~Tyr~D-Ser(Bu t )-Leu~Arg-Pro- Azgly- H a acetate

[Cs ₅ ¾4 ₅ 5jO "(C;H ₄ 0?)x where x™ I to 2.4), g serelin acetate, leuprolide acetate, tripioreira pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, niegestrol acetate, raloxifene, bicalutamide, ftutamide, nilutaraide, niegestrol acetate, CP~7247.!4; ΎΑΚ- 1.65, BKI-272, er!o kt _. , lapirtanib, canertmib, ABX-EGF antibody, erbifox, EKB-569, PK1~ 166, GW -572016, lonafarmb, BMS-214662, tipifamib; ami ferine, NVP-LAQ824, suberoyi anaKde hydroxamic acid, valproic acid, trichostatin A, FK-228, SU1 1248, sorafenib,

KRN951 , ammogluiethiraide, arnsacrme, anagrelide, L-asparagmase, Bacillus Ca!mette- Guerin (BCG) vaccine, bleomycin, buserelin, busdian, earboplatin, carmus&e,

chlorambucil, ctspktin, cladribme, dodronate, cyproterone, cyfarabine, dacarbazine, dactmomycin, daunorubkni, diethylsti!besiro!, epirubicin, fludarabme, fliidroeoriisone, fiiioxymestetone, flutaiuid , gemcitabine, gleevac, hydroxyurea,, idarubick, ifosfamide, imatimb, leuprolide, levamisole, lomustine, mechlorethamine, metphalan, 6-mercaptopurme, mesna, methotrexate, mitomycin, BUtotane, iibtoxamrone, nil atamide, octreotide, oxa!ipkihv pamkltonaie, pentosiatin, pikamycin, porfimer, procarbazine, raliitrexed, riftiximab, s reptozocin, teniposide, testosterone, thalidomide, thiogwanrae, thiotepa, tretinoin, vindesine, 13-cis-reiinoic acid, phenylalanine mustard, uracil, mustard, estramostine, aitretamke, fioxuridine, S-deooxyuridine, cytosine arabinoskie, 6-mecaptopurine, deoxycotormycin, caleitrioi, valruhicin, mithr amycin, vinblastine, vinoreibine, topoteean, razoxin, rnarimastat, COL-3, neovasiat BMS-27S291 , s uakniine, endosiat , SU54 6, SU666S, EMDI21974, interleukin-i 2, 1M862, angiostatin, vkaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, irasiuzum&b, deniteukin diftirox,gefMnib, bortezimib, pac!itaxel, irinoteean, topotecan, doxorubicin, docetaxel. vinoreibine, bevacizu ab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epiihilone B, BMS- 247550, BMS-310705, droloxifene, 4-hydroxytaittoxifen, pipendoxifene, ERA- 923, arzoxifene, Mvestxant, acolbifene, lasofoxifene, idoxifene, TSE-424, HM - 3339, 2086619, PTK787/2R 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyediy3)~rapamycin, temsirolimus, AP- 23573, RAD001 , ABT-578, BC-230, LY294002, LY292223, LY292696, LY293684, LY293646, ortmannin, ZM336372, L~779,450, PEG-filgfastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuxtmab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,

ienalidomide, gemtuzumab, hydrocortisone, interleukin-1 1 , dexrazoxane, alemtuzumab, all- transretraoic acid, ketoeonazole. interleukin-2, megestroL immune globulin, nitroge mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine,

hexamethyimelam e, bexarotene, tositumomab, arsenic trioxide. cortisone, edlttonate, mitotane, cyclosporine, liposomal daunonsbicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an -l receptor antagonists, palonosetron, aprepitanr. dipheiiiiydraraiiie, hydroxyzine, .raetoeloprarakie, lorazeparn, alprazolam, haioperidol, droperidol, dronabinol, dexaniethasone, methylpred solone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and

datbepoetin alfa, among others. la addi tion to anticancer agents, a number of other agents may be coadministered with chimeric compounds according to the present invention in the treatment of cancer. These include active agents, minerals, vitamins and nutritional supplements which have shown some efficacy in inhibiting cancer tissue or its growth or are otherwise useful in the treatment of cancer. For example, one or more of dietary selenium, vitamin E. lycopene, soy foods, curcumin (turmeric), vitamin D, green tea, omega-3 fatty acids and phytoestrogens, including beta-si tosterol, maybe utilized in combination with the present compounds to treat cancer.

Without not being limited by way of theory, anticancer compounds according to the present invention which contain a cancer cell targeting element (CCTE) and an anticancer moiety (ACM) selectively bind to cancer cells and through that binding, facilitate the introduction of the (ACM) moiety into the cancer cell selectively, where, the compound, inside the cell or during transport into the cancer cell, the cleavable linker is cleaved from the cancer cell targeting moiety, providing an agent for intercalating and/or damaging through breakage the cancer cell's DNA and causing ceil death.

Pharmaceutical compositions comprising combinations -of an effective amount of at least one compound disclosed herein, often a ditunctional chimeric compound (containing at least one ACM and at least one CCTE) according to th present invention, and one or more of the compounds as otherwise described herein, all in effective■amounts, in combination with a pharniaceutically effective amount of a earner, additive or excipieut, represents a further aspect of the present invention. These may be used in combination with at least one additional, optional anticancer agent as otherwise disclosed herein.

The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled- elease formulations. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum siearaie, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbie acid, potassium sorhate, partial glyeeride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, inc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyJcelluJose, polyaeiylates, waxes, poiyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.

The compositions of the present in vention may be administered orally, parenterally, by inhalation spray, topically, reotally, nasally, bnccally, vaginally or via an implanted reservoir, among others. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, mtra-synovial, imrastemal, intrathecal, iniraiiepatic, hitraiesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally (including via intubation through the mouth or nose into the stomach) intraperitoneally or intravenously.

Sterile injectable forms of the composi tions of this in vention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptabie diluent or solvent, for example as a solution in 1, 3-biitanediol, Among the acceptable vehicles and solvents that may be employed are water . Ringer's sol ution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland Fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyeeride derivatives are useful in the preparation of injeciahles, as are natural phanuaeeutieally- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and ^• dried com starch. When aqueous suspensions, are required for oral use, the active in redient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternati vely, the pharmaceutical compositions of this invention may be administered, in the form of suppositories for rectal administration. These ca be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but. liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa hotter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially to treat skin cancers, psoriasis or other diseases which occur in or on the skin.

Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol,

polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.

Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more

pharmaceutically acceptable carriers. Suitable carriers .include, but are not limited to, mineral oil, sorbitan moiiostearate, polysorbate 60, ceiyl esters wax, cetearyl alcohol, 2-octyidodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micromzed suspensions in isotonic, pB adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical fonmdation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability., fluorocarbons, and/or other conventional so!ubiHzmg or dispersing agents.

The amount of compound in a pharmaceutical composition of the instant invention that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host and disease treated, the particular mode of administration. Preferably, the compositions should be formulated to contain between about 0.05 milligram to about 750 milligrams or more, more preferably about 1 milligram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingredient, alone or in combination with at least one additional compound which may be used to trea cancer, prostate cancer or metastatic prostate cancer or a secondary effect or condition thereof.

Methods of treating patients or subjects in need for a particular disease state or

condition as otherwise described herein, especially cancer, comprise administration of an effective amount of a pharmaceutical composition comprising therapeutic amounts of one or more of the novel compounds described herein and optionally at least one additional bioactive (e.g. anti -cancer) agent according to the present invention. The amount of active ingredient(s) used in the methods of treatment of the instant invention that ma be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. For example, the compositions could be

formulated so that a therapeutically effective dose of between about 0.01, 0.1, 1, 5, 10, 15. 20, 25, 30 , 35, 40, 45, SO, 55, 60, 65, 70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg of the novel compounds can be administered to a patient receiving these compositions. ft should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of -excretion, ^' drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.

A patient or subject (e.g. a male human) suffering from cancer can be treated by administering to the patient (Subject) an effective amount of a chimeric compound according to the present invention including pharmaceutically acceptable salts, solvates or polymorphs, thereof optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with other known anticancer or pharmaceutical agents, preferably agents which can assist in treating cancer, including metastatic cancer or ameliorate the secondary effects and conditions associated with cancer. This treatment can also be- dministered in conjunction with other conventional cancer therapies, such as radiation treatment or surgery.

The present compounds, alone or in combination with other agents as described herein, can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subeutaneoiisSy, or topically, in liquid, cream, get, or solid form, or by aerosol form.

The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated. A preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 ng/kg to 300 mg/kg. preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient patient per day. A typical topical dosage will range from about 0,01-3% wt wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosage form _. , including but not limited to one containing less than Img, 1 mg to 3000 org. preferably 5 to 500 mg of active ingredient per unit dosage form. An oral dosage of about 25-250 mg is often

convenient.

The active ingredient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00003 -30 mM, preferably about 0.1-30 uM. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient Oral administration is also appropriate to generate effective plasma concentrations of active agent.

The concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will, also var with die severity of the condition to be alleviated, it is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the

administration of the compositions, and that the concentration ranges, set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be di vided into a number of smaller doses to be administered at varying intervals of time.

Oral compositions will generally include an inert diluent or an edible carrier. They ma be enc losed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used i the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and th like can contain any of the following ingredients, or compounds of a similar nature: a binde such as i¾icrocrysialline cellulose, gum tegacanth or gelatin; an excipient such as starch or lactose, a dispersing agent, such as algimc acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid earner such as a fatty oil In addition, dosage unit forms ca contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.

The active compound or pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certai preservatives, dyes and colorings and flavors.

The active compound or pharmaceutically acceptable salts, thereof can also be mixed with other active materials that do not impair the desired action, or with materials that

supplement the desired action, such as other anticancer agents, antibiotics, antifungals, antiinflammatories, or antiviral compounds, in certai preferred aspects of the invention, one or more chimeric antibody-recruiting compound, according to the present invention is coadministered with another anticancer agent and/or another bioactive agent, as otherwise described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propyiene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethyienediaminetetraacetic acid; buffers such as acetates, ci trates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic .

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with, carriers that will protect the compound against rapid elimination from the body, such as a controlled and/or sustained release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,

polyanhydrid.es, polygiycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

Liposomal suspensions or cholestosomes may also be pharmaceutically acceptable carriers. These may he prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat No. 4,522,81 1 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate iipkl(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyi phosphatidyl · choline, and cholesterol) in m inorganic sot vent that is then evaporated, leaving behind a thin fACM of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container. The container is then swirled by hand to free lipid raaterial front the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

General Chemistry Overview

The synthetic route begins with A½nyristoyS-D~asparagine (7), which was prepared In one step and high yield by acylation of D-asparagine with myristoyi chloride (Figure 8, Scheme I). ³⁵ EDO-mediated coupling with ( _l V)-hex~5-en~2 -amine (8, prepared in three steps, from pent-4-en-al) provided the amide 9 (84%). Oxidative cleavage of the terminal alkene (ruthenium chloride, sodium periociate), then formed the earhoxylic acid 1β (78%).

The thiazoliiiyi-thiazole side chain was prepared by the sequence shown in Figure 9, Scheme 2A, Treatment of V te ^bntoxyearbonyl)aminoacetonitrUe (1.1) with L-cysteine methyl ester provided the thiazoline 12 (85%). Conversion of the ester to a primary amide (ammonia), folio wed by treatment with Lawesson's reagent, generated the thioamide 13 (>99%). Exposure of 13 to bromopyruvie acid in the presence of triethylamine formed, the thiazolinyi-tliiazole 14 (7.1%). Cleavage of the ter^butoxycarbonyl protecti ve group (hydrochloric acid, dioxane) generated the amine IS (>99%). Silver-mediated coupling (silver trifhioroacetate, triethylamine, 63%) of the amine 15 with the p-ketothioester 16 (prepared in one step

followed by carbamate cleavage (hydrochloric acid, dtqxaue) furnished the thiaz¾Hne- thiazole fragment 17.

The isomeric thiazo!e -thiazo!inyl side chain was prepared by a modified sequence (Figure 9, Scheme 213). Treatment of Af-(fe ^butoxycai'bonyl)-2-aminoetlian-liioamide (1.8) with ethyl hromopyruvai formed the thiazole 19 (74%). Aminolysis of 19 (ammonia) followed by dehydration of the resulting primary amide (trifhioracetic anhydride,

triethylamine, 69%) generated the nitrile 20. Coupling of 20 with L-cysteine formed the bicycle 21 (97%). Removal of the carbamate protecting group (hydrochloric acid, dioxane) formed the amine 22 (>99%). Finally, coupling of 22 with the β-ketothioester 16 (silver tri&toroacetate, toethylarnine, 69%), followed by carbamate cleavage (hydrochloric acid, dioxane) provided the thiazole-thiazoline 23,

To complete the synthesis of the precoHbactla A skeleton, the carboxylic acid 10 was first converted to the β-ketothioester 24 b activation with carbon ditmidazole followed by treatment with 3-(tert-butyltiiio}~3-oxopropanoic acid in the present magnesium ethoxide. Silver-mediated coupling of 24 with the thiazoKne-t azoIe 17 or the thiazole-thiazoline 23 formed the eyclization precursors 25 and 26, respectively (high yield from 10 for 25 and 26, respectively). Altematively, the approach to 25 and 26 proceed through compound 19 as per Scheme C of Figure 9.

2S i ^* s'¾ ftonn 10J 26 ¾om Kt

Assembly of the acyclic precohbactin A precursors 25 and 26 proceeded as per Figun 9, Scheme C. The inventors then studied the cyclization of the tliiazole -thiazoline intermediate 26. Under a variety of conditions, 2 was found to rapidly cyc!ize to generate the pyridone 28. We speculated that 28 is formed.

A more detailed description of the synthetic chemistry nd related experiments associated with the present invention (e.g. a!fcy!ation of DMA, etc.) appears herein below.

Chemical Synthesis and Related Experiments

The colibactins are hybrid polyketide-nonribosomal peptide natural products produced by certain strains of commensal and extraintestinal pathogenic E. coll The metabolites are encoded by the clb gene cluster as pro-drugs termed precolihactins. clb* E. colt induce DNA double-strand breaks (DSBs) in mammalian cells in vitro and in vivo and are found in 55-67% of colorectal cancer patients, suggesting that mature colibactins could initiate tumori genesis. However, elucidation of their structures has been an arduous task as the metabolites are obtained in vanishingly small quantities {jig L) from bacterial cultures and are believed to be unstable. In the present invention, the inventors describe a flexible and convergent synthetic route to prepare advanced precolihactins and derivatives. The synthesis proceeds by late-stage union of two complex precursors (e.g., 28 + 17 ~» 29a, 90%) followed by a base-induced double dehydrative cascade reaction to form two rings of the targets in high yield (e.g., 2.9a→ 30a, 79%). The sequence has provided quantities of advanced candidate precoiibactins that exceed those obtained by fermentation, and is envisioned to be readily-scaled. These studies have guided a structural revision of the predicted metabolite precoiibaetin A (from 5a or 5b to 7) and have confirmed the structures of the isolated metabolites precoiibactins B (3) and C (6). Svirthetic precoiibaetin C (6) was converted to N- myristoyl-D-Asfl and its corresponding colibactin by colibactin peptidase ClbP. The

synthetic strategy outlined herein will facilitate mechanism of action and sinucture-function studies of these fascinating metabolites, and is en visioned to accommodate the synthesis of additional (pre)colibaciins as they are isolated.

Bacteria residing in and on humans (the human icrobiota) play an integral rote tn regulating physiolog and disease. ¹ The intestinal tract has been estimated t contain 500- 1000 species of bacteria constituting - 1 ,5 kg of biomass. ³ Certain strains of gut commensal and extraintestinal pathogenic E. coli harbour a gene cluster (clh or "pks ") that encodes a group of molecules termed precolibactins. ^"> Precoiibactins are substrates for colibactin peptidase ClpP, a protease encoded within the clh gene cluster. ClbP is anchored within ^' the inner peri lasraic membrane of the bacteria ⁴ and removes an Λ'-acyI-D-asparagine side chain from the precoiibactins. This cleavage step converts precoiibactins to cytotoxic eoSibactins and likely constitutes a prodrug resistance mechanism in the bacteria. ⁵ clh* £ ^' . coli induce DNA double-strand breaks (DSSBs) in mammalian cells in vitro ^* * and in vim Host inflamniation promotes proliferation of E. coli' and expression ofd¾>,* the clh pathwa promotes colorectal cancer in colitis-susceptible mice treated with azoxymemane ^' and two studies revealed the presence of cW E, coi in 55-67% of colorectal cancer patients, ' ' ⁹ Collectively, these data suggest that colibactms initiate tumori enesis by a mechanism in volving induction of DN A DSBs.

Fully elaborated (pre)co!ibactins have been difficult to isolate in homogenous form, and the definitive structures of the most active nietabolite(s) are not known. This has been attributed to the low levels of natural production of the metabolites, their instability under fermentation conditions, and the inflammation-dependant up-regislation of the nati ve clh gene cluster. The metabolites Ϊ 2, ^lW 3 (referred to hereafter as "precolibactin B"), ⁱ⁰ and 4 ⁱ⁰ were obtained in vanishing!)' small quantities (2.5-55 pg/L for 2 -4) from the fermentation broth of genetically-engineered cW E. coli and implicated as shunt metabolites- and/or degradation products in the colibactin biosynthetic pathwa (Figure 1 ). Using the isolation of 2, as well as HRMS analysis, isotope labelling, and bioinf rmatics based on established biosynthetic logic, the structure of precolibactin A was predicted as 5a or 5b. ^50a Key elements within the proposed structures include a hydrophobic iV-iermtrsal fragment, a spiraeyclie

aminocyelopropane, and (read from left to right) a raiazoline-thiazole chain. As the presence of the thiazolme-thiazole fragment was inferred by bioraforraatie analysis,"'* 5a and 5b could not be imequivocaily distinguished at that time, and the absolute stereochemistry of the putative tiiiazolme ring was not determined. A compound with an exact mass corresponding to 5a was observed in ittipurified extracts, but ail efforts to isolate this structure were hampered by its low levels of production and instability, ^ls The -pyridone structure 6 (referred to hereafter as "precolibactin C") was recently proposed as a candidate precolibactin based on biosynthetic considerations, isolation of precolibactin B (3), and HRMS analysis and during the preparation of this manuscript, Balskus and co-workers reported the isolation of precolibactin C (6) from a mutant strain (0.5 mg of 6 was obtained from an optimized 48-L fermentation).* ^* Although one can. envision eyclodehydration of 5a or 5b to form pyridones resembling 6, the biosynthetic relationship between these structures had not been established. 2 was shown to weakly cross-link DNA in vitro, ⁱ suggesting that, the colibactins may damage DMA by induction of replication-dependant DSBs, ^{1 '} Detailed stracture-ftmction analyses of the colibactins have been impossible to conduct owing to their low yields of natural production and the absence of a synthetic route to the targets. However , the aminocyelopropane fragments within 2 4 are reminiscent of yatakemy n, CC-1065, and the duocarmycins, which have been shown to alkylate DNA vi nucleophilic ring-opening, ¹⁴ and the biheterocyciic fragment may serve as a DNA intercalation motif. ^{3 ''}

In light of the immense difficulties associated with isolating- natural preeolibacfios. chemical synthesis provides an attractive avenue to resolve the ambiguities surrounding the composition of the active metaboiite(s) and to enable mechanism of action and structure- function studies, Studies indicate the presence of an aminomalonyl unit in the biosynthetic pathway,^' ⁵⁶ suggesting additional colibactins are formed, but no evidence relevant to the siructiires of these metabolites exists, to our knowledge. Consequently, the inventors initially focused on the synthesis of the predic ted structures of precolibactin A (5a and 5b) and precolibactin C (6), as these represent the most advanced precolibactms for which structural data had been presented. In the present application, the inventors report a convergent and high-yielding synthesis of structures 5a and 5b by eye ligation of a fully linear precursor, establish that these materials are distinct from natural precolibactin A, propose and validate by synthesis a revised structure for precolibactin A (as ?), demonstrate that acyclic precoiibaciins undergo cyclodehydration to form the pyridone residues found in precolibactin B (3), 4, and precolibactin C (6) under mild conditions, and confhtn the sunerures of precoiibaciins B (3) and C (6) b total synthesis. The inventors anticipate that this route will be amenable to synthesis of more advanced structures, including those contai ning

aminomalouyl units, as they are proposed,

ResuMs

As shown in Figure ! _s at the time the inventors began their studies, the structure of precolibactin A had been predicted as 5a or 5b, Consequently, the inventors designed their synthetic route to accommodate either heterocyclic sequence and to provide access to the bithiazole found in precolibactin C (6). The synthesis of the common left-hand fragment began with A (fc M>utoxycarbonyi)4>-aspaxagine _; which was coupled with (,S)~hex-5-en-2- araine (8, prepared in three steps, 96% yield, aad 88% ee -from pe«t~4-e«-ai) ^! using -V~(3- dimethylaminopropyl)-i^^thyicatbodiimide hydrochloride (EDOHCl). Cleavage of the tert- butoxycarbonyl protective group (hydrochloride acid) provided the amine hydrochloride 9 (84%, two steps). Acyktion of the amine 9 with myristoyl chloride, followed by oxidative cleavage of the alfcene (ruthenium chloride, sodium periodate) generated the carboxylic acid 10 (78%, two steps).

The isomeric thiazoi fte -thiazole and the related bithiazole fragments were prepared by the sequences shown in Figure 9, Scheme 2. Deuterated cysteine labelling experiments ¹⁸ supported preservation of the L-araino acid configuration in precolibactin A ¹⁰ * so we selected L-cysteine as the building block for the thiazoline ring. Treatment ofN-((er(- butoxycarbo»yl)amiftoacetonitrile (11) with L-cysteine ethyl ester provided the thiazoline 12 (85%, Figure 9, Scheme 2A). Aminoiysis of the ester, followed by heating with Lawesson's reagent, generated the thioamide 13 (>99%, two steps). Exposure of 13 to bromopyruvic acid in the presence of triethylamine formed the thiazoline - thiazole 14 (71%). The thiazoline- thiazole and subsequent intermediates were f und to be exceedingly unstable toward

hydrolylic ring-opening and, to a lesser and variable extent, oxidation to a bithiazole. Accordingly, the identiffcation of conditions to isolate and purify these intermediates without exposure to water was essential to the success of the route. Cleavage of tiie ten- butoxycarbonyl protecti ve group (hydrochloric acid. >99%) generated the amine 15.

Coupling (silver trifJuoroaeetate, txiethyS amine) of the amine 15 with the p-ketothioester 16 (prepared in one step and 56% yield "from iV^iew-bntoxycafbonyi^l-aimnocyclopfOpane-l- carboxylate)' ' followed by carbamate cleavage (hydrochloric acid, >99%) furnished the thiazolme— thiazole fragment 17.

The isomeric thiazole-thiazolme fragment was prepared by a modified sequence (Figure 9, Scheme 2B). Treatment of A^/er^kttoxyc^ (18) with ethyl bromopyruvate formed the thiazole 19 (74%). Aminolysis of 19 followed by dehydration of the resulting primary amide (trifluoroacetic anhydride, trieihylamine) generated the nitrile 20 (84%, two steps). Coupling of 20 with L-cysteine formed the thiazo!e- thiazo ne 21 (97%). In contrast to the isomeric intermediate 14, 21 was found to be stable toward aqueous workup and atmospheric oxygen. A three-step sequence analogous to that described above provided the thiazole---thiazoline 23 (69% overall).

The bithiazole fragment was prepared by the sequence shown in Scheme 2C.

Aminolysis of the ester 1 followed by heating with Lawesson's reagent formed the thioamide 24 (>99%, two steps). Treatment of the thioamide 24 with bromopyruvic acid in the presence of calcium carbonate formed the bithiazole 25 (58%). Prior efforts to prepare and isolate 25 were impeded by its instability; ¹⁹ we found that rigorous exclusion of water during work-up and purification facilitated the isolation of 25 and subsequent intermediates in homogenous form. A three-step sequence analogous to that used to prepare ! 7 and 23 then generated the bithiazole ^' fragment 27 in high yield (72% overall).

To complete the synthesis of the precolibactin A skeleton, the carboxyhc acid 10 was first converted to the β-ketothioester 28 by activation with carbonyl diimidazole followed by the addition of 3-(/er/-butylthio)-3-oxopropano*c acid and magnesium ethoxide (Scheme 3). ^* ° Silver-mediated coupling of 28 with the heterocyclic fragments 17, 23, or 27 then formed the penultimate intermediates 29a-c (90%, 87%, and 86% for 29a, 29b, and 29c, respectively). The stabilities of the fully linear precursors 29a -e paralleled those of 17, 23, or 27; the thiazole-thiazolme 29b was stable toward aqueous work-up, while the thtazolme-thiazole 29a and the bithiazole 29c were unstable toward aqueous conditions. Given the higher stability of the thiazole-t iazoline 29b, this compound was used to develop conditions to effect the key cyclization reaction (to 5b). Surprisingly, the inventors found that in preparative experiments treatment of 29b with potassium carbonate in methanol at 0 ^e C resulted in formation of the pyridone 30b (80%, Scheme 4A). Similar outcomes were obtained on exposure, of 29b to ammonium carbonate in ethanoS or aqueous sodium hydroxide. Under these conditions, accumulation of the putative monocyclized intermediate 5b was not observed (LC/MS analysis). The pyridone 30b was folly characterized and spectroscopic data for this compound were in good agreement with the isolated metabolite precoiibactin B (3; Table 1, infra)., hi particular, H-2 and H-4 of Mb resonated at 6, 16 id 5.59/5.50 ppm, respectively; these values are nearly identical to those recorded for natural precoiibactin B (3; 6.16 and 5.61 5.48 ppm, respectively)." A plausible mechanism for the formation of the pyridone 30b involves cyclodehydration to 5b, 1,2-addition of the primary amide to the adjacent cafbonyl, and aromatkation. The facile formation of 30b from 29b provides evidence that the putative and isolated colibactin metabolites 3, , and 6 may derive from related acyclic precursors, although the timing of cyciizaiions in the modular biosynthetic pathway remains unkno wn. The cyclization of the thiazoline - thiazole derivati v e 29a and the bithiazele derivative 29c proceeded i a strictly analogous manner to provide the fully cyclized derivatives 30a or precoiibactin C (6), respectively. ^{5 '} The mass spectroscopic fragmentation data and ^} H NMR data for synthetic precoiibactin C (6), as well as LC/MS co- injection with metabolite extracts, matched those of natural material (Figure 27, Figure 12A and B and Figure 1 ). ^50,12 In addition, synthetic precoiibactin C (6) was converted to N- myristoyl~D-Asn and its corresponding colibactin in a C!bP-depende manner, indicating that precoiibactin C (6) represents a suitable substrate for ClbP (Figure 29). This sequence pro vided raultimilligram quantities of 30a, 30b, and precoiibactin C (6), and is en visioned to be easily scalable.

The inventors ultimately found that treatment of 29b with potassium carbonate (-3.0 equiv) in dimethyl sulfoxide at 24 °C proceeded more slowly and allowed for detection of 5b in the reaction mixture (LC/MS analysis, Figure 1 1 , Scheme 4B). By conducting the reaction in dimethyl sulfoxide-*/*, signals consistent with the monocyclized intermediate 5b could also be observed by Ή NMR analysis (Table 1, below).

Tab!e 1. Selected Ή chemical shifts and LC/MS data for 29b, 5b, 30b, and precolibactin B (3):

Posi!ion 1 2 3 4 t _f (min)

8.83 3.60 8.94 4.56 3.52

presented in the Supporting information, Not resolved.

Mass-selective LC HR S-QTOF analysis was conducted to determine if either 5a or 5b corresponded to the structure of natural precolibactin A. As shown in Figure 12, the concentrated ethyl acetate extracts of city E. coti AelhP ⁰ * displayed a single prominent peak. of /∑ ~ 816.3788, which corresponds to [M+Ffj ^r for the proposed structure of precoHbactin A. However, the retention times of synthetic Sa and 5b were distinct and the signals did not coalesce upon co-injection with the natural sample (Figures 2A and Ί 2Β, respectively). The retention times of 5a and 5b were nearly identical (t _T ^~ .15.70 and _. .15.80 mm, .respectively), as expected, and their differences with respect to natural precolibactin A (t _v = 16,56 min) suggested a significant discrepancy in structure.

In light of the facile cyclization of our synthetic intermediates to the pyridone residues found in precolibaetins B (3) and C (6), the inventors reasoned that precolibactin A may also incorporate this substructure. The original isotope labeling, FIRMS, and MSMS data for natural precolibactin A ^u¾ could not exclude this assignmen In addition, careful inspection of the initial report revealed, that precolibactin A production was optimized by increasing the concentration of L-cysteine in the media 5-fold (to 1 g/L). Walsh and co-workers have previously repotted the formation of cysteine derailment products in the biosynthesis of yersiniabactin. ² ' Accordingly, we hypothesized that the structure of natural precolibactin A may comprise the pyridone found in pteeolibaetins B (3) and C (6) and a terminal cysteine residue appended to a single thiazole ring (Figure 1.3, Scheme 5 A). This structure (7) possesses an exact mass that is identical to the originally predicted structures 5a or 5b and would similarly match the reported amino acid isotope labeling studies. Such a change in the terminal heterocyclic fragment would also be consistent with the large differences in retention times between 5a or 5b and natural precolibactin A. The synthesis of the revised precolibactin A structure 7 was readily-accomplished using our synthetic strategy (Figure 13, Scheme SB). Treatment of-V^/er/-butoxycaibonyl)~2^inittoethanthioamide (IS) with bromopyruvic acid ²² followed by removal of the ier/-butoxycarbonyl protective group generated the thiazole 31 (74%, two steps). Silver triilimioacetate-mediated coupling of 31 and the thioester 16, followed by carbamate cleavage, formed the amine 32 (55%, two steps). Coupling of the amine 32 with the thioester 28 (Figure 10, Scheme 3; silver txtfluoroacetaie, triethylamrae). followed by double-cyelization (potassium carbonate, methanol) generated precolibactin B (3; 67% over i¾¾ steps, 36 nig). NMR spectroscopic dat for synthetic precolibaciin B (3) and LC/MS co-injection with, metabolite extracts matched those of natural material, ^{' ' '1'"'} thereby confirming the structure of the natural product ^{i !} Finally, coupling of precolibaciin B (3) with L-cysteine mediated by A-hydroxysuccinimide (NHS) and EDC-HCI generated 7 (89%), Mass-selective LC HRMS-QTOF analysis against the concentrated ethyl acetate extracts ofc £■<¾># lbP ¹¹ * revealed that 7 corresponded exactly to natural material (Figure 14). in addition, both synthetic 7 and natural precolibaciin A displayed identical mass spectral fragmentation patterns, providing further confirmation of structure (Table S2). As natural precolibactin A. is not iselabie in amounts sufficient for NMR analysis, ^K '* a direct comparison of the NMR spectra of synthetic and natural precolibactin A is not possible at ibis time. ^**

Discussion and Conclusion

The eoiibactins are a fascinating family of nat ural products that are produced by certain strains of commensal and extraintestinal £. coi ^' i, and the pathway has been implicated in the progression of colorectal cancer. ^" ' ^' Despite over a decade of intensive research, their complete simctures and mechanism of action have remained unresol ved. As highlighted in Figure i, many of these compounds have been isolated in astoundingly low yields (pg/L) by painstaking fermentation experiments. By bringi ng the power of modern bioinfoonatics, enzymoiogy, and mass spectrometry to bear on this problem, the struc tures of additional precoiibaetins, which are recalcitrant to isolation, have been predicted.

At the time the inventors began their work, 3-6 represented the most comple precolibactin structures in the literature. Precolibactm B (3) and 4 were folly characterized by isolation,'* ^' while 5» ^Ms and precolibactin C (6)' ³ were predicted. While this manuscript was in preparation, Balskus and co-workers' ² reported the isolation of precolibactin C (6; 0.5 mg horn a 48 L fennentation) from a mutant stein. Biosynthetie studies now suggest that additional precolibactms. incorporating an ^■ ammomalonyl unit exist, ^' '" ³⁶ but no evidence for their structures has been presented, to our knowledge.

The inventors have developed a high-yielding and modular synthetic route to the most advanced known precolibactin structures. Tire left band fragment 1 , which is common to all of the preeoli actins, is prepared in six steps and 63% overall yield frompe»t«4~en-al (Figure 8, Scheme 1). The inventors ha ve executed the synthesis of four distinct heterocyclic side chain fragments, in 4-7 steps and 37- 1% overall yield (Figure 9, Schemes 2 and Figure 13, Scheme 5). Finally, these intermediates are elaborated to advanced precolibactins in three steps and -50% overall yield (Figure 10, Scheme 3, Figure 1 1, Scheme 4, and Figure 13, Scheme 5), The inventors have confirmed the structures of the isolated precolibactins B (3) and C (6) by chemical synthesis, and rev ised the structure of precolibactin A, from 5a or 5b to 7. This structural revision also supports an unexpected biosynthetic route to colibaetin bithiazole formation, in which biosynthesis of the first thiazote ring may precede

he eroeyelization and oxidation of the C-terminal L-cysteine moiety. This is in contrast to bioinformatic proposals for bleomycin bi thiazole biosynthesis/ ⁴ These synthetic studies also pro vide insights into the reactivities of these striictures, and the facile cyclization of the linear precursors 29a-c to pyridones suggests this element as a common substructure. The inventors envision that the synthetic strategy we have presented will be amenable to the synthesis of precolibactins incorporating an aminomalony! substiruent or other modifications, as their structures are proposed.

The synthetic route outlined herein provides a means to procure sufficient quantities of material to study the cellular responses to isolated coHbactins and elucidate their mechanism of action for the first time. As noted in the introduction, mammalian ceils were shown to accumulate DMA DSBs when co-cultured with pks ' £. coii cells; ¹⁸ but no essential follow-up studies employing single metabolites derived from the clb pathway have been reported, to our knowledge. The inventors expect that their modular synthetic strategy will finally open the door to examining these types of questions regarding colibactin's mode of action with molecular-level resolution." ⁵

References for this Section

1. For a review, see: Do ia, M. S.; Fischhach, M. A. Science 20! 5, 349, 395. 2. Xu, 1; Gordon, J. I. Proc. Natl Acad. Set. II S. A. 200 _» 100, 10452.

3. (a) Nougayrede, j.-P.; Homburg, S.; Taieb, F.; Boury, M.; Brzuszkiewicz, £.; Gottschalk, G.; Buchrieser, C; Hacker, J.; Dobrindi, U.; Oswald, E. Science 2006, 3/5, 848. For recent reviews, see: (b) Balskus, E. P. Nat Prod. Rep. 2015. 32, 1534. (c) Trautman, E. P.;

Crawford, J. M. Curr. Top, MM. Chew, 2015, 16. I .

4. Dubois, D.; Baron, O.; Cougnonx, A.; Delmas, J.; Pradel, ; Boury, M; Bouchon, B.; Briiiger, M. A.; Nougayrede, J. P.; Oswald, E.; Bonnet, R. .1 Biol. Chem. 201 L 286, 35562,

5. (a) Btotherton, C. A.; Balskus, E. P. J. _4w. Chem. Sac. 2013, 755, 3359. (b) Bian, X.; Fa, J.; Plaza, A.; Herrmann, J.; Pistori s, D.; Stewart, A, F.; Zhang, Y,; Midler, R. Chembiochem 2013, 74, 1194. (c) Vizcaino, M, F; Engel, P.; TrairfBian, £.; Crawford, 1 M. J Am, Chem. Sac. 2014, /56 ^" , 9244,

6. Cuevas-Ramos, G.; Petit, C. R.; Marcq, 1 ; Boury, M; Oswald, E.; Nougayrede, 1-P, Proc. Nad. Acad. Sci. U, S. A. 2010, 107, 1 1537.

7. Arthur, J. C; Perez-Chanona, E.; Miihlbauer, M; Tomkovich, $.; Uronis, J. M.; Fan, T.- 1.; Campbell, B. 1.; Abujamel, T.; Dogan, B.; Rogers, A. B.; Rhodes, J. M; Stintzi, A.;

Simpson, K. W.; Hansen, J. J.; eku, T. O.; Fodor, A. A.; Jobin, C. Science 2012, 338, 120.

8. Arthur, 1.€.; Gharaibeh, R. Z.; Muhlbauer, M.; Perez-Chanona, E.; Uronis, 1. M.;

McCafferty, J.; Fodor, A. A.; Jobin, C. .Mr/. Cwmtnm, 2014, 5, 4724.

9. Buc, E.; Dubois, D.; Sauvanet, P.; Raisch, J.; Delmas, J.; DarfeuUle-Michaud, A.; Pezet, D.; Bonnet, R, 7 ^> 7..i>S <¾e 2013, 8, e56964.

10. (a) Vizcaino, M. L; Crawiord, J. M. Nat Chem. 2015, 7, 1 1. (b) Bian, X.; Plaza, A.; Zhang, Y.; Mfiller, R. (¾w. &¾< 2015, 6, 3154. (c) Brotherton, C. A.; Wilson, ,: Byrd, G.; Balskus, E. P. Org. Lett, 2015, 17, 1545.

1 1. Li, Z, R,; Li, Y.; Lai, J. Y.; Tang, J,; Wang, B.; Lu, L.; Zbu, G,; W« ₅ X.· Xu, Y. ; Qian, P. Y. Chembiochem 2015, 76 ^" , 171 .

12. Zha, L.; Wilson, M. R.; Brotherton, C. A.; Balskus, B. J*. ACS Chem. Biol 20 6, [Online early access]. DOf: 10.102i /acschembio.6b00014. Published Online: Feb. I S, 2016.

http://pubs.acs.org/doi/abs/! 0.102 l/acschembio.6b00014 p (accessed March 28, 20 6).

.13. Zhang, j.; Walter, j. C. DNA Repair 2914, .19, 135.

14. Tichenor, M. S.; Boger, D. L. Nat Prod. Rep. 2008, 25, 220.

15. Wii, W.; Vanderwall, Ό. E.; Turner. C. 1.; Boehn, S.; Chen, J.; Kozarich, J. W.; Stubbe, 1 Nucleic Acids Res. 2002, 30, 4881.

16. Brach ann, A. O.; Garcie, C; Wis, V.; Martin, P.; tleoka, R.; Oswald, E.; P el. J. Chem., Commm. 2015, 57, 13138. 17. See Supporting Information..

18. Bode, H, B.; Reinier, D.; Fuchs, S. W.; Kirehner, F.; Dauth, eglef, C; Lorenzen, W.; Brachmana, A. 0.; Grim, P. Chem. Eur. J 2012, 18, 2342.

19. Souto, J. A.; Vaz, E.; Lepore, I.; Peppier, A.-C; Fraaci, G,; Alvarez, R,; Altucci, L: de Lem _s L R. J. .MM Chem, 201.0, 55, 4654.

20. Sodeoka, M.; Sampe, R.; Kojinra, S.; Baba, Y.; Usui, T.; Ueda, K_; Osada, H. J. Med Chem. 2001, 44, 3216.

21. Gearing, A. M.; Mori, I.; Perry, R. &; Walsh, C. T. Biochemistry 1998, 57, 11637.

22. Videnov, G.; Kaiser, D,; Kempter, C,; Jung, G. Chem., Int. Ed. 1996, 55, 1503.

23. Synthetic pfecoMbactin A (7) was found, to readily undergo disulfide bond formation, which may explain its instability in complex cellular organic extracts,

24. Galm, ϋ,; Wendt-Pienkowski, E.; Wang. L.; Huang, S.-X.: Orssia C.i Tao, M;

Coughlia, J. M.; Shea, B. J. Nat. Prod. 2011, 74, 526.

25. Our data suggest that prior proposals of the colibactin mechanism of action may require revision. See the Supporting information for a discussion.

D A Alkylation and Related E e iments

The Precolibactins and coiibactins represent a family of natural products that are encoded by the clb (aknpks) gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb ^~ E, coli have been shown to induce raegalocytosis and DNA double-strand break formation in eufcaiyotic cells. Paradoxically, this gene cluster is found in the probiotic strain issle 191?, which is used for the treatment of gastrointestinal disorders. Evidence suggests that the precolibactins are converted to genotoxic colibaciins by coiibactifj peptidase (C1bP)~mediate4 cleavage of an A'-acyl-D-Asn side chain. It has been hypothesized that coiibactins exert genotoxicity by formation of an unsaturated inline and nucleotide alkylation by cyclopropane ring opening {2-~»3, Figure 1.5 A, Scheme 6).

However, no coiibactins ^' have been directly isolated from producing organisms, and this hypothesis has not been tested experimentally. In addition, many advanced precolibactins such as precolibactins A.-C (7-9, Figure .1 SB) contain a pyridone residue that cannot generate the unsaturated imines that form the basis of this mode of action hypothesis. These examples evaluate the DMA binding and alkylation activity of 13 synthetic pyridone (5) and unsaturated imine (3) colibactm derivatives and show that the unsaturated imines potently alkylate DNA, whereas the corresponding pyridone derivatives do not. The inventors define the imine. unsaturated l actam, arid cycl opropane functional groups of these molecules as essential for efficient DNA alkylation. The presence of a cationic side chain enhances DMA alkylation. These studies suggest thai precolibactins containing a pyridone residue do not form the basis for the genotoxicity of the clb gene cluster. Instead, as al l precolibactins to date have been obtained from cihP mutant strains, the inventors propose that these are off- pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The structure- nction and mechanistic models presented herein will ultimately provide a means to connect metabolite structure with the disparate phenotypes associated with clb ^y & coli,

Precolibactins and coiibactins are natural products produced by select commensal, extraintestinal, and probiotic E, coli. The metabolites are encoded by a hybrid polyketide synthase-nonribosomal peptide synthetase (P S-N PS) gene cluster termed clb otpks. ¹ clb . coli strains induce DN A damage in eukaryotic cells and are thought to promote colorectal cancer formation, ^Κ but the gene cluster is also found in the probiotic strain Nissle 1 17, which is used in Europe for the treatment of ulcerative colitis, diarrhea, and other gastrointestinal disorders Mature preeolibaetins are substrates for the 12 rans embtane multidrug and toxic compound extrusion transporter ClbM, which mediates their transfer to the bacterial periplasm." ¹ There, the eolibactin peptidase ClbP converts preeolibaetins to genotoxic colibactins via removal of an N-acyi-D-asparagine side chain/ Mutation of ClbP abolishes cellular DNA damaging-activity, ²⁸ ^ and A¾¾yrisioyl-D~asparag5.ne and closely related analogs have been identified in wild type cih ^* E. co/ cult res. ^'5d,¾ Whether the differential production of biosyntheticalJy-related. but distinct metabolites, or other factors (such as the requirement for celi-to-cell contact to observe cytopathic effects ²⁸ "*') underlie the seemingly contradictory phenotypes associated with the cih gene cluster, remains unresolved.

The inventors have focused on understanding the molecular basis of colibactin- induced DNA damage. Advanced preeolihacims arise from linear precursors of the generalized structure shown as 1 (Fibure 15, Scheme 6). The linear precursors were suggested to transform to unsaturated lactams 2 that are processed by ClbP to generate unsaturated iminium ions 3 (colibactins), which alkylate DNA by cyclopropane ring-opening (grey pathway). '" However, this mechanistic hypothesis is ostensibly incompatible with subsequent isolation ⁹ and synthesis' ⁰ efforts that lead to the identification and unequi vocal structural assignment of precoiibactms A (7), ^H B (8), and C (9), which contain a pyridone residue (Figure 15B). Preeolibaetins A-C (7-9) were obtained from cibP mutant strains; these deletion strains were employed to promote accumulation of the precoltbactm

metabolites, i f 7-9 are the genotoxic precursors, the data outlined above ⁵ suggests that amines such as 5 resulting from ClbP-mediated processing in the wild type strains are responsible for the cytopathicity of the clh cluster, as these c annot readily convert to

unsaturated iminium ions such as 3. Precolibactin C (9) was demonstrated to be a substrate for ClbP. ⁰

In earlier synthetic work, we showed that the double dehydrative cyclizaiioR of the relatively stable N-acylated linear precursors (1) to pyridones such as 7-9 was facile under mildly acidic or basic conditions (c.f, l-»2-»4. Scheme l). ⁱ⁰ The unsaturated lactam intermediates (2) could be detected by LC/ S analysis, bu they were not isolabte, arguing agains their interception by ClbP in the biosynthesis. We reasoned that the colibactins ma instead form by ClbP processing of isolahle linear precursors J. directly. Sequential

eyc!odehydration reactions proceeding through the viny!ogous ureas 6 would then provide 3 (red pathway). It follows from ibis analysis that precolibactins A-C (7-9) are non-natura! cyclization roducts deriving from the absence of ClbP in the producing organisms, and are unlikely to be genotoxic. To test this hypothesis, the inventors modified their synthetic strategy' ⁰ to allow access to the deaey!ated pyridone derivatives 5 and the analogous

'unsaturated imi utn ions 3. The show that the iminiurn ions are poten DNA alk ylation agents while the corresponding pyridone structures are not. Sn addition, we rigorously define the stnicture-fimction relationships of 3 that are required for or enhance DMA alkylation activity. Finally, the synthetic studies support the alternative biosynthetic pathway involving the intermediacy of the vinylogous amide 6 en route to 3. Collectively, the data lend further support to the hypothesis that unsaturated iminium ions 3 are responsible for the genotoxic effects of the clh gene cluster and support the conclusion that precolibactins A-C (7—9) (and other pyridone-containing isolates) are off-pathway fermentation products derived from the absence of a functional ih gene. This work constitutes the first strucrure-fimction studies of colibactin metabolites and provides a foundation to begin, to connect the disparate phenotypic effects of the cib cluster with metabolite structure.

Results

Comparison of 1> A binding and alkylation activities of unsaturated imminm ion and pyridone structures. To assess the activities of candidate coHbactm scaffolds, we required a route that would provide access to unsaturated inline and pyridone derivatives lacking the natural JV-n yristoyi-D-Asn prodru fragment Synthesis of the unsaturated imlne derivati ves was a significant challenge since we have previously shown that base- or acid-catalyzed cyciization of the linear precursors proceeded rapidly to die pyridone products. ⁵⁰ in. addition, we targeted synthetic derivatives containing a cationic residue appended to the bithiazole fragment. It has been shown that an a-aminomalonate building block is incorporated into the colibactin structure;*' ' ² we speculated that this residue could enhance DNA affinit by imparting cationic character to the metabolites, similar to other known DMA-targeting agents. ^{i J} As the presumed natural C-femiina! colibactin cationic substituent remains unknown, we initially targeted an iV-( -dimethyiaminoethyI)amide derivative, as this is a common substructure in agents that target DNA. We later prepared a series of other

derivatives to further explore this region and optimize activity (vide infra).

After some experimentation, the inventors identified the linear colibactin derivative 1 as a common intermediate ^' that could be selectively cycli ed to provide either ^' unsaturated inline or pyridone products (Figure 16, Scheme ^' ?). Sil ver-mediated coupling of the β~ ketothioester 10 (prepared hi one step and 53% yi eld by the addition of lithium ethyl fhraacetate to tert~b&ty\ («S -2-methyI-5^xopyrroIidine~l arbox.ylate, ^'14 see Supporting Information) with the β-ketoamide 1 l ^m provided the linear precursor 12 (I~3 g scale). The linear precursor 12 cycUzed to a variable extent to the vinylogous imide 13 upon purification by anion exchange cl omatograpliy. To effect quantitative cycKzation, th purified mixture of 12 and 13 was dissol ved in 0.5% formic acid-5% methanol-acetonitrile and concentrated (3x). Following this protocol, the vinylogous imide 13 was obtained in 87% isolated yield (over two steps) and >95% purity. Coupling of the carboxylic acid function with N tf- dirnethylethyienediarnine mediated by propylphosphonie anhydride (T3P) " provided the amide 14a (93%). Carbamate cleavage (trifiuoroaeetie acid) followed by neutralization with saturated aqueous sodium bicarbonate solution (to promote cyclodehydration) then provided the key unsaturated imine 15a (62%).

Alternatively, the direct addition of potassium carbonate (5 equiv) and methanol ¹ " to the solutio of the unpurified coupling product 12 induced double cyclodehydration to provide the pyridone 16 (78% from W and 11). T3P~mediated coupling with >¥,.<¥- dimethyiethyienediaiiiine provided an amide (not shown) that was treated with trifiuoroacetic acid to yield the amine 17a (40%, two steps). The -¥~methyiamide derivatives 15b and 17b were targeted to directly evaluate the significance of a cationie terminal residue on DNA binding and damaging activity. These were prepared by strictly analogous sequences .

The activity of 15a, 15b, 17a, and 17b was evaluated in a DNA alkylation assay, Plasmid pBR322 DNA was treated with EcoRl-H in Cutsmart buffer, and the resulting linearized DNA was incubated with 15a, 15b, 17a, or 17b for 15 h at 37 °C, The DNA was then denatured, and the sample was eluted on I % neutral agarose gel. DNA was visualized with SybrGold. Cisplatin (CP, 100 μΜ) and methyl methanesulfonate (MMS, 500, 100, or 10 μΜ) were used as positive controls for DNA cross- Sinking and alkylation, respectively. As shown in Figure 17 A, neither of the pyridone deri vati ves 17a or 17b displayed measurable activity in this assay at concentrations up to 500 μΜ. By comparison, both unsaturated imines 15a and 15b extensively alkylated DNA. The streaking of DNA induced by 15a and 1.5b parallels tha t observed with 500 μΜ of MM S and deri ves from the presence of shorter DNA strands that are formed by thermal o alkaline destin tion of extensively-alkylated (or in the case of M S, methylated) DNA, ³⁶ Farther evidence for direct alkylation by 15a and ISb is presented in the cross-linking study outlined below. The derivative 15a, which hears a tertiary amine substttuent that can engage in an. electrostatic interaction with the deoxyribose backbone, was significantly more active than the Λ'-raethylaraide J 5b. These data indicate that the pyridone .residue is unlikely to constitute the genotoxic phantnacophofe of the colibaetins and establis the unsaturated lactam or irnine, and cationic terminus as key functional groups in DMA alkylation activity.

A full dose response of the most potent derivative 15a was conducted. As shown in Figure 17B, extensive DNA alkylation was observed at concentrations as low as 100 n.M, and small amounts of DNA damage were detected using 50 or 10 nSVi of 15a. At 5 nM of 15a the extent of DNA damage was negligible,

A DNA melting temperature analysis was conducted to determine if the variance in DNA alkylation activity between ^' the pyridones and the unsaturated itnines was due to differences in DNA binding affinity. The hyper chromi city of calf thymus DNA (ctDNA) with increasing temperature was measured at 260 nra in the presence of varying

concentrations of 15a or 17a. The T _ro was defined as the temperature at which half of the duplex DNA was unwoimd and was determined by the maximum of the first derivative of the thermal denaturation profile. As shown in Figure I SA, treatment of ctDNA with 17a led to a dose-dependent increase in the melting temperature; an increase in T _m of 3.8 "C was observed at a ratio of 17a to base pairs (bps) of 4.0. By comparison, the unsaturated imine 15a exhibited time-dependent effects on duple stability (Figure I8B). At times less than 1 h, stabilization of the duplex was observed,, but upon longer incubations (3-15 h) duplex stability decreased. The inventors hypothesized that this was due to an initial binding of 15a to the duplex followed by slow alkylation and degradation. To probe this, they repeated the plastnkl alkylation assay using 15a and evaluated the extent of DNA alkylation as a function of time. As shown in Figure 18C, alkylation activity correlated approximately with die decrease in duplex stability observed in the melting point, experiments (Fi ure 18B).

Importantly, the affinity of 15a for DNA was less than that of 17a (1.8 and 2.8 °C increase in T _w , respectively, at ¾ _¾11( ι¾ _ρ - 2) and thus the variance in DNA alkylation activity between these two compounds cannot be attributed to decreased binding of 17a. Stracture-fanctiott relationships of unsaturated iitiiites. To gain further insights into the functional group ^' requirements for DMA alkylation and to verify the observed activities, we prepared the dirner 15c and the gem-dirnethyl. derivative I5d (Figure 19 A), As s ow in Figure 19B. incubation of linearized pBR322 DNA with 10 μ.Μ of the dimer 15c and monitoring the mixture as a function of time revealed a clear cross-linked hand at 3 h.

Consistent with the · time-dependent alkylation. assay shown in Figure 18C, this cross-linked band diminished at later ttmepoints (6 or 15 h), suggesting extensive alkylatio and degradation of the duplex. The ew-dimethyl derivative 15d did not alkylate DMA at concentrations up to 500 μΜ, These data further validate the nature of the DMA lesion as alkylation and indicate that the cyclopropane ring is essential for this activity.

To gain insights into the molecular mechanism of alkylation, we studied the reactivity of 15l in vitro using propanethiol as a model iiucleopoile (Figure 20, Scheme 8). The N- methy!amide 15b was chosen in preference to the p-(dimethy!amino)ethyl amide 15a to simplify isolation. Treatment of the methylamide 15b with excess propanethiol and/?- toluetiesulfonic acid in 6:1 acetonitrile^A V-dimetbylformajnide at 23 °C provided the ring- opened product 18 in 34% isolated yield. The adduct 18 was unstable but was characterized by NMR H, °C, gCOSY, gHSQC, gHMBC) and LC/ S analyses. Cyclopropane ring- opening was evidenced by loss of the resonances at 1.37 and 1.68 ppm (positions a and b in 15b) and generation of a four-proton system centered at 2.57 ppm (positions a, b in 18),

A series of additional compounds were prepared to further interrogate structar - function relationships and optimize DNA alkylation activity (Figure 21. A). The derivatives 15e~15i were synthesized to probe tlie influence of the nature of tlie cationic residue on DM A alkylation activity. All compounds were found to potently alkylate DMA although qualitative differences in activity were observed depending on the nature of the cati oni c residue

(guanidine < primary amine < tertiary amine). Finally, to rigorously determine if iminium ion formation is necessary' for efficient, alkylation, the inventors prepared the unsaturated lactam 19 and the unsaturated ursine 15j. Both compounds contai an Λ-methyl suhstifuent at the central amide residue. This was installed to prevent competitive cyclization of 19 to the corresponding pyridone under the assay conditions; 15j was prepared to confirm that this modification does not influence DNA alkylation activity (as compared to ISa). As shown in Figure 2 I B, the lactam 19 showed weak DM A alkylation activity at 500 μΜ,, while the iraine derivative 15j was significantly mote potent, leading to extensive decomposition of the DMA at low (10 of 1 μΜ) concentrations, as expected. These final experiments show that the monocyclized derivatives 2 do display some (albeit weak) DNA alkylation activity at high concentrations, but that the unsaturated iminium ion 3 is a significantly more potent electroptiile.

Discussion.

The eolibaetins are PKS-NRPS-derived . ^' natural products and have been implicated in the genotoxic effects of comme sal and extraintestinal K coli?** They are formed from the precoHbactms upon removal of an Λ-acyl-D-Asn side chain by the eoiibaetin peptidase ClbP. ⁵ It was proposed that colibactins may generate unsaturated iminium ions that alkylate DNA (see 2, Figure 15, Scheme 6)7 However, this hypothesis has been impossible to evaluate because no colibactins have been obtained from the producing organisms, as researchers have employed clbP mutants to facilitate isolation efforts. This modification is advantageous inasmuch as it allows for the accum ulation of candidate precolibaetions, determination of their structures, and elucidation of colibactin structures (by inference). However, direct isolation of colibactins is not possible using this strategy.

Many of the ad vanced isol ates reported to date, such as preeolibaetras A- -C (7-9), contain a pyridone nucleus, and the conversion of this aromatic substructure to an unsaturated iminium ion (5 - 3 _s Figure 15, Scheme 6) seems unlike iy. The inventors reasoned that removal of ClbP and persistence of an A^aeyi-D-Asn side chain engenders unnatural cyclization events leading to the production of the pyridones. This hypothesis was based on our previous synthetic studies, which established a facile double dehydrative cyclization of /V-acylated linear precursors Ϊ to pyridones under mildly acidic or basic conditions. ¹ " it follows from this line of reasoning that pyridone-containing structures are unlikely to be genotoxic. To test this hypothesis, the inventors prepared the linear precursor 12 (Figure 16, Scheme 7) and elaborated it to 13 pyridone and unsaturated imine derivatives, in which the suhstituents throughout the molecules were systematically modified. The successful synthesis of these unsaturated imines allowed us to evaluate their DNA alkylaiion activity for the first time. The alkylation assay shown in Figure 17 demonstrates that the unsaturated i ines, but not the corresponding pyridones, are potent DNA alkylation agents. The presence of a terminal catiooic substituent enhances DNA alkylation activity (c.f., 15a and 15b, Figure !7A} ₅ as was expected based on literature precedent. ^{1 '} These results are consistent with earlier observations ^' that an -amiiiomalonateHlerived residue is present in folly- functionalized coHbactins and is required for genotoxk effects. ¹ *'' ² Presumably this residue forms the basis for a catiomc substituent that serves the same role as the non-natural caiionic functional groups employed herein. It may also be essential for cellular efflux and trafficking to eukaryotic cells.

The data support. ClbP-mediated. deacylation as a key step to prevent formation of pyridone products and facilitate cyclization to unsaturated imines such as 3 (Figure 15, Scheme 6). While these were originally proposed to form from unsaturated, lactams 2, our synthetic studies (Figure 16, Scheme 7) suggest they maybe generated from vmyiogous ureas 6 instead. Regardless of the precise mechanism of formation, our structur -ftraction studies show thai the unsaturated lactam and cyclopropane are both necessary for DNA alkylation activity (Figure 22 ). Formation of an unsaturated imine significantly enhances DMA alkylation activity (c.f., 19 and 15j, Figure 21 and a catiomc terminal residue further increases the extent of DNA alkylati on . The observed ring-opening of 15b by propaaethiol (Figure 20, Scheme 8) is consistent with DNA alkylation by cyclopropane ring-opening.

These experiments constitute the first molecular-level analysis of candidate colibactra structure reactivity and provide support for the hypothesis previously proposed to involve D A alkylation by an unsaturated iminium ion intermediate, ^' They also suggest that the biosynf hetic relevance of precolibaetins produced by AeibP strains should be Interpreted with caution, as it is now clear that the molecules are susceptible to several different modes of reactivity, whic vary as a function of substituents within their structures. Indeed, our data indicate that precoltbaetins A-C (7-9) are unlikely to form the basis of the genotoxicity of the elb gene cluster; a macrocyclic precolibactin recently isolated from a clbP mutant may also similarly derive from unnatural .macrocycltzatton routes. ^U References

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4. Mousa, J, 1; Yang, Y. Tomkovich, S.; Sh ma,. A.; Newsorae, R. C, : Tripathi, P.; Oswald, E.; Bruner, S. D.; Jobin, C. Nat Microbiol 2016, 1, 15009.

5. (a) Dubois, D,; Baron, O.; Cougnoox, A,; Deimas, J.; PradeS, ,; Boury, .; Bouchon, B.; Bringer, M. A.; Nougayrede, J. P.; Oswald, E.; Bonnet, R. J. Biol.€hem. 201.1, 286, 35562. (b) Cougooux, A.; Gibo!d, L,; Robin, P.; Dubois, D.; Pradel, N.; Darfeuille-Miehaud, A,; Dalroasso, G.; Deimas, J.; Bonnet, R, J. Mol. Biol 2012, 424, 203. (c) Brotherton, C. A.; Balskus, E, P. J, Am. Chem. Soc. 2013, 135, 3359. (d) Bian, X.; Fu, 3.; Plaza, A.; Hernnarm, J.; Pistorius, D.; Stewart, A. F.; Zhang, Y.; Mailer, R. Chemhi ch m 2013, 14, 1 194. (e) Vizcaino, M. !.; Enge!, P.; Traiitraau, £.; Crawford, J. M. J, Am. Chem. Soc. 2014, i 36, 9244.

6. Outer membrane vesicles from Nissle 1917 undergo clathrtn-mediated endocytotosis in several human epithelial cell lines, suggesting the probiotie effects of Nissle 1917 may not require cell-to-cell contact. See: Canas, M. A.; Gimenez, R.; Fabrega, M. J.; Toloza L.; Batdoma, L.; Badia, J. PLoS One 201 , 11, ©0160374. ?. (a) Vizcaino, M I; Crawford, J. M AW. Chem. 2015, 7 _S 411. (b) BiOthorton, C. A,;

Wilson, M,; Byrd, G.; Balskus, E. P. Org. Lett. 2015, if, 1545.

8. Tlits mode of reactivity bears parallels to DNA alk tation by duocarmycin, CC-1065, and yatakemycttt. For reviews, see: (a) Boger, D. L. ; Garbaccio, . M, Acc. Chem. Res. 1999, 32, 1043. (b) Tichener, M. S.; Boger, D. L. Nat Prod. Rep. 2008, 25, 220.

9. (a) Li, 2. R.; Li, Y.; Lai, J. Y.; Tang, J..; Wang, B.; Lu, L.; Zhtt, G.: Wu, X.; Xu, Y.; Qiao, P. Y. Chemhiachem 2015, , 1715. (b) Zha, L.; Wilson, M. R.: Bratherton, C. A.; Balskus, E. P. ACS Chem. Biol 2016, 11, 1287.

10. Healy, A. R,; Vizcaino, M. i; Crawford, J. M; Herzon, S. B, J. Am. Chem. Sac. 2016, n< 5426.

1 1. The structure of precolibacttn A was originally predicted in ref. 7a and was later revised to that shown as 7 in ref. 10,

12. (a) BraclimaiHi, A. O.; Garcie, C; Wu, V.; Martin, P.; Ueoka, R.; Oswald, E.; PieL J. Chem. Commim. 2015, 5/, 13138. (b) Li, Z. R.; Li, X; On, 1 P.; Lai, J. Y.; Duggaa, B. M.; Zhang, W. P.; Li, Z. L.; Li, Y. X.; long, R. B.; Xu, Y.; Lin, D. H.; Moore, B. S.; Qian, P. Y.

Chem. Biol. 2016, 7 , 773.

13. For reviews, see: (a) Tse, W. C; Boger, D. L. Chem. Biol 2004, 11, 1607. (b) Paul, A.; Bhattacharya, S. G#r. &i. 2012, /02, 212. (c) Sirajaddm, M.; Ali, S.; Badshah, A. /

Photochem. Photobiol, B 2013, 724, 1.

14. Tanaka, A.; Usuki, T. Tetrahedron Lett. 2011, 52, 5036.

5. Basavaprablin; Vishwanatha, T. M; Panguluri, . ,; Sineshbabu, V. V.. ynthesis 2013, 1569.

16. Liffidm, C; North, M.; Erixors, K.; Walters, K.; Jenssen, D.; Goldman, A. S. H.;

Helleday, T. Nucleic Acids Res. 2005, 33, 3799.

EXAMPLES ~ First Set (First Set of References Applies)

General Experimental Methods,

Cysteine incorporation based on carbors and deuterium labeling. Nonlabeied control culture conditions, L-fU- 'CJ-Cys isotope culture conditions, and L~f 2,3, 3-D) ~Cys isotope culture conditions were employed as previously reported. ¹ LC-HRMS analysis for the in vivo cleavage of synthetic 6 by ClbP _* coli DH10B pCl P (pPEBGl ^' 8) and the bacteria harboring the empty vector pBADi 8 (as previously reported ^1* } were grown in LB medium supplemented with 100 pg/mL ampicillin. The next morning, 50 pL of these saturated cultures were used to inoculate 2.5 ruL of LB medium eontaing 100 pg niL atnpieillin. Cultures were incubated at 37 ° with shaking at 250 rpm. At an QB«jo of 0.4-0.5, cultures were induced with L-arabinose and left o grow for another 30 min. Then , the synthetic 6 substrate (10 raM stock solutio in DMSO) or DMSO (vector control) was added to each culture to a final concentration of 50 μΜ, and incubated at 37°C for 24 fa. At the designated time point, the cultures were extracted with organic solvent and analyzed, by LC-HR S on a Pbenomenex CI S-A column ( 150 x 4.6 mm, 180,4, 5 μηι particle size, Agilent) with a watenacetonitrile (AC ) gradient containing 0.1% formic acid 0,7 rnL/min; 1-2 min, 5% ACN; ramp to 98% ACN over 18 min; hold for 5 min at 100% ACN.

Extraction of oatwally-prodttced ad vanced preceJibactiits. Escherichia coli DB10B pBAC & P, generated by nonpolar gene deletion of dhP was used in co-injection experiments as natural compound comparison with synthetic compounds. A single colony of the AcibP strain was grown in M9 media as previously reported. ¹ At the designated time point, the culture was extracted with ethyl acetate (EtOAc), and the re-constituted organic- extract was utilized in comparison studies with synthetic precolibactms.

JEIRMS, and MS/MS data acquisition. All liquid chromatography high-resolution, mass spectrometry (LC-HEMS) data deseribed for this paper was collected on an Agilent iForaiel 6550 Quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray ionization (ESI) source coupled to an Agilent Infinity 1290 UHPLC scanning from m/s 50- 1200. Data was acquired using assHunter Workstation Software LC7MS Data Acquisition (Version B.05.01, Agilent Technologies) and processed with Qualitative Analysis (Version B.06.00). Co-injection experiments were analyzed on a CIS Ktnetex column (2.5 x 100 mm, 1.7 μηι) using water (0.1% formic acid) as mobile phase A. and aeetonitri!e (0. % formic acid) as mobi le phase SB. Gradient conditions were as follow; 0-2 min, 5% B; ramp to 75% B over 20 min; wash at 98% B for 6 min, and equilibrate at. 5% B for 8 min. Flow rate was set at 0.3 mlJmin, and injection volume at 5 iL. MS data was collected in ES1+ mode with source gas temp at 225°C, drying gas at 15 Vm , nebulizer at 35 psig, Vcap set at 4000V, Nozzle Voltage at 1000 V . Acquisition rate was 1 spectra s. MSMS fragmentation was acquired witli three collision energies (40, 60, 90) with an unbiased isotope model.

DFT calculations. Lowest-energy conformations of 40 and 41 were obtained by molecular mechanics optimization (500 starting conformers) using BOSS The corresponding lowest- ted to DFT optimization in Gaussian 0 [B3LYP 6-

General Experimental Procedures, All reactions were performed in single-neck, f!ame- di ied, round-bottomed ^' flasks fitted with rubber septa under a posi tive pressure of ni trogen unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe or stainless steel cannula, or were handled in a nitrogen-filled drybo (working oxygen level <10 ppm). Organic solutions were concentrated by rotary evaporation at 28-32 °C Flash- column chromatography was performed as described by Still et al./' employing silica gel (60 A, 40-63 μπ* particle size) purchased from Sorbent Technologies (Atlanta, GA). Anion- exchange chromatography was performed as described by Belaud et al.,' employing

trime&ylamine acetate-ftuictionalized silica gel (SiliaBond® TMA Acetate). Analytical thin- layered chromatography (TIC) was performed using glass plates pre-coated with silica gel (0.25 mm, 60 A pore size) impregnated with a fluorescent indicator (254 ran), TLC plates were visualized by exposure to ultraviolet light (UV).

Materials, Commercial solvents and reagents were used as received with the following exceptions. Dichloromethane, diethyl ether and AyV-dimeihylfonnamide were purified according to the method of Pangborn et al. ⁸ Triethylamine was distilled from calcium hydride under an atmosphere of argon immediately before use, Di-isro-propytamme was distilled from calcium hydride and was stored under nitrogen. Methanol was distilled from

magnesium turnings under an atmosphere of nitrogen immediately before use.

TetrahydtOturan was distilled from sodiunv-benzophenone under an atmosphere of nitrogen immediately before use. Tritluoroacetie anhydride was fractionally-distilled before use. Molecular sieves were activated by heating to 200 °C under vacuum (<1 Torr) for 12 h, and were stored in an oven at >.l 60 °C. Propylsu!fotiic acid-functionaSized silica gel (SiliaBond© SCX-2) arid trimethylamirie acetate-functionaiized silica gel (SiliaBond® TMA Acetate) were purchased from SiliCycSe (Quebec City, CA). (- ^••• )-(fi _s )-2-methyi-A'~(pent~4-en-i- ylidene)propane-2-sul$nainide (SI) ⁹ , 3~( r alyltljio)-3-oxopropanoic acid (S7) ^U) and 2- (((tert-butoxycarbonyi)amm^ acid (S12)° were prepared according to published procedures.

Instrumentation, Proton nuclear magnetic resonance spectr (1H NMR) were recorded at 400, 500, or 600 MHz at 24 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppra, δ scale) downfiek from tetramethylsilane and are referenced to residual protium in the NMR solvent (CDC¾, δ 7.26; C¾OD, 5 3.31; C ₂ D ₆ OS, δ 2.50). Data are represented as follows: chemical shift, multiplicity (s - singlet, d ^:::: doublet, t triplet, q ~ quarter, m = multtplet and/or multiple resonances, br ~ broad, app = apparent), coupling constant in Hertz, integration, and assignment. Proton-decoupled carbon nuclear magnetic resonance spectra ( ^1J C NMR) were recorded at 100, 125 or 150 MHz at 24 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfie!d from tetramethylsilane and are referenced to the carbon resonances of the solvent (CDC¾, 6 77.0; CD¾OD, δ 49.0; 0»0,08, δ 39.5). Signals of protons and carbons were assigned, as far as possible, by using the following two dimensional NMR spectroscop techniques: [ H, 1 H] COSY (Correlation Spectroscopy), I H, 13C] HSQC (Heteronuciear Single Quantum

Coherence) and long range [IB, 13C] HMBC (Heteronuciear Multiple Bond Connectivity). Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were obtained using a Thermo Electron Corporation Nicolet 6700 FTIR spectrometer referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm ¹ ), intensity of absorption (s - strong, m - medium, w - weak, br - broad). Analytical ultra high-performance liquid chromatography/mass spectrometry (UPLC/MS) was performed on a Waters UPLC/MS instrument equipped with a reverse-phase C column (1.7 pm particle size, 2,1 x 50 mm), dual atmospheric pressure chemical ionization (APiyelectrospray (ESI) mass spectrometry detector, and photodiode array detector. Samples were eluted with a linear gradient of 5% acetonitrile - water containing 0.1% formic acid ->100% acetonitrile containing 0.1% formic acid over 0,75 min, followed by 100% acetonitrile containing 0.1% formic acid for 0.75 min, at a flow rate of 800 pL/min, High-resolution mass spectrometry (HRMS) were obtained on a Waters UPLC/HRMS instrument equipped with a dual API/ESI high-resolution mass spectrometry detector and photodiode array detector. Unless otherwise noted, samples were eluted over a reverse-phase€i* column (1,7 μ ^η ι particle size, 2.1 x 50 mm) with a linear gradient of 5% acetonitrile-water containing 0.1% formic acid-»95% acetonitrile--- water containina 0.1% formic acid for I min. at a flow rate of 600 uL min.

Optical rotations were measured on a Perkin Elmer polarimeter equipped with a sodium (589 nm, D) lamp. Optical rotation data are represented as follows; specific rotation ([ctjx ¹ ) _? concentration (g/100 mL), and solvent.

Synthetic Procedures.

Synthesis of the suifinamme 82: α¼<¾ _; -48 -> 23 C * 'jH _S ' ^{1 *}

S-i «7* SZ

A solution of meihylmagnesium bromide in ether (3.0 . 3 56 mL, 10.7 mnioi, 2.00 equiv) was added dropwise via syringe pump over 30 min to a solution of the sutrmimme SI (1.00 g, 5.34 mmol, 1 equiv) in dichloromethane (35 mL) at -48 °C The resulting mixture was allowed to warm over 30 min to -30 °C. The reaction mixture was stirred for 4 h at -30 °C. The reaction mixture was then allowed to warm over 30 min to 23 °C, The product mixture was diluted sequentially with saturated aqueous ammonium chloride solution (20 mL) and ethyl acetate (20 mL), The diluted product mixture was transferred to a separatory runnel and the layers mat formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 30 mL). The organic layers were combined and the combined organic layers were washed with saturated aqueous sodium chloride solution (30 mL). The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the trepurified suiimamine S2 as a yellow oil (1.05 g, 97%).

The product S2 obtained in this way was estimated to be of >95% purity and 88% de by Ή NSviR analysis (see accompanying spectrum) and. was used without further purification. The configuration of the newly-formed stereocenter was assigned b analog to related addition products. ' ^Λ?

- H NMR (400 MHz MHz, CDC¾): δ 5.79 (ddt, J ^: ~ 16.9, 10.1, 6.5 Hz, 111 ¾), 5.08 - 4.87 (m, 2H ₅ H , 3.37 (app hept, J= 6.8 Hz, I H, ¾), 2.86 (d, J - 7.2 Hz, 1H, H ₇ ), 2.20 - 2.04 (m, 2H, H; , 1.66 - 1.42 (m, 2H, H ), 1.28 (d, J= 6.5 Hz _s 3H ₅ ¾), 1.20 (s, 9H, H§). C NMR (151 MHz, CDC ) δ 138.2 (CH), 1 15.1 (C¾), 55.8 (C), 52.3 (CH), 37.5 (C¾), 30.2 (C¾) _} 23.4 (C¾), 22,8 (CH;,). IR (ATR-FTiR), cm ^"5 : 2972 (w), 1 42 (s), 1457 (m), 1364 (s), 1050 (s), 910 (s). HRMS-CI (mJz): [M + Naf calcd for C ₅₀ ¾NNaOS ₅ 226.1242; found,

226, 1294, [a],/ ⁰ -21 ,2 (c 1.0, C¾OH).

3

A solution of hydrogen chloride in 1,4-dioxane (4.0 , 2.58 mL, J 0.3 mmol, 2.00 equiv) was added dropwise via syringe pump over 30 rain to a solution of the sulfuiaittme S2 (1.05 g, 5.16 mmol ₅ 1 equiv) in methanol (5.0 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The product mixture was concentrated to dryness. The residue obtained was suspended in ether ( 10 mL) and the resulting suspension was concentrated to dryness. This process was repeated to provide the amine 8 as white solid (700 mg, > %, CA UTJON: hygroscopic).

The product 8 obtained in this way was estimated to be of >95% purity by ^S H and ⁱ³ C NMR. analysis (see accompanying spectra) and was used without further purification.

^! H NMR (600 MHz. CDCl ₃ ) S 8.36 (bs, 3H, H ₇ ) 5.75 (ddi. J- 17.0, 10.3, 6.6 Hz, HI, H ₂ ), 5.1 1 (dd i - 17.0, 1. Hz, Hi ¾), 5.02 (dd, J = 10.3, 1.9 Hz, IH, H _{ ), 3.46 - 3.03 (m, IH, ¾), 2.34 - 2.09 (m, 211. ¾), 1.98 ···· 1.87 (in, lit II·). 1.76 ··· ^■ 1.67 (m, H, f% 1.42 (d _> J=* 6.3 Hz, IH). °C NMR (151 MHz, CDClj) δ 136.4 (CH), 1 16.5 (Cf¾), 48.0 (CH), 34.1 (C¾), 29.7 (C¾), 18.8 (CH ₃ ). IR (ATR-FTIR), cm ^" ': 2915 (w), 1645 (s), 1612 (s), 1510 (s), 1454 (s), 1390 (sh 1 1 8 (s), 996 (s), 10 (s). [ fa ² +3.5 (c 2.0, CHjOH).

S nthesis o the alkem S3:

A«-(/er/-butoxycarb.onyl)~D-aspatagine (880 mg, 3.79 mrnoL I equiv), T

hydroxybenzotria2ole hydrate (HOBt, 638 mg, 4.17 tnmol, 1,10 equiv), N,N~

diisopropylethylani e (1 ,45 mL, 8.34 mxnof 2.20 equiv), and

A ^; '-emyicarbodii ide hydrogenchloride (EDC ^» HCL 726 nig, 3.79 mrriol, 1.00 equiv) were added in sequence to a solution of the amine 8 (514 mg, 3.79 ramol, TOO equiv) in tetxaliy roi½an (40 mi.) at 23 °C. The reaction mixture was stirred for 4 h at 23 °C. The heterogeneous product mixture was concentrated and the residue obtained was diluted with saturated aqueous ammonium chloride solution (60 mL). The resulting mixture was extracted with ethyl acetate (5 χ 30 mL) and the organic layers were combined. The combined organic layers were washed sequentially with water (30 mL) and saturated aqueous sodium chloride solution (30 mL). The washed organic layer was dried over magnesium sulfate ami the dried solution was filtered. The filtrate was concentrated to provide the alkene S3 as a white solid (995 mg, 84%),

The product S3 obtained in this way was estimated to be of>95% purity by Ή and ~C NMR analysis (see accompanying spectra) and was used without further purification. ^l M NMR (400 MEz, DMSO-i¾) δ 7.46 (d, J= 8.5 Hz, 1H, ¾), 7.25 (bs, iH, ¾ $), 6.87 (bs, IH, Hj ]), 6.84 (d _; J 8.1 Hz, ί H, !¾ 5.77 (ddt, J = 16:9, 1 .2, 6.6 Hz, IH, ¾), 4.98 (dd, J = 17.2, 2.0 Hz, I B, H _{ ), 4,92 (d, J - 10.2 Hz, I H, H ₃ ), 4, 14 (td, J= 8.0, 5.6 Hz, I H, H ₇ ), 3.83 - 3.54 (m _s IH, U ₅ ), 2.43 - 2,27 (m, 2H, H _]0 ), 2.10 - 1.90 (m, 211 ¾), 1.53 - 1.37 (m, 2H, ¾), 1.37 (s, 9H, H _n ) .00 (d, J- 6,5 Hz, 3H, ¾). ^,3 C NMR (151 MHz. DMSO- V) δ 171.5 (C), 170.5 (C), 155.1 (C), 138.5 (CH), 1 14.7 (CH,), 78.1 (C), 51.5 (CH), 43.8 (CH), 37.3 (CH ₂ ), 35.2 ((¾), 29.8 {€¾), 28.2 (C%), 20.7 (CH ₃ ). I (ATR-FTIR), cm ^" : 3393 (hrh 3298 (br), 2977 (w), 2930 (w), 1688 (m), 1635 (s), 1552 (rn), I5.1 (m), 1 171 (s) _s 1054 (m), 610 (or). HRMS-C1 (m/z): M + H calcd for C _l5 H ₂ ?N ₃ 0 _4i 314.2080; found, 314.2001. [ ] _D ²⁶ +15.4 ( 0.9, CHjOH).

Syn thesis of the amine 9:

S3 ^¾ 89¾ i

A solution of hydrogen chloride in 1 ,4-dio ane (4.0 N, 4,31 mL, 17,2 mmol, 6.00 equiv) was added dropwise via syringe pump over 20 min to a solution of the alkene S3 (900 nig, 2,87 mmol, 1 eqiiiv) i dieliloromethane (30 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The product mixture was concentrated to provide the amine 9 as a white solid (717 nig, >99%). The product 9 obtained in this way was estimaled to be o.f>95% purity by 1H and *C NMR analysis (see accompanying spectra) and was used without further purification.

^! H NMR (600 MHz, DMS< ) ά 8.34 (d, J- 8.0 Hz, IH, ¾}, 8. 19 (bs, 3H, ¾), 7.72 (bs, IH, Hn), 7.22 (bs, 1.H, Hu), 5.79 (ddt J- 16.9, 10.2, 6.6 Hz, IB, ¾) _f 5.02 (dd, J= 17.2, 1.9 Hz, 1 H, H , 4.95 (di, J= 10.2, 1.7 Hz, 1.H, Hi), 4.05 - 3.85 (m, 1H, H ₇ ), 3.84 - 3.64 (m, 1 H, ¾), 2.67 (dd, J- 16.5, 5.1 Hz, 1H, H _ift ), 2.60 (dd, J- 16.5, 8.1 H , IH, H ₍₍ >), 2.13 - 1.89 (m, 2H, ¾), 1.59 ~ 1.41 (m, 2H, ¾ _. }, 1.04 (cLV = 6.6 Hz, 3H, ¾). C NMR (151 MHz, MSO- t¾) S 170,6 (C), 167,0 (C), 138.3 (CH), 1.14.9 (C¾), 49.3 (CH), 44.5 (CH), 35.6 (C¾), 34.8 (CH ₂ ) _S 29.8 (€¾), 20.4 (Q¾). 1R (AtR-FTIR), eirf ⁵ : 2932 (br), 1655 (s), 1548 (m% 1452 (m), 1427 (m), 906 (m), 812 (br), HRMS-CI (m/z): [M 4- Naf calcd for CoH^ aC^, 236.1 75; found, 236.1361. {a\ ^2<i -3.9 (c 1.0, C¾QH).

Synthesis of the aikem S4:

Trietiiyiamiiie (977 μ ,, 7,01 rnmol, 2.50 equiv) and myristoyl chloride (990 Ε, 3.64 mmof, 1.30 equiv) were added in sequence to a solution of the amine 9 (700 rag, 2.80 ramol, 1 equiv) in (35 mL) at 23 °C. The reaction mixture was stirred for 4 h at 23 °C. The heterogeneous product mixture was diluted with aqueous hydrogen chloride solution (1.0 N, 50 mL). The precipitate that formed was isolated by filtration and the isolated precipitate was washed sequentially with aqueous hydrogen chloride solution (1.0 N, 20 mL) and water (20 mL). The resulting solid was triturated with dichloromethane (20 mL) to afford the aikene S4 as a white solid (974 mg, 82%).

The product S4 obtained in this way was estimated to be of >9S% purity by Ή and

C NM R analysis (see accompanying spectra) and was used without further purification. ^l H NMR (600 MHz, DMSO-t¾) S 7.91 (d, J - 8.1 Hz, 1 H, ¾}, 7.44 (d, J~ 8.4 Hz, IH, ¾), 7.24 (bs, IB, Hal 6.83 (bs, IH, Hn), 5.77 iddi, 16.9, 10.2, 6.6 Hz, IH, ¾), 4.98 (dd, J~ 17.2, 2.0 Hz, 1 H, H _{ ), 4.92 (dd, J ^::: 10.2, 2.0 Hz, IH, ¾), 4.52 - 4.40 (m, 1 H, H ₇ ), 3.80 - 3.59 (ffl, IH, H ₅ ), 2.44 (dd, J = 15.2, 6.2 Hz, IH, H _{0. ^' h 2.32 (dd, J- 15,2, 7.7 Hz, I H, H, ₀ ), 2.08 (1 J~ 7.5 Hz, 2H, B _i2 ), 2.03 - 1.89 (m, ,2B, *½), 1.51 - 1.34 (rn, 4H, H _4> ¾ ₃ ), 1.23 (s, 20M, C¾), 1.00 (d, /= 6.6 Hz, 3H, H ₉ ), 0.85 (t, J - 6.9 Hz, 3H, H _u ). °C NMR (151 MHz, DMSC e) 8 172.1 (C), 171.4 (C), 170.3 (C), 138.5 (CH), 1 14.8 <C¾), 49.8 (CH), 43.8 (CB), 37.4 (CH ₂ ), 35.2 (2 x C¾), 31.4 <CH ₂ ), 29.9 (C¾), 29.1 (3 x CB ₂ ), 29.0 (2 x CH ₂ ), 28,9 (<¾), 28.7 (C¾), 28,6 (C¾), 25.3 (C¾), 22.2 (C¾), 20.6 (CH,), 14.1 ((¾). IS. (ATR-FUR), cm: ^"1 : 3297 (w), 2916 (w), 2850 (s), 1662 (s), 1638 (s), 1540 (s), 1394 (m), 909 (s). HRMS-CI (m/z): [M + Hf calcd for Cs^NsO , 424,3539; found, 424.3516. [a] _D ^2e +23.7 (e 0.5, CH¾Oe - DMF (1 :1)).

Synthesis of the carhoxyUc acid 10:

Water (12 mL), sodium periodate (6.1 1 Mg, 2.86 mmol, 4.10 equiv), and ratlienium chloride (3 ,9 mg, 17,0 μτηοΐ, 0.025 equiv) were added in sequence to a suspension of the alkene S4 (295 mg, 696 umol, 1 equiv) in ethyl acetate (8,0 mL) and acetonit ile (8,0 mL) at 23 °C, the reaction vessel was placed in an oil bath that had been preheated to 50 °C. The reaction mixture was stirred vigorousl for 2 h at 50 °C, The heterogeneous product mixture was partially concentrated to remove ethyl acetate and acetonitrile. The partially

concentrated solution was diluted with aqueous hydrogen chloride solution (LO N, 50 mL). The precipitate that formed was isolated by filtration and the isolated precipitate was dissolved, in ethyl, acetate-methanol (5:1 v v, 60 mL). Activated charcoal (4.5 g) was added and the resulting heterogeneous mixture was stirred for 3 h at 23 °C. The stirred

heterogeneous raixture was filtered and the filtrate was concentrated to provide the carfooxylic acid 10 as a white solid (292 mg, 95%).

The product 10 obtained in this way was estimated to be of >95% purity b 1H and C NMR analysis (see accompanying spectra) and was used without further purification, ^l H NMR (600 MHz, DMSO-t¾) S 7.90 (d, j - S.O Hz, 1H, ¾), 7,48 (<L J - 8.4 Hz, 1 H, ¾)., 7.2S (bs, 1 H, H ₇ ), 6.84 (bs ₅ IB, H ₇ ), 4.46 (td, J - 7.8, 6.0 Hz, 1H, H ₅ ), 3.77 - 3.64 im, 1 E, H ,), 2.43 (dd, J - 1 .2, 6.0 Hz, 1H, ¾), 2.32 (dd, J - 15.2, 7.7 Hz, 11I, i¾), 2.24 - 2.09 (m, 2H, H ₁₂ ), 2.08 (t, J - 7.5 Hz, 2H, ¾), 1.65 - 1.51 (m, 2H, H _u ), 1.51 ·· ^■ 1.36 (m, 2H, H ₂ ), 1.23 (bs, 20H, Ci¾), 1.00 (d _s J - 6.6 ¾ 3B, ¾>>, 0.85 (t, J - 7.0 ¾ 3H, ¾). ^l3 C NMR (15 ! MHz. DMSCM ) 5 174.4 (Q, 172.1 (C), 171.4 (C), 170.4 (C), 49.8 (CH), 43.9 (CH), 37.4 (C¾), 35.2 (C¾X 31.3 (C¾) ₅ 3t.2 (CH ₂ ), 30.5 (CH ₂ ), 29.09 <C¾), 29.07 (2 ^χ CH ₂ ), 29.04 (CH ₂ ), 28.97 (C¾), 28.86 (CH ₂ ), 28.73 (CH ₂ ), 28.65 (C¾), 25.2 (CH ₂ ), 22.1 (CH ₂ ), 20.5 (C )% 14.0 (Ci¾). IR (ATR-FTIR), cm ^{' 5} : 3295 (w), 2920 (w), 2851 (s), 1666 (s), 1631 (s), 1541 (s), 1 40 (m). HRMS-CI (m/z): [M ÷ B} ^: ca cd for C^HuNaCb, 42.3281 ; found, 442.3251. [ajo ²⁰ +16.6 (c 0.9, CH ₃ OH).

Synthesis of the thiozoline 1.2:

Triet yla.ffll»e (540 μΐϋ, 3.84 nn¾ol, 0.20 equiv) was added to a deoxygenated solution of ^if^- ttt ycaitNsn ^-amkoacetosiliil (11, 3.00 g, 19.2 rnmol, 1 equiv) and h-(+}~ cysteine ethyl ester hydrogenchloride (5.35 g, 28.8 rnmol, 1.50 equiv) in methanol (35 mi.) at 23 °C. The resulting mixture was stirred for 14 h at 23 °C, The product mixture was concentrated and the residue obtained was purified by flash-column chromatography (eluting with 20% ethyl acetate-bexanes initially, grading to 40% ethyl acetate-bexaaes, linear gradient) to provide the thiazolme 12 as a colorless oil (4.71 g, 85%).

The product 12 obtained in this way was estimated to be of >95% purity by 1H and ⁿ C NMR analysis (see accompanying spectra) and was used without further purification.

³ H and C NMR data for the thiazoline 12 prepared in this way were in agreement with the literature. ¹⁴

^} H NMR (600 MHz, DMS€ ₆ > δ 7.46 (t, J- 6.3 Hz, ΪΗ), 5.12 (app t, J = 9.3 Hz, IH, ¾), 4.23 - 4.06 (m, 2B, H ₅ ), 3.94 - 3.90 (m, 2H, ¾), 3.53 (app t, J- 10.5 Hz, 1 H, ¾), 3.39 (dd, /= U .3, 8.9 Hz, IR H ₃ ), 1 -39 (s, 9H, H _t ), 1.22 ( /= 7.1 Hz, 3H, ¾). ^{! 3} C NMR (151 MHz, DMSO- δ 173.8 (C), 170.4 (C), 155.6 (C), 78.4 (C), 78,0 (CH), 61.0 (C¾), 42,3 (C¾), 33.9 (CH ₂ ), 28.2 ( ^' <¾), 14.0 (C¾).

Synthesis of the amide $ ^■ $:

Aqueous ammonium hydroxide solution (28% w v, 50 mL) was added to a solution of the ester 12 (3.86 g, 13,5 mrooJ, 1 equiv) in methanol (.100 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The product mixture was concentrated and the residue obtained was dried by azeotrepic distillation from toluene (2 κ 30 mL) to provide the amide SS as a white solid (3.49 g, >9 %).

The product S5 obtained in this way was estimated to be of >95% purity by ^! H and ^{5 ,} C NMR analysis (see accompanying spectra) and was used without further purification.

Ή NMR (600 MHz., DMSO ) δ 7.45 (t, J™ 6.1 Hz, 1 H), 7.33 (bs, 1 H, H _s ), 7.14 (bs, 1 H, ¾), 4.99 - 4,91 (in, 1 H ¾X 4,02 ~ 3.82 (m, 2H _? ¾), 3.54 - 3.39 (m, 2H, ¾) _; 1.39 (s _; 9H, Hi). ¹³ C MR (151 MHz, DMSO-i¾) 8 172.5 (C), 172.3 (C), 155.7 (C), 78.5 {CM), 78.4 (C) ₅ 42.5 (C¾X 34.6 (C¾) _; 28.2 (C¾), 1R (ATR-FTIR), em ^"3 ; 3321 (w), 1684 (s), 1615 (m), 1503 (s), 1251 (s), 1156 (w), m z (ES+) 260.15 ([M+!¾ _; 100 %), [ ]» ²⁰ +6.5 (c 0.8,

C¾OH).

Synthesis of the thioamide 13

Lawesson's reagent (4.03 g, 9.96 rnmoL 0.75 equiv) was added to a sol ution of the amide S5 (3.44 g, 13.3 mmoi, 1 equiv) in dic otoxnetaane (80 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The product mixture was filtered through a pad of celite (2.5 x 4.5 cm). The filter cake was washed with dichioroinethane (20 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was dissolved in ethyl acetate (50 mL) and the resulting solution was washed sequentially with saturated aqueous sodium bicarbonate solution (2 x 30 mL) and saturated aqueous sodium chloride solution (30 mL). The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the thioamide .13 as a pale yellow solid (3.66 g, >99%). The thioatnide 13 obtained in this way was estimated to be of>95% purity by E and ^l C NMR analysis (see accompanying spectra) and was used without further purification.

^! H NMR (600 MHz, C Ci ₃ ) S 8.37 (bs, I B, ¾), 7.74 (bs, !H, ¾), 5,35 (t, J= 9.1 Hz, IH, ¾), 5.29 - 5.12 (in, I H), 4.18 - 4.05 (m, 2H, !¾}, 3.90 - 3.83 (m, IB, ¾), 3.90 - 3.83 (in, IH, H ), 1.45 (s, 9H, H,). °C NMR (151 MHz, CDC1 ₃ ) δ 206.7 (C), 174.5 (C), 156.2 (C), 84.3 (CH), 80.7 fC), 43.6 (CH ₂ ), 39.3 (CH ₂ ), 28.4 (C¾). IR (ATR~PT1R), cm ^"1 : 3300 (w), 1688 (s), 1596 (s), 1501 (s), 1248 (s), 1157 (w). HRMS-Cl (ra/z): [M + Naf calcd for C _{ oH. _l7 N3 a02$2, 98.0660; found, 298.0621. [a],/ ⁰ -1.4 (c 1.0, CH3OH).

Synthesis of (he (hiazoline—thiazo

Ϊ1¾>

Triethylamine (759 pL, 5.48 tnmol, 3.00 equiv) was added dropwise via syringe to a solution of the thioamide 13 (500 mg, 1.82 mmol, 1 ecpiv) and bromopyruvic acid (364 mg, 2.18 mmol, 1.20 eqistv) in methanol (13 niL) at 23 °C. The reaction vessel was fitted with a reflux condenser and then was placed in an oil bath that had been preheated to 72 °€. The reaction mixture was stirred and heated for 3 b at 72 °C. The product mixture was concentrated and the residue obtained was purified using trimethylamine acetate- functionalized silica gel (Si-TM A acetate; elating with 2% acetic acid-methanoS) to provide the thiazoline-thiazole 14 as a white solid (440 mg, 71 %).

³ B NMR (600 MHz, CDt¾) S 8.20 (s, I B, H ₆ ), 5.89 (app t, ~ 8.6 Hz, IH, H ₄ ), 5.35 (t, J- 5.7 Hz, I H), 4.30 - 4.16 (m, 2H, ¾), 3.89 (dd, = 1 1.4, 9.2 Hz, IH, ¾}, 3,64 (d J- 1,1.4, 7.6 Hz, I H, H ₃ ), 1.46 (s, 9H, H _$ ). C NMR (151 MHz, CDCy δ 175.7 (C), 172.4 (C), 164.2 (C), 155.7 (C), 147.0 (C), 129.0 (CH), 80.5 (C) ₅ 77.0 (CH), 43.3 (C¾) _s 39.1 (C%), 28.5 (C¾). IR (ATR-FTIR), cm ^"1 : 1696 (w), 1 07 (s), 1248 (s), 1 156 (s). HRMS-Cl (m z): [M + Ma]' calcd for C} 3Hi ₇ N ₃ Na0 ₄ ¾, 366.0558; found, 366.0508. [α]» ²⁰ +6.6 (c 2 , CH3OH).

Synthesis of the amine 15:

o o

BoeHN'^ -. ^N N -^OH HO C,'S*f¾h V.,-,N

r i} _ ,. j. _™ f I

14 15 A solution of hydrogen chloride in l,4-<3ioxane (4.0 N, 3.50 mL, 14,0 mmol, 16.7 equiv) was added dropwise via syringe pump over 20 min to a solution of the thiazoline- thiazoie 14 (287 tag, 836 μηαοΐ, 1 equiv) in . dichloromethane (14 mL) at 23 °C. The resulting mixture was stirred for I h at 23 °C. The product mixtuie was concentrated to provide the amine IS as a white solid (234 mg, >99%).

The product 15 obtained ra this way was used directly in the following step without further purification.

Ή N R (600 MHz, DMSO- ) 5 B.65 - 8.54 (bs, 3H), 8.47 (s, 1 R H$), 6.02 ···· 5.83 (m, 1H, ¾), 4.05 (s, 2H, ¾), 4.04 - 4.00 (m, 1H, ¾), 3.70 (dd, J = 1 1 ,3, 8.5 Hz, 1H, ¾). ^! ¾ NMR (151 MHz, DMSO-£¾) d 170.3 (C) _s 167.9 (C), 162.0 (C), 147.1 (C), 129.3 (CH), 763 (CH), 40,2 (C¾). 38.9 (Cf¾).

Synthesis of the β-ketothioester 16:

£6 S7 56%. 16

1 ,1 '-Carbon yldiimidazole (846 mg, 5.22 mmol, 1.50 equiv) was added to a solution of

equiv) in tetrahydrofuran ( 18 mL) at 23 °C. The resulting mixture was stirred for 6 h at 23 °C. in a second round-bottomed flask, magnesium ethoxide (299 mg, 2,61 mmot, 0.75 equiv) was added to a solution of 3-( e /-biitylthio)-3-oxopropanoic acid (S7, 920 mg, 5.22 mmol, 1.50 equiv) in tetrahydrofuran (9.0 mL) at 23 °C. The resulting mixture was stirred for 6 h at 23 ^; C. and then was concentrated to dryness. The activated carboxylic acid prepared hi the first flask was transferred via cannula to the dried magnesium salt prepared in the second flask. The resulting mixture was stirred for 14 h at 23 °C. The product mixture was diluted sequentially with saturated aqueous ammonium chloride solution (20 mL) and ethyl acetate (30 m.L), The diluted product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 3 mL). The orgastic layers were combined and the combined organic layers were washed with saturated aqueous sodium chloride solution (30 mL), The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified ^' b fiash-eohimn chromatography (ehrting with hexanes initially, grading to 10% ethyl acetate-hexancs, linear gradient) to provide the β-keroihioester 1.6 as a colorless oil (61 nig, 56%).

R/~ 0.36 (20% ethyl acetate-hexanes; !JV), 'fl NMR (600 MHz, CDCt ₃ ) δ 5.25 (fas, 1H) ₅ 3.80 (s, 2H, ¾), 1.62 U{. J= 4.5 Hz, 2H, H ₂ ), 1.49 (s. 9H, I L _; ) _; . 1.47 (s _s 9H, ¾), 1.22 - 1.15 (m, 2H, ¾). °C NM (151 MHz, CDCb) δ 203.7 (C), 193.9 (C), 155.8 (C), 80.7 (C), 55.3 (C¾), 49.1 (C), 41.6 (C), 29.8 (C¾), 28.5 (C¾), 21.8 (C¾). IR (ATR-FTIR), cm ^" ' : 3382 (m), 3333 (m), 2969 (br), 1699 (s), 1658 (m), 1597 (m), 1487 (s), 1249 ( _s ), 1 161 (s), 1065 (s). HRMS-CI (m/z): [M - a]* ca!ed for C _i3 H ₂₅ Na<¾S, 338.1402; found, 338.1384.

S nthesis of the fi-ketoamkk S :

w « ^S! ss

Three equal portions of silver trifluoroacetate (26.8 mg, 122 μιηοΐ, 0.40 equiv each) were added over 1 h to a solution of ir ethySaniiiie (170 μΙ , 1.22 mmoi, 4.00 equiv), 15 (85.0 nig, 304 μι ^η οΐ, 1 equiv), and 16 (1 15 mg, 365 ,uraol, 1.20 equiv) in N,N~di ^' tnethylformamide (3.5 mL) at 0 °C The reaction mixture was stirred for 1 h at 0 °C. The product mixture was directly applied to a column containing trimeihylaniiiie acetate-f nctionalked silica gel (Si- TMA acetate; elutmg with 2% acetic add-methanol). The tractions containing product were collected, combined, and concentrated. The residue obtained was furtiier purified by automated flash-column chromatography (elating with 2% acetic acid-dichloromethane initially, grading to 2% acetic ac.id ^» 6% methanol-dichloromethane, linear gradient) to afford the β-ketoamide S8 as a white solid (90,0 mg, 63%),

^J H NMR (600 MHz, DMSQ k) 8 12.99 (bs, IH), 8.67 (t, J - 6,0 Hz, I II), 8.40 (s. 111 H,}. 7,73 (bs, IH), 5,91 (t, j ~ 8.7 Hz, IH, H ₄ ) ₅ 4.31 - 4.08 (n , 2H, H ₂ ), 3.86 (dd, J- l l .3, 9.3 Hz, I B, ¾) 3,55 - 3,49 (m, 311, ]¾, ¾), 1,40 (s, 9H, ¾), 1.35 (d, J ^:::: 3. Ϊ Hz, 2H, H ₇ ), 1.06 (d, J~ 3.6 Hz, 2H, H ₇ ). C NMR ( 151 MHz, OMSO-d*) δ 204.6 (C), 173.9 (C), 1.71.5 (C), 166.6 (C), 162.2 (C), 156.3 (C), 147 J (Q, 128.9 (CH), 78.6 (C), 76.9 (CH), 45.9 (CH ₂ ), 41.2 (C), 41.1. (CB ₃ ), 37.7 (CH ₂ ), 28.2 (CH ₃ ), 1 .5 (€¾). IR (ATR-FTIR), cm ^" ': 3000 (br), 1702 (s), 1507 (m), 1249 (m), 1164 (3), 1068 (m). HRMS-CI (m/z): [M + H calcd for

C _J& H ₂ sNM¾S _3i 469.1216; found, 469.1257. [α]ι> ^2ί +3.0 (c 2Xl CHiOH). Synthesis of the amine 1

A solution of hydrogen chloride in 1 ,4-dioxane (4.0 N, 1.00 ml.., 4.00 mmol, 22,6 equiv) was added dropwise via syringe pump over 20 rain to a solution of the β-ketoamide S8 (83.0 mg, 1 77 umol, 1 equiv) in dichloromethane (4.0 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C The reaction mixture was concentrated to provide the amine ^" 17 as a white solid (71.7 nig, >99%).

The product 17 obtained in this way was used, directly i the following step,

1H NMR (600 ¾ DMSQ-</ ₆ ) 8 8,87 (t, J - 6.0 Hz, Hi Hi), 8.81 (s, 3H), 8.41 (s, IH, ¾), 5.96 - 5.86 ( , IB, ¾}, 4.25 - 4.01 (m, 2H, ¾), 3.87 (dd, J~ 1.1.3, 9.2 H , ! E, ¾), 3.54 (dd, J - 1 L8, 8 _* 2 Hz, .1 H, ¾}, 3.35 (s, 2H, ¾), LS6 - 1.67 (m, 2H, H?) _s 1.56 - 1.37 (m, 2H, H ₇ ). ^B C HMR (151 MHz, DMS<W _fr ) 8 199.2 (CL 173.4 (C) _? 171.3 (C), 165.7 (C), 162.0 (C), 147.1 (C), 129.0 (CH), 76.8 (CH), 42.3 (CH ₂ ), 42.0 (C), 41.2 (C¾) _f 37.8 (C%), 13.1 (C¾).

S nthesis of the ihiazoie Ϊ9:

Ethyl bromopyruvate (2.37 mL, 1.8.9 mrnoi, 1.20 equiv) and calcium carbonate (L58 g, 15,8 mniol, LOO equiv) were added in sequence to a solution of teri-batyl 2-amino-2- thioxoethylcarbamate (18, 3.00 g, 15.8 niniol, 1 equiv) in ethanol (60 mL) at 23 °C, The reaction mixture was stirred for 6 h at 23 °C. The product mixture was concentrated and the residu obtained was purified by ilash-cohitnn chromatography (eluting with 1.0% ethyl acetate-hexanes initially, grading to 30% ethyl acetate-hexanes, linear gradient) to furnish the thiazole 19 as a white solid (3.34 g, 74%).

³ H and ^U C NMR data for thiazole 19 prepared in this way were in agreement with the literature. ¹⁵ R; ^{■■■■■■} 039 (40% ^· ethyl acetate-hexanes; UV). Ή NMR (600 MHz, CDCI ₃ ) δ 8.12 (s, 1 ΪΤ H ₃ ), 4.65 (d, J = 6.4 Hz. 2H, H ₂ ) _} 4.42 (q, = 7.1 Hz, 2H, H ₄ ), 1.46 (8, 9H, H _¾ ), 1.40 (t, /= 7.1 Hz, 3H, ¾). C NMR (151 MHz, CE ¾) δ 170.1 (C), 161.4 (C), 155.8 (C), 147.1 (C), 128.1 (CH) _S 80.7 (C), 61.7 (C%), 42.5 (C¾), 28.5 (C¾) _s 14.5 (C¾l

S rtthesi* of ike amide $9: 19

A solution: of aqueous ammonia (28% w/v, 42 niL) was added to a solution of the thiazole 19 (2.38 g; 8.29 mmol, I equiv) in anhydrous methanol (84 raJL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The product mixture was concentrated and the residue obtained was dried by azeotropie distillation from toluene (2 x 30 mL) to afford the product S9 as a yellow solid (2.13 g, >99%).

The amide S9 obtained in this way was estimated to be of >95% purity by ^l H and ' ^' € NMR analysis (see accompanying spectra) and was used without further purification.

Ή and ^' € NMR data for amide 59 prepared in this way were in agreement with the literature. ^s

Hi NMR (600 MHz, CDCU) 6 8.08 (s, 1 R ¾), 7.12 (bs, 1H, ¾), 5.83 (bs, 1H, H ₄ ), 5.30 (t, / - 6.2 Hz, 1H), 4.60 (d, J= 6.2 Hz, 2H, H ₂ ), 1.47 (s, 9H, Hj). ¹³ C NMR (151 MHz, CDC¾) ό 169.5 (C), 163.0 (C), 155.7 (C), 149.3 (C) ₅ 124.8 (CH), 80.7 (C), 42.5 (C¾) ₅ 28.5 (CH ₃ ).

Synthesis o f the nitrite 20:

^ \ H , i

S9 20

Trifluoroaceiic anhydride (1.24 mL, 8.94 mtno!, 1.10 equiv) was added dropwise via syringe pump over 20 rai to a solution of the amide S9 (2.09 g, 8.12 mmol, 1 equiv) and triethyiamine (2.49 mL, 17,9 mmol, 2.20 equiv) in dichloromethane (120 mL) at 0 °C. The resulting mixture was stirred for 30 rain at 0 °C. The reaction mixture was then allowed to warns over 30 mm to 23 °C. The warmed, reaction mixture was stirred for 2 h at 23 °C. The product mixture was concentrated and the residue obtained was purified by flash-column chromatography (eluting with hexaaes initially, grading to 20% etiiyl acetate-hexanes, linear gradient) to furnish the nitrile 20 as a white solid (1.63 g, 84%).

³ H and ^U C NMR data for nitrile 20 prepared in this way were in agreement with the literature.'*

R _f < - 0.51 (40% ethyl acetate-hex a»es; U V). ^f Ή NMR. (600 Hz, CDC ) § 7.95 (s ₅ 1 H, H ₃ ), 5.31 (t, J - 6.4 Hz, IH), 462 (d, J = 6.4 Hz, 2H, ¾}, 1.47 (s, 9iL ¾). C NMR (1.51 MHz, CDC ) δ 171.5 (C), 155.8 (C), 131.0 (CH), 126.6 (C), 113.9 (C), 81.0 (C), 42.5 (C¾), 28.4 (CH ₃ ). IR (ATR-FTIR), cm ^""1 : 3334 (w), 3073 (wl 1684 (s), 1520 (s), 1297 (s), 1161 (m), 618 (tn).

Synthesis of the thiazote-thiazolwe 21:

20 SI

Triethylamine (1.01 mL, 7.21 mmol, 1.10 equiv) was added dropwise via syringe to a solution of the nitrile 20 (1.57 g, 6.55 mmol, 1 equiv) and I, -cysteine (870 mg, 7,21 mmol, 1.10 equiv) in methanol (60 mL) at 23 °C. The reaction vessel was fitted with a reflux condenser and then placed in an oil bath that had been preheated to 73 °C. The reaction mixture was stirred and heated for 3 h at reflux. The product mixture was cooled to 23 °C and the cooled product mixture was concentrated. The residue obtained was dissolved in saturated aqueous sodium bicarbonate solution (40 mL) and the resulting solution was washed with ether (30 mL), The aqueous layer was acidified to pH -3-4 by the dropwise addition of 3.0 N aqueous hydrochloric acid solution. The resulting mixture was extracted with ethyl acetate (3 x 30 mL) and the organic layers were combined. The combined organic layers were dried over sodium sulfate and the dried solution was filtered. The filtrate was concentrated to provide the thiazole-thiazoime 21 as a white solid (2.18 g, 97%).

The thiazole-thiazoline 21 obtained in this way was estimated to be of >95% purity by ' and ¹ 'C NMR analysts (see accompanying spectra) and was used without further purification.

Ή ^" and ^iJ C NMR data for thiazole -thiazoline 21 prepared in this way were in agreement with the literature. ^{1 *} Ή NMR (600 MHz, C¾OD) 5 8.1 ( , 1.H, H ₃ ) _> 5.30 (t, J = 9.1 Hz, I II, l is), 4.52 ($, 2H, ¾), 3.85 - 3.51 (m, 2H, ¾), 1.47 (s, 9H, Hi). ^B C NMR (151 MHz, C¾OD) § 173.8 (C), 173.3 (C), 167.9 (C), 158.3 (C), .1493 (C), 123.0 (CH), 81.0 (C), 79.1 (CH), 43.1 < ¾ _, ), 35.6 (C¾), 28.7 (C¾). IR ( ATR-FTIR), cm ^"1 : 3351 (w), 2978 (w), 1.701 (w) ₅ 1519 (w), 1250 (s), 1165 (s).

A solution of hydrogen chloride In 1 ,4-dioxane (4.0 N, .1.5 mL, 6.00 mmol, 6.90 equi v) was added dropwise via syringe pump over 20 mm to a solution of the tkiazole -- thtazotine 21 (300 nig, 870 μιηοί, 1 equiv) in dichloromethane (12 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The reaction mixture was concentrated to provide the amine 22 as a white solid (244 mg, >99%),

The product 22 obtained in this way was used directly in the following step.

! H NMR (600 MHz, CD _s OD) S 9.02 (s, 1 H, ¾) _f 5.66 (dd _s 10.5, 5.8 Ez, I E,. H ₅ ), 4.64 (¾ 2H, f¾), 4.17 (dd, J- 12.1, 1.0.5 Hz, 1H, H ₄ ), 4.10 (dd, J - 12, 1 , 5.8 Hz, Hi B ₄ ). ^!3 C NMR (151 MHz. CD3OD) ά 1803 (C), 1703 (C), 166.0 (C), 143.5 (C), 134.2 (CH), 69.9 (CH), 40,9 (CH ₂ ), 35.7 (CH ₂ ).

Synthesis of the β-ketoamkie $10:

Three equal portions of silver trifluoroacetate (31.6 mg, 143 μτηοί, 0.40 equiv each) were added over 1 h to a solution of triethylamine ( 199 μΐ, 1.43 mmol, 4.00 equiv), the β- ketothioester 16 (135 mg, 42.9 μηιοΐ, 1.20 equiv). and the amine 22 (100 mg, 35.7 μηιοΐ, 1 equiv) hi A^A'-dimeihylfonnamide (3.5 mL) at 0 °C. The reaction mixture was stined for I h at 0 °C. The product mixture was directly applied to a column containing trimethylamine acetate-tnnctionalized silica gel (Si-TMA acetate; eluting with 2% acetic acid-methanoi). The fractions containing product were collected, combined, and concentrated. The residue obtained was further purified by automated flash-column chromatography (eluting with 2% acetic aeid-di ttorornethane initially, grading to 2% acetic acid-6% methanol- dichlofomethane, linear gradient). The fractions containing the product Sift were collected, combined, and concentrated to provide the β-ketoamide SIO as a white solid (115 mg, 69%). ^! H NM (600 MHz, DMS< ) d 8.95 (t, J - 6.0 Hz, IH, Hi), 8.23 (s, IH, %), 5,26 (dd, J- 9.7, S.2 Hz, IH, ¾), 4.55 (d, . J= 4.5 Hz, IH, ¾), 3.63 (dd, /= 11.3, 9.7 Hz, ^' ΪΗ, H ), 3.56 (s, 2H, ¾ ^' 3.56 - 3.51 (m, IH, H ₄ ), 1.41 (s, 9H, B _g ) 1.40 ·- 1.3 J (η 2Η, Ητ), 1.07 (q, J - 4.3 Hz, 2H, H ₇ ). ^W C NMR (151 MHz, DMSC O δ 204.7 (C), 171.8 (C), 170.1 (C), 166.8 (C), 163.3 (C), 156.0 (C), 147.4 (C), 122.1 (CH), 78.6 (C), 78.3 (CH), 46.1 (CH ₂ ), 41.1 (C), 40.3 (CH ₂ ), 34.4 (C¾), 28,2 (CH ₃ ), 19.5 (C¾). IR (ATR-FTIR), cm ¹ : 3317 ibr), 1702 (s), 1506 (m), 1248 (ml 1 162 (s), 1063 (m). HRMS-CS (mfz): [M + H ealed for C _1¾ e ₂₅ 0sSa, 469.1216; found, 469, 1257, [ofo* +5.6 (c 2,2, CH ₃ OH).

A solution of hydrogen chloride in 1 ,4-dioxane (4,0 N, 1 .0 mi, 4,00 raraoi, 21.5 eqwiv) was added dropwise via syringe pomp over 20 min to a solution of the p ¾toamide S10 (87,0 mg, 186 μηιοΐ, 1 equiv) m dichloromethane (4,0 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The reaction mixture was concentrated to provide the amine 23 as a white solid (75.2 mg. >99%),

The product 23 obtained in this w ay was used directly in the folio win step.

¾H NMR (600 MHz, DMSO-<&} δ 9.17 (t, J = 6.0 Hz, IH, Hi), 8.83 (hs, 3H), 8.30 is, IH, H ₃ ), 5.28 (dd, J - .7, 8, 1 Hz, IH, ¾}, 4.58 (d, /= 5.9 Ez, 2H, ¾), 3,66 (dd, J - 11.2, 9,9 Hz, I B, H ₄ ), 3.60 - 3.51 (ra, IH, H ₄ ), 3.38 (s, 2H, ¾), 1.81 - 1.72 (m, 2H, H _? >, 1.57 ~ 1.49 (m, 2H, H ₇ ). C NMR (151 MHz, DMSO-< ) δ 199,39 (C), 171 ,66 (C), 169.76 (C), 165.92 (C), 164.04 (C), 147.09 (C), 122.66 (CH), 77.72 (CH), 42.38 (C¾), 42.00 (C), 40.43 (CH ₂ ),

34,40 (C¾), 13.12 (C¾).

Synthesis of ike thimmide 24:

Lawesson's reagent (1.51 g„ 7.73 mmol, 0.75 equiv) was added to a solution of the amide S3 (1.28 g, 4,98 mmol, I equiv) in dichloromethane (30 mL) at 23 °C, The resulting mixture was stirred for 1 h at 23 °C. The product mixture was filtered through a pad of ceiite (2,5 x 4.5 cm). The -filter cake was washed with dichloroinethat e (10 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was dissol ved in ethyl acetate (40 mL) arid the resulting solution was washed sequentially with saturated aqueous sodium bicarbonate solution (2 x 20 mL) and saturated aqueous sodium chloride solution (20 mL). The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated, to provide the thioamide 24 as a pale yellow solid (1.36 g, >99%).

The thioamide 24 obtained in thi s way was estimated to be of >95% purity by ¾ and C NMR analysis (see accompanying spectra) and was used without further purification. lH and C NMR data for the thioamide 24 prepared in this way were in agreement with the literature. ¹ '

- 0.37 (2% methanoI-C¾Gl ₂ ; UV). Ή NMR (600 MHz, (CD ₃ )CQ ₂ ) 8 9.04 (hs, 1 H, ¾}, 8.38 (s, 1 H, !¾ 6,96 (t, J™ 6.3 Hz, I Hk 4.55 id. ,/- 6. 1 Hz, 111 H ₂ ), 1.44 (s. 9H, ¾). C NMR (151 MHz, (CD ₃ )0¾) 8 191.3 (C), 172.0 (C), 156.7 (C), 154.6 (C), 127.5 (CH), 79.8 (€), 43.2 (€¾), 28.5 (C¾).

Syntk&iis of/he hkhi mie 25:

o

r _→ · ,- > i i ^" l\

Hit; EiGH. 23 C s-' ^' *

24 ^¾i¾ - _. 25

Bromopyruvic acid (170 rag, 1,02 mmol, 1.50 equiv) and calcium carbonate (136 mg, 1.3 mmol, 2.00 equiv) were added in sequence to a solution of the thioamide 24 (186 rag, 680 μηιοί, 1 equiv) in ethanol (6.0 mL) at 23 °C. The reaction mixture was stirred for 16 h at 23 °C. The product mixture was concentrated and the residue obtained was applied to a trimemylaraine acetate-functionaHzed silica column (S Si-TMA acetate; eluting with 2% acetic acid-methanol) to provide the bithiazole 25 as a white solid (135 mg, 58%). Ή NMR (600 MHz, OM O k) B 8.42 (s, 10, ¾) _s 8.22 (s, 1H, ¾), 7.87 (t, J~ 6.1 Hz, I H), 4.45 (d, J - 6.1 Hz. 2H, ¾), 1.42 (s, 9H, Hj). ^t5 C NMR (151 MHz, DMSO- ) δ 1 2.82 (C), 162.25 (C), 161.92 (C\ 155.80 (C), 149.09 (C), 147.34 (Q, 128.29 (CH), 17.78 (CH), 78.72 (C), 41.93 (C%), 28.16 (CH ₃ ). IR (ATR-FTIR), cm ^"1 : 3322 (w), 128 (w), 1685 (s), 1534 (m% 1290 (m), 1233 (m), 1 168 (m), 772 (m), 751 (in), H MS-CI (rn/z): pVH- Hf calcd or Ct3H ₁₆ N ₃ 0 S2, 342.0582; found, 342.0577.

Synthesis of ike amine 26:

A solutiott of hydrogen chloride in 1,4-dioxane (4.0 N, 1.5 mL, 6.00 mmol, 16.8 equiv) was added dropwise via syringe pump o ver 20 mm to a solution of the btthiazole 25 (122 mg, 357 μηιοΐ, 1 equiv) in dichloromethane (6.0 mL) at 23 ^e C. The resulting mixture was stirred for 1 h at 23 °C The reaction mixture was concentrated to provide tile amine 26 as a white solid (99.3 mg, >99%).

The product 26 obtained in this way was used directly in the follo wing step.

^} H NMR (600 MHz, DMSG- 's) δ 9.57 (bs, 3H), 9.33 ( _S> IH, ¾), 9,24 ( _S> I H, ¾), 5.32 (s, 211 ¾). ^¾ ¾ NMR (151 MHz, DMSO-*¾) δ 164.2 (C), 162.4 (C), 162.1 (C), 148.6 (C), 147.7 (C), 129.7 (CH), 120.7 (CH), 39.7 (CH ₂ ),

S nth sis qfthe fi-keioawiid $11:

Three equal portions of silver trifluoroacetate (27.0 mg, 12.2 μηιοΐ, 0.40 equiv) were added over 1 h to a. solution of ixiethyl amine (171 μϊ_, 1.22 mmol, 4.00 equiv), the β- ketothioester 16 (1 16 mg, 36.7 μιΐΐθΐ, 1.20 equiv), and the amine 26 (85.0 mg, 30.6 μηιοΙ, 1 equiv) in A ^r ,.¥-dimethylformamide (3.SO mL) at 0 °C. The reaction mixture was stirred for .1 h at 0 °C. The product mixture was directly applied to a column containing trinielhylamine acetate- f nctionalized silica gel (Si-TMA acetate; eluting with 2% acetic acid-methanol). The fractions containing product were collected, -combined _* and concentrated. The residue obtained was further purified by automated flash-column chromatography (eiuting wit 2% acetic acid~dichlorometbane initially, grading to 2% acetic aci<M>% dichloromet«ane~- nietbanol, linear gradient). The fractions containing the product Sll were collected, combined, and concentrated to provide the p~ketoa ide SI 1 as a white solid (102 nig, 72%). ^l H NMR (600 MHz, DMSC s _. ) § 13.14 (bs, I.H), 8.98 (t, =* 6.0 Hz, IB, ¾), 8.47 (s, 1H, ¾), 8.25 (s, 1 H, ¾), 7.77 (s, 1H), 4.60 (d, J = 6.0 Hz, 2H, ¾), 3.57 (s, 2H, ¾), 1.41 (s, 9H, ¾ 1.40 ~ 1.36 (m, 2H, H ₇ 1.08 (q, / ^» 4.6 Hz, 2H, H ₇ ). ^{! 3} C NMR (151 MHz, DMSO-4 ) δ 204.7 (C) _s 171.3 (CI 166.9 (C), 162.1 (C), 162.0 (C), 156.1 (C), 148,2 (C), 147.1 (C), 128.9 (CM), 118.4 (CH), 7S.6 (C), 46.1 (CH ₂ ), 41.2 (C), 40.5 (CH ₂ ), 28.2 (t%), 19.6 (CH ₂ ). 1 (ATR-FT1R), cm ^"1 : 3322 (m), 2931 (br), 1709 (s), 1686 (s), 1511 (in), 1282 (m), 1237 (m), 1 164 (m), 758 (m). HRMS-CI (m/z): [M + aj" calcd for 489.0878; found, 489.0794,

Synthesis of the amine 27:

A solution of hydrogen chloride in 1 ,4-dioxane (4.0 N, 0.5 mL, 2,00 mraol, 33,3 equiv) was added drop wise vi syringe pump over 20 min io a solution of the p-ketoan de SI 1 (28.0 mg, 6Q.0 uniol, 1 equiv) in dichioromethane (2.0 ml.) at 23 °€. The resulting mixture was stirred for 3 h at 23 °C. The reaction mixture was concentrated to provide the amine 27 as a white solid (24.2 mg, >99%).

The product 27 obtained in this way was used directly in the following step.

Ή NMR (600 MHz, DMSCW ₆ ) S 13.15 (bs, 1 H) _f 9, 19 (t, J= 6.0 Ez, 1H _S ¾), 8.79 (bs, 3H), 8.49 (s, IB, H ₄ ), 8.28 (s, 1H, ¾), 4.63 (d, J™ 5.9 Hz, 2H, ¾), 3.40 (s, 2H, ¾), 1.78 (q, J - 5.9 Hz, 2H, H _? ), 1.53 (q, J = 6.0 Hz, 2H, H ₇ ), C NMR (151 MHz, DMSCW<s) 5 199.4 (C), 170.7 (C), 166.0 (C), 162 J (C), 162.0 (C), 148.1 (C), 147.2 (C), 129.0 (CH), 1 18.4 (CH), 42.4 (CB ₂ ), 42.0 (C), 40.5 (CH ₂ ), 13.1 (CB ₂ ).

Synthesis of the th ioester 28:

1 J'-Carbonyldiimidazole (CDI; 165 nig, 1.02 mmoi, 1.50 equiv) was added to a solution of the carboxylic acid 10 (300 rag, 679 μηιοΐ, 1 equiv) in Ayv'-dimet ylformamide (7.0 mL). The resulting solution was stirred for 8 li at 23 °C. In a second roiind-bottomed flask, magnesium ethoxtde (77.7 rag, 679 mol, 1.00 equiv) was added to a solution of 3- (ter/-butylthio)-3-oxopK)p8noic acid (ST, 238 mg, 1.36 mmol, 2,00 equiv) in tetrahydroiuiiaii (3.0 mL). The resulting mixture was stirred for 10 h at 23 °C. The reaction mixture was concentrated to provide the magnesium salt of the β-ketothioester S7 (271 mg, >99%) as a colorless solid. A solution of the magnesium salt of the β-ketothioester S7 in N,N~

dimethylformaffiide (1.0 mL) was transferred via cannula to the activated carboxylic acid and the resulting mixture was stirred for 16 h at 23 °C. The product mixture was diluted with aqueous hydrogen chloride solution (1.0 N, 20 .mL). The resulting solid precipitate was isolated b filtration and the isolated filtrate was applied to trimethylamine acetate- funefionalized silica column (Si-T A acetate; eluting with methanol) to provide the β~ ketott oester 28 as a white solid (359 mg, 95%).

^! H NM (400 MHz, DMS< ) d 7.88 (d, J- 7.8 Hz, IH, ¾), 7.50 (d, J= 8.6 Hz, IB, .¾), 7.25 (bs, IH, H ₇ ), 6.85 (bs, I H, H ₇ ), 4,41 (q, J - 7.4 Hz, I B, %), 3.74 - 3.59 (m, 3H, B ₅ , H _i3 ), 2.50 - 2.42 (m, 2H, H _i2 ), 2.4l (dd, J- 14.1 , 5.0 Hz, 1H, %), 2.33 (dd ₅ J- 15.1 , 7.7 Hz, 1 H, ¾), 2.09 (dd, J~ 9.4, 6.7 Hz, 2H, ¾), 1.66 - 1.52 (m, 1 H, H _u ), 1.54 - 1.42 (m, 3H, H _n , ¾), 1.41 (s, 9H, Hul 1.23 (bs, 20H, 10 x CH ₂ \ 0.98 (d, /= 6.6 Hz, 3H, H _} ), 0.85 (t _? J - 6.5 Hz, 3H, Hj). ¾ NMR (126 MHz, DMSO-<¾) δ 202.5 (C), 192.8 (C), 172.1 (C), 171.4 (C), 170.5 (C), 57.5 (C¾), 49.9 (CH), 48.1 (C), 43.5 (CH), 39.0 (CH ₂ ), 373 (C¾), 35.2 (CH ₂ ), 31.3 (C%), 29.6 (¾}, 29.2 (C¾\ 29.06 (CH ₃ ), 29.04 (2 χ C¾ 29.01 (C¾), 28.95 (CH ₂ ), 28.87 «¾), 28.84 {€¾}, 28.7 #¾), 25.2 (CH ₂ ), 22.1 «¾), 20.5 (C¾), 14.0 (<¾ ^' ). IR (ATR-FTiR), cm ^"1 : 3297 (m\ 2955 (m), 1660 (s) _s 1633 (s), 1542 (in), 1 121 (br), 1029 ( ), 587 (m). HRMS-Cl (m/z): found, 556.3759. [a] _D ^2i} +4.8 (c 1.7, C¾OH).

Synthesis of (he linear precursor 29a:

Silver trifluoroacetate (1.8.3 mg, 83.0 μηιοΐ, 2.00 equiv) was added to a solution of triet y!amine (23.0 pL, 166 μηιοί, 4.00 equiv), the β-ketothioester 28 (23.0 mg, 4L0 μιηοΐ, 1 equiv), and the amine 17 (20,1 mg, 50.0 μιηοΙ, 1.20 equiv) in (0.8 mL) at 0 °C. The reaction mixture was sirred for 1 h at 0 °C. The heterogeneous product mixture was diluted with aqueous citric acid solution (5%, 4.0 mL). The resulting precipitate was isolated by filtration. The solid was dried in vacuo to provide the linear precursor 29a as a white solid (31 ,0 mg, 90%).

Ή NMR (600 MHz, DMSO- ) δ 8.80 (s, IH, H ₁₄ ), 8,67 ( J - 6.1 ί-fz, IB, H*?), 8,37 (s, IH, ¾i), 7.90 (bs, I H, H ₄ ), 7.52 d, J= 8.5 Hz, 1H, ¾), 7.28 (¼, IH, ft), 6.86 (bs, IH, H?) ₅ 5.91 (t, J - 8.6 Hz, IH, I½), 4.43 (q, 7.2 Ik, 111 H ₅ ), 4.17 (d, 5.5 Hz. 2H, IU 3.86 (t J - 10.2 Hz, IH, Hi 3.75—3.63 (m, IB, ¾), 3.55 (s, 2Ή, Bus , 3.52 11.5, 8.4 Hz, lB, H _i9 3.34 (s, IB, H, ₃ ), 2.55 - 2.43 (m, 2 1 H ₁₂ ), 2.42 (dd, J™ 15.2, 6.1 Hz, IB. H ₆ ), 2.36 (dd, J= 15.5, 7.9 Hz, IH, H ₆ ), 2.13 - 2.03 (m, 2H, H ₃ ), 1.67 - 1.53 (m, IH, H _n ), 1.54- 1.47 (m, IH, Hn), 1.50 - 1.40 (m, 2H, ¾), 1.38 (d, J - 4.4 Hz, 2H, H _{] 5} ), 1 -22 (bs, 20H, 10 ^χ

C¾), 1.05 - 1.01 (m, 2H, H _{i 5} ), 0.99 (d, J- 6.6 Hz, 3H, H _i( ,), 0.85 (t, J- 6.9 Hz, 3H, H,). ^W C NMR (151 MHz, DMSO< ₆ ) S 204.8 (C), 204.0 (C), 173.8 (C), 172.2 (C), 172.1 (C), 171.4 (C), 171.3 (C), 170.5 (C), 168.0 (C), 166.7 (C), 162.2 (C), 128.4 (CH), 76.9 (CH), 50.2 (Cft), 50.0 (CH), 46.4 (Cft), 43.6 (CH), 41.1 (Cft), 40.5 (C), 39.1 (C¾), 37.7 (Cft), 37.4 (CH ₂ ), 35.2 (Cft), 31.3 (CH ₂ ), 29.7 (CH ₂ ), 29.09 (CH ₂ ), 29.07 (2 x CH,), 29.03 (C¾), 28.97 (CH ₂ ), 28.87 (Cft), 28.73 (Cft), 28.67 (CH ₂ ), 25.2 (Cft), 22.1 (CH ₂ ), 20.6 (Cft), 1 .4 (C%), 14.0 (CH ₃ ). HRMS-C! (m/z): [M + Hp calcd for ¾¾Ν ₇ 0<¾, 834.3894; found, 834.3833. [aj _D ² -17.0 (c 0.8, DMSO).

Synthesis of the linear precursor 29b:

Silver triflnoroacetate (23.8 mg, 0.1 1 rnmo , 2,00 equiv) was added to a solution of triethyi amine (30.0 pL, 0.22 nimol, 4.00 equiv), the p-ketoi oester 28 (30.0 nig, 54,0 μηιοΐ, 1 equiv), and the amine 23 (26.2 mg, 65.0 μτηοΐ 1.20 equiv) in A^N-dimethylfomratmde (2.0 ffiL) at ø °C, The reaction mixture was stirred for 1 h at 0 °C, The heterogeneous product, mixture was diluted with aqueous citric acid solution (5%, 30 a»L), The resulting precipitate was isolated by filtration. The solid was dried in vacuo to provide the linear precursor 29b as a white solid (39,0 mg _? 87%),

Ή NMR (600 MHz, DMSCWs) 8 8.94 (t, J~ 6.0 Hz, 1 H, E _{l 7} ), 8.83 (s, 1 B, H _u ), 8.22 (s, H, K _l9 ), 7M (d, 7.7 Hz, 1.H, ¾), 7.52 (d, J™ 8.5 Hz, 3 H, ¾), 7.26 (bs, 1 H, H ₇ ), 6.87 (bs _? IH, H ₇ ), 5.26 (t, 9.0 Ez, IB, ¾), 4.5 (d, .. ^::: 6.0 Has, 231, l i _)¾ K 4.43 (q, J~ 7.2 Hz, 1 H. 1:1s), 3.70 (id, J~ 8.6, 4.4 Hz, IH, H*), 3.67 - 3.60 (m, 1H, H _2I ), 3.60 (s, 2H, H _J6 ), 3.54 (dd, J = 11.3, 8.2 Hz, IH, H ₂₁ ), 3.34 (s, 2H, H _}3 ), 2.56 - 2.44 (m, 2H, H ₁₂ ), 2.42 (dd,./= 15.4, 6.0 Hz, IH, ¾), 2.35 (dd, /= 15.4, 7.7 Hz, IH, ¾), 2.09 (t, J- 7.5 Hz, 2H, H ₃ ) _> 1.68 ~ 1.53 (ra, IH, Ha), 1.55 - L47 (m, 1 E, H _H ), 1-49 - 1.41 (m, 2H, H ₂ ) 1.42 - 1.31 (m, 2H, H ₁₅ ), 1.23 (bs, 20H, 10 CH ₂ ), 1.05 (q, ~ 3.3 Hz, 2H, H _i3 ), 0.99 (d, J- 6.7 Hz, 3H, H _i() ), 0.85 (t, J- 6.9 Hz, 3B, Hj). C NMR (151 MHz, DMSO-fife) δ 204.8 (C), 204.1 (C), 172.2 (C), 171.8 (C), 171.3 (C), 170.5 (C), 170.2 (C), 168, 1 (C), 167.0 (C), 163.3 (C), 347.3 (C), 322.2 (CH),

78.2 (CH), 50.2 (CH ₂ ), 49.9 (CH), 46.5 (CH ₂ ), 43.6 (CH), 40.5 (CH ₂ ), 40.5 {€), 39.1 (C¾), 37.4 (C%X 35.2 (CH ₂ ), 34.4 (C¾), 31.3 (€¾), 29.7 (CH ₂ ), 29.09 (CH ₂ ), 29.07 (2 x CH ₂ ),

29.03 0¾ 28.97 (CH ₂ ), 28 J7 (CH ₂ ), 28.73 (C¾), 28.67 (CH ₂ ), 25.2 (C¾), 22.1 (CH ₂ ), 20.6 (C¾), 19.4 (C¾X 3 .0 (C¾). HRMS-C! (miz): [M + HJ* calcd for C3 _¾ B _¾ N ₇ 0 ₉ S ₂ , 834.3894; found, 834.3835. [α]» ²⁰ -10,0 (c 0.8, DMSO).

Synthesis of the linear precursor 29c;

Silver trifluoroacetate ( 9.1 mg, 86.0 μΜοί, 2.00 equiv) was added to a solution of tri ethyl amine (24.0 pL, 0.17 mmol, 4.00 equiv), the β-ketoi oester 28 (24.0 mg, 43,0 μηιοΐ, 1 equiv), and the amine 27 (21.0 mg, 52.0 pmoL 1.20 equiv) in A^N-dimethylfomtatmde (0.8 mt) at ø °C, The reaction mixture was stirred for 1 h at 0 *C. The heterogeneous product mixture was diluted with aqueous citric acid solution (5%, 8.0 inL). The resulting precipitate was isolated by filtration. The solid was dried in vacuo to provide the linear precursor 29c as a white solid (31 ,0 mg, 86%),

^! H NMR (600 MHz, DMSCWg) 8 8.97 (t, J~ 6.0 Hz, 1 H, H _n ), 8.84 (s, 1 B, H _H ), 8.47 (s, H, ¾>}, 8.25 (e, IH, If s), 7.90 (tf, J - 7.7 Hz, 1 II !¾), 7.52 (d, J™ 8.6 Hz, 1 H, ¾), 7.27 (bs, IH, H ₇ ), 6.87 (bs, IH, B ₇ ), 4.60 (d, J 6.0 Hz, 211, H _tg ), 4.43 (q, J 7.2 Hz, IH, ¾), 3.76 - 3.64 (m, IH, ¾), 3.62 (s, 2H, H ₁₆ ), 3.35 (s, 2H, H _i3 ), 2.55 - 2.44 (m, 2H, E _u ), 2.42 (dd, J- 15.3, 5.8 Hz, IH, ¾), 2.35 (dd, J = 15.3, 7.8 Hz, IH, ¾), 2.14 - 1.94 (m, 2H, ¾), 1.67 ~ 1.53 (m, IH, Hu), 1.54 - 1.46 (ra, IH, H _n ) _? 1 -48 ~ 1.42 (m, 2H, ¾), 1.40 (q,J= 3.2 Hz, 2H, H _i5 ), 1-31 - 1.13 (m, 20H, 10 x C¾), 1.06 (q, J- .5 Hz, 2B, H, ₅ ), 0-99 (d, J- 6.6 Hz, 3H, H _if> ), 0.84 (t, /= 6.9 Hz, 3H, Hi). ⁿ C NMR ( 151 MHz, Chloroform-ii) 5 204.8 (C), 204.1 (C), 1.72.2 (C), 171.4 (C), 171.3 (C), 170.5 (C), 168.1 (C), 167.0 (C), 162.1 (C), 162.0 (C), 148.1 (C), 147.1 (C), 129.0 (CH), 118.3 (CH), 50.2 (C¾), 50.0 (CH), 46.5 (C¾), 43.6 (CH), 40.6 (C¾), 40.5 (C>, 39.1 (CH ₂ ), 37.4 (C¾), 35.2 (C¾), 31.3 (C¾), 29.7 <CH ₂ ), 29.08 (CH ₂ ), 29.07 (2 x Cl 29.03 (CH ₂ ), 28.97 (CH ₂ ), 28.87 (C¾), 28.73 (C¾), 28.67 (C¾), 25.2 (C¾) _s 22.1 (C¾X 20.6 (CH ₃ ), 19.4 (CH ₂ ), 14.0 (CH, _? ). HRMS-CI (m/z): [M + Hf calcd f r C _35i H ₅₈ N _? ¾S2 _» 832.3737; found, 832.3693. [a] _D ²⁰ +28.0 (c 0.8, DMSO).

Synthesis ofihepyrk ne 30a:

29a Ma

Potassium carbonate (9.94 mg, 72.0 μηιοΐ, 3.00 equiv) was added to a solution of the linear precursor 29a (20.0 mg _s 24.0 iraol _f 1 equiv) in methanol (1.5 mL) at 0 *C. Tire reaction mixture was stirred for 3 h at 0 °C. The heterogeneous product mixture was filtered through a plug of propylsulfonic acid iuoctionaiized silica gel. The filter cake was washed with methanol (10 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was applied to a trimethylami&e acetate- fimctionaiized silica column (Si-T A acetate; eruting with 0.5% formic acid -acetoftitrile). The fractions containing the product 30a were collected, combined, and concentrated to provide the pyridone 30a as a white solid (15.1 rng, 79%).

^} H NMR (500 MHz, DMSO- -CD ₃ OD (3:1)) 5 8.19* (bs, I H, Η»), 8.17 (s, IH, H _l9 ), 7.83* (app t J- 7.7 Hz, IH, H ), 7.64* (app dd, /= 8.0, 5.2 Hz, IH, %), 7.29* (bs, IH, H ₇ ), 6.79* (bs, 1 H, H ₇ ), 6.07 (s, 1 e, H _{i 5} ), 5.86 (app t, J= 8.4 Hz, IH, H _}8 ), 5.24 - 5.06 (m, 2H, B _Hi ), 4.58 ~ 4.46 (m, IH, H ₅ ), 3.92 (app t, 10.3 Hz, IB. H _n ), 3.89 ~ 3.77 (m, IH, H<>), 3.68 - 3.57 (m, IH, Hp), 3.34 ~ 3.16 (m, 2H, H _{2 ), 2.49 - 2.37 (m, 2H, ¾) ₅ 2.16 ~ 2.02 (m, 2H, ¾), 1.80 - 1.68 (m, I B, ¾) _> 1.68 - L57 (m, 1 H, ¾), 1.51. - 1.41 (m, 2H, ¾), 1.39 (q, - 4.8, 3.9 Hz, 2H _; EUX 1.32 (app t, J - 3.5 Hz, 2H, H _.l4 ), 1.28 - 1.12 (m, 20H, 10 * CH ₂ ), 1.05 (app dd, J 9.0, 6.5 Hz, 3H, ¾), 0.83 (t, J ^::: 6.9 Hz, 3H, H,). ¹³ C NMR (1 1 MHz, DMSO- ™ CD ₃ OD (3:1)) 8 572.7 (C), 172.5 (C), 171.5 (C), 170.8 (C), 167.2 (C), 164.1 (C) _s 163.1 (C), 162.1 (C), 160.0 (Q, 153.6 (C), 149.7 (C), 127.4 (CH), 109.5 (C), 103.2 (CH), 76.7 (CH), 50.2 (CH), 45.1 (CH), 45.0 (CH ₂ ), 40.1 (C), 39.0 (CH ₂ ), 37.6 (C¾), 35.6 (C¾), 35.5 (CB ₂ ), 31.7 (CH ₂ ), 29,45 (CH ₂ ), 29.43 (2 CH ₂ ), 29.41 (C¾), 29.34 (C¾), 29.25 (C¾), 29.1

(CH ₂ ), 29.0 (CH ₂ ), 25.6 (CH ₂ ), 24.8 (CH ₂ ), 22.5 (CH ₂ ), 20.3 (C¾), 15.4 (CH ₂ ), 14.1 (C¾). HRMS-CI (ra/z): [M + H] * calcd for 798.3683; found, 798.3623. [a]r _> ²⁰ -5.0 (c 0.8, DMSO).

*H-D exchange occurred slowly m solution.

Synthesis of the pyridone 30b:

Potassium carbonate (4,97 mg, 36.0 μηιοΐ, 3.00 equiv) was added to a solution of the linear precursor 29b (10,0 rag, 12,0 μηιοΐ, I equiv) in methanol (800 μ-L) at 0 °C. The reaction mixture was stirred for 3 h at 0 °C. The heterogeneous product mixture was filtered through a plug of propylsulfonic acid funciionalized silica gel. The filter cake was washed with methanol (8,0 mL), The filtrates wer combined and the combined filtrates were concentrated. The residue obtained was ap lied to a trimethylam ie acetate- fiinctionai zed silica column (Si-T A acetate; eluting with 0.5% formic acid -acetonitrile). The fractions containing the product 30b were collected, combined, and concentrated to provide the pyridone 30b as a white solid (7.7 mg, 80%).

^} H NM (400 MHz, DMSO- -CD ₃ OD (3:1)) δ 8.17 (bs, 2H, ¾ ^* , H _!7 ), 7,89* (d, J = 7.9 Hz, IH, ¾), 7.65* (d ₅ /= 7.9 Hz, !H, ¾), 730* (bs, IH, H ₇ ) _s 6.77* (bs, IH, H ₇ ), 6,1 1 (s, IH, H ₁₅ ), 5,70 - 5,39 (m, 2H, H _½ ), 5,24 - 4.98 (m, 1 H, E \ 4.53 (app q, J ~ 7.3 Hz, IH, ¾}, 3.97 ~ 3.77 (m, IH, ¾), 3.64 ~ 3.45 (¾, 2H, His), 3.44 - 3,26 (m, 2H, H ₁₂ ), 2.53 (dd, J = 15.5. 6.3 Hz, IH, %), 2.43 (dd, J = 15.2, 7.4 Hz, IH, %), 2.23 ~ 1.98 (m, 2H, ¾ 1.82 ~ 1.66 (m, IH, Hi i), 1.66 - L55 (m, I H, H _A ), 1.50 - 1.41 (m, 2H, H ₂ ), 1.39 (q, J- 5.1 , 4.0 Hz, 2H, E _u ), 1 ,32 (q, J- 6.1 , 5.0 Hz, 2H, H _.L4 ), 1 -28 - 1.11 (m, 20H, 10 CH ₂ ), 1 ,07 (d, J= 6.5 Hz, 3H, Hio), 0.82 (t, J - 6.7 Hz, 311 Hj). C NMR. (3:1.)) δ 173.2 (C), 172,5 (C) _s . 172.4 (C) _s . 171.1 (<¾ 167.3 (C), 166.1 (C), 163.4 (C), 162.5 (G), 160,5 (C), 553.7 (C), 147.8 (C), 123.7 (CH), 1.10.2 (C), .103.6 (CB), 79.6 (CH>, 50.5 (CH), 45.2 (CH), 44.8 (C¾), 40.1 (C), 37.5 (CH ₂ ), 35.9 (C¾), 35.8 (C¾), 35.2 (<¾), 3 1.9 (CH ₂ ), 29.61 (2 ^χ C¾), 29.58 (CH ₂ ), 29.56 (C¾), 29.50 (CH?), 29,4 (CH ₂ ), 29.3 (C¾), 29.1 (C¾), 25.7 (CH ₂ ), 24.7 (CH ₂ ), 22.6 (CH ₂ ), 20.5 (CH ₃ ), 15.5 (CH ₂ ), 4.1 (C¾). HRMS-CI (m z): [M + Bj calcd for 798.3683; found, 798.3625. [α]» ²⁰ +39.0 (c 0.8, DMSO).

*H-D exchange occurred slowly in solution.

Synthesis of precoUhactin C (6):

Potassium carbonate (7.48 mg, 54,0 μ*ηοί, 3.00 equtv) was added to a solution of tfee linear precursor 29c (15.0 mg, 18.0 iirnol, 1 equiv) in methanol (L0 mL) at 0 °C. The reaction mixture was stirred for 3 h at 0 ^iJ C. The heterogeneous product mixture was filtered through a plug of propyl sulfonic acid functional ized silica gel (1.0 x 0.5 cm). The filter cake was washed with methanol (10 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was applied to a trimethylamine acetate- functionalized silica column (Si-TMA acetate; elating with 0.5% formic acid-acetonitrile). The tractions containing the product.6 were collected, combined, and concentrated to provide preeolibaetin C (6) as a white solid (11.9 mg, 83%).

^! H NMR (600 MHz, DMSO-i¾) d 8.51 (s, IH, H ), 8.24 (s, 1H, H i 8.18 (s, I B. Η _ϊ8 ), 7.99 (d ₅ J - 7,8 Hz, i H, ¾), 7.72 (d, 8.0 Hz, 1H, ¾), 7,29 (bs, 1H, H ₇ ), 6.82 (bs, 1 H, H ₇ ), 6.16 (s, IH, His), 5.57 (q, J = 15.9 Hz, 2H, Η _ί6 ), 4.48 (q, .7- 7.2 Hz, IB, H ₅ ), 3.88 (dq, J- 13.8. 6.8 Hz, 1H, ¾>), 3.50 ~ 3.31 (ra, 2H, H. _l2 ), 2.47 (dd, J 15.2, .2 Hz, H, ¾), 2,37 (dd, J- 1.5.0, 7.4 Hz, I H, l¾ 2.11 - 1.98 (m, 2H, H ₃ ), 1.78 - 1.64 (m, 2H, H _n ), 1.44 - 1.35 (m, 4H, ¾, H _}4 ), 1 ,36 - 1.31 (m, 2H, H ₁₄ ), 1.2 - 1.11 (m, 20H, 10 CH ₂ ), 1.06 (d, J - 6,5 Hz, 3H, i½), 0.83 (t, 7.0 Hz, 3 Hi, Hj). ¹³ C MR (151 MHz, DMSO- 05 172.2 (C), 171.6 (C), 170.5 (C), 1 6.7 (C), 1 6.5 (C), 164.0 (C), ί 63.2 (C), 161.8 (C), 161.0 (C) ₅ 159.8 (C¾ 153.1 (C), 147.2 (C), 126 J (CH), 118.6 (CH), 109.6 (C), 103.3 (CH), 50.1 (CH), 44,7 (CH), 44.4 (CH ₂ ), 39.7 (C), 37.4 (C¾), 35.4 (CH ₂ ), 35.2 (<¾), 31.3 (C¾ 29.07 (CH ₂ ) ₅ 29.04 (2 CH _¾ ), 29.02 (C¾), 28,96 (C¾), 28.87 (CH _¾ ), 28.7 (CH ₂ ), 28,6 (CH ₂ ), 25.2 (CH ₂ ), 24,1 (CH ₂ ), 22.1 (CH ₂ ), 20.3 (C¾), 5.2 (CH ₂ ), 14.0 (CH ₃ ).

^} H NMR (500 MHz, C¾OD) 8 8.28 (s, IH, H _i7 ), 8.16 (s, IH, H _}8 ), 6.16 (s, IH, H _{] 5} ), 5,75 (d, J = 15.7 Hz, 1 H, H _K< ), 5.70 (d, J - 15.7 Hz, IH, H _i6 ), 4.73 (t, J = 6.5 Hz, IH, H ₅ ), 4.12 - 3.99 (m, IH, ¾), 3.71 ~ 3.57 (m, IH, Η _ί2 ), 3.55 ~ 3.42 (m, IH, H _i2 ), 2.71 (t, J - 6.1 Hz, 2H, ¾), 2.23 (t ₅ J - 7.4 Hz, 2H, ¾), L98 - 1.86 (m, I H, H _u ), 1 -81 - 1.68 (m, IH, H,,}, 1.66 - 1.53 (m, 2H, ¾), 1.54 - 1.50 (m, 2H, H _¾4 ) _? 1.47 - 1.38 (m, 2H, H ₁₄ ), 1,32 - 1.20 (m, 20H, 10 x CH ₂ ), 1.18 (d, J - 6.6 Hz, 3H, H ₁₀ ), 0.89 (t, J - 7.0 Hz, 3H, H,). C NMR (126 MHz, Metha»ol~d4) 5 176.3 (C), 175.0 (C), 172,9 (C) ₅ 1693 (C), 167.2 (C), 164.9 (C 163.6 (C) _s 163.4 (C). 162.5 (Q, 155.2 (C\ 149.3 (C), 148,1 (Q, 126,9 (CH), 120.2 (CM), 1 12.2 {€), 104.2 (CH), 52.0 (CH , 47.0 (CH), 46.0 (CH ₂ .X 41.5 (Q, 38.0 (CH ₂ ), 37.0 (C¾), 36.8 (CH ₂ ), 33.1 (C¾X 30.80 (C¾X 30.77 (CH ₂ ) ₅ 30.76 (2 x CH ₂ ), 30.65 (C¾), 30.5 (CB ₂ X 30.48 (C¾X 30.3 (Ci¾), 26.8 (C¾X 26.0 (CH ₂ } _; 23.7 (CHs), 20.8 (CH ₃ ), 16.3 (0¾X 14.4 (C¾). HRMS-CI (mte): [M + Hf caled for Cs^ - ^S-., 796.3526; found, 796.3466. fafo ²⁶ -21.0 (c 0.8, DMSO).

Synthesis of the amine 31:

A solution of hydrogen chloride in 1,4-dioxane (4,0 N, 6,0 mL, 24.0 mnioi, 13,3 equiv) was added dropwise via syringe pump over 20 rain to a solution of the thiazoie S.12 (467 mg, 1.81 nimol, 1 equiv) in dichloromethane (18,0 mL) at 23 °C. The resulting mixture was stirred for 1 h at 23 °C. The reaction mixture was concentrated to provide the amine 31 as a white solid (352 mg, >99%).

The product 31 obtained in ibis way was used directly in. the following step.

*H NMR (600 MHz, D SO-(¾ δ 8.80 (bs, 3H), 8.52 (s, 1H, ¾), 4.44 (q, - 5.8 Hz, 2B, ¾). ^{S 3} C NMR (151 MHz, D S(W _6. ) 5 162.8 (C), 161 ,8 (C), 3 6.8 <C\ 130.6 (CH ). 39.5 ( H ₂ ).

Synthesis of the β-ketoamid Si 3:

Three equal portions of silver trifluoroaeetate (72.6 mg, 328 ηιοΧ 0.40 equiv) were added over I h to. a solution of triethylamine (459 pL, 3 ,29 mrnoL 4,00 equiv), the β- ketothioester 16 (31 1 nig, 986 umol, 1.20 equiv), and the amine 31 ( 1 0 mg, 822 μηιοΐ, I equiv) in jV^V-dimethyLfonnamide (9.0 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C. The product mixture was directly applied to a column containing trimetli lannne acetate-functiona!ized silica gel (Si-Ί ^' ΜΑ acetate; eluting with 2% acetic acid-methanol). The fractions containing product wore collected, combined, and concentrated. The residue obtained was further purified by automated flash-column chromatography (eluting with 2% acetic acid -dichloromethane initially, grading to 2% acetic acid™ 10% dichloromethane™ methanol, linear gradient). The fractions containing the product SI 3 were collected, _, combined, an concentrated to provide the p-ketoamide S13 as a white solid (173 ig, 55%). Hi NMR (600 MHz, DMSO- ) δ 12.95 bs, 1H), 8.93 (t, - 6.0 Hz, 1H, Η _¾ ), 8.36 (s, 1H, ¾), 7.76 (s, 1H), 4.54 (d, J = 6.0 Hz, 2H, ¾), 3.55 (s, 2H, ¾), 1.40 (a, 9H, ¾), 1.38 ~ 1.33 (m, 2H, H ₇ ), 1.07 (q, J - 4.2 Hz, 2H, H ₇ ). ⁱ³ C NMR (151 MHz, DMSO-i¾) 0 204.7 (C), 169.8 (C), 166.8 (Q, 62.0 (C), 156.0 (C), 146.6 (C), 128.9 (CH), 78.6 (C), 46.1 (CH ₂ ), 41.2 (C), 40,5 (CH ₂ ), 28.2 (CH ₃ ), 19,5 (CH ₂ ). IR (ATR-FTIR), cm ^"'1 : 3316 (br), 2977 (w), 1701 (s), 1684 (s), 1509 (s), 1249 (s), 1 161 (s), 1069 (s), 751 (m). HRMS-Cl (m/st): [M 4· Hf calcd for CitfHsa iiOsS, 384.1229; found, 384.1224.

Synthesis of the amine » ' r , OH

A solution of hydrogen chloride in 1 ,4-dioxane (4.0 N, 2.0 mL, 8,0 mmol, 55.9 equiv) was added dropwise via syringe pump over 20 min to a solution of die β-ketoaraide SO (55.0 mg, 143 μηιοΐ, 1 equiv) in dicMoromethane (6.0 mL) at 23 *C. The resulting mixture was stirred for 1 h at 23 °C. The reaction mixture was concentrated to provide the amine 32 as a white solid (45.9 mg, >99%).

The product 32 obtained in this way was used directly in the following step,

*H NMR (600 MHz, DMSO- ) δ 9.13 (t, J- 6.0 Hz, 1H, Hi), 8.79 (bs, 3H), 8.38 (s, 1H, H ₃ ), 4.57 (d, J - 5.9 Hz, 2H, ¾), 3.38 (s, 2H, H ₄ ), 1.83 - L68 (m, 2H, H ₅ ), 1.57 - 1.26 (m, 2H, H ₅ ). ⁿ C NMR (151 MHz, DMSQ- ) δ 1 9.40 (C), 169.29 (C), 165.86 (C) ₅ 161,99 (C), 146.67 (C), 128.94 (CH), 42.35 (C¾), 42.01 (C), 40. 1 ((¾), 13.10 (CH ₂ ).

Synthesis of the linear precursor S3:

Silver triftnoroacetate (33.4 nig, .151 μκιοΐ, 2.00 equiv) was added to a solution of triemyi amine (42.1 pL, 302 μηιοΐ, 4.00 equiv), the p-ketotliioester 28 (42,0 mg, 75.6 μιηοΐ, 1 equiv), and the amine 32 (24.2 mg, 75.6 μτηοΐ, 1.00 equiv) in AyWime lfonnam de (1.5 ffiL) at ø °C, The reaction mixture was stirred for 1 h at 0 °C, The heterogeneous product, mixture was diluted with aqueous citric acid solution (5%, 32 ruL), The resulting precipitate was isolated by filtration and was dried in vacuo to provide 33.

The product 33 obtained in this way was used directly in the following step.

!H NMR (600 MHz, DMSCWg) 8 8.92 (t, J~ 6.0 Hz, 1 H, E _{l 7} ), 8.83 (s, 1 B, H _H ), 8.36 (s, H, K _l9 ), 7M (d, 8,0 Hz, I.H, H ₄ ), 7.52 (d, J™ 8.6 Hz, 1 B, ¾) _s 7.29 - 7.18 (in, 1.H, H?) _.( 6.91 ··· 6.78. (ni, IH, H ₇ ),4.55 (d, J~ 5.9 Hz, 2H, H ₁₈ ), 4.49 - 4.36 ( , IH, H _s ), 3.78 - 3.62 (m, I H, ¾) _; 3,59 (s, 2H, H ₁₆ ), 3.34 (s, 2H, H,), 2.56 - 2.45 (m, 2H, H ₁₂ ), 2.48 - 2.37 (m, 1 H, H«), 2.39 - 2.27 (m, IH, ¾), 2.08 (q, /== 7.7 Hz, 2H, H ₃ ), 1.64 - 1.53 (m, 1H, H _U ), 1.51— 1.43 (m, 3H, H _n , H ₂ ), 1.39 (q, 7= 3.4 Hz, 2H _? H _J5 ) _f 1.29 - U S (m, 20H, 10 CH ₂ ), 1.05 (q, ,/= 3.6 Hz, 2H, His), 1 -04 - 0.93 (m, 3H, H _Kl ), 0.85 (t, 7= 7.0 Hz, 3H, H _} ). ⁵³ C NMR (151 MHz. DMSO- ) δ 204.8 (C), 204.1 (C), 172.2 (C), 171.4 (C), 171.3 (C), 170.5 (C), 168.1 (C), 1.66.9 (C), 162.0 (C), 146.6 (C), 128.9 (CH), 50.2 (CH ₂ ), 49.9 (CH), 46.6 (C¾), 43.6 (CH), 40.53 (CH ₂ ), 40.50 (C), 39.1 (CH ₂ ), 37.4 (C¾), 35,2 (CH ₂ ) _S 31.3 (CH ₂ ), 29.7 (CH ₂ ), 29.08 (CH ₂ ), 29.07 (2 x C¾), 29.03 (CH ₂ ), 28.97 <<¾), 28-87 (CH ₂ ), 28.73 (CH ₂ ), 28.67 (CH ₂ ), 25.2 (CH ₂ ), 22.1 (C¾), 20.6 (CH ₃ ), 1 .4 (CH ₂ ), 14.0 (C¾).

Synthesis of precoUbactin B 0}:

Potassium carbonate (31.3 mg, 227 pmol, 3.00 equiv) was added to a solution of the unp rified linear precursor 33 (nominally 56.6 mg, 75.6 μηιοΙ, 1 equiv) in methanol (3.5 mL) at 0 °C. The reaction mixture was stirred for 2 h at 0 ^C C. The heterogeneous product mixture was filtered through a pad of propyls lfonic acid tunctionalized silica gel (1.0 * 0.5 cm) . The filter cake was washed with methanol (10 mL). The {titrates were combined and the combined filtrates were concentrated. The residue obtained was applied to a. tximethylamme aeetate-funetionalized silica column (Si-T A acetate; eluting with 0.5% formic acid- aeetonitrile). The f actions containing the product 3 were collected, combined, and concentrated to provide precolibactin B (3) as a white solid (36,0 mg, 67% over 2 steps),

³ H NMR (600 MHz, OM$0~d ₆ ) δ - 13.09 (bs, IH), 8.47 (s, IH, ¾), 8,39 (s, IH, E ₇ % 7.83 (d, J - ST Hz, IH, ¾), 7.72 (d, J= 8.3 Hz. IH, ¾), 7.25 (hs, IH, H ₇ ), 6.83 (bs, IH, H ₇ ), 6.16 is, IH, H ₅ ), 5.62 (d, J~ 15.7 Hz, I H, H _}6 ), 5.51 (d,./- 15.8 Hz, I H, H _½ }, 4.52 (q, J- 7.2 Hz, IH, Hs), 3.93 - 3.79 (m, 1 H, ¾), 3.44 - 3.35 (m, IH, H ₅₂ ), 3.33 ·· ^■ 3.22 (m, IH, 1½), 2.50 ··· ^■ 2.45 (m, IH, H ₆ ), 2.41 (dd, J ~ 15.2, 7.4 Hz, H, H ₆ ), 2.07 ·· ^■ 1.97 (m, 2H, ¾), 1.77 - 1.61 (m, IH, ¾), 1.62 - 1.51 (m, I H, H _{I F} ), 1.48 - 1.39 (m, 2H, H ₂ ), 1.39 - 1.30 (m, 4H, HH), 1.27 - 1.12 (m _s 20H, 10 χ CH ₂ ), 1.03 (d, J - 6.6 Hz, 3H, H _!0 ), 0.84 (t J = 7.0 Hz, 3H, Hi). ^!3 C NMR (151 MHz, DMSCW*) S 172.2 (C), 171.7 (C), 170.6 (C), 166.7 (C), 165.7 (C), 162.0 (C), 161.9 (C), 159.9 (C), 153.2 (C), 146.3 (C), 130.1 (CH), 109.7 (C), 103.3 (CH), 50.0 (CH), 44.6 (CH), 44.3 (CH ₂ ), 39.9 (C), 37.3 (C¾), 35.5 (CH ₂ ), 35.3 (CH ₂ ), 31. (C¾X 29.4 (C¾), 29.13 (2 (¾ !, 29.1 1 (Ofe), 29.04 (C¾) ₅ 28.9 (C¾), 28.81 (CH ₂ ), 28.7 (CH ₂ ), 25.2 (C¾), 24.2 (0¾), 22.2 (C¾), 20.6 (€¾), 15.3 (CH ₂ ), 1.4.1 (CH ₃ ).

H NMR (500 MHz, CDsOD) 5 8.17 (s, I H, H , ₇ ), 6. 7 (s, I H, H ₁₅ ), 5.82 (d, J = 15,3 Hz, IH, E _¼ 5.68 (d, J - 15.2 Hz, 1 H, H _½ ), 4.82 (t, J - 6.5 Hz, 1 H, H ₅ ), 4.20 - 4.03 (m, 1 H, ¾}, 3.71 - 3.53 (m, IH, H ₁₂ ), 3.52 3,42 (m, I H, H _l2 ), 2.81 - 2.72 (m, 2H, ¾), 2.30 - 2.16 (m, 2H, ¾), 1.99 - 1.82 (m _s IH, H _H ) _S 1.62 - 1.54 (m, 3B, H _U† ¾), L56 - 1.47 (m, 2H, H _j4 ), 1.45 - 1.35 (m, 2H, H _H ), 1.34 - 1.19 (ra, 20H, 10 * C¾), 1.16 (d, J - 6.6 Hz, 3H, H ₁₀ ), 0.90 (t J - 6.9 Hz, 3H, H ₅ ). ^l3 C NMR (126 MHz, C¾OD) § 176.4 (C), 175.2 (C), 172.9 (C), 169.2 (C), 166.4 (C), 166.0 (C), 164.9 (C), 162.4 (C) ₅ 155.2 (C), 151.5 (C), 128.9 (CH), 1 12.2 (C), 104.1 (CH), 52.3 (CH), 46.7 (CH), 46.0 (CH ₂ ), 41.4 (C), 37.9 (C%), 37.0 (C¾), 36.8 (CH ₂ ), 33.0 (CH ₂ ), 30.74 (CH ₂ ), 30.71 (3 x CH ₂ ), 30.6 (C¾), 30.4 (2 * C¾), 30.3 (CH ₂ ), 26.8 (CH ₂ ), 26.0 (CH ₂ ), 23.7 (C¾), 21.1 (C¾), 16.3 (CH ₂ ), S4.4 (C¾). HRMS-CI ( h): [M + H calcd for CAHsOA 713.3696; found 713.3689. afo ²⁰ -15.0 (c 0.7, C¾OH).

Synthesis ^' o r c Ubactm A (7):

chloride (EDC*HC1 _? 2.6 mg, 13.5 μηιοΐ, 1.20 equiv) was added to a degassed solution of the pyridone 3 (8.00 mg, 11.2 μηιοΐ. 1 equiv) and A¾ydroxysuccinimide (NHS, LSI mg, 15.7 pminol, 1.40 equiv) in ΛζΛ-dimethylfbrffiaoiide (300 ,» L) at 0 °C. After 30 min the reaction mixture was warmed to 23 ^¾ C and stirred for 8 h. L-eysieirie (2.72 mg. 22.4 μκιοΐ, 2.00 equiv) and triethylararae (9.26 pL, 44.9 μηιοΐ, 4.00 equiv) were added to the reaction .mixture. Th reaction mixture was stirred for a further .14 h at 23 °C. The product mixture was filtered through a plug of propylsttlfonic acid functionalized. silica gel (1 ,0 ^x 0,5 em) nnder an atmosphere of

dimtrogen. The filter cake was washed with methanol (5.0 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was diluted with water (10 mL) and the resulting precipitate was isolated by filtration through a plug of

propy!sulfonic acid lunctionalized silica gel (2.0 ^x 1.0 cm) under a N? atmosphere. The filter cake was washed with methanol (10 mL). The filtrates were combined and the combined filtrates were concentrated to provide preeolibactra A (7) as a white solid (8,2 rag, 89%).

1H NMK (600 MHz, CD ₃ OD) δ 8.23 (s, 1H, H _!? ), 6.16 (s, 1H, ¾), 5.70 (s, 2H, Hi 4.79 (t, J ~ 5.2 Hz, 1H, ¾ , 4.73 (t, J = 6.5 Hz, HI, H ₅ ), 4.10 - 4.02 (m, 1H, %), 3.70 - 3.63 (m ₅ IB, Mn 3.48 ~ 3.41 (m, IH, H _{i 2} ), 3.1 1 (d, J - 5.3 Hz, 2H, ¾), 2.70 (app t J - 6.2 Hz, 2H, ¾), 2.23 ( J - 7.4 Hz, 2H, ¾), 1.99 - 1 ,90 (m, 1H, ¾ ,), 1.77 - 1.67 (ra, I B, H _u ), 1.62 - 1.54 (ra, 2H _S ¾), 1.54 - 1.49 (m, 2B, Hu), L44 ~ 1.38 (m _s 2H, Bui IM- 1,20 (m, 2QB, 10 x C¾), 1.21 (d, J - 6.9 Hz _s ,3H, R _w ) _t 0.89 (t, J = 7.1 Hz, 3H, Hi). C NMR (151 MHz, CD ₃ OD) δ 1.76.3 (C), 175.1 (C) _s 172.83 (C), 172.81 (C) _s 169.2 (C), 166.8 (C), 164.9 (C), 162.7 (C). 162.6 (Q, 155.2 (C\ 149.6 (C), 127.2 (CH), 1 12.2 (C), 104.2 (CM), 55.6 (CH), 5.1.9 (CH), 46.9 (CH), 46.0 (CH ₂ ), 41.5 (C), 38.1 ((¾), 37.0 (CH ₂ ), 36.7 (C¾X 33.1 (CH ₂ ), 30.81 (CH ₂ X 30.79 (C¾), 30.77 (2 * C¾X 30.66 (CH ₂ ), 30.5 i (CH ₂ ), 30.49 (CH ₂ ), 30.3 (C¾X 26.83 (€¾}, 26,79 (CH ₂ ), 25.8 (CH ₂ ), 23.8 (0¾) ₍ ^' 20.7 (CH ₃ X 16.3 (Cl¾), 14.5 (CHs HRMS-O (m/z): [ ÷ H cakd for C^s^O^, 816.3788; fbnad, 816.3787.

[a3» ^2e -1.0 (c l .0 ₅ C¾OH)

Further Examples (Second Set of References Apply) General Experimental Methods.

IW Spectroscopy. tlV thermal denatux ation samples were prepared by mixing calf thymus DMA [32.0 mM base pairs (bps)] m 2.09 mM NaH ₂ P0 ₄ , 7.13 mM Na ₂ HP0 ₄ , 928 μΜ

Na ₂ EDTA, 1 .01 raM DM SO, pH 7.18 to a final volume of 1 ,0 mL. Samples were subjected to sonication (6 b) at 25 °C to effect complete dissolution. After incubation with ISa, 15b, 17a, and 17¾ for 5 min, 1 h, 3 h, 6 h, or 1 h, the OV thermal denaturatlon spectra of the samples were recorded at 260 nm as a function, of temperature (55~>80 °C _S heating rate: 0.5 °C/min). First derivative plots were used to determine the denaturation temperature.

Electrophoretic gel assay. The 4,163 bp plasmid pBR322 was propogated in DH5a, isolated by MaxiPiep (Qiagen), and linearized with 5ϋ/μ§ EeoRI (NEB). The cut plasmid was column purified and eluted into 10 mM Tris p 8.0. For each reaction, 130 ng of DNA (20 μ. base pairs) was incubated with compound in a 10 ΐ, total volume. Reactions proceeded for i 5 h at 37 °C. unless otherwise noted. Compounds were diluted in D SO such that each reaction consisted of a fixed 5% DMSO concentration. Pure MMS (Alfa Aesar) and cisp!atin (Biovision) stock solutions were diluted into DMSO immediately prior to use. After incubation, 35 μΐ- o.f denaturatlon bufier (6% sucrose, 1% sodium hydroxide, 0.04% bromophenol. blue) was added to each reaction. Non-denatured control samples were diluted with 6% sucrose, 0.04% bromophenol blue. Samples were votiexed for 1 mm, left at room temperature for 15 min, and then immediately frozen at -80 °C. Thawed samples were then loaded onto a 1 % agarose Tris-Borate-EDTA (TBE) gel stained with SybrGoId (Molecular Probes) and run in TBE bailer for 1 hour at 120 V. General Expe imental Procedures. AS ! .reactions were performed in single-neck, flame- dried, round-bottomed flasks fitted with rubber septa under a positive pressure of nitrogen unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe or stainless steel cannula, or were handled in a nitrogen-filled drybox (working oxygen level <10 ppm). Organic solutions were concentrate by rotary evaporation at 28-32 °C. Flash- column chromatography was performed as described by Still et at, ¹ employing silica gel (60 A, 40-63 μπι particle size) purchased from Sorbent Technologies (Atlanta, GA). Anion- exchange chromatography was performed as described by Belaud, et al. employing triniethylaniine acetate-functionaltzed silica gel (SiliaBond® TMA Acetate). Analytical thin- layered chromatography (TLC) was performed using glass plates pre-coated with silica gel (0.25 nan, 60 A pore size) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light (UV).

Materials. Commercial solvents and reagents were used as received with the following exceptions. DieMoromemane, ether and A^ T-dhnethylformamide were purified according to the method of Pangborn et l Triethylamine was distilled from calcium hydride under an atmosphere of argon immediately before use. Di s/>propyiamine was distilled from, calcium hydride and was stored under nitrogen. Methanol was distilled from magnesium turnings under an atmosphere of nitrogen immediately before use. Tetrahydrofuran was distilled from sodiuiTJ-benzophenone under an atmosphere of nitrogen immediately before use.

Deoxyribonucleic acid sodium salt from calf thymu (Type I, fibers) was purchased front Sigma Aldiicli. Trimethytainine acetate-functionalized silica gel (SiliaBond® TMA Acetate) was purchased from SiliCycle (Quebec City, CA). er -butyI-( 2-methyI-5-oxopyrroIidine- I -carboxyiate (SI), ⁴ 2'-((3-(l-aminocyclo ropyl -3-oxo panamtdo)methyl -[2,4 ^, - bithiazoIeJ-4-carboxylic acid hydrochloride (11), ^' 3-(terr-butylthio)-3-oxopropanoic acid (S5) ' 2'~(anunomethyl)- 2,4'-bitlnazole]-4-carboxylie acid hydrochloride (S7), ⁵ A,N-bis- (/e /-butoxycarbonyl)-.¥'H2-aminoefhyl)-guanidine (Sll), ^' AT-bis-(teri-butoxycar0onyl)- iV-(4-aminobutyl)-guantdine (S.12),' ½r/-butyl-((4-carbamothioylthiazol-2- yl)methyl)carbamate (SI 3). ⁵ and S-(fer/~hutyl)~3-( 1 -((ier/- btttox.ycarlx>nyl)amino _. )cyclopropyl)-3-oxopropanethioa.te (SI?) ^" were prepared according to published procedures. i«$tr«inent ie«, Proton nuclear magnetic resonance spectra ( H NMR) were recorded at 500 or 600 MHz at 24 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfieW from tetramethylsilane and are referenced to residual protium in the NMR solvent (CD ₂ C! ₂ , 5 5.32; CD ₃ OD, S 3.31 : C ₂ D ₆ OS _t δ 2.50). Data are represented as follows: chemical shift, multiplicity (s ~ singlet, d ~ doublet, t ~ triplet, q ~ quartet ro ~ midtiplet and/or multiple resonances, br ~ broad, app = apparent), coupling constant in Hertz, integration, and assignment. Proton-decoupled carbon nuclear magnetic resonance spectra ( ^,3 C NMR) were recorded at 125 MHz at 24 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, 6 scale) downfleld from

tetramethylsilane and are referenced to the carbo resonances of the solvent (CDaCla, δ 54.0; CD-30D, δ 49.0; CiDsOS, δ 39.5). Signals of protons and carbons were assigned, as far as possible, by using the following two dimensional NMR spectroscopy techniques: [ H, H j COSY (Correlation Spectroscopy), [ ⁵ H, C] HSQC (Heteroniiclear Single Quantum

Coherence) and long range[Ή, ^{! ,} C] HMBC (Heteronuclear Multiple Bond Connectivity). Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were obtained using a Thermo Electron Coiporaiion Nico!et 6700 FT1R spectrometer referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm ^'" *}, intensi ty of absorption (s ~ strong, m - medium, w =~ weak, br =~ broad). Analytical ultra high-performance liquid chromatography/mass spectrometry (UFLC/MS) was performed on a Waters UPLC/MS instrument equipped with a reverse-phase CH$ column (1.7 μτη particle size, 2.1 x 50 mm), dual atmospheric pressure chemical ionization {APi)/eleetrospray (ESI) mass spectrometry detector, and photodiode array detector. Samples were elated with a linear gradient of 5% acetonitrile - water containing 0.1% formic acid- ->100% acetonitrile containing 0.1% formic acid over 0.75 .rain, followed by 100% acetonitrile containing 0. 1% formic acid for 0.75 mm, at a flow rate of 800 pL/tnin, High-resolution mass spectrometry (HRMS) were obtained on a Waters UPLC/HRMS instrument equipped with a dual API/ESI high-resolution mass spectrometry detector and photodiode array detector. Unless otherwise noted, samples were elated, over a reverse-phase is column (1.7 μ ^η ι particle size, 2.1 x 50 mm) with a linear gradient of 5% acetonitri!e-water containing 0.1% formic -acid-*95% acetonitrile-water containing 0,1 % formic acid for I min, at a flow rate of 600 pL r in, Optical rotations were measured on a Perki Elmer poJarimeter equipped with a sodium (589 nra, D) lamp. Optical rotation data are represented as follows; specific rotation ([ajx ¹ ), concentration (g/100 mL), and solvent. UV spectra were recorded on a Cary 3E UV/Vis spectrophotometer equipped with a the »oeleclT.ica!ly controlled 12-cell holder. High precision quartz SUPRAS ^' IL cells with a 1 em path length were used for all ahsorbance studies.

Sy n thetic Proced .ares,

Synthesis qfthe β-ketothioesfer Hi

Ethyl thioaeetafe (2.08 iaL, 19.5 nimoL 1.30 equiv) was added dropwise via syringe to a solution of lithium di-iw-propylamide (19.5 mmol, 1.30 equiv) in tetrahydrofiiraii (75 mL) at -78 °C. The reaction mixture was stirred for 30 min at— 78 °C, A solution of the imide SI (3,00 g, 15, 1 mmol, 1 equiv) in tetrahydrofhran (28 mL) was added dropwise via cannula to the reaction mixture. The resulting mixture was stirred for 3 h at -78 °C. The product mixture was diluted sequentially with saturated aqueous ammonium chloride solution (30 mL) and ethyl acetate (50 mL). The diluted product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 50 mL). The organic layers were combined and the combined organic layers were washed with saturated aqueous sodium chloride solution (30 mL). The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified by flash-column chromatography (etuting with 5% ethyl acetate-hex anes initially, grading to 20% ethyl acetate- hex anes, linear gradient) to provide the β-ketothioester 10 as a light pink solid (2.40 g, 53%).

⁵ H NM (500 ¾ CD ₂ C1 ₂ ) S 4.38 (bs, 1 H), 3.66 (s,.2ΪΙ ¾) _» 3 8 (m, 1R H ₃ ), 2:90 (q, J™ 6,9 Hz, 2H, H ₇ ), 2.65 - 2.50 (m _s 2H, ¾) _. , 1.77 - 1.67 (m, IH, ¾) _. , 1.62 - 1 ,52 (m, 1 ^" H, ¾), 1.41 (s, 9H, Hj), 1.25 (t, . /- 7.8 Hz, 3.H, ¾), 1.10 (d,J = 6.6 Hz, 3H, ¾). C NM (126 MHz, C 2CI2) δ 202.4 (C), 192.6 (C), 155.9 (C), 79.3 (C), 58.2 (CH ₂ ), 46.4 (CH), 40.4

(C%), 31 ,2 (CH ₂ ), 28.7 (Ci¾), 24.5 (Cl¾ 21.8 ((¾), 14.9 (Ci¾), IR (ATR-FTIR), cm ^"1 : 3387 (m), 2797 (w), 2929 (w), 1717 (w), 1683 (s), 1512 (s), 1310 (m), 1170 (m), 1051 (s), 541 (m), HRMS-Ci (m/z): [M +· Na calcd for C^ N C^S, 326.1397; found, 326.1399. [a] _D ^2i> +8.0 (<· 1 Λ, CH _{3 2} ). Synthesis qfth acki 13:

a

- S' M ₃

Silver trifluoroacetate (.1 4 mg, 742 μηιοΐ, 2.00 equiv) was added to a solution of

tr.iethylam.be (207 μΤ, 1.48 mraol, 4.00 equiv) and the amine 11 (149 mg, 371 uraol, 1 equiv) in A^-dimethyiformarmde (2.7 mL) at 0 °C. A solution of the β-keiothioester 10 (146 mg, 482 μηιοΐ, 1.30 equiv) in A^A-dimethyiformamide (1.2 mL) was added dropwise via syringe to the reaction mixture. The reaction vessel was covered with foil to exclude light and the reaction mixture was stirred for 1 h at 0 °C. The heterogeneous product mixture was filtered through a fritted funnel the filtrate was concentrated. The residue obtained was applied to a column containing irimethylamine aeetate-funetionalized silic gel (Si-TMA acetate; eliiting with 0.5% formic acid-acetomtrtle). The fractions containing product were collected, combined, and concentrated. The concentrated product was diluted with a solution containing 0,5% formic aeid-5% me anol-acetonitrile (600 mL), The diluted product solution was concentrated. This process was repeated until LC/MS analysis indicated full conversion to the acid 13 (white solid, 185 mg, 87%).

^¾ H NMR (500 MHz, O SO-i¾) § 13.12 (bs, IH), 8.94 ft, J- 6.0 Ήζ, IH ₅ H _} »), 8.47 (bs, IB, H ₇ ), 8.46 (s, 1 ¾ ¾), 8.23 (s, 1H, H _n ), 6.55 (s, 1H, ¾), 4.59 (d, J - 5.9 Hz, 211 H _n ), 4.17 (app p, J = 6.6 Hz, IB, H ₃ ), 3.55 (s, 2H, ¾), 3.41 (dd, J = 18.2, 8.7 Hz, 1H, H ₅ ), 2.82 (dt, ,/= 18.6, 9.9 Hz, I H, ¾), 1.89 (ddd, J - 20,6, 12.2, 8.6 Hz, 1 H, B ₄ ) _s 1.55 (app t, J ~ 10.4 Hz, I B, ¾), 1.47 (s, 9H, H , 1.39 - 1.34 (m, 2H, %), 1.15 (d, J~ 6.3 Hz, 3H, ¾), 1.05 - 0.99 (m, 2H, ¾}. ^K 'C NMR (126 MHz, DMSO-c¾) 8 204.7 (C), 171.4 (C) _s 169.0 (C), 167,0 (C), 163, 1 (C), 162.1 (C), 153.3 (C), 151.2 (C), 148.3 (C), 147.1 (C), 128.8 (CH), 1 8.2 (CH), 9S.4 (CH), 80.9 (C), 56.0 (CH), 46.3 (C¾), 40,6 (C>, 40,5 (CH ₂ ), 28.9 (C¾), 27,8.(<¾), 27.8 (CH _;? 19.5 (€¾), 19,5 (€¾), 19.3 (CH ₅ ). IR (ATR-FTI ), cm ^"1 : 3329 (m), 2978 <w), 1722 (w), 1711 (w), 1673 (s), 1641 (w), 1586 (m), 1543 (m), 1518 (m), 1286 (s), 1230 (s), 1 I SO (s), 1 1 7 (s), 142 (s), 780 (m). HRMS-CI (m/z): [M ÷ Hf ca!cd for C2«H _{32 s} O?S2,

590.1738; found, 590.1731. [aj _D ² +13.0 (c 1.0, DMSO). Synthesis o f the amide 14a: NMM, T3f>; OMEN ~i«oe „, „ ¾-¾ S„

A solution of T3P in ethyl acetate (50 wt%, 10.6 μϊ,, 17.8 umol, 1.50 equiv) and 4- memyhnorphol ne (6.5 μΐ,, 59.4 ιηοΐ, 5.00 equiv) were- added in sequence to a solution of the acid .13 (7.0 mg _s 1 A 3 u ol, 1 equiv) in tetrahy rolmao (240 uL) at 23 °C. The reaction mixture was stirred for 20 min at 23 °C. A solution of A ^f _tJ ¥-dimethyiethyIenediamine (3.2 uL, 29.7 μίϊΐοΐ, 2.50 equiv) in tetrahydrofuran (50 μΐ.) was added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The concentrated product mtx.ture-.was diluted with ethyl acetate (10 mL). The diluted product, mixture was poured into a separatorv funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers that formed, were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 14a as a whit solid (7.3 mg, 93%). The product so obtained was used without further purification . ^l H NM (400 MHz, DMSO-t¾) S 8.96 (t, J- 5.7 Hz, IH, _¼), 8.48 (bs, 1H, H ₇ ) _s 8.26 (s, 1 H, Hj j , 8.24 (bs, 1 B, HJ 8.19 (s, IH, ¾}, 6,55 (s, IH, ¾), 4,59 (d, J= 5.4 Hz, 2H, H _n ), 4.17 (app p, J 5.7 Hz, 1 H _t ¾), 3.55 (s, 2H, ¾), 3.47 - 3.35 (ra, 3H, ¾, ¾), 2.82 (dt, J ^~ 19.0, 9.9 Hz, IH, Hs), 2.41 (t, J= 6.2 Hz, 2H, H ), 2.18 (s, 6H, H. _l7 ), 1 -97 - 1.81 (m, IH, H ₄ ), 1.55 (ap t, J- 1.0.4 Hz, IH, ¾), 1.47 (s, 9Η, ¾), 1.39 - 1.33 (m, 2H, H _g ), 1.15 (d.J- 5.8 Hz, 3H, ¾1 1.06 - 0.96 (m, 2H _; ¾). C NMR (101 MHz, DMSO- ) δ 204.7 (C), 171.6 (C), 169.0 (C), 167.1 (C), 161.9 (C), 160.2 (C), 153.3 (C), 151.2 (C), 150.8 (C), 147.2 (C), 124.1 (CH), 1 18.2 (CH), 98.4 (CH), 80.9 (C), 58.1 (CH ₂ ), 56.0 (CH), 46.4 (CH ₂ ), 45.2 (C¾), 40.7 (C), 40.1 (CH ₂ ), 36.7 (CH ₂ ), 28,9 ((%), 27.8 (C¾), 27,8 (CH,), 19.5 (CH ₂ ), 1 .5 (CH ₂ ), 19.3 (C¾). 1R (ATR-FTIR), cm ^"1 : 3278 (b w), 2975 (w), 2931 (w), 1703 (m), 1648 (s), 1603 (m), 1549 (m), 1291 (nil 1158 (s). HRMS-CI (m/z): M ÷ H calcd for

660.2633; found, 660.2631. [ ] _D ²⁰ +25.0 (c 1.0, O OH).

Synthesis of the lactam 15a:

Triiiuoroaceiic acid (751 μΤ, 9.82 mmol, 120 equiv) was added dropwise via syringe to a solution of the amide 14a (54.0 mg, 81.8 μο οΐ, 1 equiv) in dicWoromethane (1.6 mL) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C. The reaction mixture was concentrated and the concentrated reaction mixture w s diluted with saturated aqueous sodium bicarbonate solution (4.0 mL). The diluted reaction mixture was stirred for h at 23 ^,0 C. The product mixture was diluted sequentially with water (10 mL) and ethyl acetate (30 mL). The diluted product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 ^χ 30 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and die filtrate was concentrated to provide the lactam 15a as a light yellow solid (27.5 org, 62%). The product so obtained was used without further purification.

^! H NM (500 MHz, DMSO-c¾) d 10.28 (t /- 6,0 Hz, IB, .¾) _> 8.60 (fas, IH, ¾), 8.28 ~ 8.23 (m, 2H, Ηπ, I½), 8-1 (s, 1H, ¾), 4.66 - 4.57 (m, 2B, ¾) _f 4. ! 8 - 4.07 (m, H, IK). 3.39 (app q, - 6.5 Hz. 211, His), 3.35 - 3.30 (ni, 211. H?) _r 3.15 - 3.06 <m, IH, ¾ , 2.87 (dl J- 17.8, 8.8 Hz, IH, H ₅ 2.45 ( J = 6.7 Hz, 2H, H ), 2.21 (s, 6H, H _{i S} ), 2. 12 - 2.06 (m, IE, ¾), 1.76 - 1.61 (m, 2H, ¾), 1.42 - 1.33 (n 3H, ¾, ¾), L 19 (d, J - 6.6 Hz, 3H, B _¾ ). ^U C NMR (126 MHz, DMSO k} 6 171.1 (C), 169.6 (C), 168.3 (C), 168.2 (C), 161.8 (C), 160.2 (C) _; 157.6 (C), 150.8 (C), 147.4 (C), 127.4 (C), 124.2 (CH), 1 18. (CH), 66.5 (CH), 58,0 (C¾) _s 45.3 (C) ₅ 45.1 (CBs), 40.4 (C¾) _s 36.6 (C¾), 36.5 (CH ₂ \ 33.5 (CH ₂ ), 29.7 (CH ₂ ), 21.9 (Cf¾), 11.8 (C¾), 1 1.7 (Q¾). HRMS-O ( /z): [M + H calcd for CS^NTO^, 542.2003; found, 542.2016. [α] ₀ ^2β -<>.0 (<? 1.5, DMSO-

Synthesis ofthepyridone 16:

Sliver trifluoroacetate (410 mg, 1.86 nimol, 2,00 equiv) was added to a solution of triethylaniiae (518 μΐ,, 3.71 mniol, 4.00 equiv) and. the amine 11 (374 mg. 930 μτηοΐ, 1 equiv) in NJ -dimethyiforaiamide (6.0 mL) at 0 °C. A solution of the β-ketothioester 10 (366 mg, 1.21 rniitel 1.30 eqiiiv) in ?V,.AMimethylfoouamide (2.0 mL) was added dropwise via syringes to the reaction mixture. The reaction vessel was covered with foil to ex-elude. light and the reaction mixture was stirred for 1 li at 0 °C. Potassium carbonate (385 mg, 2.79 mrnol, 3.00 equiv) and methanol (8.0 mL) were then added in sequence to the reaction mixture at 0 °C. The reaction mixture was stirred for 30 mm at 0 °C, The heterogeneous product mixture was filtered through a fritted funnel. The filter cake was washed with methanol (10 mL). The filtrates were combined, and the combined, filtrates were concentrated. The residue obtained was applied to a trimethylarome acetaie-funciionaMzed silica column (Si-TMA acetate;

eluting with 0.5% formic acid-acetomtrile). The fractions containing the product 16 were collected, combined, and concentrated to provide the pyridone 16 as a white solid (414 mg, 78%).

^! H NMR (600 MHz, DMSO-£¾) d 8.48 (bs, 1H, ¾), 8.39 (s _> IH, H _n ), S.25 (s, H i. M _w , 6.80 (d, J - 8.0 Bz, IB), 6.17 (s, IH, ¾), 5.60 (d, J - 16.0 Hz, IH, ¾), 5.49 (d, J ~ 1.5.9 Hz, IH, H X 3.63 - 3.52 (m, ΪΗ, ¾), 3.53 ~ 3.45 (m, IH, H ₅ ) _s 3.32 - 3.14 (in, I H, ¾), 1.75 - 1.60 (m, 2H, H ₄ ), 1.37 (app t _s J - 2.7 Hz, 2H, H ₇ ), 1.35 (app t, J - 2,8 Hz, I H, H ₇ ), 1,30 (s, 9H, H _f ), 1.06 J - 6.6 Hz, 3H, H ₂ ). C NMR (151 MH , DMSO-i¾) δ 166.9 «¾ 166.7 (C), 162.3 (C), 161.8 (C\ 161.6 (C), 159.9 (C), 155.1 (C), 153.0 (C), 149.3 (C), 147.2 (C), 128.3 (CH), 1.18.7 (CH), 109.7 (C), 103.3 (CH), 77.4 (C), 46.1. (CH), 44.2 (CH ₂ ), 39.7 (C), 35.5 (C¾), 28.2 (C¾), 24.1 (C¾), 20.7 (<¾), 15.2 (C¾). IR (ATR-FTIR), cm ^"1 : 3327 (w) _s 3121 (w), 2971 (w), 2355 or w), 1720 (w), 1702 (w), 1674 (m), 649 (s), 1571 (m), 1518 (m), 1 171 (m\ 578 (s). HRMS-Ci (ra z): [M + Hf calcd for C _2$ %o ₅ 0«S _2> 572.1632: found, 572.1630. [a] _D ²⁰ ~64.0 (c 0.5, DMSO).

Synthesis of the amide 82:

A solution of T3P in ethyl acetate (50 wt%, 54.7 fiL, 9i ,S μχηοΐ, 1 ,50 equiv) and 4- methylinoiphoiine (33.7 μΐ,, 306 μ ^η ιοΐ, 5.00 equiv) were added in sequence to a solution of the acid 16 (35.0 mg, (SI .2 μιηοΐ, 1 equiv) in tetrahydrofuran (790 μΤ) at 23 °C. N _f N~

Dimethylethyienediamine ( 16.7 153 μιηοΐ. 2.50 equiv) was then added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The concentrated product mixture was diluted wit ethyl acetate (10 mL). The diluted product mixture was poured into separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide S2 as a white solid ( 18.1 rag, 46%). The product so obtained was used without further purification. ^! H NMR (500 MHz, CD ₂ C1 ₂ ) δ 8.06 (s, IH, B _n ) _> 8.01 (s, I E, H ₅₀ ), 7.64 (hs, IE, H _J2 ), 6.33 (bs, 1H _S ¾), 6.01 (s, I H, E _H ), 5.62 (d, J - 15.0 Hz, I H, ¾), 5.55 (d, J- 14.2 Hz, 1 H, ¾), 5.38 (d, J - 7.4 Hz, I H), 3.82 - 3.74 (m _s IH, I¾), 3.6 - 3.58 (m, 1 Η, Η ₅ ), 3.51 (ap , ,/ - 5,9 Hz, 2H , His), 3.48 - 3.41 (m, IH, ¾), 2,51 (t, J = 6,1 Hz _s 211 Hj*) _s 2.27 (s, 6H, Η _ϊ5 ), 1.89 - 1.81 (m, 1 H, H ), 1.80 - 1.69 (m, H, B ₄ ) _> 1.52 - 1.45 (m, 2B, H ₇ ), 1.41 (s, 9H, B,}, 1.37 - 1 J2 (m, 2H, H _? ) _t 1.19 (d, J~ 6.5 Ez, 3H ₅ ¾). C NMR (126 MHz, CD ₂ C12) δ 1 8,4 (C), 166.0 (C) _} 163.1 (C) _} 162.7 (C), 161.2 (C), 160.5 (C) _s 156.1 (C) _s 154.4 (C), 151.8 (C), 148.5 (C), 123.8 (CH), 1 19.2 (CH), 1 10.3 (C), 103.9 (CH), 79.2 (C), 58.7 (CH ₂ ), 47.1 (CH), 45.7 (CI¾), 45.1 (Cl¾ 40.6 (C), 37.5 (Ci¾h 36.1 (Cf¾ 28.7 (CI¾), 25,0 (C¾), 21.3 (C¾), 16.2 (C¾). 1R (ATR-PTIR), cm ¹ : 3327 (br w), 2972 (w), 1694 (w), 1651 (s), 1541 (in), 1250 (m), 1 165 (m), 568 (m). HRMS-CI (m z): [M + Hf caicd for C _S0 H _{40 7} O ₅ S ₂ , 642.2527; found, 642.2532. [«] _D ^2<! -125.8 (c .93, CH ₃ OH).

Synthesis of the amide 17a:

sz

Trifluoroacetic acid (206 μ-L, 2.69 raraol, 120 equiv) was added dropwise via syringe to a solution of the amide S2 (14,4 mg, 22.4 mol, 1 equi v) in dichlorometfaane (560 μί,) at 0 °C. The reaction mixture was stirred for 14 It at 0 °C. The reaction mixture was concentrated. The concentrated product mixture was diluted with chloroform (10 mL). The diluted product mixture was poured into a separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers that formed were separated. The aqueous layer was extracted with chloroform (2 χ .10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 17a as a white solid (10.5 mg, 86%). The product so obtained was used without further purification.

1H NMR (400 MHz, CD3OD) δ 8.24 is, 1H, ¾»), 8.18 (s, Hi ¾>, 6.19 (s, 1 R B-), 5.73 (d, J = 15.4 Hz, Η, ΗχΧ 5.68 (d, J - 15.5 Hz, 1H, ¾}, 3.75 - 3.62 (m, 1H, H ₄ ), 3.60 - .50 (m, 3H, ¾ H _t2 ), 3.10 - 3.02 (m, IB, ¾), 2.59 (t, J - 6.7 Hz, 2H, _n ), 2.32 (s, 6H, M ₁ ), I -87 - 1.72 (m, 2H, ¾), L58 - 1.50 (m, 2H, ¾) ₅ 1 -47 - 1.40 (m, 2H, ¾}, 1.18 (d, J= 6.3 Hz, 3H, Hi). ¹⁵ C MR (151. MHz, CD3OD) 6 169.5 (C), 167.5 (Q, 165.0 (Q, 163.7 (C), 163.4 (C), 162.5 (C), 154.9 (C), 1 1.7 (C), 149.1 (Q, 125.2 (CH), 120.2 (CH), 1 2.3 (C), 104.4 (CH), 59.3 (CH ₂ ), 47.7 (CH), 46.1 ({¾), 45.5 (C¾), 41.5 (Q, 39.0 (CH ₂ ), 38.0 (C¾), 25.5 (Cl¾), 22.5 (C¾), 16,3 (C¾). IR (ATR-FTJR), cm ¹ : 3355 (or w), 2956 (w), 6 1 (w), 1648 (s), 1572 (w), 1545 (w), 1288 (m), 568 (m). HRMS-CI (m/z): [M +· Hf calcd for C ₂₅ ¾ ₂ N _? O _s $ _2> 542.2003; found, 542.2004. [afc ² -13.0 (c L0, O¾0H).

Synthesis of the amide 14h:

A solution of T3P ra ethyl acetate (50 wt%, 22.7 &, 38.2 μηιοί, 1.5 equiv) and 4- methylmorpholine (14,0 μί,, 127 μηιοΐ, 5.00 eqoiv) were added in sequence to a solution of the acid 13 (15.0 mg, 25,4 μηιοΙ, 1 equiv) in tetrahydrofuran (330 uL) at 23 ^a C. The reaction mixture was stirred for 20 min at 23 °C, A solution of methy!amme in tetrahydrofuran (2,00 M, 23 £, 63.6 μι»ο1, 2.50 equiv) was then added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The residue obtained was purified by tl.ash-col.umn chromatography (eluttng with dichloromethane initially, grading to 10% methanol~d.ichlorometh.ane, linear gradient) to provide the amide 14b as an off-white solid (12.1 mg, 79%).

^} H NM (500 MHz, CD ₂ C1 ₂ ) δ 8.05 (s, IH, H ), 8.04 (t s, !H, H _i0 ) 7.93 (s, iH, H _J3 ), 7,38 (bs, I H, Hul 6.55 (s, IH, ¾ ^' ) _> 6.23 (bs, IH, H _? ), 4.76 (d, ./= 6.0 Hz, 2H ₅ H _n ), 4.23 (app p, J ^{■■■■■■} 6.8 Hz, 1H, ¾), 3.66 (s, 2H, ¾), 3.47 (dd, J ^:::: 18.3, 8.7 Hz, IH, H ₅ ) _; 2,98 (d, J - 5.1 Hz, 3H, His), 2.89 (dddd, J - 18.3, 1 1.1 , 8.3, 2.2 Hz, IH, H ₅ ) _; 1.92 (ti _; J - 12.1, 8.5 Hz, 1H, ¾), i .62 (app q, J = 4.4 Hz, 2H, H ₈ ), 1.56 (dd, J = 12.2, 8.6 Hz, 1 H, H ₄ ), 1.18 (d, J -= 6.5 Hz, 3 H, ¾), 1.17 - 1.13 (in, 2H, ¾). C NMR (126 MHz, CD ₂ CI ₃ ) δ 206.1 (C), 170.6 (C), 170.4 (C) _: 167.2 (C), 163.0 (C), 161.9 (C), 156.9 (C), 152.4 (C), 151.6 (C), 148,9 (C), 123.5 (CH), 17.6 (CH), 97.2 (CH), 82.2 (C), 57.5 (CH), 45.6 (CH ₂ ), 42.0 (C), 41.7 (CH ₂ ), 30.2 (CH ₂ ), 28.9 (CHs), 28.5 (CH.,), 26.3 (CH,), 21.6 (CH ₂ ), 21.6 (CH ₂ ), 1 .9 (CH ₃ ). IR (ATR-FTIR), cm ^"3 : 3295 (br w), 2975 (w), 293.1 (w), 1706 (m), 1651 (s), 1606 (m), 1547 (m), 1289 (m), 1 156 (s), 771 (,m). HRMS-O (rn/z): [M + H] ^* found, 603,2053, [a]o ²⁰ +36.1 (c 0.83, C¾C1 ₂ ).

Tritluoroacetic acid (21 μΤ, 2,83 mmol, 120 equiv) was added dropwise via syringe to a solution of the amide 14b (14.2 ig, 23.6 μηιοΐ, 1 equiv) in dichloromethane (590 μ£) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C, The reaction mixture was concentrated. The conce ntrat ed reacti on mixture was diluted with sal orated aqueo us sodium bicarbonate solution (400 μ£). The diluted reaction mixture was stirred for I h at 23 °C. The product mixture was diluted sequentially with water (1.0 mL) and ethyl acetate (3.0 mL). The diliited product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 x 3.0 mL). The organic layers were combined and the combined organic layers were dried ove sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the lactam 15b as a light yellow solid (4,5 mg, 39%), The product so obtained was used without further purification.

Ή NMR (500 MHz. D SO- ) 8 10.28 (t J- 6.0 Ex, 111 ¾), 8.60 (bs _? 1 H, ¾}, 8.41 - 8.34 (m, IB, H _i2 ), 8.25 (s, 1H, H _u ), 8.17 (s, 1H, H _lf »), 4.65 - 4.57 (m, 2H, ¾), 4,17 - 4.07 (n% 1H, .%), 3.38 ~ 3.26 (m, 2R H ₇ ), 3.15 ~ 3.05 (m, 1H, H ₄ ), 2.91 - 2.85 (m, 1H, H ₄ ) _} 2.81 (d, J ^{■■■■■■} 4.8 Hz, 3H, En 2 1 - 2.03 (m, 1M, ¾), 1.73 - L61. (m, 2H, ¾), 1.42 - 1.29 (m, 3H, ¾ ¾), 1. 19 (d, J= 6.7 Hz, 3H, H ₅ ), ¹³ C NMR (126 MHz, DMSO-i¾) δ 171.1 (C), 169.6 (C), 168.4 (C), 168.2 (C), 161.7 (C), .160.9 (€), 157.7 (C), 1.50.9 (C), 147.5 (C), 127.4 (C), 123.8 (CH), 1 17.8 (CH), 66.5 (CH), 45,3 (C), 40,4 (€¾), 36,5 (CH ₃ ), 33.5 (C¾), 29.7 (CH ₂ ), 25.8 (Ct¾ 21.9 (Ci¾), .1.1.8 (C i), 1 1.8 (<¾). H MS-CT (tn/z) [M + Hf calcd for

C ₂₂ H ₂ 5Ns0 ₃ S ₂ , 485.1424; found 485.1418. | ]ΐ) ^2α --2.3 (c 1.3, DMSO-i¾).

-(6

A solution of T3P in ethyl acetate (50 wt%, 54.7 μ£, 91.8 μιηοΐ, 1.50 equiv) and 4- methylmorpholine (33.7 μΐ,, 306 μτηοΐ, 5.00 equiv) were added in sequence to a solutio of the acid 16 (35.0 tng, 61.2 utno!, 1 equiv) in tetehydrofaran (790 μΤ) at 23 °C. A solution of metliylamine in tetrahydrofuran (2,00 M, 77 uL, i 53 μιηοΙ, 2.50 equiv) was then added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The concentrated product mixture was diluted with ethyl acetate (10 mL). The diluted product mixture was poured into a separator)' ^" funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers thai formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 ¾L). The organic layers were conibiiied and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide S3 as a white solid (20.8 mg, 58%). The product so obtained was used without further purification. ^l H NMR (600 MHz, CD _:¾ OD) δ 8.22 (s, I B, Μ _η ), 8.16 (&, I H, ¾<>}, 6,16 (s, 1H, ¾}, 5.71 (d, J- 15.8 Hz, I H, ¾ 5.65 (4 J- 15.8. Hz, I B, ¾), 3.74 (app ft, ,/- 6,4 Hz, I H, ¾), 3.60 - 3,50 (m, 2H, ¾), 2.96 (s, 3H, Η»), 1.89 - 1 ,75 (m, 2H, ¾}, 1.54 - 1.51 (m, 2H, H ₇ ), 1.43 - 1,40 (m, 2H, ¾}, 1.37 (s, 9H, ¾), 1.17 (d, ,/ = 6,7 Hz, 3H, H ₂ ). ^{i 3} C NMR (151 MHz, CD3OD) 8 169.4 (C), 167.4 (C), 164.9 (C), 164.0 (C), 163.7 (C), 162.5 (C), 157.9 (C), 155.1 (C), 151.8 (C), 149.3 (C), 124.9 (CH), 1 19.8 (CH), ! 12.2 (C), 104.3 (CH), 79.9 (€), 47.8 (CH), 45.9 ((¾ 41.5 (C), 36.8 (CB ₂ ), 28.9 (C¾). 26.3 (CH ₃ ), 25.9 (C¾} ₅ 21.2 (CH ₃ ), 16.3 (C¾). IR (ATR-FTIR), cm ^"1 : 3284 (br w), 2971 (w), 1694 (m), 1652 (s), 1573 (m), 1550 (m), 1 167 (ml 570 (s). HRMS-C1 (m/z): [ + if]' ^" caicd for C27¾ ₃ N ₆ 0 ₅ S ₂ , 585.1 48; found, 585.1948. [α]» ²⁰ -101.2 (c 0.85, C¾OH).

Synthesis of the amide 17b:

S3 17»

Trifluoroacetic acid (273 μ , 3.57 mniol, 120 equiv) was added dropwise via swinge to a solution of the amide S3 (1 7.4 rug, 29.8 p ol. 1 equiv) in dichloromethane (740 ,uL) at 0 °C, The reaction mixture was stirred for 14 h at 0 °C, The reaction mixture was concentrated. The concentrated product mixture was diluted with chloroform ( 10 mL), The diluted product mixture was poured into a separatorv funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers thai formed were separated. The aqueous layer was extracted with chloroform (2 x 10 raL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 17b as a white solid (12.6 mg, 87%). The product so obtained was used without further purification.

5B NMR (600 MHz, CftOD) 8 8.24 (s, 1 B, H ₉ ), 8.16 (s, i¾ H _j0 ), 6.2.1 (s, IH, H ₇ ), 5.72 (d, J - 15.7 Hz, 1 H, ¾), 5.67 (d, j - 15.7 Hz, 1H, Bg), 3.74 ··· ^■ 3.65 (m, 1H, H ₄ ), 3.62 - 3.51 (m, 1H, K 3.31 - 3.26 («!, IB, ¾), 2.96 ( , 3H, ¾) _> 2.04- 1.86 (rm 2H, ¾), 1.58 - 1.50 (m, 2H, ¾), 1.48 ~ Ί .41 (m, 2H, ¾), 1.30 (d, J - 6.6 Hz, 3H, Hi). ^,3 C NMR (151 MHz, C¾OD) 8 169.6 (C), 167.3 (C), 164.8 (C), 164.0 {€,), 163.7 (C), 162.3 (C), 153.7 (Q, 151.8 (Q, 149.1 (C), 124.8 (CH), 120.3 (CH), 112.5 (C), 104.8 (CH), 48. 1 (CH), 46.0 (CH ₂ ), 41.6 (C), 36.4 (C¾) _} 26.4 (C¾) _s 25.0 (Ci¾), 20.3 (CBj), 16.4 (€¾). IR (ATR~F 1R), cm ^"1 : 3419 <br w), 2926 (w), 28S7 (w), 1688 (w), 1647 (s), 1556 (ra). 1289 (w), 568 (m). HRMS-CI <m&): [M + H calcd for QdH W^Sa, 485.1424; found, 485.1425. Γα] ₀ ^2δ -29.0 (c 1.0, CH ₃ OH).

Synthesis of ike dimeric amide 14c:

A solution of T3P in ethyl acetate (50 t%, 50.5 μ.Ι, 84.8 μιηβΐ, 2.50 equiv) and 4- raethylmorphoiine (37,3 pL, 339 μη¾ο1, 10.00 equi v) wer added in sequence to a solution of the acid 1.3 (20.0 rng _s 33.9 utnol, i equiv) in tetrahydrofuran (680 μΤ) at 23 °C. The reaction mixture was stirred for 20 min at 23 °C. A solution of ¾A¾is(3-amiuopropyl)methy1amirie (2.7 ί _? 17.0 umol, 0.50 equiv) in tetrahydrofuran (50 μΤ) was then added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The concentrated product mixture was diluted with ethyl acetate (10 mL), The diluted product mixture was poured into separatory funnel, that had been charged with saturated aqueous sodium bicarbonate solution (5.0 ml..) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to. provide the dimeric -amide 1 c as a white solid (1-2.5 nig, 58%). The product so obtained was used without further purification.

H NMR (500 MHz, CD ₂ CI ₂ ) δ 8.21 - 8.14 (m, 2H, !¾), 8.07 - 8.01 (m, 2H, H _M ), 8.01 (s, 2H, HH), 7.80 (s, 2H, H _i2 ), 6.76 (bs, 2H, H ₇ ), 6.57 (s, 2H, ¾), 4.68 (d, J= 5.9 Hz, 4H _; B„), 4.28 - 4.16 (m, 211 ¾ , 3.68 (s, 4H, ¾) 3,55 - 3.49 (m, 411 H, ₅ ), 3.50 - 3.42 (m, 2H, ¾), 2.95 - 2.77 (m, 2H _} H ₅ ), 2.51 (t, J= 6.6 Hz, 4H, H _{7 ), 2.28 ($, 3H, H ₎₈ ), 1.98 - 1.86 (ra, 2H ₅ >¾), 1.87 ··· ^■ 1.80 (m, 4H, ¾& 1 -6 - L58 (m, 4H, ¾), 1.60 - 1.52 (m ₅ 2H, H ₄ ), 1.49 ( _S> 18H, H _f ), 1.17 (d, J = 6.5 Hz, 6H. %), 1.14 - 1.01 (m, 4H ₅ ¾). ^{! 3} C NMR (126 MHz. CD ₂ C1 ₂ ) δ 206.2 (C), 170.6 (C 170.2 (C), 167.4 (C), 162.8 (Q, 161.3 (C) ₍ 156.6 (C), 152.4 (C), 15LS (C), 148.7 (C), 123.6 (CM), 17.6 (CH), 97.5 (CH), 82.1 (C), 57.4 (CH), 56,7 (CM ₂ ), 46.0 (CH ₂ ), 42.5 (<¾), 41.9 (C), 41.6 (Cl¾), 38.9 (CH ₂ ). 30.1 (C¾X 28.9 (CH ₂ ), 28.5 (C¾), 27.5 (CH ₂ ), 21.6 (C¾) ₅ 21.6 (CH ₂ ), 19.8 (C¾). I (ATR-FT1R), cm ^"1 : 3282 ( r w), 2974 (w), 2933 (w), 1708 (m), 1 51 (s), 1608 (m), 1541 (s), 1315 (m), 1290 (s), 1 156 is), 102 ( ), 770 (m), 755 (ra), 620 (w). HRMS-CI (m z):

1288.4770; found, 1288.4768. [ctfe ²⁰ +19,0 (c 1.0, C¾C1 ₂ ).

Synthesis of the im ric lactam 15c:

Trill uoroacetic acid (57.0 μ]1, 745 μτηοί, 120 eqiiiv) was added dropwise via syringe to a solution of the amide 14c (8.0 nig, 6.21 μηιοΙ, 1 eqiii v) in dichioromethane (200 μΐ.) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C. The reaction mixture was concentrated. The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (650 μΐ,). The di kited reaction mixture was stirred for 1 h at 23 °C, The product mixture was diluted sequentially with water (1.0 mL) and ethyl acetate (3.0 mL). The diluted product mixture was transferred to a separately funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 χ 3,0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the dimeric lactam 15c as a Sight yellow solid (4.8 nig, 73%). The product so obtained was used without further purification.

^! H NMR (500 MHz, DMSO~i¾) § 10.28 (t, J- 5.9 Hz, 2H _S ¾), 8.6.1 (bs, 2H, H _s ) ₅ 8.55 (t, J ^::: 5.9 Hz, 2H, Hi 8.22 (s, 2B, ¾,), 8.15 (s, 2H, H _Si> } ₅ 4.60 (d ₅ J- 6.0 Hz, 4H, ¾), 4.16 - 4.07 (m, 2H, ¾), 3.38 - 3.29 (m, 8H, ¾, H ), 3-16 - 3.05 (m, 2H, H ₄ ), 2.86 (dt, J- 18.1, .1 H , 2H, ¾), 2.39 (t, J~ 6.8 Hz, 4H, Hi ₅ ), 2.19 (s, 3H, H _> ), 2.10 -2.04 (m, 2H, ¾), 1.77 ~ 1.65 (m, 8H, ¾, Hu), 1.38 - 1.34 (m, 6H, ¾ ¾), 1.17 (d, J- 7.0 Hz, 6H, H _f ). "C NMR (126 MHz, DMSO-cfe) οΊ71,0 (Q, 369.6 (C), 168,4 (C), 168.2 (C), 161.7 (C), 160.2 (C) ₅ 157.7 (C), 151.0 (C), 147.4 (C), 127.4 (C), 123.9 (CH), 1 17,8 (CH), 66.5 (CH), 55,4 (C¾), 45.3 (C), 41.8 <<¾), 40.4 (C¾), 37.7 (C¾), 36.5 (C¾), 33.5 (C¾), 29.7 (C¾), 26.7 (C¾), 21.9 (C¾), 11.8 (<¾), 11.8 (C¾). HRMS-CI (m/z): [M + U† caicd for C^Hss nO^, 1052.3510; found, 1052.3514. [α]» ²⁰ +1.3 ( 1.5, DMSO-i¾).

Synthesis of the p-ketoth ^' ioester $6 :

ss

1 ,Γ'-Carbonyldiimidazole (1.20 g, 7.38 mmoL 1.50 equiv) was added to a solution of 2-(( en- bi}toxycarbonyl)amino)-2-meihylpiOpanoic acid (S4, 1.00 g, 4.92 mmol, 1 equiv) in tettahydrofuran (25 mL) at 23 °C. The resulting mixture was stirred for 6 h at 23 °C. In a second round-bottomed flask, magnesium ethoxide (845 mg, 7.38 mmol, 1.50 equiv) was added to a solution of 3-(/er/-birtykhio)-3-o opropanoic acid (S5, 2.60 g, 14.8 mmol, 3.00 equiv) In teiiahydrofuran (13 mL) at 23 °C. The resulting mixture was stirred for 6 h at 23 °C, and then was concentrated to dryness . The activated earhoxyiie acid prepared in the first flask was transferred via cannula to the dried magnesium salt prepared in the second flask. The resulting mixture was stirred for 14 h at 23 °C. The product mixture was diluted sequentially with saturated aqueous ammonium chloride solution (20 mL) and ethyl acetate (30 ml,). The diluted product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 30 mL). The organic layers were combined and the combined organic layers were washed with satorated aqueous sodium chloride solution (30 mL). The washed organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified- b llasli-eobnin chromatography (ehrting with 75%

dic oromethane-hexanes initially, grading to dichloromethane, linear gradient) to provide the p-ketoihioesier S6 as a white solid (275 mg, 18%).

Ή NMR (500 MHz, CD ₂ C1 ₂ ) δ 5.04 (bs, 1 B), 3.72 (s, 2H. ¾), 1.46 (&, 9H, H ), 1.42 (s, 9H, H _} ), 1.34 (s, 6H, ¾). ⁿ C NMR (126 MHz, CD ₂ a ₂ ) d 203.7 (C), 193.7 (C), 155.2 (C), 80.7 (C), 61.6 (C), 51.7 (C¾), 49,1 (C), 29,9 (CH _S ), 28.6 (C¾), 24,2 (CH ₃ ). IR (ATR-FTIR), cm ^l : 3348 (ml 2974 (m), 2942 (w), 1723 (s), 1697 (s), 1668 (s), 1522 (s), 1452 (m), 1366 (ra), 1273 (s), 1 166 (s) _s 1080 (s), 1022 (m), 995 (s), 686 (s). HRMS-CI (m/z): [M + Na calcd for CuHay NaC^S, 340.1553; found, 340.1553.

SB

A solution of the tfaoester Sfi (821 mg, 2.59 mmol, 1.30 eqoiv) in A^N-dimethylformamide (5.4 mi.) was added dropwise via syringe over 20 mm to a solution of silver trifiuoroacetate (879 mg, 3.98 mmol, 2.00 equiv), triethylamme (1 ,1 1 mL, 7,96 mmol, 4.00 equiv), and the amine S7 (480 mg _f 1.99 mmol, 1 equiv) in A^AT-d nethylformainide (21 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 ° . The product mixture was filtered through a fritted funnel and the filtrate was concentrated. The concentrated product mixture, was applied to a column containing trimethylmnine acetate-ftinctionaltzed silica gel (Si-TMA acetate; etuting with 2% acetic acid-metihano ). The .fractions containing product were collected, combined, and concentrated. The residue obtained was triturated, with

dichloromethane (50 mL) to provide the β-ketoamide S8 as a white solid (368 mg, 40%).

*H NMR (500 MHz, DMSO- ) δ 13.01 (bs, 1H) _S 8.94 - 8.86 (m, 1 H, ¾), 8.47 (s, I B, »?), 8.25 (s, til. B 7.49 (bs, 1 H), 4.60 (d, 5.9 Hz, 2Ή, H ₅ ), 3.4 (s, 2H, ¾), 1.39 (s, 911, Hj), 1.23 (s, 6H, ¾). °C NMR (126 MHz, DMSC O δ 205.4 (C), 171.3 (C), 167.2 (C), 162.1 (C), 162.0 (C), 155.0 (C), 148.2 (C), 147.1 (C) ₍ .128.9 (CH), 118.3 (CH), 78.6 (C), 60.2 (C), 43,0 (CH ₂ ), 40.5 (C¾), 28.2 (CH ₃ ), 23.2 (C¾X IR (ATR-FTIR), cm ^'" *: 3308 (br m), 2962 (w), 2933 (w), 1713 (m), 1670 (s), 1650 (s), 1516 (s), 1293 (s), 1 163 (s) _? 1053 (m), 754 (ml HRMS-CI (m/z): [M + Hf calcd for 469.1210; found, 469.1212. Synthesis of the hydrochloride sail S9:

Si S9

A solution of hydrogen chloride in 1 ,4-dioxane (4.0 N, 3.74 mL, 14.5 mmol, 70,2 equiv) was added dropwise via swinge to a solution of the β-keioamide S8 (100 mg, 213 μιι οΐ, 1 eqiiiv) in dichloromethane (4.0 mL) at 0 °C. The reaction mixture was aliowed to warm to 23 °C and stirred at this temperature for I h. The product mixture was concentrated to provide the hydrochloride salt S9 as a white solid (86.4 mg, >99%). The product S9 obtained in this way was used directly in the following step.

^} H NMR (400 MHz, DMSO-i ) δ 9.22 (t, J = 6.1 Hz, 1H, %), 8.49 (s, 1H, ¾), 8.41 (bs, 3H), 8.29 (s, ί 11 H ₅ ), 4.65 (d, J - 5.8 Hz, 2H, ¾), 3.77 (s, 2H, H ₂ ), 1.49 (s, 6H, H,)- ^iJ C NMR (126 Hz, DMSO-£¾) δ 202.7 (C), 170.7 (C), 165.9 (C), 162.0 (C), 162.0 (C), 148.1 (C), 147.1 (C), 128.9 (CH), 1 18.4 (CH), 61.5 (C), 43.7 (CH ₂ ), 40.5 (CH ₂ ), 21.9 (CH ₃ ).

Synthesis of the acid SI 9:

Silver trifi uoroacetate (164 mg, 742 μηιοί, 2.00 eqaiv) was added to a solution of

tfrethyl amine (207 uL, 1.48 mmol, 4.00 eqtiiv) and the amine $9 (149 mg, 3 1 μβϊθί, 1 equiv) in AyV-dimethylfonnamide (2.7 mL) at 0 °C, A solution of the β-ketothioester 10 (146 mg, 482 μ ^η αοΐ, 1.30 equi v) in (1.2 mL) was added dropwise via syringe to the reaction mixture. The reaction vessel was covered with foil to exclude light and the reaction mixture was stirred for 1 h at 0 °C. The heterogeneous product mixture was filtered through a fritted funnel and the filtrate was concentrated. The residue obtained was applied to a column containing trimemylamine aeetate-fonctionaiized silic gel (St-TMA acetate; ehiting with.0.5% formic acid- acetonitriie). The fractions containing product were collected, -combined, and concentrated to provide a 23: J mixture of the acids S.t0 and S.iO* as a white solid (1 6 mg, 34%). The product mixture so obtained was used without further purification.

H NMR (SI0, 400 MHz, DMSCW ₆ ) δ 8.81 (t. J™ 6.0 ft, IH, H _i0 ), 8.47 (s, IH, H-, ₃ ), 8.29 (bs, IH, H ₇ ), 8.25 is, IH, H _{2 ), 6.59 (s, IH, ¾), 4.60 (<L ,/= 5.9 Hz, 2H, H _n ), 4.15 (app p, /= 6,5 Hz, I H, ¾), 3.43 (s, 2H, H ₉ ), 3.40 - 3.26 fm, I H, ¾), 2.75 (dt, 7 = 18.9 _S 10.0 Hz, I B, Hs), 1.95 - 1.77 (m, IH, ¾), 1.56 ~ 1.47 (m, IH, H ), 1.47 (s, 9H, Hi), 1.23 (s, 6H, ¾), 1.13 (d, J - 6.4 Hz, 3H, H ₂ ). *H NMR (S!0', 400 MHz, DMSO- ) δ 8.87 (t, J = 6.1 Hz, IH, H ₁ -), 8.61 (bs, I H, Hr), 8.47 (s, I H, H\r), 835 (s, IH, H _i ) _? 6.71 - 6.64 (m, IH), 4.60 (d, J- 5.9 Hz, 2H, Hn- 3.51 (s, 2H, ¾-), 3.39 - 3.27 (m, 3H, H _r , ¾*), 2.54 - 2.44 (m, 2H, ¾>), 1.56 - 1.45 (m, 2H, H ₄ >), 1.37 (s, 9H, H _r ), 1.27 (s, 6H, H ), 0.98 (d, J- 6.5 Hz, 3H, ¾·}. °C NMR (SI0, 101 MHz, DMSC ₆ ) δ 204.9 (C), 171.4 (C), 1 7.6 (C), 1 7.5 (C), 163.1 (C), 162.0 (G), 153.3 (C), 151.2 (C), 148.1 (C), 147.1 (C), 128.9 (CH), 118.3 (CH), 98.2 (CH), 80.8 (C), 59.9 (C), 56.0 (CH), 43.0 { ¾}, 40.6 (CH ₂ ), 28.8 _. (CH ₂ ), 27.8 (CH ₂ ), 27.8 (C¾), 23.4 «¾.), 23.3 (C¾), 19.3 (0¾). ^l3 C NMR (Si 0*, 101 MHz, DMSC δ 204.7 (C), 204.6 (C), 171.4 (C), 167.3 (C), 166.5 (C), 163.1 (C), 162.0 (C), 155.1 (C), 148.1 (C), 147.1 (C), 128.9 (CH), 1 18.3 (CH), 77.4 (C), 60.4 (C), 50.0 (CH ₂ ), 45,2 (CH), 43.4 (CH ₂ ), 40,6 (C¾), 38.8 (CH ₂ ), 29.9 (<¾), 28.3 (CBsl 23.4 (C¾), 23.3 (C¾), 20.9 (C¾). IR (ATR-FTIR), cm ^{" 1} : 3297 (br w), 2976 (w) _f 2933 (w) ₅ 171 1 (s), _. 1648 (s), 1244 (m) _s 1 158 (s), 754 (m), 635 (w). HRMS-CI (m/z): [M + H calcd for 592.1894; found, 592.1891. [a] _D ²⁰ +15.0 (c 1.0,

CH ₃ OH),

Synthesis of the amide 14d:

A solution of T3P in ethyl acetate (50 wt%, 154 uL, 259 μοιοΐ, 1 ,50 equiv) and 4- methylmorpholine (94,9 jiL, 863 μιηο , 5.00 equiv) were added in sequence to a solution of the acid SI® (102,1 rag, 173 μηιοϊ, 1 equiv) in teirahydrofnrnn (1.2 mL) at 23 °C. The reaction mixture was stirred for 20 min t 23 °C. i^A^Dimeihyle ^' thylenediamine (47, 1 ί., 431 μιηοΐ, 2.50 equiv) was added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C, The product mixture was concentrated. The concentrated product mixture was diluted with ethyl acetate (1.0 mL). The diluted product -mixture was poured into a separaiory funnel that had been charged with satuiated aqueous sodium bicarbonate solution (5.0 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x .10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified by flash-column chromatography (elating with 1 % .methanol-dichloromeihane initially, grading to 40% methanol-dichioromethane, linear gradient) to provide the amide 14d as a white solid (94.7 nig, 83%).

⁵ H NMR (500 MHz, CD ₂ CI ₂ ) δ 8.05 (bs, 2H, B _n , H _I3 ), 7.92 - 7.85 (in, 2H, H _J0 , E _x4 ), 6.55 ( _s> 1H, ¾), 5.97 (bs, I H, H ₇ ), 4.75 (d, J= 6.0 Hz, 2H, H _u ), 4.20 (app p, J= 6.7 Hz, 1 H, ¾),

3.67 - 3.60 (m, 2H, His), 3.54 (s, 2H, H _s ), 3.37 (dd _s ./== 18.3, 9.0 Hz, 1 H, H ₅ ), 2.86 2.71 (m,

3H, ¾, H _J6 ), 2,50 (s, 6H, E _i7 ), 1 ,86 (ddd, J = 20.7, 12.1, 8.6 Hz, I H, ¾), 1.50 (s, 10H, E _x , ¾), 1.34 (s, 6H, ¾), 1.14 (d, J~ 6.4 Hz, 3H, ¾). ^{I 3} C NMR (126 Hz, CD ₃ C1 ₂ ) δ 206.1 (C), 170.7 (C), 169.3 (C), 168.0 (C), 163.2 (C), 161.7 (C), 156.9 (C), 152.4 (C), 151.4 (C), 148.8 (C), 123.9 (CM), 118.1 (CH), 96.9 (CH), 82.2 (C), 61.2 (C), 58.7 (Cl¾), 57.4 (CH), 45.2 (C¾), 43.3 (CH ₂ ), 41 .7 «¾), 36.7 (CH ₂ ), 30.1 (CH ₂ ), 28.8 (C¾) _s 28.5 (CHj), 24.3 (C¾), 24.3 (CH ₃ ), 19.8 (<¾). ffi. (AT!l-FTIR), cm ^""1 : 3306 (br w), 2974 (w), 2934 (w), 1712 (m), 1654 (s), 1601 (w), 1540 (m), 1245 (mh 1 156 (s), 620 (w). HRMS-C1 (mix): [M + fff calcd for C3ol¾N70 _Ct S ₂ , 662.2789; found, 662.2787. α^ -θ.Ο (c 1.0, CH ₂ C1 ₂ ).

Synthesis of the lactam tS4:

Triiltioroacetic acid (31.9 p.L, 417 uraol, 120 equiv) was added dropwise via syringe to a solution of the amide 14d (2,3 mg, 3.48 μηιοΐ, 1 equiv) in dichloromethane (200 μί.) at 0 °C, The reaction mixture was stirred for 14 at 0 °C. The reaction mixture was concentrated. The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (200 uL). The diluted reactio mixture was stirred for 1 h at 23 °C. The product mixture was diluted sequentially with water (1.0 mL) and ethyl acetate (3.0 mL). The diluted product mixture was transferred to a separaiory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 x 3.0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. To induce complete cycHzation, the residue obtained was diluted with dkhloromethane (1.0 mL) and left to stand for 3 fa at 23 °C. The diluted product mixture was concentrated to provide the lactam ISd as a off-white solid (1.3 rag, 67%).

lH NMR (500 MHz, CD ₂ C1 ₂ ) δ 10.84 (t, J - 6.0 Hz, 1 H, :¾), 8.05 (s, 1 H, H _n ), 7.92 (s, IH, Hjo), 7.66 (bs, IH, ¾), 6.23 (bs, IE, H ₅ ), 4.71 - 4.65 (m, 2H, ¾), 4.23 - 4.15 (m, IH, H ₂ ), 3.59 - 3.45 (m, 4H _s H7, Hn), 3.21 - 3.11 (m, 1 H, H ₄ ), 3.03 - 2.90 (m, IH, ¾), 2.54 - 2.47 (m, 2H, H _i4 ), 2.27 (s, 6H, H _t5 ), 2.21 - 2.13 (m, IH, ¾), 1 ,44 - 1.40 (m, 7H, ¾, ¾), 1 ,25 (d, J= 6.8 Hz, 3H Hj). "C NMR (126 MHz, CD ₂ CS ₃ ) S 171.2 {£¾ 169.8 (C), 169.8 (C), 169,2 (C), 163.6 (C), 163.0 (C), 161.3 (C), 151.7 (C), 149.0 (C), 127.9 (C) _s 123.7 (CH), 117.4 (CM), 67.9 (CH), 62.1 (C), 58.8 ((¾), 45.7 (CH ₃ ), 41.4 (C¾), 37.5 (C¾), 37.5 (C¾), 36.5 (C¾), 30.8 (C¾). 25.5 (CH ₃ ), 25.5 (CH ₃ ). 22.4 (C¾). I (ATR-FTIR), cm ^"1 : 3277 (br w), 2961 (w), 1655 (s), 1604 (w), 1543 (s), 1 187 (m), 619 (m). HRMS-Ci ( z): [M + Hf calcd or C ₂₅ H ₃ N70 ₃ S ₂ , 544.2159; found, 544.2164. [α]ο ²⁰ ·· 15.0 c 1.0, CH ₂ C! ₂ ).

Synthesis of the ihwether IS:

1Sb 1S

Propanediol (100 uL) and jo-toluenesulfonic acid raonohydrate (2.0 rag, 10.3 μιηοΐ. 1,00 equiv) were added in sequence to a solution of the amide 15b (5.0 mg, 10.3 prnol, 1 equiv) in acetonitrile (300 pL) at 23 °C, The reaction mixture was stirred for 30 min at 23 °C. N,N- Dimethylformamide (50 pL) was added to the reaction mixture at 23 °C. The reaction mixture was stirred for 3 h at 23 °C, The product mixture was diluted sequentially with toluene (5.0 mL) and hexanes (3.0 mL). The diluted product mixture was filtered and the filtrate was concentrated. The residue obtained was dried by azeotropic distillation with toluene (10 mL) to provide the thioether 18 as a light yellow solid (1.7 mg, 34%).

H NMR (500 MHz. DMSC ₆ ) 5 9.34 - 9.26 (ra. IH, H ₇ ), 8.41 - 8,36 (m, 2H, H _3} Hn), 8,25

(s, IH, H, ₀ ), 8, 18 (s, I H, ¾), 4.60 - 4,55 (m, 2H, ¾}, 3,93 - 3.85 (m, IH, ¾), 3,32 (d, J~ 17.0 Hz, I H, ¾), 3.28 (d, J = 16.8 Hz, IE, ¾), 3.02 - 2,92 (m, I H, ¾), 2.91 - 2.84 (m, IH, ¾), 2,82 (bs, 3H, E _u ), 2.62 - 2,54 (m, 4H, H _{3 , H _H ), 2,49 - 2.42 (m, 2H/H ₁₅ ), 2.14 - 2.07 IH, ¾), 1.55 - 1.41 (m, 3B, ¾, ¾), 1.17 (d,J- 6.8 Hz, 3H, B,), 0. 0 (t, J - 7.2 Hz, 3H, H _i7 ). ^J H NMR (400 MHz, Cl¾OD) d 8.39 - 8.14 (m, 2H, ¾ ¾ ₀ ), 4.71 (d, J ^' = 5.2 Hz, 2H, ¾), 4.12 - 3.99 (m, IH, H ₃ ), 3.49 (s, 2H, ¾), 3.04 - 2.89 (m, 5H, B ₄ , H _I2 ), 2.77 - 2.65 (n , 4H, Hi ,, HH , 2.44 ( /= 7.2 Hz, 2¾ His), 2.32 - 2. 19 (m, IH, ¾), 1.67 - 1.56(ra, 1 H, H ₃ j, 1.54 (app ¾ J ^™ 7.3 Hz, 2H, H _½ ), 1.34 - 1.24 (m, 3B, H,), 0.93 (t, J ^::: 7.3 Hz, 3H, H, _? ). ^L3 C NMR (126 MHz, DMSO- ) § 171.8 (C), 171.3 (C), 16.1.7 (C), 160.9 (C), 160.8 (C), 150.9 (C), 147.2 (C), 125.8 (C), 123.7 (CH), 1 17.9 (CH), 104.3 (C), 96.1 (C), 54.6 (CH), 40.6 (C¾X 33.1 (C%), 32.0 (C¾), 30.4 (CH ₂ ), 30.2 (CH ₂ ), 29.2 (CH ₂ ), 25.8 (C¾) ₅ 25.7 (CH ₂ ), 22.5 «¾), 21 ,3 (CH ₃ ) ₅ 13.3 «¾). HRMS-CI (m/z): [ ÷ Ef calcd for C ₂ s¾N ₆ 0 _;¾ $: ₅ , 561.1771; .found, 561.1776. [a] _D ^¾i +60.5 (c 0.22, DMSG-i¾).

Synthesis of ^' th amide

A solution of T3P hi ethyl acetate (50 wt%, 30.3 JAL, 50.9 μχηοΐ 1.50 equiv) and 4~ tnethylmorpholme (18.6 uL, 70 μηιο ^' Ι, 5.00 equiv) were added in sequence to a solution of the acid 13 (20.0 mg, 33.9 jrtnol, 1 equiv) in tetrahydroiuran (680 μί_) at 23 °C. The reaction mixture was stirred for 20 mill at 23 °C. A A'-Dmietliyi-l ^^diammopropane (1 .7 _t uL, 84.8 umo!, 2.50 equiv) was then added to the reaction mixture. The resulting mixture was stirred for 7 ii at 23 °C. The product mixture was concentrated. The concentrated product mixture was diluted with ethyl acetate (10 raL). The diluted product mixture was poured into a separators' funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 mL) and the layers that formed were separated. The aqueous layer was extracted wit ethyl acetate (2 x 10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 14e as a white solid (21,8 mg, 95%), The product so obtained was used without further purification.

Ή NM (500 MHz, CD ₂ Ci ₂ ) 5 8.33 - ^' 8.22 (ra, 1H,¾ S. l 1 - 8.05 (m, I B, H _m % 8.03 (s, IH, En 7.90 (s, I H, H _i2 ), 6.55 (s, I B, ¾), 6,29 (bs ₅ IH, H ₇ ), 4.76 (d, i- 5,9 Hz, 2H, E ), 4.30 - 4.16 (m, I H, H ₃ ), 3.66 (s, 2H, ¾), 3,53 - 3.45 (m, 2B, H _I5 ), 3.48 ···· 3.42 (ra, IH, ¾), 2.99 - 2,80 (m _s I B, H ₅ ), 2.41 (t, J- 6.5 Hz, 2B, !½), 2.25 (s, 6H, Η·, ₈ ), 1.92 (tt, J- 11.9, 8.5 Hz, IH, H ₄ >, 1.83 - 1.70 (m, 2H, H _¾ ), 1.62 (app q, J- 4.4 Hz, 2H, ¾), 1.56 (dd, J- 12.4, 8.4 Hz, 111 ¾), 1.49 (s, 9E, Hi), 1.17 (d, J ^::: 6.4 Hz, 3H, ¾), 1.18 - 1 .32 (m, 2B, ¾). C NMR ( 126 MHz, CDsCk) δ 206.1 (€), 170.6 (C), 170.4 (C), 167.2 (C), 162.8 (C), 161.3 (C), 156.8 (Q, 152.4 (Q, 152.1 (C), 149.0 (C), 123.4 (CH), 1 17.3 (CH), 97.3 (CH), 82.2 (C), 58.7 (CH ₂ ), 57.4 (CH). 45.8 (C%X 45.6 (CH ₂ ), 42.0 (C), 41.7 (C¾), 39.2 (CH ₂ ), 30.2 (C¾), 28.5 (C¾ 28.5 (Ci¾), 27.3 (C¾), 21.6 (CH ₂ ), 21.6 (CBs), 19.8 (CH ₃ ). IR (ATR-FTIR), cm ; 3282 (w), 2976 (w), 1709 ^' (m), 1650 (s), 1606 (w), 1542 (m), 1290 (m), 1155 (s), 770 (w). HRMS-Ci (m/z): [M + H calcd for H ₄ 4N ₇ f¾S _2j 674.2789; found, 674.2795. [a]o ^2f) +33,0 (c 1.0, C¾C ).

Synthesis of the amide 14f:

A solution of T3P in ethyl acetate (50 t%, 22.7 μΐ, 38.2 μηιοΐ, 1 ,50 equiv) and 4- roethylnrarpRoline (14.0 μΐ,, 127 μιηοΐ, 5,00 equiv) were added in sequence to a solution of the acid 13 (15.0 mg, 25.4 μπιοΐ, 1. equiv) hi tebahydrofaran (510 μ£) at 23 °C. The reaction mixture was stirred for 20 mm at 23 °C. tert-Bitty1~A ⁱ -(2-amiiioethyl)carbamate (9.1 μ1. ₅ 57.2 μηκ>1 2.25 equiv) was added to the reaction mixture. The resulting mixture was stirred for 7 b at 23 °C. The product mixture was concentrated. The residue obtained was purified by flash-column chromatography (elating with dichloromethane initially, grading to 10%

methanol "dichloronietliane, linear gradient) to provide the amide 14f as an off-white solid (15.1 mg, 81%).

^■ H NMR (600 MHz. CD ₂ Cl ₂ ) S 8,10 - 8,04 (m _} 211 H _!8 , ¾), 7,96 ( , IH, H , ₂ ), 7 76 (bs, I B, H, ₄ ), 6.55 (s, 1H, ¾), 6,24 (bs, IH, B ₇ ), 5,08 (bs, I H), 4.76 (d, /= 4.1 Hz, 2H, H _n ), 4.32 - 4.19 (m, IH, ¾), 3.67 (s, 2H, ¾), 3.57 ··· ^■ 3.50 (m, 2H, H _{5 ), 3.48 (dd, J- 18.3, 8.8 Hz, IH, H ₅ ), 3.38 - 3.33 (m, 2H, His), 2.96 - 2.81 (m, 1 H, H ₅ ), 2.03 - 1.85 (m, 1 H, H ), 1.65 ·· ^■ 1.59 (m, 2H, lis), .1.61 - 1.52 (m, 1 H, lh% L50 (s, 9H, Hi), 1.40 (s, 911 H _l7 ), 1.20 - 1.13 (m, 5H, ¾, ¾). ^,3 C NMR ( 126 MHz, D ₂ CI ₂ ) δ 206.2 (C), 170.6 (C), 170.4 (C), 167.3 (C), 163.0 (C), 162.1 (C), 156.8 (C), 156.8 (C), 152.4 (C), 151.3 (C) _; 148.8 (C), 123.9 (CH), 117.8

(CH), 97.3 (CH), 82.2 (C), 79.7 (C), 57.4 (CH), 45.7 (CM,), 42.0 (C), 41.6 (CH ₂ ) _S 41.2 (CH ₂ ), 40.5 (CH ₂ ) _f 30.2 (CH,), 28.8 (CH ₂ ), 28.6 (C¾), 28.5 (CH ₃ ) _f 21.6 (CH ₂ ) _S 21.6 (C¾), 19.8 (C¾). IR (ATR-FT!R), cm ⁴ : 3313 (br w), 2970 (w), 2930 <w), 1704 (m), 1650 (m), 1524 (w), 1 154 (s 1 25 (w), 802 (m). HRMS-CI (m/z): |M + Hf calcd for

732.2844; found, 732.2852. [ct] _D ^2e +4.0 (c 1.0, CH ₂ C. ₂ ).

Synthesis of the amide I4g:

A solution of T3P in ethyl acetate (50 wt%, 30.3 μΐ,, 50.9 μηιοΙ, 1.50 equiv) and 4~

methylmorphoime (18.6 μΐ,, 370 μιηοΐ, 5.00 equiv) were added in sequence to a solution of the acid 13 (20.0 nig, 33,9 μηιοΐ, 1 eqaiv) in tetrafiydtofuran (670 uJL) at 23 °C. The reaction mixture was stirred for 20 rein at 23 °C. ½/t~Butyl~iV-(2-aminopenty])car amate (17.7 μΤ, 84.8 μπίοΐ, 2.50 equiv) was added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The residue obtained was purified by fiash-co!innri chromatography (elating with dich oromethane initially, grading to 3 % methanol-dicbloromethane, linear gradient) to provide the amide 14g as an off-white solid (12,6 mg, 48%).

Ή NMR (500 MHz, CD ₂ C1 ₂ ) δ 8.09 - 8.04 (ra, IH, H _J0 ), 8.05 (s, IH, ¾}, 7.96 ( _s> I H, H _l2 ),

7.42 (t, J- 6.2 Hz. I H, H. ₁₄ ), 6.55 (s, I H, ¾), 6.27 (bs, 1 H, H-). 4.76 (d, ,/= 6.0 Hz, 2H, H , ,) _s 4.64 (bs, IH), 4.23 (app p, J - 6.7 Hz, IH, ¾), 3.66 (s _s 2H, ¾), 3.51 - 3.44 (m, H, ¾),

3.43 (app q, J - 6.7 Hz, 2H, His), 3.13 ··· ^■ 3.05 (m, 2H, ¾), 2.95 - 2.84 (m, 1 H, H ₃ ), 1.92 (it, J- 12.6, 8.6 Hz, IH, ¾), .68 - 1.60 (ni, 4H, ¾, H _¾e ), 1.60 - 1.53 (m, IH, H ₄ ), 1.54 - 1.50 (m, 2H, His), 1.50 (s, 9H, H,), 1.43 ~ 1.37 (m, 3 I H, H, ₇ , ¾<,), 1.18 (d, J- 6.5 Hz, 3H, ¾), 1.18 - 1 .12 (m, 2H, ¾), C NMR (126 MHz, CD ₂ C1 ₂ ) δ 206.1 (C), 170.6 (C), 170.4 (C), 167.2 (C), 163.0 (C), 161.3 (C), 156.8 (C), 156.4 (C), 1 2.4 (C), 151.7 (C), 348.8 (C), 323.6 (CH), 1 17.7 (CH), 97.3 (CH), 82.2 (C), 79.2 (C), 57.4 (CH), 45.6 (C¾) _s 42.0 (C), 41.7 (C¾), 43.0 (C¾), 39.7 (C¾), 30.3 (C¾), 30.2 (CH ₂ ), 30.3 ((¾), 28.9 (CH ₂ ) ₅ 28.7 ((¾), 28.5 (CH ₃ ), 24,7 (C¾), 23.6 (CH ₂ ), 1.6 (C¾), 39.9 ((¾). IR (ATR-PTIR), era ^"1 : 3305 (br ml 2970 (w), 2932 (w), 1699 (s), 1653 (s), 1543 (of), 1242 (m), 1 156 (s), 1026 (m), 621 (w). HRMS-CI (m/z): [M + H calcd for C _3< ¾ 70sS ₂ , 774.3313; found, 774.3309. [a] _D ² ° +30.0 (c 0.9, c¾a ₂ ).

A solution of T3P in ethyl acetate (50 wt%, 22.7 μ£, 38.2 μηιοΐ, 1.50 eqoiv) and 4- mefhylmoipholine (14.0 jiL, 127 μι ^η οΐ, 5.00 equiv) were added in sequence to a solution of the acid 13 (15.0 mg, 25.4 μκιοΐ, 1 equiv) in te rahydrofuran (510 uL) at 23 °C. The reaction mixture was stirred for 20 min at 23 °C. The amine Sll (17.3 mg, 57.2 μ«κ»1, 2.25 equiv) was added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The residue obtained was purified by flash-column chromatography (elating with dichloromethane initially, grading to 10% methanol™ diehloromethane, linear gradient) to provide the amide .141» as an oft- while solid (14.9 mg, 67%).

⁵ B NMR (500 MHz, CD ₂ CI ₂ ) § 1 1.50 (bs, !.H), 8.49 (bs, 1.H), 8.1 1 8.03 (Bi 2H, H _Ki , H _i3 ),

7.96 (s. IB, Hn), 7.70 - 7.64 (m, 1 11, H _u ), 6.55 is, !H, ¾}, 6.25 (bs, IH, H _? ), 4.75 (4, J 5.9 Hz, 2H HH), 4.29 -- 4.14 (m, 1H, H ₃ ) _> 3.66 (s, 2H, ¾), 3.65 ··· ^■ 3.58 ( , 4H, H _IS , Η _1δ ), 3.47 (dd, J= 18.3, 8.7 Hz, IK, Hs), 2.94 - 2.82 (m, Hi ¾), 1.98 - 1 ,86 (ro ₅ 1H ₉ \ i 1.65 - 1.59 (re, 2H, Bg), 1.60- 1.52 (m, IH, ¾), 1.49 (s, 9H, ¾). 1.48 (s, 9H, H _l7 ). 1.44 (s, 9H, H«), 1.17 (d, J = 6.2 Hz, 3H, ¾), 1.18 - 1.12 (m, 2H, ¾). C NMR (12 MHz, CD ₂ C ) § 206.1 (C), 170.6 (C), 170.3 (C), 167.2 (C), 164.0 (C), 163.0 (C), 161 ,8 (C), 157.2 (C), 156,9 (C), 153.6 (C), 152.4 (C), 151.3 (C), 148.8 (C), 123.9 (CH), Ϊ 17.8 (CH), 97,2 (CH), 83.7 (C), 82.2 (C), 79.5 (C), 57.4 (CH), 45.6 (C¾), 42.0 (C), 41.6 (CH ₂ ), 40.9 (C¾) _s 39.2 (CH ₂ ), 30.2 (C¾X 28.8 (CH ₃ ), 28.8 (C¾), 28.6 (CH,), 28.5 (CH ₃ ), 21.6 (CH ₂ ), 21.6 (CH ₂ ), 19.8 (C¾). IR (ATR-FTI ), cm ^""1 : 3323 (br w), 2976 (w), 2934 (w), 1716 (m), 1639 (s), 161 (s). 1544 (w), 1316 (m), 1289 (m), 1 133 is), 1024 (m), 771 (w). HRMS-CI (mix): [M + Hf cakd for S74.3586; found, 874.3583. [α]» ²⁰ +27.0 (c 1.0, C¾C1 ₂ ).

A solution of T3P in ethyl acetate (50 t%, 303 jxL, 50.9 μηιοΙ, 1.50 equiv) and 4~

erayhnorphofme (18.6 uL, 170 μηιο ^' Ι, 5.00 equiv) were added in sequence to a solution of the acid 1 (20.0 i¾g, 33.9 μιηο!. 1 eqttiv) hi tetrabydrafwran (680 L) at 23 ^a C. The reaction mixture was stirred for 20 mm at 23 °C. The amine Si 2 (39.2 mg, 119 μηιοί, 3.50 equiv) was added to the reaction mixture. The resulting mixture was stirred for 7 h at 23 °C. The product mixture was concentrated. The residue obtained was purified by flash-column chromatography (elating with dichlorometliane initially, grading to 10% methanol- dichioroniethane _s linear gradient) to provide the amide .14 as an off-white solid (22.1 rag, 72%),

^} H NMR (600 MHz, CD ₂ Ci ₂ ) δ 1 1.50 (bs, 1H), 8.29 { J- 5.5 Hz, ΪΗ), 8.1 1 (1 J - 6.0 H¾, IB, Hio), 8.06 (s, IH, H _l3 ), 7.95 (s, 1H, Hu), 7.49 (t, J - 6.2 Hz, IB, H _J ), 6.56 (s, 1H, ¾), 6.39 (bs, 1 H, H ₇ ), 4.76 (d, J - 6.2 Hz, 2H, H _n ), 4.34 - 4.16 (m, 1H, H ₃ ), 3.66 (s, 2H, H»), 3.56 - 3.43 (m, 3H, H ₅ , H _{3 ), 3.41 (app q, J= 6.1 Hz, 2R H, ₈ ), 2.89 (dt, /= 18.6, 9.8 Hz, 1H, ¾), 1.92 (ddd J- 20.9, 12.0, 8.4 Hz, 1 H, H ₄ ), 1.71 - 1.63 (m, 4H, H _!6 , H _i7 ) _s 1.64 - 1.59 (m, 2H, H ₈ ), 1.60 - 1.52 (m, IH, ¾}, 1.49 (s, 9H, H _{ ), 1.49 (s, 9H, ¾»), L44 (s, 9H, ¾ ₀ l 1.17 (d, J- .6 Hz. 3H ₅ H ₂ ), 1.1 - 1.13 (m, 2H, ¾). ¹³ C NMR (126 MHz, CP2O2) ό 206.1 (C), 170.6 (C) ₅ 170.3 (C) ₅ 167.2 (C), 164.1 (C), 163.0 (C), 161.4 (C) _s 156.9 (C), 156.7 (C), 153.8 (C), 1.52.4 (C), 151.7 (C), 148.8 (C), 123.7 (CH), 1.1.7.7 (CH), 97.2 (CH), 83.6 (C), 82,2 (C), 79.3 (C), 57.5 (CH), 45.6 (CH*), 42.0 (C), 41.7 (CH¾), 41.0 (C%), 39.5 (C¾). 30.2 (CI¾), 28.9 (CH ₂ ). 28.6 (C¾), 28.5 (C¾), 28.4 (C¾), 27.8 (CH ₂ ), 27.2 (CH ₂ ) _S 21.6 (CH ₂ ), 21.6 (C¾), 19.9 (C¾). IR (ATR-FTIR), era ^"5 : 3324 (br w), 2974 (w), 2935 (w), 1716 (ra), 1639 ($), 1612 (s), 1366 (m), 1316 (m), 1290 (m), 1155 (s), S 132 (s), 1051 (m), 102 (m), 771 (w). HRMS-Ci (m/z): [M + ^■ H calcd for C _4l ¾oN*O S ₂ , 902.3899; tad, 902.3901. [afc* +15.0 (c 1.0, CH ₂ CI ₂ ). Synthesis of the lactam }Se:

I ifluoroacetic acid (219 iL, 2.8? mmoi, 120 equiv) was added dropwise via syringe to a soktios ^' of the amide 14e (16.1 mg ₅ 23.9 ηιοΐ, I equiv) m dicMoromethane (600 uL) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C The reaction mixture was concentrated. The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (1.3 nil.). The diluted reaction .mixture was stirred for 1 h at 23 °C. The product mixture was diluted sequentially with water (1.0 niL) and ethyl acetate (3.0 mL). The diluted product mixture was transferred to a separatOry tunnel and the layers thai farmed were separated. Hie aqueous layer was extracted with ethyl acetate (5 x 3,0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the lactam 15e as a light yellow solid (10.1 mg, 76%). The product so obtained was used without further purification.

^! H NMR (500 MHz, DMSO-i¾) d 10.29 (t, /= 5.9 Hz, 1H, ¾), 8.70 - 8.58 ( , 2H, ¾, H ₎₂ X 8.24 (s, 1H, Hn) _> 8.17 is, IH, Η _{0 Χ 4.65 ··· 4.57 (ni, 2E, ¾), 4.16 - 4.08 (m, J H, ¾), 3.35 ··· ^■ 3.29 (m, 4H, ¾ H _i3 ), 3.15 - 3.02 (m, 1H, Η 2.86 (dt, J = 17.7, 8.8 Hz, 1H ₅ H ₄ X 2.28 (t, 6.9 Hz, 2H, H _l5 ), 2.15 (s, 6H, ¾), 2.12 - 2.04 (m _? 111 ¾ L7I. - 1.63 (m ₅ 4H, H _u %

I .41 - 1.31 (m, 3H ₅ ¾ ¾), 1.18 (4 J- 6.6 Hz _; 3B, Hj)- ^S ¾ NMR (126 MHz, DMSC s) δ 171.2 (CX 169.7 (C), 168.4 (C), 168.3 (C), 161.7 (C) _s 160.3 (C) _; 157.7 (C), 151.0 (C), 147.5 (C 127.4 (C), 123.9 (CH), 117.9 (CH), 66.6 (CH), 57.3 (CH _2. X 45.3 (C), 45.2 < ¾), 40.4 (C¾X 37.7 (CH ₂ ), 36.6 (C¾), 33. (CI¾X 29.7 (CH?), 26. (C¾), 21.9 (CH ₃ ), 1.1.9 <€¾),

I I .8 (C:¾). HRMS-CI (m/z): [M + H]* caled for 556.2159; found, 556.2151. [α]η ^2β -5.5 (c 3.1. DMSO-<¾). Synthesis of the lactam }Sf:

Trifltioroacetic acid (125 μί,, 1 ,64 mmoi, 120 equiv) was added dropwise via syringe to a solution of the amide I4f (10.0 rag, 13.7 μαιοί, i equiv) in . dichloromethane (340 .L) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C. The reaction mixture was concentrated. The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (700 μ ,). The diluted reactio mixture was stirred, for 1 h at. 23 °C. The product mixture was diluted sequentially with water (1.0 inL) and ethyl acetate (3.0 uiL). The diluted product mixture was transferred to a separatory tunnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 x 3.0 ml). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the lactam I5f as a light yellow solid (3.4 mg, 49%). The product so obtained was used without further purification.

⁵ H NMR (500 MHz, DMSO-i¾) 8 10.30 ( J - 6.0 Hz, .1 fl ¾), 8.62 (bs, 1 E, H ₅ ), 8.44 (t, J - 5.9 Hz, IH, H _l2 ), 8.28 (s, ΙΗ, Ηπ), 8.19 (8, IH, H _J0 ), 4.64 ~- 4.58 (m. 2H _s . H ₉ ), 4. Ϊ 7 - 4.08 (m, 1H, ¾), 3.38 ^» 3.29 (m ₅ a H ₇ , E _n ), 3.14 - 3.06 (m, 1H, ¾), 2.87 {*, ./= 17.8, 8,7 Hz, 1H, 11 _; ), 2.77 (t, J = 6.5 Hz, 2H, H _! ,l 2.1 - 2.04 (m, IH, H > h 1.71 - 1.66 (ra, 2H ₅ ¾), 1.41 - 1.30 (m, 3H, ¾ ¾), 1.19 (d, J= 6.7 Hz, 3H, H ₅ ), °C NMR (126 MHz, DMSO-^) δ 171.2 (C), 169.7 (C), 168.4 (C), 168.3 (C), 161.7 (C), 160.6 (C), 157.7 (C), 150.9 (C), 147.4 (C), 127.4 (C), 124.2 (CH), 18.0 (CH), 66.6 (CH), 45.3 (C), 40.9 (<¾), 40.7 (C¾), 40.4 (CH ₂ ). 36.6 (CH ₂ ), 33.6 (CH ₂ ), 29.7 (C¾), 21.9 (CH ₃ ), 11.9 (CH ₂ ), 1 1.8 (C¾). HRMS-Cl (m/z):

[M + Hf calcd for CaAsN-C Sa, 514. 690; found, 514.1690. [a] _D ²⁰ -1.7 (c 1.2, DMSO- ).

S nthesis of the lactam JSg:

Trifluoroacefcic acid (108 μΤ, 1.41 mtnol, 120 equiv) was added drop wise via syringe to a solution of the amide 14g (9.1 n g, 1 1.8 μ ^η οΐ, 1 eqiiiv) in dichloromethane (290 pL) at 0 °C. The reaction mixture was stirred for 14 h at 0 °C. The reaction mixture was concentrated. The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (650 μΐ,). The diluted reaction mixture- was stirred for 1 h at 23 °C. The product mixture, was diluted sequentially with water (1.0 mL) and ethyl acetate (3.0 rnL). The diluted product mixture was transferred to separatory ^' funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 x 3.0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the lactam lSg as a light yellow solid (3.4 mg, 45%). The product so obtained was used without further purification.

^! H NMR (500 MHz, DMSO-£¾) δ 10.34 - 10.26 (m, 1 H, ¾), 8.61 (bs _s IH, H ₅ ), 8.44 - 8.38 ( , IH, i½), 8.25 (s, I H, Hu}, ^' 8.1.9 (s, 1 ^: H, H _.w ), -4.64 - 4.59 (in, 2H, ¾}, 4.17 - 4.07 (m, IH, H ₂ ) ₅ 3,35 - 3.26 (m, 4H, H _7s H _n ), 3.15 - 3.05 (m, IH, H ₄ ), 2.87 (dt, = 18.0, 8. Hz, IH, ¾), 2.73 (t, J = 7.5 Hz, 2H, H ₁₇ ), 2.15 ··· 2.05 (m, I H, ¾), 1.72 - 1.66 (m, 2H, ¾), 1.58 ··· ^■ 1.50 (m, 4H, Hi4, Η _> 1 -39 ··· ^■ 1.32 (m, 5H, H _3> ¾, H, ₅ ), 1.19 (<L ,/= 6.7 H , 3H, H,). ^,3 C NMR (126 MHz, DMSO-i¾) 5 171.1 (C), 169.6 (C), 168.4 (C), 168.3 (C), 161.7 (C), 160.3 (C), 157.7 (C), 151.0 (C), 147.5 (C), 127.4 (C), 124.0 (CH), i 17.9 (CH), 66.5 (CH), 45.3 (C), 40.4 (CH,) _S 39.3 (C¾), 38.5 (CH ₂ ), 36.5 (CH ₂ ), 33.5 (CH ₂ ), 29.7 (CH*), 28.8 (CH,), 27.9 (C¾), 23.4 (C¾), 21.9 (C¾), .1 1.8 (C¾), 1 i .8 (CH ₂ ). HRMS-CI (m/z): [M + H] ^* calcd for C26H3 7O3S2, 556.2159; found, 556,2167, [α]» ²⁰ +2.5 (<? 1.2, DMSQ- ).

Synthesis of the lactam ISh:

TrifJuoroacetic acid (105 μί, 1 ,37 nimoi, 120 equiv) was added dropwise via syringe to a solution of the amide I4h (10.0 nig, i 1.4 μηιοΐ, 1 equiv) in dichloromethane (290 μΙΤ at 0 °C. The reaction mixture was stirred for 14 h at 0 °C. The reaction ixture was

concentrated. The concentrated reaction mixture was diluted with sat amted aqoeoa sodium bicarbonate solution (650 uL). The diluted reaction mixture was stirred for 1. h at 23 °C. The product mixture was diluted sequentially with water (1.0 mL) and ethyl acetate (3,0 oil.). The diluted product mixture was transferred to a. separatory funnel and the layers that formed were separated. The aqueous layer was extracted with, ethyl acetate (5 x 3.0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provid the lactam. I Sh as a light yellow solid (2.2 mg, 34%). The -product so obtained was used without further purification.

lH NMR (600 MHz, DMSO~<¾) δ 10.31 (t, /- 6.1 Hz, IH, ¾), 8,58 (¼, 2H, H ₅ , H _l2 ), 8.31

(s, 111 Hi j), 8.16 (s. 1 H, H _l0 ), 7.42 (bs, 1 H), 4.65 4.60 (m, 211. ¾), 4.17 - 4.08 ( , IH,

¾), 3.47 - 3.41 (m, 2H, H _u ), 3.36 - 3.30 (m _s 4H, H ₇ , H, ), 3.14 - 3.07 (m, I H, ¾), 2.87 (dt, J - 18.2, 8.9 Hz, IH, H ₄ ), 2.12 - 2.04 (m, 1H, %), 1.71 - 1 ,65 (m, 2H, ¾) ₅ 1.53 - 1.43 (m, IH, f¾), 1.38 ~ 1.33 (m, 2H, ¾), 1.19 (<L J= 6.6 Hz, 3H ₅ H,)- C NMR (126 MHz, DMSO- £¾) 6 171.2 (C), 169.6 (C), 168.4 (C), 168.3 (C), 161.9 (Q, .160.9 (C), .157.6 (C), 1.57.0 (C), 150.5 (C), 147.4 (C), 127.4 (C), 124.7 (CH), 1 17.9 (CH), 66,5 (CH), 45.3 (C), 40.4 ((¾), 40.4 (CH ₂ ), 38.0 (Ci¾), 36.5 (CH ₂ ), 33.5 (CH ₂ ), 29.7 (CH ₂ ), 21.9 (CH ₃ ), 1 1.8 (CH ₂ ) ₉ 1 1.7 (CH ₂ ), HRMS-CI (ffl/z); [M ÷ Hf caicd for CsAoN^ASa _. , 556,1908; found, 556.1913.

[a] _t > ²⁰ -5.5 (c 1.3. DMSO~t/ _e ).

Triflttoroacettc acid (164 μΤ, 2.14 mmol, 120 equiv) was added dropwise via syringe to a solution of the amide 14i (16.1 mg, 17.8 μηιοΐ, 1 equiv) in dicMoromethane (450 μΤ) at 0 °C. The reaction .mixture was stirred for 1.4 h at 0 °C. The reaction mixture was concentrated.

The concentrated reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (1 ,0 mL), The diluted reaction mixture was stirred for 1 h at 23 °C, The product mixture was diluted sequentially with water (1.0 mL) and ethyl acetate (3.0 mL). The diluted, product mixture was transferred to a separator funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (5 x 3.0 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the lactam ISi as a light yellow solid (5.0 nig, 48%). The product so obtained was used without farther purification.

Ή NMR (500 MHz, OMSO~d ₆ ) δ 10.34 ··· .10.28. (m, 1 E, ¾), 8.63 (bs, IB,. H _s ) ₅ 8,50 (t, J™ 6.2 Hz, 1H, H _i2 ), 8.26 (s, 1H, B _n ), 8.19 (s, 1 H, Η _ιβ ). 7.88 (bs, 1 H), 4.64 - 4.58 (m, 2H, H ₉ ), 4.16 - 4.08 (n 1H, ¾), 3.34 ~ 3.27 (m, 4H, ¾, H _i3 ), 3.15 - 3.05 (m ₅ 3H, ¾, ¾), 2.87 (dt, J~ 17.8, 8.8 Hz, iH, ¾), 2.1 1 - 2.04 (m, 1H, H ₃ )- 1.70 - 1.63 (m, 2H, ¾), 1.60 - 1.52 (m, 2H, Hw), 1 -53 - 1.46 (m, 2B, Η«), 1.38 - 1.33 (m, 3H, ¾, ¾), 1.18 (d, ./= 6.6 Hz, 3H, Hi). ^}S C NMR (126 MHz, DMSO- ) δ 171.2 (C), 1 9.7 (C), 168.4 (C), 1 8.3 (Q, 161.7 (C), 160.5 (C), 157.7 (Q, 156.9 (C), 150.9 (C), 1 7.5 (C), 127.4 (C), 124.1 (CH), 1 17.9 (CM), 66.6 (CH), 45.3 (C), 40.4 (C¾), 40.4 (C¾), 38.2 (C¾), 36.6 (C¾), 33.6 (CH ₂ ), 29.7 <(¾), 26.5 (C¾X 26.1 (CH ₂ ), 21.9 (<¾), 1 1.9 (€¾), 1 1.8 (CH,). HRMS-CI (m/z): [M + Bf calcd for C ₂ sl¾N ⁽ ¾S _2j 584.2221 ; found, 584.2221. [ajo ²⁰ +3.1 (c 2.6, DMSC s).

Synthesis of the ethyl ester $14:

sw

Ethyl bromopyruvate (3,01 g, 1 ,4 mmol, 1 ,50 equiv) was added to a solution of the

t ioamtde S13 (2.8 g, 10.3 mmol, 1 equiv) in Ive-propanoi (100 mL) at 23 °C. The reaction mi ture was stirred tor 16 h at S3 °C, The reaction mixture was concentrated, ¾ residue obtained was dissolved in 1 ,4-dioxane (45 mL) and the resulting solution was cooled to 0 "C.

irf-boty] dicarbonate (3.60 g, i 6.5 mmol, 1.60 equiv) and a solutio of aqueous potassium bicarbonate (1 N, 15 mL) were then added sequentially to the cooled solution. The reaction mixture was stirred for 16 h at 0 °C. The product mixture was concentrated and the residue obtained was applied to a oimemylamine acetate-functioualized silica column (Si~ TMA acetate; eluting with 2% acetic acid-methanol) to provide the bithiazole S14 as a white solid (2.51 g, 66%).

lH NMR (600 MHz. DMSO-i ) δ 8.55 (s, I B, H ), 8.27 (s, 1H, %) _> 7.87 (t, 1 = 6.2 Hz, IH), 4.45 (d, J = 6.1 Hz, 2H, ¾), 4.34 (q, J - 7.1 Hz, 2H, ¾), 1.42 (s, 9H, ¾) _> 1 3 ( j = 7.1 Hz, 3B, ¾). C NMR (151 MHz, DMSC ¾) 5 172.9 (<¾ 162.5 (C), 160.7 (C), 155.8 (C), 147.1 (C), 147,0 (C), 129,4 (CH), 118.2 (CH), 78.7 (C), 60,9 (C¾), 41.9 (C¾), 28,2 (C¾) _? 14,2 (C!¾ IR (ATR-FIIR), cm ^"1 : 3343 (m\ 3130 (w), 3109 (w), 2983 (w), 1721 (s) _s 1686 (s), 1526 ($), 1298 (m), 1284 (m), 1204 (s), 1 164 (s), 1 100 (s), 81 (m), 770 (m) _s 621 (m).

HRMS-CI (m/z): [M + H caicd for C15H20 3O ₄ S2, 370.0890; found, 370.0882.

Synthesis oftheacM SJ5: *C

A dispersion of sodium hydride in mineral oil (60%. 109 mg, 2.83 mmoL 2.00 equiv) was added slowly to absolution of the hithiazole S14 (523 mg, 1.42 mmol, I equiv) and

iodomethane (793 pL, 12.7 mmol, 9.00 equiv) in A^A-dimethytfomiamide (6.0 mL) at -5 °C. The reaction mixture was stirred for 30 min at -5 °C and then was wanned to 15 . The warmed mixture was stirred for .14 h at 15 °C. Lithium hydroxide (891 mg, 21.2 mmol, 15.0 equiv) and water (6.0 mL) were then added in sequence to the reaction mixture. The reaction mixture was stirred for 3 h at 15 °C. The heterogeneous product mixture was filtered through a fritted funnel. The filter cake was washed with methanol (10 mL), The filtrates were combined and the combined filtrates were concentrated. The product mixture was diluted sequentially with saturated aqueous ammonium chloride solution (10 mL) and ethyl acetate (10 mL). The diluted product mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (3 x 5.0 mL). The organic layers were combined and the combined organic layers were dried over sodium ^• sulfate. The dried solution was filtered and the filtrate was concentrated. The concentrated product mixture was applied to a column containing trimethylamine acetate- fimctionalized silica gel (Si-TMA acetate; eluting with 2% acetic acid-methanol). The tractions containing product were collected, combined, and concentrated to provide the acid S15 as a white solid (495 mg, 98%).

In DMSO-£¾ at room temperature the title compound exists as an approximate 1 : mixture of amide-bond rotamers.

*H NMR (600 MHz, DMSO-i¾) S 8.36 (s, 1 H, ¾}, 8.28 (s, 1H, ¾), 4.71 (s, 2H, ¾), 2.92 (d, 3H, ¾), 1.42 (d, 9B, Hj). ¹³ C NMR (151 MHz, DMSO-dk) δ 169.9 (C), 162.4 (C), 161.6 (C), 15.4 J (¾ Cj, 150, 1 (C), 147.5 (C), 127.6 (CH), Ϊ 18.1 (CH), 79,6 (C), 49.S (d, CH ₂ ), 34,7 (d, Cll _< ), 273 (d. CHs), IR (ATR-FTIR), cm ^! 31 13 (w), 2975 (w) ₃ 1681 (s), 1484 (m), 1392 (ιτι), 1295 (ro), 1240 (m), 1 167 (s), 751 (m), 564 (w). HRMS-C3 (m z): [M + Hf calcd for Ci ₄ HnN _s H 0 S2, 378.0553; found, 378.0554.

Synthesis of the hydrochloride salt $16:

A solution of hydrogen chloride in l ,4-dioxane (4.0 N, 8.0 mL. 32.0 mraol, 24.1 equiv) was added dropwise via syringe to a solution of the bithiazole SI 5 (472 mg, 1 , 33 mmol, 1 equiv) In diehloromethane (24 mL) at 23 °C. The resulting /mixture was stirred for J h at 23 °C. The product mixture was concentrated to provide the hydrochloride salt SI 6 as a white solid (387 nig, >99%). The product S16 obtained in this way was used directly in the following step.

H NMR (600 MHz, DMSO~t¼) δ 9.72 (bs, 2H), 8.52 (s, 1 H, H ₄ ). 8.45 (s, 1 H, ¾), 4.61 (t _s J ^{■■■■■■■■} 5.8 Hz, 2H, H ₂ ), 2.66 (t, J- 5.3 Hz, 3H, ¾). ⁱ³ C NMR (151 MHz, DMSO- ,) 5 162.0 (C), 161.9 (C), 161.6 (C), 148.2 (C), 147.5 (C), 129.3 (CH), 120.7 (CH), 47.3 (CH ₂ ), 32.5 (C¾).

Synthesis qf fhe acia $18:

0 0

^ " tSMSu

A solution of the P-ketothioester SI 7 (506 mg, Ϊ .60 mmol,..1.30 equiv) in N _f N~

dimethylfomramide (3.0 mL) was added dropwise via syringe over 20 mm to a mixture of silver trifluoroaeetate (545 mg, 2.47 mmol, 2.00 equiv), triethylan ie (688 L, 4.94 mmol, 4.00 equiv). and the acid SJ6 (360 rag, 1.23 mmol, 1 equiv) in A ^; ,A-dimethyIfommraide (12 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C. The heterogeneous product mixture was filtered through a fritted funnel. The filter cake was washed with methanol (12 mL). The filtrates were combined and the combined, filtrates were concentrated. The concentrated product mixture was applied to a column containing trimethyl amine acetate- functionalized silica gel (Si-TMA acetate; elating with 2% acetic acid-niefhanot). The fractions containing product were collected, combined, and concentrated. The residue obtained was recxystailized from di ^' chlorometha e-hexanes (1 :4; 50 mL) to provide the acid S18 as a white solid. (565 nig, 95%).

In DMSO-i/ή at room temperature the title compoiuid exists as a mixture (3.5:1) of amide- bond rotamers,. The NMR .signals are reported fo the major isomer.

lH NMR (600 MHz, DMS0~ ) § .13.1 (bs, IHX 8.48 (s, 1 H, B ₇ % 8.30 (s, 1 H, ¾), 7.75 (bs, 1H), 4.83 (s, 2H, H ₅ ), 3.82 (s, 2H, ¾}, 2.98 (s, 3H, ¾), 1.41 (s, 9 , ¾), 1 .41 - 1.36 (m, 2H, %}, 1.09 (q, J = 4.4 Hz, 2H, ¾). C NMR (151 MHz, DMSO^.) 5 204.8 (C), 168.9 (C), 167.7 (C), 162.1 (Q _s 162.0 (C), 156.2 (C), 148.1 (C), 147.1 (C), 129.0 (CH), 118.9 (CM), 78.8 (C), 48.4 (CH 45.3 ({¾), 41.2 (0, 36.0 (C¾), 28.1 (C¾) ₅ 19.8 (€¾). IR CATR- FTIR), cm ^"1 : 3328 (m), 2935 (w), 1704 (s), 1680 (s), 1643 (s), 1506 (s), 1287 (m), 1236 (m), 1 160 (s), 746.2 (m), 457 (m). HRMS-CJ mJz): [M + Hf caicd for C ₂ o¾jN ₄ C¾S ₂ , 481.1210; found, 481.1215.

A solution of hydrogen chloride in 1 ,4~dioxane (4.0 N, 15.0 mL, 60.0 mmol, 54.5 equiv) was added dropwise via syringe to a solution of the bithiazoie SIS (527 rog, 1.10 mmol, 1 equiv) in dichloromethaiie (45,0 mL) at 23 '€. The resulting mixture was stirred for 3 h at 23 °C. The product mixture was concentrated to provide the hydrochloride salt SI 9 as a white solid (457 nig, >99%), The product S19 obtained in this way was used directly in the folio wing step.

In DMSO-<:¾ at room temperature ^' the title compoiuid exists as a mixture (3: 1) of amide-bond rotamers. The NMR signals are reported for the major isomer.

Ή NMR (600 MHz, DMSO- ) δ 8.85 (bs, 3H), 8.49 (s, IH, ¾}, 8,32 (s, I H, H ₅ ), 4.84 (s, 2H, ¾), 3.78 (s, 2H, ¾>, 3.05 (s, 3H, H ₃ ) 1.83 - 1.74 (m, 2H, ¾), 1.56 ··· 1.44 (m, 2H, H _} ). ^K 'C NMR (15 1 MHz, DMSO-ffc) δ 199.8 (C), 168.5 (C), 167.2 (C), 162.0 (C), 162,0 (C), 148.1 (C), 147.1 (C), 129.0 (CH), 1 18.9 (CH), 48.5 (C¾), 42.0 (C), 41.1 (CH,), 36.1 (CH ₃ ), 13.6 (CH ₂ ). S nthesis o f the lactam S2§:

Si9

A solution of the thioester 10 (397 nig, 1 1 mmol, 1.30 equiv)

(2.0 mL) was added dropwise via syringe over 20 rain to a mixture of silver trifluoroacetate (445 nig, 2.03 mmol, 2.00 equiv), ttiethyiarnkie (562 μΐ.., 4.03 mol, 4.00 equiv), and the ^' amine S19 (420 rag, 1 .01 mmo!, 1 equiv) hi A A'-dimetl Ifbrmamtde (10 mL) at 0 °C. The reaction mixture was stirred for 1 h at 0 *C. Potassium carbonate (418 nig, 3.02 mmol, 3.00 equiv) and methanol (10 mL) were then added in sequence to the reaction mixture at 0 °€. The reaction mixture was allowed to warm to 23 °C and stirred at this temperature for 6 h. The heterogeneous product mixture was filtered through a fritted funnel. The filte cake was washed with methanol (10 mL). The filtrates were combined and the combined filtrates were concentrated. The residue obtained was applied to a trimetiiyianune acetate- ftmctionaiized silica column (Si-TMA acetate; eluting with 0.5% formic acid-acelonitrile). The fractions containing the product S20 were collected, combined, and concentrated to provide the lactam S20 as a white solid (380 mg, 63%).

In DMSOi¾ at room temperature the title compound exists as a mixture (3.1) of amide-bond rotamers. The NMR signals are reported for the major isomer.

⁵ H NMR (600 MHz, DMSCWg) δ 8.62 (bs, 1H, ¾), 8.44 (s, 1H, H _}2 ), 8.28 (s, I H, H _u ), 6.63 (bs, I H), 4.82 (d, J - 15,7 Hz, I H, B _u >), 4.78 (d ₅ J- 15.8 Hz, I E, H _.u> ), 3.62 (d, = 16.2 Hz, 1H, 3.55 (d, 16.2 Ik, 1 II S¾ 3.46 - 3.38 (m _f 1H, ¾), 3.18 (s, 3Ϊ-Ι, i¾) _; 2.91 - 2.83 (m, 21L ¾), 1.62 - 1.49 (in, 4H _} H , H ₇ ), 1.50 ~ 1.40 (m, 2H, lh), 1.36 ^' (s _s 9H, H- 1.00 id. J - 6.3 Hz, 3H, ¾). ¹³ C NMR (1 ST MHz, DMSO-4) 8 197.2 (C), 169.0 (C), 168.9 (C) ₅ 1 8.4 (C), 167.7 (C) ₅ 166.8 (C) ₅ 162.2 (C), 155.0 (C), 147.2 (C), 130.5 (€), 128.5 (CH), 118.7 (CH), 77.3 (C), 48.8 (C¾), 45.5 (C), 45.4 (CH), 38.4 (CH ₂ ) _? 35.9 (CH ₃ ), 29.9 (C¾), 29.7 (CHzl 28.3 (Ci¾), 20.9 (CH ₃ ), 13.3 (CH ₂ ). IR. (ATR-FTIR), cm ^" ': 3328 (b w), 2975 (w), 1792 (w), 676 (s), 1631 (s), 1501 (m), 1391 (m), 1 169 (s), 1152 (s), 674 (w), 556 (m). HRMS-Cl (m/z): {M - Naf ealed for CsiHssW OTSa, 626.1714; fomid, 626.1716. [ ] _D ²⁰ +8.0 (c 1.0, DMSO).

S nthesis of the amide 19;

A solution of T3P in ethyl acetate (50 wt%, 1 18 pL, 199 μηιοί, 1.50 equiv) and 4~ metiiyhnorpholme (72,8 μ£, 663 μηιοΙ, 5.00 equiv) were added in sequence to a solution of the acid S20 (80.0 tug, 133 μηιοΐ, I equiv) in tetrahydrofuran (2.7 mL) at 23 °C. The reaction mixture was stirred for 20 mm at 23 °C. A A'-Dinietlniethylenediamine (36.2 μΤ, 331 μηιοΙ, 2.50 equiv) was added to the reaction mixture. The resulting mixture was stirred tor 14 h at 23 °C. The product mixture was concentrated. The concentrated product mixture was diluted wit ethyl acetate (10 mL). The diluted product mixture was poured into a separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 inL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 x 10 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 19 as a white solid (52,0 rag, 58%).

The product 19 obtained ia this way was estimated to be of >95% purity by ⁵ H and ^U C NM analysis (see accompanying spectra) and was used without further purification. In DMSO- ; at room temperature the title compound exists as a mixture (3:1) of amide-bond retainers. The NMR signals are reported for the major isomer.

^S H NMR (600 ¾DMSO- d § 8.62 (s _s 1H, ¾} _? 8.27 (hs, 1 H, H _l3 ), 8.24 (s, 1 H, ¾,), 8.23 (bs _t Hi Hjs), 6.63 (bs, LH), 4.82 (d _; J - 15.9 Hz, IH, Hio), 4.79 (d, J- 15.9 Hz, 1H, H _l0 ), 3.62 (d, J 16.2 Hz, 1H, ¾), 3.55 (d, J- 16.1 Hz, 1.H, ¾), 3.50 - 3.40 (m, IH, H ₃ ), 3.39 (app q, J~ 6.4 Hz, 2H, H ₁₄ ), 3.18 is, 3H, H ₉ ), 2.91 ··· ^■ 2.80 (m, 2H, H ₅ ) ₅ 2.42 <td, J- 6.6, 1.8 Hz, 2H, Hj$), 2.18 (s, 6H, ¾ ₆ ), 1.6 - 1.48 (m, 4H, H ₍ H ₇ ), 1.47 - 1.38 (m, 2H, H?), 1.36 (s, 9H, M ₃ ), LOO (d, /- ,5 Hz, 3H, ¾). C NMR (151 MHz, DMSO~ _£ :¾ S 197.2 (C), 169.1 (C), 169.0 (C). 168.4 (C), 166,8 (C), 161.8 (C), 160,2 {€), 155,0 (€), 150.8 (C), 147.1 (C), 130.5 (C), 124.1 (CH), 1 18.7 (CH), 77.3 (C), 58.1 (CH ₂ ), 48.8 (<¾), 45.5 (C), 45.4 (CH), 45.2 (C¾), 38.4 (¾), 36.7 (CH ₂ ), 36.0 (CH ₃ ), 29.9 (CH ₂ ), 29.7 (CH ₂ ), 28.3 (CH ₃ ), 20. (C!¾) _s 13.3 CH ₂ ). lR(Am-FT!R), cm ¹ : 3325 (br w), 2975 (w), 2931 (w), 1681 (si 1654 (s), 1543 (m), 1453 (w), 1 165 (m), 766 ( ), 621 (w). HRMS-Cl (m/z); [M + Hf calcd for CsjHw TOeSa, 674.2789; found, 674.2795. [α]» ²⁰ -33.0 (c 1.0, C¾CI ₂ ).

19 15}

TriiTuoroacetic acid (200 uL, 2,61 iurooJ, 176 equiv) w s added dropwise via syringe to a solution of the amide 19 (10.0 mg, 14.8 μιυοΙ, 1 equiv) in dichlorometliaiie (400 μΐ,) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C. The product mixture was concentrated.. The concentrated product mixture was diluted with dichlororaethane (10 niL). The diluted product mixture was poured into a separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (5.0 m.L) and the layers that formed were separated. The aqueous layer was extracted with dich!oromethane (2 x 10 mL), The organic layers wer combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide the amide 15j as a white solid (6.9 tag, 81%).

hi DMSO-rf _¾ at room temperature the title compound exists as a mixture (3: 1) of amide-bond fotaraers. The NMR signals are reported for the major isomer.

*H NMR (600 MHz, DMSO- ) δ 8.46 <bs, HI ¾}, 8.27 (s, IH, H _n ), 8.25 (bs, IH, H _l2 ),

8.24 (s, 11, Hi ( _f 4.84 (& J - 15.6 Hz, IH, 4.70 (d, J - 15.6 Hz, E, ¾), 3.95 - 3.88 (m, IH, ¾), 3.71 (d, J - 15.8 Hz, 1 H, H ₇ ), 3.60 (d, J - 15.8 Hz, .1 II Hi), 3.39 (ap q, J - 6.4 Hz, 2H, His), 3.17 (s, 3H, ¾), 3.01 ··· ^■ 2.92 (m, IH, ¾X 2.81 ··· 2.69 (m, 1 H, ¾), 2.41 (L J - 6.6 Hz, 2H, e _{i 4} ), 2.18 (s, 6H, H _1S ), 2.02 - 1.92 (ra, IH, H ₃ ), 1.51 - 1.43 (n 2H, ¾), 1.39 - 1.30 (m, 2E, ¾), 1.28 - 1.21 (m, 111 H ₃ ), 1.07 (d, J 6.7 Hz, 3H, Hj). C NMR (151 MHz, DMSCW*) 6 170.3 (C). 169.0 (C), 168.9 (C), 166,8 (C), 161.8 (C), 160,2 (C), 59.3 (C), 150.8 (C), 147.1 (C), 127.2 (C), 124.1 (CM), 1.18.7 (CH), 66.9 (CH), 58.1 <€¾), 48.8 (CH ₂ ), 45.4 (C), 45.2 (0¾) _s 36.7 (¾), 36.1 (C¾), 29.7 (C¾) ₅ 29.6 (<¾), 22.0 (C%), 12.2 (Ci¾), 12.0 (<¾). HRMS-Cl (m/z): [M + Hf 556.2159 found, 556.2167. [a] _D ^2ft -38.0 (c 1.0, <¾<¾). First Set of References (First Set of Examples)

( !) Vizcaino, M. I; Crawford, J. M. A¾. Chem. ·2015 ₅ 7, 41 L

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Cossi, M.; Rega, N.; Millani, . .?.; Kiene, M.; Knox, J. E.; Cross, I B.; Bakken, V.; Adamo, C; Jaramillo, X; Gomperts, R.; Stratmami, R, E.; Yazyev, O.; Austin, A. J,; Cammi, R,;

Poraelli, C.; Ochterski, J. W,; Martin, R. L; Morokuma, K.; Zakrzewski V. G.: Voth, G. A.; Salvador, P.; Dannenberg, I, j.; Dapprich, S.; Daniels, A. D,; Farkas, 0,; Foresman, J. B.; Ortiz, J. ¥,; Cioslowski, j.; Fox, D, j..; Gaussian, Inc. ! Gaussian 09; WaS!ingford, CT, USA, 2009.

(6) Still, W. C; Kahn, M,; Miira, A, J. Org, Chem. 1978, 43, 2923.

(7) Siii Botid® TMA Acetate: a NEW strong anion exchange sorb nt and it applications in SPE format, Francpls Beland, N. H., Sieeves Potvin and Lynda Tremblay. Ed.; SiiiCyde, 2009.

(8) Pangbom, A. B.; Giardello, M. A.; Grubbs, R, 8.;. Rosen, R. K.; Timmeis, F. j. Organometallics 1996, 15, 1518.

(9) Babij, N. R.; Wolfe, I P. Angew, Chem., Int. Ed 2012, 57, 128.

(10) Bae, 8. Y,; Sim, j. H,; Lee, J.-W.; List, B.; Song, C. E, Angew. Chem,, Int. Ed. 2013, 52, 12143.

(11) Videnov, G.: Kaiser, D.; Kempter, C; Jung, G. Angew. Chem,, In Ed, 1996, 55, 1503.

(12) Fustero, S.; Monteagudo, S,; Sanchez-RoseHo, M.; Flores, S. Barrio, P.; de Pozo, C. Chem, -Eur, J. 2010, 16, 9835. (13) L¼ G. ; Cogan, D. A El!maiL J. A. 1 Am. Chem. Soc. 1997, / i 9, 13.

(14) Kaiser, P.; Koerber, .; Von Deyn, W.; Deshmukk P.; Marine, A.; Dickhaut, J.; Bandar, N. G.; Lange ald, J.; Culbertson, D. L.; Anspaugh, D. D.; BASF SB, German .

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(15) Tliompson, . E.;JoIlf.¾ . A..; Payne, R. I. Org. Lett. 2011, 13, 680.

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Second Set of References (Second Set of Examples)

(!) Still, W. C; Kahn, M.; Mifra, A. J. Org. Chem, 1978, 43, 2923.

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(4) Tanaka, A.; Usuki, T. Tetrahedron left. 2011, 52, 5036,

(5) Healy, A. R.; Vizcaino, M. I; Crawford, J. M; Herzon, S. B. J. Am. Chem. Soc. 2016, 138, 5426.

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15. Supporting Information- please see attached.