SYNTHESIS OF CYCLODEPSIPEPTIDE COMPOUNDS HAVING ANTINEOPLASTIC AND/OR ANTIMICROBIAL ACTIVITY

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is based on and claims the benefit of U.S. provisional application Ser. No. 60/942,197, filed June 5, 2007, the content of which is incorporated herein in its entirety by reference thereto.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

Financial assistance for this invention was provided by the United States Government, through the Division of Cancer Treatment and Diagnosis, National Cancer Institute, DHHS by Outstanding Investigator Grant CA44344-03-12 and ROl CA90441-01-05. Therefore, the United States Government may own certain rights to this invention.

FIELD OF THE INVENTION

The present invention is directed to effective methods of synthesizing cyclodepsipeptide compounds having antineoplastic and/or antimicrobial activity. Preferably the methods are directed to synthesis of kitastatin 1 (1a), respirantin (1b) or a valeryl modification (Ic). The present invention is further directed to important intermediates used in the disclosed methods.

BACKGROUND OF THE INVENTION

The rapidly intensifying search for biologically and medicinally important marine organism constituents has attracted a great deal of interest worldwide. Not unexpectedly, a reassuring number of marine animals, plants and microorganisms are being found to produce promising anticancer substances of unprecedented structural types.

Discovery and synthesis of potentially useful compounds from naturally occurring materials comprises one of the most essential and promising approaches for new anticancer drugs. Of special interest include cyclodepsipeptide compounds having antineoplastic and/or antimicrobial activity. The isolation and structures of three exceptional cancer cell growth inhibitory cyclodepsipeptides from the bacteria Kitasatospora spp. found on the Beaufort Sea coast of the Alaska North Slope ^1a are of particular interest, including newly discovered kitastatin 1 (1a) (Figure 1), while the other two cyclodepsipeptides were identified as respirantin (1b) and a valeryl modification (1c). ^1b

A need exists for a method of effectively and efficiently synthesizing these compounds in order to provide sufficient amounts necessary to meet the projected public need. Without an economically viable method of synthesizing these powerful naturally occurring cyclodepsipeptides, it will be nearly impossible to provide these compounds to patients on a commercial level. In order to produce the compounds on a commercial scale, they must be synthesized under strict protocols as the entrainment of even a slight amount of unidentifiable impurities in the extracted product could create problems which would prevent the natural substance from meeting the strict uniformity required for the approval by the United States Food, Drug and Cosmetic Administration (FDA) and corresponding regulatory agencies of other nations.

Accordingly, an important need exists for the development of an effective and economically viable and truly replicable procedure for synthetically producing substantially pure cyclodepsipeptide compounds, preferably kitastatin 1 (1a) respirantin (1b) or a valeryl modification (Ic) in sufficient quantities to meet the public demand. It is toward the fulfillment of that need that the present invention is directed.

SUMMARY OF THE INVENTION

The present invention is directed to methods of synthesis of potentially useful cyclodepsipeptide compounds having antineoplastic and/or antimicrobial activity. In a preferred embodiment, the methods of synthesis are directed to production of three exceptional cancer cell growth inhibitory cyclodepsipeptides from the bacteria Kitasatospora spp. found on the Beaufort Sea coast of the Alaska North Slope ^1a , including newly discovered kitastatin 1 (1a) (Figure 1), and two other cyclodepsipeptides, namely respirantin (1b) and a valeryl modification (1c). ^1b

One preferred method of preparing a cyclodepsipeptide comprises coupling a compound having the structure

and a fragment having the structure

to yield a cyclodepsipeptide having the structure

wherein R is H or CHO;

R ₁ is CH ₂ CH(CH ₃ ) ₂ or CH(CH ₃ ) ₂ ; and

X is CHO or Cbz.

In one preferred embodiment, the intermediate compound having the structure

is prepared according to the following scheme:

wherein (a) is MeI, K ₂ CO ₃ , DMSO, and (b) is H ₂ , Pd/C, EtOAc.

Furthermore, the compound having the structure of

is preferably prepared by adding a catalytic salt to the compound of

The β-keto ester alcohol intermediate compound having the structure of:

is preferably prepared by coupling a compound having the structure

and the fragment having the structure

to yield the β-keto ester alcohol compound.

In another embodiment, the preferred method of synthesizing a cyclodepsipeptide compound having antineoplastic and/or antimicrobial activity, comprises the steps set forth in the following scheme:

wherein R is R is H or CHO; and X is CHO or Cbz.

The present invention is further directed to the important intermediates used in the synthesis of these cyclodepsipeptide having antineoplastic and/or antimicrobial activity. Preferably the intermediate the compounds are selected from the group consisting of:

Preferably the intermediate compound is in a substantially pure form.

The invention further encompasses methods of preparing the intermediates. Preferred methods of preparing intermediates include for example, coupling the compounds below:

Another example includes selective carboxyl deprotection by treatment of a £-butyl ester compound having the following structure:

wherein R = TBDMS or R = H, and refluxing in toluene; coupling a compound having the structure

and the fragment having the structure

to yield a β-keto ester alcohol having the structure

A final example of preparing a preferred intermediate having the following structure

comprises the step of adding a catalytic salt to the following structure:

Preferably the catalytic salt is CuSO ₄ and the reaction is carried out in refluxing toluene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cyclodepsipeptides, specifically, kitastatin (1a), respirantin (1b) and valeryl modification (1c); (+)-Antimycin A _3b (2).

DETAILED DESCRIPTION OF THE INVENTION

Reference to a compound of the invention herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. Salts of the compounds of the invention may be formed, for example, by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Formation salts is well within the ability of one skilled in the art. Examples of specific salts of the compound of the invention are provided herein, but are not intended to be limiting.

Preferred compounds synthesized by the methods disclosed herein inhibit the growth of cancer cells and/or parasitic microbial growth. In a preferred embodiment, the compounds synthesized can be used to inhibit cancer cell growth and microbial activity.

The present invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Analysis and inspection of the kitastatin 1 (1a) and respirantin (1b) macrocycle reveals that these compounds are composed of common amino acids, or α-hydroxycarboxylic acids derived from them, along with the β-ketoester unit. ¹ Since the absolute stereochemistry of Ia and 1b was undetermined at the onset of this study, our initial target Ib was selected by assuming the most common S-configuration for the constituent amino acids and their presumed α-hydroxy derivatives. Fortunately, that proved to be the correct choice among the 256 possible optical isomers. A retrosynthetic analysis of the ultimately successful route to respirantin (1b) is presented in Scheme 1. Antimycin syntheses ² techniques were used for appending the protected benzoic acid 3 to amino substituted macrocycles. Other issues that needed to be addressed in developing our approach to Ib included introduction of the β- ketoester unit, selection of appropriate esterification and peptide bond forming methods, protecting-group strategy, and the method and site of its macrocyclic lactonization.

Scheme 1. Retrosynthetic Analysis of Respirantin

10

Our initial approach is outlined in Scheme 2 where β-ketoester 7 ⁴ represented a good starting material for incorporating carbons 6-9. Introduction of the gem-dimethyl groups was not trivial, but after some experimentation α, α-dimethyl ester 11 was obtained in reasonable yield. While β-ketoacids are well known to be susceptible to decarboxylation, carboxylic acid 12 was acquired via carefully controlled saponification. However, all attempts to esterify 12 with alcohol 13 were deflected either by decarboxylation to ketone 15 or intramolecular cyclization to the pyrrolidine-2,4-dione 16. The method of choice for the preparation of complex β-ketoesters is via transesterification. However, as this reaction proceeds through a ketene intermediate, ^6,7 ester 11 is not a suitable substrate. Nevertheless, this approach was

pursued in the belief that the introduction of the gem-dimethyl groups could be postponed until the needed β-ketoester linkage was formed. After extensive experimentation we were able to obtain ester 14 via reaction of ester 7 with excess alcohol 13 in refluxing cyclohexane ⁸ in the presence of a catalytic amount of activated zinc. ⁹ However, the modest yield and the need for excess alcohol 13 limited this approach. A preferred method was discovered and implemented based on a report relating to high yield intramolecular β-ketoester transesterification used to form a 15-membered macrocycle ¹⁰ . Scheme 2 ^a

^a Reagents and conditions: (a) K ₂ CO ₃ , MeI, DMSO, 23°C, 48 h, 65%. (b) KOH, aq CH ₃ OH,

23°C, 0.25 h, 81%. (c) 13, Zn, cyclohexane, 80°C, 33%.

The initial approach designed to utilize the intramolecular β-ketoester transesterification method of macrocyclization is outlined in Scheme 3.

Scheme 3a

^a Reagents and conditions: (a) (i) Oxalyl chloride, catalytic DMF, DCM, 0-23°C, 2h. (ii) 19, pyridine, 23°C, 16 h, 75%. (b) LiI, pyridine, H0°C, 40 h, 89%. (c) (i) 7, 1:1 TFA-DCM, 0.5 h. (ii) PyBroP, DIPEA, DCM, product from (i), 4 h, 65%. (d) BF ₃ .Et ₂ O, DCM, 0.5 h, 87% for 23, 83% for 27-28. (e) 25, MNBA, DMAP, TEA, DCM, 23°C, 16 h, 77%.

The diester 20 was obtained via reaction of the acid chloride derived from silyl ester 18 under neutral conditions ^11,12 with alcohol 19. ⁵ Selective hydrolytic cleavage of methyl ester 20 could not be achieved as extensive cleavage of the internal ester linkage occurred. The desired carboxylic acid 21 was obtained via nucleophilic alkyl cleavage with LiI in pyridine. ¹³ Formation of the amide linkage leading to amide 22 proved to be problematic. Reaction of carboxylic acid 21 with the amine derived from TFA deprotection of Boc protected 7 under a variety of peptide coupling procedures (BOP ¹⁴ , PyBroP ¹⁵ , DEPC ¹⁶ ) afforded at best low yields of amide 22 along with the pyrazine 24. The formation of 24 can

be explained by dimerization of the amine free base via Schiff base formation followed by oxidative aromatization. Subsequent experimentation revealed that while the trifluoroacetate salt of the parent amine from 7 could be isolated we were not able to isolate the corresponding free base. In attempts to prepare amide 22, dimerization of the free base occurred in preference to reaction with the activated carboxylic acid 21. To avoid this problem a solution of the TFA salt in DCM was added to a solution of 21, PyBroP, and three equivalents of DIPEA in DCM. By this method, the free base was only generated in the presence of excess activated carboxylic acid and a reasonable yield of amide 22 was reproducibly obtained.

Deprotection of amide 22 was also nontrivial. Standard TBAF treatment afforded a fairly complex mixture of which the desired alcohol 23 was the major component. Better results were obtained using BF ₃ .Et ₂ O ¹⁷ which provided alcohol 23 cleanly and in high yield. Condensation of 23 with carboxylic acid 25 ¹⁸ mediated with 2-methyl-6-nitrobenzoic anhydride (MNBA) ¹⁹ afforded ester 26 in a reasonable yield. Desilylation of 26 using the BF ₃ .Et ₂ O procedure cleanly provided a 1:1 mixture of isomeric alcohols 27-28. The spectral and analytical properties of both were consistent with the expected product and characterized as 27-28, a mixture of diastereomers arising from racemization of the carbon bearing the terminal hydroxyl.

An explanation for the epimerization evident during the deprotection of silyl ether 26 remains obscure. Model studies (Scheme 4) did not indicate evidence of any obvious problem. The epimerization problem and the somewhat variable results in the presence of the β-ketoester suggested that presence of this potentially base labile moiety could be problematic and that delaying its introduction should be beneficial. Concurrently, additional model studies indicated another area of concern. The C 1-5 fragment 34 was prepared by condensation of the acid chloride derived from silyl ester 32 ²⁰ and alcohol 33 ²¹ with a view toward increasing the convergency of the synthesis.

Scheme 4 ^a

^a Reagents and conditions: (a) TBAF, THF, 0°C, 1 h, 85%. (b) 25, MNBA, DMAP, TEA, DCM, 23°C, 16 h, 75%. (c) BF ₃ -Et ₂ O, DCM, 23°C, 1.5 h, 92%.

However, efforts to deprotect either the carboxyl (mild base or Lil/pyridine) or the hydroxyl (TBAF) groups led to β-elimination of the leucic acid moiety leading to olefin 36 as the major product (Scheme 5). These results introduced additional constraints upon the reagents available for this synthetic approach. The desired desilylated alcohol 35 was eventually obtained by BF ₃ .Et ₂ O deprotection.

Scheme 5 ^a

^a Reagents and conditions: (a) (i) Oxalyl chloride, catalytic DMF, DCM, 0-23°C, 2h. (ii) 33, pyridine, 23°C, 16 h, 55%. (b) BF ₃ -Et ₂ O, DCM, 23°C, 1 h, 51%. (c) K ₂ CO ₃ , aqueous CH ₃ OH or TBAF.

Scheme 6 outlines our approach to the respirantin macrocyclic lactone 5.

Scheme 6 ^a

^a Reagents and conditions: (a) (i) Oxalyl chloride, catalytic DMF, DCM, 0-23°C, 2h. (ii) 37, pyridine, 23°C, 16 h, 81%. (b) TBAF, THF, 23°C, 1 h, 100%. (c) 25, MNBA, DMAP, TEA, DCM, 23°C, 16 h, 87%. (d) TBAF, THF, 23°C, 1 h, 100%. (e) (i) TBDPSCl or TBDMSCl, imidazole, DMF, 23°C, 16 h. (ii) LiOH, aq THF/CH ₃ OH, 0 - 23°C, 24 h, 80% for 40, 85%

for 41. (f) 40 or 41, MNBA, DMAP, TEA, DCM, 23°C, 16 h, 85% for 42, 83% for 43. (g) ZnBr ₂ , DCM, 23°C, 24 h, 80% for 44; SiO ₂ , toluene, H0°C, 4 h, 59% for 8. (h) (i) 7, 1:1 TFA-DCM, 0.5 h. (ii) PyBroP, DIPEA, DCM, product from (i), 4 h, 52% for 45, 65% for 46. (i) CH ₃ OH, AcCl, 0.5 h, 63%. (j) Toluene, anhydrous CuSO ₄ , 125°C, 4 h, 80%.

Condensation of the acid chloride derived from 18 with alcohol 37 ²² provided ester 38. The t-butyl ester was chosen for carboxyl protection due to the lability of the leucic acid portion to the conditions required for methyl ester cleavage. Desilylation with TBAF followed by MNBA mediated condensation of the resulting alcohol 10 with carboxylic acid 25 provided ester 39 and ultimately alcohol 9 following TBAF deprotection. Anticipating the need for acidic conditions to achieve deprotection of the t-butyl ester it was considered prudent to utilize the more stable TBDPS protecting group rather than the usual TBDMS group for the terminal hydroxyl protection. Condensation of carboxylic acid 40, (available from ester 13), ⁵ with alcohol 9 using the MNBA procedure provided tetraester 42. Cleavage of the t-butyl group was achieved with ZnBr ₂ in DCM ²³ providing carboxylic acid 44, which was coupled with the amine derived from β-ketoester 7 employing the PyBroP coupling procedure previously described to afford amide 45. At this point we were challenged by attempts to remove the TBDPS group which resulted in eliminating the leucic acid unit.

For example, amide 45 was inert to BF ₃ .Et ₂ O at ambient temperature, as well as several other acidic reagents, and TBAF caused the expected elimination of the leucic acid residue. Consequently, we chose to proceed with a MNBA promoted coupling of alcohol 9 with carboxylic acid 41 to provide ester 43. To achieve good results with this esterfication, it was necessary to use freshly prepared acid 41. Apparently the acidity of 41 is sufficient to cause decomposition to the corresponding α-hydroxyacid. As anticipated, cleavage of the t- butyl ester in the presence of the TBDMS group proved to be problematic. The ZnBr ₂ procedure successful with silyl ether 42, resulted in simultaneous cleavage of the TBDMS group and the t-butyl ester. Selective carboxyl deprotection was achieved by treatment of t- butyl ester 43 with flash silica gel ²⁴ in refluxing toluene to afford 8. PyBroP promoted condensation of 8 with the amine derived from β-ketoester 7 provided the key intermediate ester 46. Desilylation once again proved to be a nontrivial operation. Similar to the results observed with silyl ether 26, reaction of silyl ether 46 with BF ₃ .Et ₂ O afforded a 1:1 mixture of compounds with spectral and analytical properties consistent with epimeric alcohols 6 and 47. Better results were obtained by effecting desilylation using acetyl chloride in CH ₃ OH ²⁵ which provided predominantly a single product. While we were unable to unequivocally

distinguish between epimers 6 and 47 for the desilylation product, we tentatively assigned isomer 6 as the structure for the predominant product. The stage was now set for the key macrocyclization step. Gratifyingly, treatment of alcohol 6 in refluxing toluene ¹⁰ in the presence of catalytic anhydrous CuSO ₄ ²⁶ smoothly afforded macrocyclic lactone 5. The synthesis of the aromatic synthon 3 was achieved in four steps from the commercially available intermediate 48 as outlined in Scheme 7. Scheme 7 ^a

"Reagents and conditions: (a) Formamide, 150°C, 0.5 h, 100%. ²⁷ (b) MeI, NaHCO ₃ , DMF,

23°C,, 18 h, 83%. ²⁸ (c) BzIBr, K ₂ CO ₃ , DMF, 60°C, 18 h, 95%. ²⁹ (d) LiOH, aq THF/CH ₃ OH, 23°C, 18 h, 79%.

The completion of the synthesis is outlined in Scheme 8.

Scheme 8 ^a

^a Reagents and conditions: (a) MeI, K ₂ CO ₃ , DMSO, 23°C, 3 h, 28%. (b) H ₂ , Pd/C, EtOAc, 23°C, 2 h, 73%. (c) 3, EDCI, HOBt, NMM, DMF, 23°C, 11 h, 61%. (d) H ₂ , Pd/C, EtOAc,

23°C, 2 h, 82%.

Introduction of the gem-dimethyl groups at C7 (5 — > 52) was problematic. Insertion of one methyl group occurred readily, while addition of the second methyl group to afford lactone 52 was more difficult and occurred in only a modest yield. Hydrogenolysis of the Cbz protecting group afforded amine 4 which was condensed with benzoic acid 3 employing EDCI to provide amide 53. Removal (hydrogenolysis) of the benzyl ether protecting group provided the cyclodepsipeptide presumed on the basis of spectral data to be respirantin (1b).

When amine 4 was allowed to react (Scheme 9) with benzoic acid derivative 55 using EDCI for amide bond formation, the principal synthetic objective kitastatin 1 (1a) was obtained after deprotection of 56. The synthetic specimens of cyclodepsipeptides Ia and 1b were found to be identical with the corresponding natural products. The spectral properties ( ¹ H-, ¹³ C-NMR, IR, HRMS) of Ib matched perfectly with the published values for respirantin. Ib

Scheme 9 ^a

^a Reagents and conditions: (a) CbzNHS, DMF, 100°C, 6 h. (b) (i) BzIBr, K ₂ CO ₃ , DMF, 120°C, 3 h. (ii) LiOH, aq THF/CH ₃ OH, 23°C, 18 h. (c) 4, EDCI, HOBt, NMM, DMF, 23°C, 11 h. (d) H2, Pd/C, EtOAc, 23°C, 2 h.

The absolute stereochemistry of depsipeptide Ib at carbons 2, 3, 9, 11, and 13 follows from the chirality of the starting materials. Presumably, the 2(S), 3 (R)- stereochemistry of natural threonine and the 2(S), 3(S)-stereochemistry of natural isoleucine have been retained in the biosynthesis of kitastatin 1 (1a) and respirantin (1b). In accord with that assumption, C- 5 was tentatively assigned the R configuration (cf. 1b) as the synthetic and natural specimens were identical. The modular nature of this approach should offer ready access to the scaleup synthesis of respirantin, kitastatin, and a variety of structural modifications to develop structure-activity relationships in this interesting class of powerful cancer cell growth inhibitors.

Kitastatin 1 (1a), respirantin (1b), and the valeryl analog Ic were evaluated as inhibitors of cancer cell growth versus the murine P388 leukemia cell line ³⁰ and a panel of human cancer cell lines. ³¹ The data are reported in Table 1.

Table 1. Comparison of the Cancer Cell Growth Inhibition (GI ₅₀ , μg/ml) of Kitastatin 1 (1a), Respirantin (1b), and the Valeryl Analog Ic against a Panel of Murine (P388, Lymphocytic Leukemia) and Human Cancer Cell Lines.

All three compounds displayed an impressive spectrum of activity. An interesting observation is the substantially better activity of kitastatin 1 (1a) against the pancreas BXPC- 3 human cancer cell line relative to the other panel members. Whether this indicates a special selectivity against this cancer is a question which must be explored. Pancreatic cancer is one of the most deadly types and is notoriously refractory to current modes of treatment. In addition to the human cancer cell line activity cyclodepsipeptide Ib had activity against the pathogenic fungus Cryptococcus neoformans (minimum inhibitory activity, MIC = 2).

Solvents were redistilled prior to use. Reagents were used as received. MNBA was obtained from TCI America. Thin layer chromatography (tic) was carried out with Analtech 250 μ thick silica gel GHLF plates and visualized with H ₂ SO ₄ , phosphomolybdic acid, iodine, or UV. Organic extracts were dried over anhydrous NaSO ₄ and evaporated under reduced pressure using a rotary evaporator. The crude products were separated by flash column chromatography on flash (230-400 mesh ASTM) silica from E. Merck.

Melting points are uncorrected and were determined employing an Electrothermal Mel-Temp apparatus. Optical rotations were measured using a Perkin-Elmer 241 polarimeter. The [α] _D values are given in 10 ^-1 deg cm ² g ^-1 . IR spectra were obtained with a Thermo Nicolet Avatar 360 FT-IR instrument equipped with a Single Reflection Horizontal ATR sampling device from PIKE Technologies. HRMS data were recorded with a JEOL LCmate mass spectrometer. The ¹ H and ¹³ C spectra were recorded employing Varian Gemini 300, Varian Unity 400, or Varian Unity 500 instruments in CDCl ₃ unless otherwise noted and were referenced to either TMS or the solvent. Elemental analyses were determined by Galbraith Laboratories, Inc., Knoxville, TN.

Methyl 4-(t-butoxycarbonyl)amino-2,2,6-trimethyl-3-oxoheptanoate (11). Ketone 7 (0.47 g, 1.63 mmol), K ₂ CO ₃ (2.26 g, 16.3 mmol) and MeI (0.31 mL, 0.71 g, 4.98 mmol)

were placed in DMSO (7 mL) under N ₂ and stirred at ambient temperature for 48 h. The reaction mixture was diluted with H ₂ O (30 mL) and extracted with Et ₂ O (3 x 20 mL). The extracts were combined, washed with H ₂ O (10 mL), 5 M NaCl (5 mL), dried and evaporated. The residue was flash chromatographed (15 g, SiO ₂ , 95:5 hexane-EtOAc) to afford 0.34g (65%) of ketone 11 as a colorless oil: tlc R _f 0.63 (4:1 hexane-EtOAc); IR 3377, 1705 cm ^-1 ; ¹ H NMR δ (4.78 1H, br d), 4.65 (1H, m), 3.73 (3H, s), 1.70 (1H, m), 1.43 (17H, m), 0.95 and 0.92 (6H, 2 d, J = 6.6 Hz); ¹³ C NMR δ 208.7, 173.5, 155.0, 79.7, 54.7, 53.8, 52.5, 41.8, 28.3, 24.6, 23.5, 22.2, 22.0, 21.33.

4-(t-Butoxycarbonyl)amino-2,2,6-trimethyl-3-oxoheptanoic acid (12). To ester 11 (65.7 mg, 0.21 mmol) in CH ₃ OH (0.35 mL) under N ₂ was added 3.5 N KOH (0.25 mL, 0.88 mmol) and the solution stirred at ambient for 15 min. The reaction mixture was diluted with H ₂ O (15 mL) and washed with Et ₂ O (2 x 15 mL). The aqueous layer was acidified (pH 2) with 1 N H ₂ SO ₄ and extracted with Et ₂ O (3 x 15 mL). The combined extract was washed with H ₂ O (5 mL), 5 M NaCl (5 mL), dried and evaporated to afford 50.8 mg (81%) of 12 as a viscous oil which solidified on standing: mp 121°C; tlc R _f 0.52 (95:5:1 DCM-CH ₃ OH- HOAc); IR 3268, 1714, 1655 cm ^-1 ; ¹ H νMR (d ₆ -DMSO) δ 7.05 (1H, d, J = 9 Hz), 4.51 (1H, td, J = 9, 7 Hz), 1.87 (1H, m), 1.38 (9H, s), 1.34 (3H, s), 1.26 (4H, s and m), 0.99 (1H, dd, J = 9, 7 Hz), 0.87 (6H, d); ¹³ C νMR (d ₆ -DMSO) δ 214.1, 155.5, 78.0, 56.8, 38.3, 35.3, 28.1, 23.0, 21.1, 18.7, 18.2. Anal. C 60.12%, H 9.27%, ν 4.68%, calcd for C ₁₅ H ₂₇ NO ₅ , C 59.78%, H 9.03%, N 4.65%. 1-Methoxycarbonyl-3-methylbutyl 4-t-butoxycarbonylamino-6-methyl-3- oxoheptanoate (14). Ketone 7 (0.274 g, 0.95 mmol), alcohol 13 (0.17 g, 1.13 mmol), and activated Zn (30 mg, 0.46 mmol) were placed in cyclohexane (4 mL) under N ₂ and heated at reflux for 16 h with a Dean-Stark separator. Additional 13 (0.17 g, 1.13 mmol) in cyclohexane (1 mL) was added and heating at reflux continued for 24 h. The solution was diluted with EtOAc (40 mL), filtered through Celite, washed with 6% NaHCO ₃ (3 x 10 mL), H ₂ O (10 mL), 5 M NaCl (10 mL), dried, and evaporated to give 0.38 g of a pale yellow oil. This was flash chromatographed (10 g, SiO ₂ , 93:7 hexane-EtOAc) to afford 0.127 g (33%) of ester 14 as a colorless oil: tlc R _f 0.50 (4:1 hexane-EtOAc); IR 3368, 1751, 1712 cm ^-1 ; ¹ H NMR δ 5.48 (1H, br d), 5.08 (1H, m), 4.28 (1H, br t), 3.77 and 3.46-3.80 (5H, s and m), 1.56- 1.82 (6H, m), 1.45 (9H, s), 0.95 (12H, m); ¹³ C NMR δ 203.3, 171.1, 166.2, 155.8, 79.8, 71.6, 58.5, 52.4, 46.1, 39.7, 39.6, 28.2, 24.7, 24.4, 23.2, 22.8, 21.4, 21.3. Anal. C 59.88%, H 9.02%, N 3.49%, calcd for C ₂₀ H ₃₅ NO ₇ , C 59.83%, H 8.79%, N 3.49%.

Methyl 2-[2-(t-butyldimethylsilyl)oxypropionyloxy]-3-methylpentanoa te (20).

Silyl ether 18 (3.23 g, 10.16 mmol) was dissolved in CH ₂ Cl ₂ (10 mL) containing DMF (280 μL, 0.26 g, 3.62 mmol) under N ₂ and cooled to 0°C. Oxalyl chloride (5.6 mL of 2M solution in CH ₂ Cl ₂ , 11.2 mmol) was added dropwise over 5 min. The solution was stirred at 0°C for 1.5 h and at ambient temperature for 0.5 h. The solvent was evaporated. To the residue was added dropwise a solution of alcohol 19 (1.27 g, 8.71 mmol) in pyridine (5 mL). The solution was stirred under N ₂ for 16 h, diluted with THF (100 mL) and filtered through celite. The filtrate was evaporated and the residue was partitioned between EtOAc (200 mL) and H ₂ O (20 mL). The organic phase was separated, washed with H ₂ O (20 mL), 6% NaHCO ₃ (2 x 30 mL), H ₂ O (20 mL), and 5M NaCl (10 mL), dried and evaporated. The residue was flash chromatographed (90 g, SiO ₂ , 96:4 hexane-EtOAc) to yield 2.16 g (75%) of ester 20: tlc R _f 0.37 (95:5 hexane-EtOAc); IR 1757 cm ^-1 ; ¹ H NMR δ 4.93 (1H, d, J = 4.8 Hz), 4.41 (1H, q, J = 6.6 Hz), 3.72 (3H, s), 2.01 (1H, m), 1.45 (3H, d), 1.33 (2H, m), 0.97 (3H, d), 0.92 (3H, t), 0.91 (9H, s), 0.11 and 0.09 (6H, 2s); ¹³ C NMR δ 179.1, 175.2, 81.7, 73.4, 57.3, 41.9, 31.0, 29.9, 26.7, 23.6, 20.6, 16.8, 0.4, 0.0. Anal. C 58.07%, H 9.87%, calcd for C ₁₆ H ₃₂ O ₅ Si, C 57.79%, H 9.70%.

2-[2-(t-Butyldimethylsilanyloxy)propionyloxy]-3-methylpentan oic acid (21). Ester 20 (1.01 g, 3.04 mmol) and LiI (1.24 g, 9.25 mmol) were placed in pyridine (8.0 mL) under N ₂ and stirred at 105°C for 40 h. The reaction mixture was allowed to cool, diluted with toluene (30 mL), evaporated, and coevaporated with toluene (20 mL). The residue was diluted with H ₂ O (50 mL), acidified (pH 4) with KHSO ₄ , and extracted with EtOAc (3 x 30 mL). The extracts were combined, washed with 10% Na ₂ S ₂ O ₃ (10 mL), H ₂ O (10 mL), and 5 M NaCl (10 mL), dried, and evaporated. The residue was flash chromatographed (30 g, SiO ₂ , 99:1:0.5 DCM-CH ₃ OH-HOAC) to provide 0.86g (89%) of carboxylic acid 21 as a pale yellow oil: tlc R _f 0.58 (95:5:1 DCM-CH ₃ OH-HOAC); IR 1726 cm ^-1 ; ¹ H NMR δ 4.98 (1H, d, J = 4.4 Hz), 4.42 (1H, q, J = 7.6 Hz), 2.04 (1H, m), 1.56 (1H, m), 1.45 (3H, d, J = 6.6 Hz), 1.37 (1H, m), 1.01 (3H, d, J = 7.2 Hz), 0.94 (3H, t, J = 7.1 Hz), 0.91 (9H, s), 0.11 and 0.08 (6H, 2s); ¹³ C NMR δ 175.3, 173.8, 75.9, 68.1, 36.5, 25.7, 24.4, 21.3, 18.2, 15.3, 11.5, -5.0, -5.4.

Methyl 4-{2- [2- (t- butyldimethylsilyloxy)propionyloxy ] -3- methylpentanoylamino}-6-methyl-3-oxoheptanoate (22). Ketone 7 (0.88g, 3.05 mmol) was placed in 1:1 TFA DCM (12.0 mL) under N ₂ and stirred at ambient temperature for 1 h. The solvent was removed and the residue coevaporated with toluene (2 x 10 mL). Carboxylic acid 21 (0.88g, 2.76 mmol) and PyBroP (1.29 g, 2.76 mmol) in DCM (6.0 mL) under N ₂ was cooled to 0°C. Diisopropylethylamine (1.07 g, 1.4 mL, 8.28 mmol) was added over 5 min.

The residue from the TFA cleavage reaction was dissolved in DCM (10 mL) and added over 15 min. The solution was stirred at 0°C for 4.5 h. The reaction mixture was diluted with EtOAc (100 mL), washed with 5% citric acid (2 x 10 mL), H ₂ O (10 mL), 6% NaHCO ₃ (2 x 10 mL), H ₂ O (10 mL), and 5 M NaCl (10 mL), dried and evaporated. The residue was flash chromatographed (60 g, SiO ₂ , 90:10 → 80:20 hexane-EtOAc) to afford 0.88g (65%) of amide 22 as a yellow oil: tlc R _f 0.39 (4:1 hexane- EtOAc); IR 3341, 1750, 1665 cm ^-1 ; ¹ H NMR δ 11.98 (0.1H, s), 6.46 (1H, d, J = 7.5 Hz), 5.14 (1H, d, J = 4.5 Hz), 4.72 (1H, m), 4.43 (1H, q), 3.86 (0.5H, s), 3.72 (3H, s), 3.57 (1H, d, J = 16.5 Hz), 3.49 (1H, d, J= 15.3 Hz), 2.04 (1H, m), 1.65 (2H, m), 1.46 (6H, m and d), 1.26 (1H, m), 0.91 (21H, m), 0.12 and 0.10 (6H, 2 d); ¹³ C NMR δ 201.5, 172.8, 169.1, 167.2, 89.5, 77.6, 68.3, 56.3, 52.4, 46.1, 39.7, 37.0, 25.7, 24.8, 24.2, 23.2, 22.4, 21.4, 21.3, 18.1, 14.9, 11.4, -4.9, -5.2; MS APCI ⁺ 488.30444 [M+H] ⁺ Calcd 488.3044. Anal. C 58.94%, H 9.54%, N 2.94%, calcd for C ₂₄ H ₄₅ NO ₇ Si, C 59.11%, H 9.30%, N 2.87%.

Methyl 4-[2-(2-hyroxypropionyloxy)-3-methylpentanoylamino]-6-methyl -3-oxo- heptanoate (23). To amide 22 (0.5 Ig, 1.04 mmol) in DCM (30 mL) under N ₂ was added BF ₃ -Et ₂ O (1.42 g, 1.27 mL, 10 mmol) and the solution stirred at ambient for 2 h. The solution was poured into 6% NaHCO ₃ -ice (100 mL). The organic phase was separated and the aqueous phase extracted with DCM (40 mL). The combined organic extract was washed with 6% NaHCO ₃ (30 mL), H ₂ O (20 mL), 5 M NaCl (20 mL), dried and evaporated. The residue was flash chromatographed (15g, SiO ₂ , 60:40 hexane-EtOAc) to give 0.34g (87%) of carboxylic acid 23 as a colorless oil: tlc R _f 0.34 (50:50 hexane-EtOAc); ¹ H NMR δ 12.02 (0.1H, s), 6.53 (1H, d, J = 8.2 Hz), 5.14 (1H, d, J = 4.9 Hz), 4.72 (1H, m), 4.40 (1H, m), 3.74 (3H, s), 3.60 (1H, d, J = 16 Hz), 3.50 (1H, d, J = 16 Hz), 2.95 (1H, d, J = 5.5 Hz), 2.05 (1H, m), 1.61 (2H, m), 1.50 (4H, d and m, J = 7.1 Hz), 1.28 (1H, m), 0.95 (12H, m); ¹³ C NMR δ 201.6, 174.4, 168.7, 167.4, 78.5, 67.1, 56.5, 52.6, 46.2, 40.0, 37.0, 25.0, 24.3, 23.7, 21.5, 20.2, 15.0, 11.4; FAB MS 374.2189 [M + H] ⁺ . Calcd 374.2179. Anal. C 57.16%, H 8.56%, N 3.70%, calcd for C ₁₈ H ₃₁ NO ₇ 0.2H ₂ O, C 57.33%, H 8.41%, N 3.71%.

Methyl (3,6-diisobutyl-5-methoxycarbonylmethyl-pyrazin-2-yl)acetate (24). Ketone 7 (0.28g, 0.97 mmol) was placed in 1:1 TFA-DCM (4.0 mL) under N ₂ and stirred at ambient for 45 min. The solvent was evaporated and the residue coevaporated with toluene (2 x 10 mL). The residue was dissolved in DCM (3.0 mL) and cooled to 0°C. TEA (0.41 mL, 293.3 mg, 2.91 mmol) was added dropwise and the solution stirred at 0°C for 4 h. The reaction mixture was diluted with EtOAc (50 mL), washed with 5% citric acid (2 x 10 mL), H ₂ O (10 mL), 6% NaHCO ₃ (2 x 10 mL), H ₂ O (10 mL), 5 M NaCl (10 mL), dried and

evaporated. The residue was flash chromatographed (10 g, SiO _2, 95:5 → 90:10 hexane- EtOAc) to afford 78.2 mg (49%) of 24 as a pale yellow solid, which was recrystallyzed from hexane (1 mL): tlc R _f 0.34 (4:1 hexane- EtOAc); mp 72-74°C; IR 1734 cm ^-1 ; ¹ H NMR δ 3.87 (4H, s), 3.70 (6H, s), 2.61 (4H, d, J = 7.1 Hz), 2.14 (2H, m), 0.92 (12H, d); ¹³ C NMR δ 170.6, 151.7, 146.2, 52.1, 42.6, 40.6, 28.2, 22.4. Anal. C 63.82%, H 8.52%, N 8.20%, calcd for C ₁₈ H ₂₈ N ₂ O ₄ , C 64.26%, H 8.39%, N 8.33%.

Methyl 4-(2-{2-[2-benyloxycarbonylamino-3-(t-butyldimethylsilyloxy) butyryloxy]propionyloxy}-3-methylpentanoylamino)6-methyl-3-o xoheptanoate (26). Carboxylic acid 25 (0.26g, 0.72 mmol), alcohol 23 (241.2 mg, 0.65 mmol), MNBA (0.25g, 0.73 mmol), DMAP (20.0 mg, 0.16 mmol) and TEA (0.30 mL, 0.22 g, 2.13 mmol) were placed in DCM (3.5 mL) under N ₂ and stirred at ambient for 16 h. The reaction mixture was diluted with EtOAc (50 mL), washed with H ₂ O (10 mL), 6% NaHCO ₃ (2 x 10 mL), H ₂ O (10 mL), 5% citric acid (2 x 10 mL), H ₂ O (10 mL), 5 M NaCl (10 mL), dried, and evaporated. The residue was flash chromatographed (15 g, SiO ₂ , 85:15 hexane-EtOAc) to yield 0.36g (77%) of ester 26 as a colorless oil which crystallized on standing: mp 84-86°C; tlc R _f 0.22 (4:1 hexane-EtOAc); ¹ H NMR δ 7.37 (5H, m), 6.94 (1H, d, J = 8.3 Hz), 5.47 (1H, d, J = 9.4 Hz), 5.16 (4H, m), 4.67 (1H, m), 4.48 (1H, q), 4.27 (1H, m), 3.72 (3H, s), 3.52-3.62 (2H, m), 2.00 (1H, m), 1.63 (2H, m), 1.53 (4H, d and m, J = 7.1 Hz), 1.25 (5H, m), 0.93 (12H, m), 0.84 (9H, s), 0.06 and -0.01 (6H, 2 s); ¹³ C NMR δ 202.0, 171.7, 168.9, 168.8, 167.5, 156.7, 136.2, 128.6, 128.3, 128.0, 78.5, 70.0, 68.7, 67.1, 60.2, 56.4, 52.3, 45.9, 38.8, 37.1, 25.6, 24.7, 24.2, 23.3, 21.2, 21.1, 17.8, 16.9, 14.8, 11.4, -4.4, -5.4; MS FAB ⁺ 723.3890 (M + H). Calcd 723.3889. Anal. C 59.95%, H 8.44%, N 3.91%, calcd for C ₃₆ H ₅₈ N ₂ OnSi, C 59.81%, H 8.09%, N 3.87%.

Methyl 4-{2-[2-(2-benzyloxycarbonylamino-3-hydroxybutyryloxy)propio nyloxy]- 3-methylpentanoylamino}-6-methyl-3-oxo-heptanoate (27-28). To silyl ether 26 (0.73g, 1.02 mmol) in DCM (30 mL) under N ₂ was added BF ₃ -Et ₂ O (1.44 g, 1.3 mL, 10.2 mmol) and the solution stirred at ambient for 1.5 h. The solution was poured into 6% NaHCO ₃ -ice (100 mL). The organic phase was separated and the aqueous phase extracted with DCM (50 mL). The combined extract was washed with 6% NaHCO ₃ (30 mL), H ₂ O (20 mL), and 5 M NaCl (20 mL), dried and evaporated. The residue was flash chromatographed (20 g, SiO ₂ , 70:30 hexane- EtOAc) to afford 0.155g, (25%) of alcohol 27 as a single isomer: tlc R _f 0.62 (50:50 hexane- EtOAc); ¹ H NMR δ 12.11 (0.1H, s), 7.35 (5H, m), 6.72 (1H, d, J = 8.2 Hz), 5.60 (1H, d, 9.9 Hz), 5.18 and 5.14 (4H, m and s), 4.67 (1H, m), 4.53 (1H, m), 4.39 (1H, d, J = 9.3 Hz), 3.72 (3H, s), 3.64 (1H, d, J = 16.2 Hz), 3.50 (1H, d, J = 16.5 Hz), 3.13 (1H, d, J = 5.8 Hz),

2.05 (1H, m), 1.59 and 1.46-1.64 (7H, d and m), 1.32 (5H, m), 0.92 (12H, m); ¹³ C NMR δ 202.5, 171.5, 169.9, 168.9, 167.9, 156.9, 136.2, 128.5, 128.2, 128.0, 78.5, 69.9, 67.6, 67.2, 58.8, 56.4, 52.6, 46.2, 38.8, 37.1, 24.7, 24.0, 23.2, 21.3, 20.1, 17.2, 15.0, 11.4. Anal. C 59.09%, H 7.45%, N 4.49%, calcd for C ₃₀ H ₄₄ N ₂ O ₁₁ , C 59.20%, H 7.29%, N 4.60%. Continued elution led to 0.16g (31%) of alcohols 27-28 as a mixture of isomers. Further elution provided 0.20g, (39%) of alcohol 28 as a single isomer: tlc R _f 0.58 (50:50 hexane- EtOAc); ¹ H NMR δ 12.04 (0.1 H, s), 7.35 (5H, m), 6.50 (1H, d, J = 8.3 Hz), 5.65 (1H, d, J = 9.3 Hz), 5.18 and 5.14 (3H, m and s), 5.03 (1H, m), 4.72 (1H, m), 4.53 (1H, m), 4.42 (1H, d, J = 9.4 Hz), 3.72 (3H, s), 3.55 (2H, m), 3.03 (1H, d, J = 5.5 Hz), 1.99 (1H, m), 1.61 (6H, d and m, J = 7.2 Hz), 1.29 (5H, m and d, J = 6.6 Hz), 0.93 (12 H, m); ¹³ C NMR δ 201.7, 171.1, 170.2, 168.5, 167.3, 156.8, 136.2, 128.5, 128.2, 128.0, 78.8, 69.7, 67.8, 67.1, 59.2, 56.5, 52.5, 46.2, 39.6, 36.9, 24.9, 24.4, 23.2, 21.6, 21.3, 19.7, 16.8, 14.8, 11.2. Anal. C 59.28%, H 7.58%, N 4.24%, calcd for C ₃₀ H ₄₄ N ₂ O ₁₁ , C 59.20%, H 7.29%, N 4.60%.

Methyl 2-(2-hydroxypropionyloxy)-3-methylpentanoate (29). To a solution cooled to 0°C of silyl ether 20 (0.52 g, 1.56 mmol) in THF (10 mL) under N ₂ was added a IM THF solution (3.2 mL) of TBAF dropwise and the resulting solution stirred at 0°C for 20 min. The solution was poured into H ₂ O and extracted with EtOAc (3 x 25 mL). The combined extract was washed with H ₂ O (10 mL), and 5 M NaCl (10 mL), dried and evaporated. The residue was flash chromato graphed (15 g, SiO ₂ , 85:15 hexane-EtOAc) to afford 0.29 g (85%) of alcohol 29 as a colorless oil: tlc R _f 0.29 (80:20 hexane-EtOAc); IR 3488, 1744 cm ^-1 ; ¹ H NMR δ 5.00 (1H, d, J = 4.4 Hz), 4.37 (1H, q, J = 6.6 Hz), 3.75 (3H, s), 2.82 (1H, d, J = 6.0 Hz), 2.04 (1H, m), 1.50 (4H, d and m, J = 6.0 Hz ), 1.33 (1H, m), 0.98 (3H, d, J = 7.1 Hz), 0.93 (3H, t, J = 7.7 Hz); ¹³ C NMR δ 175.44, 169.58, 66.66, 52.13, 36.51, 24.41, 20.50, 15.31, 11.49.

Methyl 2-{2-[2-benyloxycarbonylamino-3-(t-butyldimethylsilyloxy)but yryloxy]- propionyloxy}-3-methyl-pentanoate (30). Alcohol 29 (0.115 g, 0.53 mmol) and carboxylic acid 25 (0.213 g, 0.58 mmol) were allowed to react using the MNBA esterification procedure described for 26 to afford 0.23g (75%) of ester 30 as a colorless oil: tlc Rf 0.43 (80:20 hexane- EtOAc); ¹ H NMR δ 7.37 (5H, m), 5.47 (1H, d, J = 9.3 Hz), 5.24 (1H, q, J = 7.1 Hz), 5.14 (2H, s), 4.98 (1H, d, J = 4.4 Hz), 4.47 (1H, m), 4.30 (1H, dd, J = 9.3, 1.6 Hz), 3.73 (3H, s), 2.01 (1H, m), 1.57 (3H, d, J = 6.6 Hz), 1.50 (1H, m), 1.30 (1H, m), 1.26 (3H, d, J = 6.0 Hz), 0.97 (3H, d, J = 6.5 Hz), 0.91 (3H, t, J = 7.7 Hz), 0.83 (9H, s), 0.05 and 0.00 (6H, 2 s); ¹³ C NMR δ 170.35, 169.85, 169.63, 159.61, 136.32, 128.56, 128.19, 68.91, 68.63, 67.13, 60.39, 59.71, 52.10, 36.52, 25.70, 24.43, 21.25, 17.88, 17.10, 15.31, 11.50, -4.31, -5.34.

Methyl 2-{2- [2-benyloxycarbonylamino-3-hydroxybutyryloxy] -propionyloxy }-3- methyl-pentanoate (31). Ester 30 (0.60g, 1.05 mmol) was converted employing the BF ₃ .Et ₂ O desilylation procedure described for 23 to provide 0.44g (92%) of alcohol 31 as a colorless oil: tlc R _f 0.13 (80:20 hexane-EtOAc); ¹ H NMR δ 7.35 (5H, m), 5.56 (1H, d, J = 9.9 Hz, ), 5.27 (1H, q, J = 7.1 Hz), 5.13 (2H, s), 5.02 (1H, d, J = 4.4 Hz), 4.58 (1H, m), 4.43 (1H, dd, J = 9.0, 1.6 Hz), 3.74 (3H, s), 3.17 (1H, d, J = 4.4 Hz), 2.02 (1H, m), 1.63 (3H, d), 1.47 (1H, m), 1.32 (1H, m), 1.26 (3H, d, J = 6.0 Hz), 0.98 (3H, d, J = 6.5 Hz), 0.92 (3H, t, J = 7.7 Hz); ¹³ C NMR δ 171.14, 170.88, 169.59, 156.78, 136.25, 128.49, 128.08, 127.91, 77.07, 69.14, 67.73, 67.05, 59.38, 52.31, 36.59, 24.41, 18.97, 16.58, 15.20, 11.46.

2-Benzyloxycarbonylamino-2-methoxycarbonyl-1-methylethyl 2-(t- butyldimethylsilyloxy)-4-methylpentanoate (34). Silyl ester 32 (4.5 g, 12.5 mmol) and DMF (300 μL, 3.7 mmol) were placed in DCM (20 mL) under N ₂ and cooled to 0°C. Oxalyl chloride (12.5 mL of a 2 M solution in DCM, 25 mmol) was added dropwise. The mixture was warmed to ambient temperature, stirred for 4.5 h and solvent evaporated. To the residue under N ₂ was added a solution of alcohol 33 (2.02 g, 8 mmol), DMAP (2.9 g, 24 mmol), and TEA (2.3 mL, 20 mmol) in DCM ( 15 mL) at 0°C. The mixture was stirred at ambient temperature for 2 h, the reaction was terminated with 6% NaHCO ₃ (50 mL), and extracted with Et ₂ O (3 x 50 mL). The extracts were combined, dried, and evaporated. The residue was flash chromatographed (100 g, SiO ₂ , 6:1 hexane-EtOAc) to afford 2.17 g (56%) of ester 34 as a colorless oil: tlc R _f 0.33 (80:20 hexane-EtOAc); ¹ H NMR δ 7.37 (5H, m), 5.44 (2H, m), 5.15 (2H, s), 4.56 (1H, d, J = 8.1 Hz), 4.15 (1H, dd, J = 4.2, 8.1 Hz), 3.72 (3H, s), 1.78 (1H, m), 1.59 (1H, m), 1.40 (1H, m), 1.32 (3H, d, J = 6.6 Hz), 0.87 (18H, m), 0.22 (6H, m); FAB MS [M + H] ⁺ 496.2735. Calcd for C ₂₅ H ₄₂ NO ₇ Si: 496.2731.

2-Benzyloxycarbonyl-2-methoxycarbonyl-1-methylethyl 2-hydroxy-4-methyl- pentanoate (35). The BF ₃ .Et ₂ O desilylation procedure described for 23 was applied to silyl ester 34 (265.3 mg, 0.54 mmol) to provide 0.11 g (51%) of alcohol 35 as a colorless oil: tlc R _f 0.18 (80:20 hexane-EtOAc); ¹ H NMR δ 7.37 (5H, m), 5.56 (1H, d, J = 9.3 Hz), 5.49 (1H, qd, J = 6.6, 2.2 Hz), 5.14 (2H, s), 4.55 (1H, dd, J = 9.3, 2.5 Hz), 4.12 (1H, q, J = 6.6 Hz), 3.73 (3H, s), 2.71 (1H, d, J = 6.1 Hz), 1.83 (1H, hept, J = 6.6 Hz), 1.49 (2H, t, J = 6.6 Hz), 1.33 (3H, d, J = 6.6 Hz), 0.94 and 0.92 (6H, 2 d); ¹³ C NMR δ 174.5, 170.2, 156.5, 128.6, 128.3, 128.2, 71.6, 69.0, 67.4, 57.4, 52.8, 43.2, 24.3, 23.1, 21.5, 16.8. t-Butyl 2-[2-(t-butyldimethylsilyloxy)propionyloxy]-3-methylpentanoa te (38). The acid chloride derivative of silyl ester 18 (8.73g, 27.4 mmol) and alcohol 37 (3.90 g, 20.7 mmol) were allowed to react using the catalytic DMF, oxalyl chloride esterification

procedure described for ester 20 to give 6.24 g (81%) of ester 38 as a colorless oil: tlc R _f 0.60 (4:1 hexane-EtOAc); IR 1738, 1651 cm ^-1 ; ¹ H NMR δ 4.78 (1H, d, J = 4.5 Hz), 4.38 (1H, dd, J = 13.8, 6.6 Hz), 1.95 (1H, m), 1.24-1.54 (15H, m), 0.84-0.97 (15H, m), 0.10 (6H, m); MS APCI ⁺ 375.2567 [M + H] ⁺ . Calcd for C ₁₉ H ₃₈ O ₅ Si: 375.2567. Anal. C 61.42%, H 10.36%, calcd for C ₁₉ H ₃₈ O ₅ Si, C 60.92%, H 10.23%. t-Butyl 2-(2-hydroxypropionyloxy)-3-methylpentanoate (10). Ester 38 (0.77 g,

2.05 mmol) was transformed employing the TBAF desilylation procedure described for alcohol 29 to provide 0.54 g (100%) of alcohol 10 as a colorless oil: tlc R _f 0.41 (80:20 hexane-EtOAc); IR 1745 cm ^-1 ; ¹ H NMR δ 4.85 (1H, d, J = 4.5 Hz), 4.35 (1H, dd, J = 13.8,

6.6 Hz), 2.72 (1H, m), 2.01 (1H, m), 1.23-1.68 (15H, m), 0.95 (6H, m). Anal. C 59.93%, H 9.35%, calcd for C ₁₃ H ₂₄ O ₅ , C 59.98%, H 9.29%. t-Butyl 2-{2-[2-benzyloxycarbonylamino-3-(t-butyldimethylsilyloxy)bu tyryloxy] propionyloxy}-3-methylpentanoate (39). Alcohol 10 (0.50 g, 1.92 mmol) was esterified with carboxylic acid 25 (768 mg, 2.09 mmol) by means of the MNBA procedure described for ester 26 to yield 1.02 g (87%) of ester 39 as a colorless oil: tlc R _f 0.55 (80:20 hexane- EtOAc); IR 3453, 1745, 1625 cm ^-1 ; ¹ H NMR δ 7.36 (5H, m), 5.46 (1H, m), 5.24 (1H, m), 5.13 (2H, s), 4.81 (1H, d, J = 4.2 Hz), 4.46 (1H, d, J = 6.0 Hz), 1.96 (1H, m), 1.22-1.55 (18H, m), 0.95 (6H, m), 0.82 (9H, s), 0.02 (6H, m); MS APCI ⁺ 610.3456 [M + H] ⁺ . Calcd for C ₃₁ H ₅₂ NO ₉ Si: 610.3411. t-Butyl 2-[2-(2-benzyloxycarbonylamino-3-hydroxybutyryloxy)propionyl oxy]-3- methylpentanoate (9). Ester 39 (1.02 g, 1.68 mmol) was converted using the TBAF desilylation procedure described for alcohol 29 to afford 0.83 g (100%) of alcohol 9 as a colorless oil: tlc R _f 0.46 (6:1 hexane-EtOAc); IR 1745 cm ^-1 ; ¹ H NMR δ 7.32 (5H, m), 5.54 (1H, d, J = 9.9 Hz), 5.16-5.23 (2H, m), 5.12 (2H, s), 4.86 (1H, d, J = 4.5 Hz), 4.58 (1H, m), 4.41 (1H, dd, J = 9.3, 3.0), 3.29 (1H, d, J = 4.2 Hz), 1.98 (1H, m), 1.21-1.60 (18H, m), 0.84- 0.99 (9H, m); MS APCI ⁺ 496.2541 [M + H] ⁺ . Calcd for C ₂₅ H ₃₈ NO ₉ : 496.2547. Anal. C 60.50%, H 7.67%, N 2.66%, calcd for C ₂₅ H ₃₇ NO ₉ , C 60.59%, H 7.53%, N 2.83%.

2-(t-Butyldiphenylsilyloxy)-4-methylpentanoic acid (40). Alcohol 13 (3.00 g, 20.5 mmol), imidazole (2.79 g, 41.0 mmol), and TBDPSCl (7.93 g, 28.8 mmol) were dissolved in DMF (30 mL under N ₂ ) and the solution stirred at ambient temperature for 18 h. The reaction was terminated with 5 M NaCl (100 mL) and extracted with EtOAc (2 x 100 mL). The extracts were combined, washed with cold 5% citric acid (50 mL), H ₂ O (20 mL), 5 M NaCl (20 mL), dried and evaporated, and the residue coevaporated with toluene (2 x 75 mL). The residue was flash chromatographed (270 g, SiO ₂ , 95:5 hexane-EtOAc) to afford 6.92 g

(88%) of the methyl ester: tlc R _f 0.37 (95:5 hexane-EtOAc); IR 1750, 1649 cm ^-1 ; ¹ H NMR δ 7.67 (4H, m), 7.40 (6H, m), 4.23 (1H, dd, J = 4.2, 7.2 Hz), 3.44 (3H, s), 1.43-1.76 (3H, m), 1.09 (9H, s), 0.81 (6H, dd, J = 6.0, 16.5 Hz); ¹³ C NMR δ 174.0, 136.0, 135.9, 133.9, 133.3, 129.73, 129.66, 127.6, 127.4, 71.5, 51.3, 44.3, 26.9, 24.1, 22.9, 22.2, 19.4.

A portion of this material (1.76 g, 4.58 mmol) was placed in 1:1 THF- CH ₃ OH (40 mL) at 0°C under N ₂ and LiOH (14 mL of 0.5 M cold solution, 7.0 mmol) was added over 20 min. The mixture was stirred at ambient temperature for 28 h, cooled to 0°C, acidified (pH 3) with 1 M KHSO ₄ , and extracted with EtOAc (2 x 50 mL). The combined extract was washed with H ₂ O (20 mL), 5 M NaCl (20 mL), dried, and solvent evaporated. The residue was flash chromato graphed (60 g, SiO ₂ , 8:1 hexane-EtOAc) to provide 1.73 g (98%) of carboxylic acid 40 as a colorless oil: tlc R _f 0.67 (95:5:1 DCM-CH ₃ OH-HOAC); IR 1721 cm ^-1 ; ¹ H NMR δ 7.64 (4H, m), 7.41 (6H, m), 4.26 (1H, t, J = 6.0 Hz), 1.48-1.74 (3H, m), 1.08 (9H, s), 0.69 (6H, dd, J = 6.6, 9.3 Hz).

2-(t-Butyldimethylsilyloxy)-4-methylpentanoic acid (41). Alcohol 13 (1.06 g, 6.84 mmol) was treated with TBDMSCl (1.61 g, 10.26 mmol) according to the procedure described for obtaining 40 and silyl ester that lead to 1.74 g (98%) of the TBDMS ether as a colorless oil: IR 1761 cm ^-1 ; ¹ H NMR δ 4.22 (1H, dd, J = 3.9, 8.4 Hz), 3.70 (3H, s), 1.76 (1H, m), 1.55 (2H, m), 0.93-0.98 (15H, m), 0.04 (6H, dd, J = 4.2, 18.6). A portion of this methyl ester (1.30 g, 5.0 mmol) was hydrolyzed as described for carboxylic acid 40 and that led to 1.07 g (87%) of carboxylic acid 41 as a somewhat unstable colorless oil: ¹ H NMR δ 4.27 (1H, dd, J = 4.2, 7.2 Hz), 1.84 (1H, m), 1.62 (2H, m), 0.82-0.95 (15H, m), 0.06-0.12 (6H, m).

2-Benzyloxycarbonylamino-2- [ 1 -( 1 -t- butoxycarbonyl-2- methylbutoxycarbonyl)ethoxycarbonyl]-1-methylethyl 2-(t-butyldiphenylsilyloxy)-4- methylpentanoate (42). Alcohol 9 (0.83 g, 1.68 mmol) was esterified with carboxylic acid 40 (0.78 g, 2.1 mmol) employing the MNBA procedure described for diester 26 to afford 1.21 g (85%) of ester 42 as a colorless oil: IR 1745 cm ^-1 ; ¹ H NMR δ 7.61 (4H, m), 7.35 (HH, m), 5.22 (2H, m), 5.09 (3H, m), 4.80 (1H, d, J = 4.5 Hz), 4.43 (1H, dd, J = 3.3, 9.3 Hz), 4.30 (1H, t, J = 6.1 Hz), 1.94 (1H, m), 1.24-1.69 (16H, m), 1.05 (9H, s), 0.94 (6H, m), 0.74 (6H, dd, J = 4.2, 15.3 Hz); MS APCI ⁺ 848.4402 [M + H] ⁺ . Calcd for C ₄₇ H ₆₆ NO ₁₁ Si: 848.4406.

2-Benzyloxycarbonylamino-2- [ 1 -( 1 -t- butoxycarbonyl-2- methylbutoxycarbonyl)ethoxycarbonyl]-1-methylethyl 2-(t-butyldimethylsilyloxy)-4- methylpentanoate (43). By applying the preceding method (cf. 26 and 42) alcohol 9 (3.17 g, 6.40 mmol) was esterified with carboxylic acid 41 (1.84 g, 7.60 mmol) using MNBA and that reaction led to 3.85 g (83%) of ester 43 as a colorless oil: tlc R _f 0.38 (6:1 hexane-EtOAc); IR

3446, 3336, 1747 cm ^-1 ; ¹ H NMR δ 7.35 (5H, m), 5.43 (2H, m), 5.11 (3H, m), 4.82 (1H, d, J = 4.5 Hz), 4.55 (1H, m), 4.22 (1H, m), 1.94 (1H, m), 1.24-1.59 (21H, m), 0.85-0.98 (21H, m), 0.11 (6H, m); ¹³ C NMR δ 173.0, 169.2, 169.1, 128.5, 128.2, 128.1, 82.2, 76.9, 70.5, 69.5,

67.3, 57.6, 43.9, 36.6, 28.0, 25.7, 24.5, 23.4, 16.9, 15.3, 11.6, -4.8, -.5.5. Anal. C 61.57%, H 8.62%, N 1.87%, calcd for C ₃₇ H ₆₁ NO ₁₁ Si, C 61.38%, H 8.49%, N 1.93%.

2-Benzyloxycarbonylamino-2-[1-(1-carboxy-2- methylbutoxycarbonyl)ethoxycarbonyl]-1-methylethyl 2-(t-butyldiphenylsilyloxy)-4- methylpentanoate (44). To t-butyl ester 42 (1.09 g, 1.29 mmol) in DCM (5 mL) was added ZnBr ₂ (1.45 g, 6.43 mmol), the solution was stirred for 48 h, H ₂ O (20 mL) was added and stirring continued for 2 h. The organic phase was separated and the aqueous phase extracted with DCM (2 x 20 mL). The organic solutions were combined, dried and evaporated to furnish 0.82 g (80%) of carboxylic acid 44 as a colorless oil: tlc R _f 0.50 (50:1 DCM-CH ₃ OH); IR 1752 cm ^-1 ; ¹ H NMR δ 7.62 (4H, s), 7.30 (HH, m), 4.91-5.21 (5H, m), 4.44 (1H, m), 4.30 (2H, m), 1.98 (1H, m), 0.73-1.66 (33H, m); FAB MS 792.3786 [M + H] ⁺ . Calcd for C ₄₃ H ₅₈ NO ₁₁ Si: 792.3780.

2-Benzyloxycarbonylamino-2-[1-(1-carboxy-2- methylbutoxycarbonyl)ethoxycarbonyl]-1-methylethyl 2-(t-butyldimethylsilyloxy)-4- methylpentanoate (8). To t-butyl ester 43 (2.52 g, 3.10 mmol) in toluene (70 mL) was added 230-400 mesh silica gel (5 g). The mixture was heated at reflux under N ₂ for 6 h, allowed to cool, and diluted with 4:1 DCM-CH ₃ OH (200 mL). The solution was filtered and the solid phase washed with 4:1 DCM-CH ₃ OH (50 mL). The combined DCM filtrate and washings were evaporated to dryness. The residue was flash chromatographed (60g, SiO ₂ , 50:1 DCM- CH ₃ OH to afford 1.54 g (66%) of carboxylic acid 8 as a colorless oil: tlc R _f 0.51 (50:1 DCM- CH ₃ OH); [α] ²⁶ _D -33.9 (c 1.1, CHCl ₃ ); IR 3319, 1755 cm ^-1 ; ¹ H NMR δ 7.34 (5H, m), 5.31 (2H, m), 4.98-5.13 (5H, m), 4.57 (1H, dd, J = 3.3, 9.9 Hz), 4.20 (2H, dd, J = 3.6, 8.7 Hz), 2.01 (1H, m), 1.33-1.76 (9H, m), 0.81-1.24 (22H, m), 0.01-0.05 (6H, m); ¹³ C NMR δ 173.1, 169.3, 156.5, 136.0, 128.6, 128.3, 128.1, 76.2, 70.7, 70.5, 69.5, 67.4, 57.6, 43.9, 36.5, 25.7,

24.4, 24.0, 23.4, 21.5, 18.1, 16.9, 16.7, 15.3, 11.5, -4.8, -5.5. Anal. C 59.49%, H 8.32%, N 1.97%, calcd for C ₃₃ H ₅₃ NOnSi, C 59.35%, H 8.00%, N 2.10%.

Methyl 4-[2-(2-{2-Benzyloxycarbonylamino-3-[2-(t-butyldiphenylsilyl oxy)-4- methylpentanoyloxy]butyryloxy}propionyloxy)-3-methylpentanoy lamino]-6-methyl-3- oxoheptanoate (45). B oc-protected ketone 7 (0.287 g 1.0 mmol) was deprotected (TFA- DCM) and allowed to react with carboxylic acid 44 (0.640 g, 0.81 mmol) were reacted employing the PyBroP mediated amide formation procedure described for 22 to afford 0.375

g (52%) of amide 45 as a colorless oil: tlc R _f 0.45 (80:20 hexane-EtOAc); IR 3367, 1755, 1682 cm ^-1 ; ¹ H NMR δ 7.59 (4H, m), 7.33 (HH, m), 6.70 (1H, d, J = 7.8 Hz), 5.00-5.13 (6H, m), 4.70 (1H, m), 4.41 (1H, m), 4.27 (1H, m), 3.69 (3H, d, J = 3.0 Hz), 3.52 (2H, d, J = 2.7 Hz); ¹³ C NMR 5 201.7, 172.4, 169.7, 168.7, 167.3, 156.3, 136.0, 135.7, 133.1, 129.9, 128.6, 128.3, 128.1, 127.7, 78.6, 71.7, 70.7, 70.1, 67.3, 57.9, 56.4, 52.4, 46.0, 44.5, 39.2, 37.0, 29.7, 26.8, 24.8, 24.2, 24.1, 23.3, 22.9, 22.3, 21.4, 19.4, 16.9, 14.9, 11.4; FAB MS 961.4854 [M + H] ⁺ . Calcd for C ₅₂ H ₇₃ N ₂ O ₁₃ Si: 961.4882. Anal. C 64.76%, H 7.77%, N 2.72%, calcd for C ₅₂ H ₇₂ N ₂ O ₁₃ Si, C 64.98%, H 7.55%, N 2.91%.

Methyl 4-[2-(2-{2-benzyloxycarbonylamino-3-[2-(t-butylmethylsilylox y)-4- methylpentanoyloxy]butyryloxy}propionyloxy)-3-methylpentanoy lamino]-6-methyl-3- oxo-heptanoate (46). Ketone 7 (1.16 g, 4.00 mmol) following cleavage of the Boc group and carboxylic acid 8 (2.25 g, 3.37 mmol) were coupled by PyBroP promoted amide formation as described for amide 22 to supply 1.83 g (65%) of amide 46 as a colorless oil: tlc R _f 0.46 (80:20 hexane-EtOAc); [α] ²⁵ _D -38.5 (c 0.98, CHCl ₃ ); IR 3359, 1753, 1682 cm ^-1 ; ¹ H NMR δ 7.35 (5H, m), 6.70 (1H, d, J = 7.8 Hz), 5.43 (2H, d, J = 9.3 Hz), 5.13 (4H, m), 4.68 (1H, m), 4.54 (1H, m), 4.18 (1H, dd, J = 3.6, 9.3 Hz), 3.70 (3H, t, J = 3.0 Hz), 3.52 (2H, s), 2.00 (1H, s), 1.27-1.74 (18H, m), 0.88-0.94 (24H, m), 0.02 (6H, m). Anal. C 60.40%, H 8.52%, N 3.31%, calcd for C ₄₂ H ₆₈ N ₂ O ₁₃ Si, C 60.26%, H 8.19%, N 3.35%.

Methyl 4-(2-{2-[2-Benzyloxycarbonylamino-3-(2-hydroxy-4-methylpenta noyloxy) butyryloxy]propionyloxy}-3-methylpentanoylamino)-6-methyl-3- oxoheptanoate (6). To amide 46 (0.367 g, 0.44 mmol) in CH ₃ OH (5 mL) under N ₂ at 0°C was added AcCl (50 μL, 69.5 mg, 0.88 mmol). The solution was stirred at ambient temperature for 30 min, diluted with DCM (40 mL), washed with 6% NaHCO ₃ (20 mL) and H ₂ O (10 mL), dried and evaporated. The residue was flash chromato graphed (10 g, SiO ₂ , 2:1 hexane-EtOAc) to afford 0.198 g (63%) of epimer 6 as a colorless oil: tlc R _f 0.40 (50:50 hexane-EtOAc); [α] ²⁵ _D -30.4 (c 0.92, CHCl ₃ ); ¹ H NMR δ 7.37 (5H, m), 6.63 (1H, d, J = 8.4 Hz), 5.45 (2H, m), 5.53 (2H, s), 5.03 (1H, d, J = 4.8 Hz), 4.65 (2H, m), 4.10 (1H, m), 3.72 (3H, d, J = 2.7 Hz), 3.50 (2H, m), 1.86-2.02 (2H, m), 0.68-1.83 (33H, m); ¹³ C NMR δ 201.7, 174.8, 169.3, 169.2, 168.6, 135.8, 128.6, 128.4, 128.2, 79.0, 78.8, 71.2, 69.9, 69.0, 68.9, 67.5, 57.5, 56.5, 56.4, 46.1, 42.8, 39.7, 36.8, 29.7, 24.9, 24.3, 24.3, 23.2, 21.5, 16.7, 14.8, 11.2; FAB MS 723.3735 [M + H] ⁺ . Calcd for C ₃₆ H ₅₅ N ₂ O ₁₃ : 723.3704. Anal. C 59.64%, H 7.81%, N 3.79%, calcd for C ₃₆ H ₅₄ N ₂ O ₁₃ , C 59.82%, H 7.53%, N 3.88%.

Benzyl (5-s-butyl-8,13-diisobutyl-2,16-dimethyl-3,6,9,11,14,18-hexa oxo-1,4,12,15- tetraoxa-7-azacyclooctadec-17-yl)carbamate (5). A mixture of alcohol 6 (0.12 g, 0.17

mmol) and anhydrous CuSO ₄ (0.60 g, 3.75 mmol) in toluene (150 mL, under N ₂ ) was stirred at 120°C for 12 h. The mixture was allowed to cool, the mixture filtered and the solvent evaporated. The residue was flash chromato graphed (10 g, SiO ₂ , to afford 92 mg (80%) of lactone 5 as a colorless solid: tlc R _f 0.61 (75:25 hexane-EtOAc); [α] ²⁵ _D +13.5 (c 0.68, CHCl ₃ ); IR 3336, 1735, 1717, 1684 cm ^-1 ; ¹ H NMR δ 7.46 (1H, d, J = 8.7 Hz), 7.38 (5H, m), 5.91 (1H, m), 5.70 (1H, dd, J = 7.2, 13.8 Hz), 5.54 (1H, d, J = 9.6 Hz), 5.20 (3H, m), 4.82 (1H, d, J = 9.0 Hz), 4.71 (3H, m), 3.45 (1H, d, J = 15.9 Hz), 3.23 (1H, d, J = 15.6 Hz), 2.03 (1H, m), 1.42-1.86 (10H, m), 1.36 (6H, d, J = 6.9 Hz), 0.87-1.02 (15H, m); ¹³ C NMR δ 204.5, 171.7, 170.1, 169.8, 167.7, 166.4, 156.7, 135.8, 128.6, 128.4, 128.2, 81.2, 72.4, 72.2, 71.3, 67.6, 57.8, 57.2, 47.0, 41.2, 39.2, 36.7, 25.3, 24.9, 24.4, 23.5, 22.8, 21.8, 20.8, 18.5, 16.4, 14.4, 10.6; FAB MS 691.3450 [M + H] ⁺ . Calcd for C ₃₅ H ₅₁ N ₂ O ₁₂ : 691.3442. Anal. C 61.23%, H 7.30%, N 4.06%, calcd for C ₃₅ H ₅₀ N ₂ O ₁₂ , C 60.86%, H 7.30%, N 4.06%.

2-Hydroxy-3-formylaminobenzoic acid (49). Aniline 48 (0.51 g, 3.31 mmol) was suspended in formamide (3.0 mL under N ₂ ) and the mixture stirred at 150° for 0.5 h. The resulting solution was allowed to cool, dissolved in 6% NaHCO ₃ (50 mL), acidified with 1 M KHSO ₄ , and extracted with EtOAc (3 x 50 mL). The combined extract was washed with 5 M NaCl (10 mL), dried, evaporated, and the residue coevaporated with toluene (10 mL) to furnish 90% of phenol 49 as a greenish gray solid: mp 168-169°; tlc R _f 0.20 (95:5:1 DCM- MeOH-HOAc); ¹ H NMR (d ₆ -DMSO) δ 9.82 (1H, s), 8.38 (1H, d, J = 9.3 Hz), 7.55 (1H, d, J = 7.7 Hz), 6.92 (1H, t, J = 7.7 Hz); ¹³ C NMR (d ₆ -DMSO) δ 172.3, 160.3, 151.2, 126.5, 125.7, 124.5, 118.6, 112.6.

Methyl 2-hydroxy 3-formylaminobenzoate (50). Benzoic acid derivative 49 (1.37 g, 7.57 mmol) and NaHCO ₃ (1.40 g, 16.65 mmol) were placed in DMF (20 mL) under N ₂ . MeI (5.37 g, 2.36 mL, 37.85 mmol) in DMF (20 mL) was added and the mixture stirred at ambient temperature for 15 h. The mixture was diluted with EtOAc (250 mL), washed with H ₂ O (50 mL), 6% NaHCO ₃ (50 mL), H ₂ O (20 mL), 5 M NaCl (20 mL), dried, solvent evaporated and the residue coevaporated with toluene (50 mL). The residue was flash chromatographed (36 g, SiO ₂ , 70:30 hexane-EtOAc) to supply 1.17 g (79%) of ester 50 as an off-white solid: mp 99°C; tlc R _f 0.66 (95:5 DCM-MeOH); IR 3248, 1693, 1651 cm ^-1 ; ¹ H NMR δ 11.29 and 11.18 (1H, 2 s), 8.76 and 8.56 (1H, 2 dd, J = 7.9, 1.7 Hz), 8.51 (1H, d, J = 1.7 Hz), 7.97 (1H, br s), 7.65 and 7.57 (1H, 2 dd, J = 8.2, 1.6 Hz), 6.90 and 6.88 (1, 2 t, J = 8.2 Hz), 3.97 (3H, s); ¹³ C NMR δ 170.8, 170.4, 161.2, 158.9, 151.2, 150.3, 126.5, 125.8, 125.4, 124.3, 121.7, 119.2, 113.1, 111.9, 52.7, 52.6.

Methyl 2-benzyloxy-3-formylaminobenzoate (51). To methyl ester 50 (1.01 g, 5.20 mmol) and benzyl bromide (1.44 g, 1 mL, 8.42 mmol) in DMF (20 mL under N ₂ ) was added K ₂ CO ₃ (1.44 g, 10.40 mmol) and the mixture stirred at 60°C for 15 h. The mixture was diluted with EtOAc (100 mL), washed with H ₂ O (2 x 20 mL), 5 M NaCl (10 mL), dried, solvent evaporated and the residue coevaporated with toluene (20 mL). The residue was separated by flash chromatography (50 g, SiO ₂ , 80:20 hexane-EtOAc) to afford 1.41 g (95%) of benzyl ester 51 as a pinkish oil which solidified on standing. A portion was recrystallized from toluene-hexane: mp 52-53°C; tlc R _f 0.59 (50:50 hexane-EtOAc); IR 3284, 1720, 1674 cm ^-1 ; ¹ H NMR δ 8.53 (1H, dd, J = 8.3, 1.6 Hz), 8.19 (1H, d, J = 1.7 Hz), 7.65 (2H, dd, J = 8.2, 1.6 Hz), 7.41 (5H, m), 7.18 (1H, t, J = 8.3 Hz), 5.03 (2H, s), 3.93 (3H, s); ¹³ C NMR δ 165.7, 158.7, 147.7, 136.4, 132.0, 128.9, 128.9, 128.5, 126.7, 124.9, 124.4, 77.8, 52.4. Anal. C 67.51%, H 5.42%, N 4.88%, calcd for C ₁₆ H ₁₅ NO ₄ , C 67.36%, H 5.30%, N 4.91%.

2-Benzyloxy-3-formylaminobenzoic acid (3). To methyl ester 51 (1.28 g, 4.48 mmol) in 3: 1 THF-MeOH (20 mL) under N ₂ was added LiOH (11.6 mL of 0.5 M aqueous solution, 5.8 mmol) and the mixture stirred at ambient temperature for 18 h. The reaction mixture was acidified (pH 3) with 1 M KHSO ₄ , diluted with H ₂ O (200 mL), and extracted with EtOAc (3 x 65 mL). The combined extract was washed with H ₂ O (10 mL), and 5 M NaCl (20 mL), dried, and evaporated. The residue was crystallized from EtOAc-hexane to afford 0.96 g (79%) of benzoic acid 3 as an off-white solid: mp 133°C; tlc R _f 0.51 (95:5:1 DCM-CH ₃ OH-HOAC); IR 3343, 1697, 1636 cm ^-1 ; ¹ H NMR (d ₆ -DMSO) δ 13.09 (1H, s), 9.76 (1H, s), 8.34 (1H, s), 8.30 (1H, d, J = 8.2 Hz), 7.25-7.60 (6H, m), 7.18 (1H, t, J = 7.7 Hz), 4.95 (2H, s); ¹³ C NMR (d ₆ -DMSO) δ 167.0, 160.5, 147.3, 136.8, 132.3, 128.4, 128.0, 127.9, 126.4, 125.6, 124.7, 124.0, 75.7. Anal. C 65.84%, H 4.91%, N 5.06%, calcd for C15H13NO4O.I H ₂ O, C 65.97%, H 4.88%, N 5.12%.

Benzyl (5-s-butyl-8,13-diisobutyl-2,10,10,16-tetramethyl-3,6,9,11,1 4,18-hexaoxo- 1,4,12,15-tetraoxa-7-azacyclooctadec-17-yl)carbamate (52). To a stirred solution of lactone 5 (76 mg, 0.11 mmol) in DMSO (3 mL) under N ₂ were added K ₂ CO ₃ (153 mg, 1.01 mmol) and MeI (20 μL, 0.33 mmol). The mixture was stirred at ambient temperature for 3 h, diluted with H ₂ O (12 mL), and extracted with EtOAc (3 x 20 mL). The extracts were combined, washed with H ₂ O (10 mL), and 5 M NaCl (5 mL), dried and evaporated. The residue was flash chromatographed (10 g, SiO ₂ , 3:1 hexane-EtOAc) to afford 22 mg (28%) of Cbz-protected lactone 52 as an oil: tlc R _f 0.55 (75:25 hexane-EtOAc); [α] ²⁵ _D -17.7 (c 0.90, CHCl ₃ ); IR 3330, 1738, 1718 cm ^-1 ; ¹ H NMR δ 7.50 (1H, d, J = 9.3 Hz), 7.37 (5H, m), 5.91 (1H, m), 5.80 (1H, dd, J = 6.6, 13.8 Hz), 5.56 (1H, d, J = 9.0 Hz), 5.18 (2H, dd, J = 12.0, 17.1

Hz), 4.86 (2H, m), 4.72 (1H, d, J = 9.0 Hz), 4.59 (1H, dd, J = 4.2, 10.2 Hz), 2.06 (1H, m), 1.13-1.83 (20H, m), 0.77-0.98 (19H, m); ¹³ C NMR δ 208.3, 173.1, 171.8, 170.0, 169.7, 167.8, 156.6, 135.8, 128.7, 128.5, 128.2, 80.8, 72.3, 71.9, 71.1, 67.6, 57.7, 56.4, 53.0, 43.0, 39.4, 36.6, 31.9; MS APCI ⁺ 719.3767 [M + H] ⁺ . Calcd for C ₃₇ H ₅₅ N ₂ O ₁₂ : 719.3755.

17-Amino-5-s-butyl-8,13-diisobutyl-2,10,10,16-tetramethyl-1, 4,12,15-tetraoxa-7- azacyclooctadecane-3,6,9,ll,14,18-hexaone (4). Benzyl carbamate 52 (16 mg, 0.022 mmol) and 10% Pd/C (15 mg) in EtOAc (3 mL) was stirred under a H ₂ atmosphere at ambient temperature for 2 h. The solution phase was filtered through Celite and the solid phase washed with CH ₃ OH (20 mL). The combined solvent filtrate and washings were evaporated and the residue flash chromatographed (10 g, SiO ₂ , 3:1 hexane-EtOAc) to furnish 9.5 mg (73%) of amine 4 as a colorless oil: tlc R _f 0.40 (75:25 EtOAc-hexane); [α] ²⁷ _D -55.1 (c 0.67, CHCl ₃ ); IR 3222, 1749, 1712, 1686 cm ^-1 ; ¹ H NMR δ 7.60 (1H, d, J = 9.3 Hz), 5.90 (1H, d, J = 6.6 Hz), 5.78 (1H, dd, J = 6.3, 13.5 Hz), 4.86 (2H, m), 4.63 (1H, dd, J = 4.2, 9.9 Hz), 3.62 (1H, br s), 2.08 (1H, m), 1.26-1.82 (22H, m), 0.86-1.18 (18H, m); ¹³ C NMR δ 208.5, 173.1, 172.1, 171.7, 170.2, 170.1, 80.7, 72.8, 72.1, 70.6, 58.3, 56.5, 53.1, 43.0, 39.5, 36.6, 29.7, 25.3, 24.7, 24.5, 24.0, 23.6, 22.9, 21.4, 21.1, 19.8, 18.2, 16.5, 14.5, 10.5; FAB MS 585.3360 [M + H] ⁺ . Calcd for C ₂₉ H ₄₉ N ₂ O ₁₀ : 585.3387.

2-Benzyloxy-N-(5-s-butyl-8,13-diisobutyl-2,10,10,16-tetramet hyl-3,6,9,11,14,18- hexaoxo-1,4,12,15-tetraoxa-7-azacyclooctadec-17-yl)-3-formyl aminobenzamide (53). Benzoic acid 3 (14.0 mg, 0.051 mmol), 1-hydroxybenzotriazole (7.0 mg, 0.051 mmol), EDCI (7.4 mg, 0.038 mmol), and N-methylmorpholine (20μL, 0.18 mmol) were added successively to a solution of amine 4 (15.0 mg, 0.026 mmol) in DMF (1.5 mL) under N ₂ . The reaction mixture was stirred at ambient temperature for H h, the reaction was terminated by addition of saturated NaHSO ₄ (20 mL), and extracted with EtOAc (30 mL). The extract was dried, evaporated, and the residue flash chromatographed (10 g, SiO ₂ , 2.2:1 hexane-EtOAc) to provide 13 mg (61%) of amide 53 as a colorless oil: tlc R _f 0.42 (2:1 hexane-EtOAc); [α] ²⁵ _D - 45.7 (c 0.65, CHCl ₃ ); IR 3321, 1745, 1678 cm ^-1 ; ¹ H NMR δ 8.45 (1H, d, J = 8.1 Hz), 8.20 (1H, d, J = 9.3 Hz), 8.10 (1H, s), 7.79 (1H, d, J = 6.0 Hz), 7.52 (1H, d, J = 9.3 Hz), 7.26-7.38 (7H, m), 6.05 (1H, d, J = 6.6 Hz), 5.86 (1H, dd, J = 8.2, 13.8 Hz), 5.45 (1H, d, J = 11.7), 5.36 (1H, m), 4.88 (3H, m), 4.56 (1H, d, J = 9.9 Hz), 2.10 (1H, m), 1.47-1.85 (8H, m), 1.13-1.42 (12H, m), 0.75-1.02 (18H, m); ¹³ C NMR δ 208.2, 173.3, 171.8, 170.0, 169.8, 167.9, 165.8, 146.0, 135.3, 131.5, 129.5, 129.2, 129.1, 126.5, 126.2, 125.5, 124.9, 80.9, 79.1, 72.6, 72.0, 71.2, 56.4, 55.9, 53.1, 43.1, 39.5, 36.6, 25.3, 24.6, 24.4, 24.1, 21.0, 19.8, 18.3, 16.6, 14.5, 10.4; MS APCI ⁺ 838.4131 [M + H] ⁺ . Calcd for C ₄₄ H ₆₀ N ₃ O ₁₃ : 838.4126.

N-(5-s-Butyl-8,13-diisobutyl-2,10,10,16-tetramethyl-3,6,9,ll ,14,18-hexaoxo- 1,4,12,15-tetraoxa-7-azacyclooctadec-17-yl)-3-formylamino-2- hydroxybenzamide (Respirantin 1b). Amide 53 (15 mg, 0.018 mmol) and 10% Pd/C (17 mg) in EtOAc (3 mL) was stirred under a H ₂ atmosphere at ambient temperature for 2 h. The solution phase was filtered through Celite and the solid phase washed with 1:1 EtOAc-CH ₃ OH (20 mL). The combined solvent filtrate and washings was evaporated and the residue flash chromato graphed (10 g, SiO ₂ , 1:1 hexane-EtOAc) to afford 11 mg (82%) of Ib as a glassy solid: tlc R _f 0.38 (50:50 hexane-EtOAc); [α] ²⁵ _D -6.0 (c 0.53, CH ₃ OH); IR 3325, 1749, 1708, 1687 cm ^-1 ; ¹ H NMR δ 12.51 (1H, br), 8.58 (1H, d, J = 8.0 Hz), 8.52 (1H, d, J = 1.5 Hz), 7.94 (1H, s), 7.47 (1H, d, J = 9.5 Hz), 7.36 (1H, d, J = 9.5 Hz), 7.15 (1H, d, J = 9.0 Hz), 6.97 (1H, t, J = 8.0 Hz), 6.03 (1H, dd, J = 2.7, 6.6 Hz), 5.86 (1H, q, J = 6.8 Hz), 5.21 (1H, dd, J = 2.7, 8.7 Hz), 4.94 (1H, ddd, J = 3.8, 9.9, 11.0 Hz), 4.86 (1H, d, J= 9.6 Hz), 4.68 (1H, dd, J = 4.5, 9.9 Hz), 2.12 (1H, m), 1.50-1.90 (10H, m), 1.24-1.44 (12H, m), 0.90-1.02 (16H, m); ¹³ C NMR δ 208.1, 173.4, 171.8, 170.4, 169.9, 169.5, 167.5, 159.0, 150.6, 127.5, 125.0, 120.3, 119.1, 112.8, 80.9, 72.3, 72.0, 71.5, 56.4, 55.6, 53.0, 43.1, 39.4, 36.5, 25.3, 24.7, 24.5, 24.1, 23.6, 22.8, 21.4, 21.0, 19.8, 18.2, 16.6, 14.4, 10.4; MS APCI ⁺ 748.3631 [M + H] ⁺ . Calcd for C ₃₇ H ₅₄ N ₃ O ₁₃ : 748.3657.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention.

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