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Title:
SYNTHESIS OF THE ARYLOMYCIN MACROCYCLIC CORE
Document Type and Number:
WIPO Patent Application WO/2017/214534
Kind Code:
A1
Abstract:
The invention provides a copper-mediated process for making compounds according to Formula (I): that are the macrocyclic scaffolds of the arylomycin class of biologically active compounds, where R1, R2, R3, RA1, RA2, RA3, B, G1, G2, PG, n2, and n3 are defined in the specification.

Inventors:
ROMESBERG FLOYD E (US)
BARAN PHIL (US)
PETERS DAVID (US)
Application Number:
PCT/US2017/036797
Publication Date:
December 14, 2017
Filing Date:
June 09, 2017
Export Citation:
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Assignee:
SCRIPPS RESEARCH INST (US)
International Classes:
C07K5/11; C07K5/062; C07K5/087
Domestic Patent References:
WO2013138187A12013-09-19
WO2012036907A22012-03-22
WO2012166665A22012-12-06
Other References:
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EDWARDS, J.T.; MERCHANT, R.R.; KYLE S.; MCCLYMONT, K.S.; KNOUSE, K.W.; QIN, T.; MALINS, L.R.; VOKITS,B.; SHAW, S.A.; BAO, D., NATURE, 2017
LI, C; WANG, J.; BARTON, L.M.; YU, S.; TIAN, M.; PETERS, D.S.; KUMAR, M; YU, AW.; ; JOHNSON, K.A.; CHATTERJEE, A.K., SCIENCE, 2017
QIN T.; MALINS, L.R.; EDWARDS, J.T.; MERCHANT, R.R.; NOVAK, A.J.E.; ZHONG J.Z.; MILLS, R.B.; YAN, M.; YUAN, C.; EASTGATE, M.D., ANGEW. CHEM., vol. 129, 2017, pages 266
WANG, J.; QIN, T.; CHEN, T.-G.; WIMMER, L.; EDWARDS, J.T.; CORNELLA, J.; VOKITS, B.; SHAW, S.A.; BARAN, P.S., ANGEW. CHEM. INT. ED., vol. 55, 2016, pages 9676
QIN, T.; CORNELIA, J.; LI, C.; MALINS, L.R.; EDWARDS, J.T.; KAWAMURA, S.; MAXWELL, B.D.; EASTGATE, M.D.; BARAN, P.S., SCIENCE, vol. 352, 2016, pages 801
CORNELLA, J.; EDWARDS, J.T.; QIN,T.; KAWAMURA, S.; WANG, J.; PAN, C.-M.; GIANATASSIO, R.; SCHMIDT, M; EASTGATE, M.D.; BARAN, P.S., JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 138, 2015, pages 2174
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Attorney, Agent or Firm:
PERDOK, Monique M. et al. (US)
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Claims:
WE CLAIM: 1. A process for synthesizing a compound according to Formula (I):

comprising contacting a compound according to Formula (II):

with a copper pre-catalyst, an amine, and an oxidant, wherein PG is an amine protecting group; R1 is selected from the group consisting of H, (C1-C6)alkyl, (C3-C7)cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R1 can be substituted with 1 to 3 J; each instance of R2 and R3 are independently selected from the group consisting of (C1- C6)alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH2, (C1-C4)alkoxy, and (C1- C4)acyloxy, wherein any carbon atom in R2 and R3 can be substituted with J, wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R2 groups taken together, and/or two R3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J; RA1, RA2, and RA3 are independently selected from the group consisting of H, (C1- C6)alkyl, (C3-C7)cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J; B is CO2H, CH2CO2H, C(=O)NHCH2C(=O)H, CH2C(=O)H, C(=O)NHCH2B(ORB)2 or C(=O)NHCH2P(=O)(ORB)2, wherein RB is H, (C1-C6)alkyl, or (C6-C10)aryl G1 and G2 are each independently selected from the group consisting of H, (C1-C6)alkyl, (CH2)1-pN(R')2, a glycosyl residue, and a group cleavable under physiological conditions to provide a compound of formula (I) wherein G1 or G2 respectively is H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF3, OCF3, O, S, C(O), S(O), methylenedioxy, ethylenedioxy, (CH2)0-pN(R')2, (CH2)0-pSR', (CH2)0-pS(O)R', (CH2)0-pS(O)2R', (CH2)0-pS(O)2N(R')2, (CH2)0-pSO3R', (CH2)0-pC(O)R', (CH2)0-pC(O)CH2C(O)R', (CH2)0-pC(S)R', (CH2)0-pC(O)OR', (CH2)0- pOC(O)R', (CH2)0-pC(O)N(R')2, (CH2)0-pOC(O)N(R')2, (CH2)0-pC(S)N(R')2, (CH2)0- pNH-C(O)R', (CH2)0-pN(R')N(R')C(O)R', (CH2)0-pN(R')N(R')C(O)OR', (CH2)0- pN(R')N(R')CON(R')2, (CH2)0-pN(R')SO2R', (CH2)0-pN(R')SO2N(R')2, (CH2)0- pN(R')C(O)OR', (CH2)0-pN(R')C(O)R', (CH2)0-pN(R')C(S)R', (CH2)0- pN(R')C(O)N(R')2, (CH2)0-pN(R')C(S)N(R')2, (CH2)0-pN(COR')COR', (CH2)0- pN(OR')R', (CH2)0-pC(=NH)N(R')2, (CH2)0-pC(O)N(OR')R', or (CH2)0-pC(=NOR')R'; wherein p is 4; and each instance of R' is independently selected from the group consisting of hydrogen, (C1- C12)-alkyl, (C2-C12)-alkenyl, (C2-C12)-alkynyl, (C3-C10)-cycloalkyl, (C3-C10)- cycloalkenyl, [(C3-C10)cycloalkyl or (C3-C10)-cycloalkenyl]-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], (C6-C10)-aryl, (C6-C10)-aryl-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O)2. 2. The process according to claim 1, wherein the compound according to Formula (I) is a compound according to Formula (IA):

3. The process according to claim 2, wherein each of n2 and n3 is 0; and each of G1 and G2 is H. 4. The process according to claim 3, wherein each of RA1 and RA2 is H; and RA3 is (C1-C6)alkyl. 5. The process according to claim 4, wherein RA3 is methyl. 6. The process according to claim 5, wherein the compound according to Formula (IA) is:

7. The process according to claim 1, wherein the pre-catalyst is a Cu(I) compound. 8. The process according to claim 7, wherein the Cu(I) compound is selected from the group consisting of [Cu(MeCN)4]PF6, Cu(OAc), CuI, CuBr, CuCl, Cu(OS(O)2CF3), and Cu(BF4). 9. The process according to claim 1, wherein the amine is a bidentate diamine. 10. The process according to claim 9, wherein the bidentate diamine is selected from the group consisting of (Ra)2N(CH2)aN(Ra)2 (wherein each Ra is independently selected from H, (C1-C6)alkyl, and (C6-C10)aryl, and a is an integer selected from 1, 2, 3, and 4), 2,2’- bipyridine, and 1,10-phenanthroline. 11. The process according to claim 10, wherein the bidentate diamine is

(Ra)2N(CH2)aN(Ra)2. 12. The process according to claim 11, wherein each Ra is (C1-C6)alkyl and a is 2. 13. The process according to claim 12, wherein the bidentate diamine is

Me2NCH2CH2NMe2. 14. The process according to claim 1, wherein the molar ratio of pre-catalyst to compound according to Formula (I) is 5:1 to 2:1. 15. The process according to claim 14, wherein the molar ratio is 2:1. 16. The process according to claim 1, wherein the molar ratio of nitrogen atoms in the amine to the copper pre-catalyst is 4:1 to 1:1. 17. The process according to claim 16, wherein the molar ratio is 2:1. 18. The process according to claim 1, wherein the oxidant is O2.

19. A process for synthesizing a compound according to Formula (III) or Formula (III’):

comprising contacting a compound according to Formula (I):

with a compound according to the formula [R5C(O)]2O or LG-R6 in the presence of at least one base, wherein PG is an amine protecting group; R1 is selected from the group consisting of H, (C1-C6)alkyl, (C3-C7)cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R1 can be substituted with 1 to 3 J; each instance of R2 and R3 are independently selected from the group consisting of (C1- C6)alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH2, (C1-C4)alkoxy, and (C1- C4)acyloxy, wherein any carbon atom in R2 and R3 can be substituted with J, wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R2 groups taken together, and/or two R3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J; R4 is selected from (C1-C6)alkyl, (C3-C7)cycloalkyl, -C(O)(C1-C6)alkyl, -C(O)(C3- C7)cycloalkyl, and–C(O)aryl, wherein R4 is optionally substituted with 1 to 3 J; when R4 is -C(O)(C1-C6)alkyl, -C(O)(C3-C7)cycloalkyl, or–C(O)aryl, then the compound according to Formula (I) is contacted with a compound according to [R5C(O)]2O, wherein R5 is (C1-C6)alkyl, (C3-C7)cycloalkyl, or aryl, respectively; when R4 is (C1-C6)alkyl or (C3-C7)cycloalkyl, then the compound according to Formula (I) is contacted with a compound according to LG-R6, wherein R6 is (C1-C6)alkyl or (C3-C7)cycloalkyl, respectively; and LG is a leaving group; RA1, RA2, and RA3 are independently selected from the group consisting of H, (C1- C6)alkyl, (C3-C7)cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J; B is CO2H, CH2CO2H, C(=O)NHCH2C(=O)H, CH2C(=O)H, C(=O)NHCH2B(ORB)2 or C(=O)NHCH2P(=O)(ORB)2, wherein RB is H, (C1-C6)alkyl, or (C6-C10)aryl G1 and G2 are each H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF3, OCF3, O, S, C(O), S(O), N3, methylenedioxy, ethylenedioxy, (CH2)0- pN(R')2, (CH2)0-pSR', (CH2)0-pS(O)R', (CH2)0-pS(O)2R', (CH2)0-pS(O)2N(R')2, (CH2)0- pSO3R', (CH2)0-pC(O)R', (CH2)0-pC(O)CH2C(O)R', (CH2)0-pC(S)R', (CH2)0-pC(O)OR', (CH2)0-pOC(O)R', (CH2)0-pC(O)N(R')2, (CH2)0-pOC(O)N(R')2, (CH2)0-pC(S)N(R')2, (CH2)0-pNH-C(O)R', (CH2)0-pN(R')N(R')C(O)R', (CH2)0-pN(R')N(R')C(O)OR', (CH2)0- pN(R')N(R')CON(R')2, (CH2)0-pN(R')SO2R', (CH2)0-pN(R')SO2N(R')2, (CH2)0- pN(R')C(O)OR', (CH2)0-pN(R')C(O)R', (CH2)0-pN(R')C(S)R', (CH2)0- pN(R')C(O)N(R')2, (CH2)0-pN(R')C(S)N(R')2, (CH2)0-pN(COR')COR', (CH2)0- pN(OR')R', (CH2)0-pC(=NH)N(R')2, (CH2)0-pC(O)N(OR')R', or (CH2)0-pC(=NOR')R'; wherein p is 4; and each instance of R' is independently selected from the group consisting of hydrogen, (C1- C12)-alkyl, (C2-C12)-alkenyl, (C2-C12)-alkynyl, (C3-C10)-cycloalkyl, (C3-C10)- cycloalkenyl, [(C3-C10)cycloalkyl or (C3-C10)-cycloalkenyl]-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], (C6-C10)-aryl, (C6-C10)-aryl-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O)2. 20. The process according to claim 19, wherein each of n2 and n3 is 0; each of RA1 and RA2 is H; and RA3 is (C1-C6)alkyl. 21. The process according to claim 19 or 20, wherein R4 is -C(O)(C1-C6)alkyl, -C(O)(C3- C7)cycloalkyl, or–C(O)aryl, and the compound according to Formula (I) is contacted with a compound according to [R5C(O)]2O, wherein R5 is (C1-C6)alkyl, (C3-C7)cycloalkyl, or aryl, respectively. 22. The process according to claim 21, wherein R4 is -C(O)(C1-C6)alkyl. 23. The process according to claim 21 or 22, wherein R4 is -C(O)isopropyl.

24. The process according to claim 19, wherein R4 is (C1-C6)alkyl or (C3-C7)cycloalkyl, and the compound according to Formula (I) is contacted with a compound according to LG- R6, wherein R6 is (C1-C6)alkyl or (C3-C7)cycloalkyl, respectively, and LG is a leaving group. 25. The process according to claim 24, wherein R4 is (C1-C6)alkyl. 26. The process according to any one of claims 19– 25, wherein the base is a tertiary amine according to the formula NR3, wherein each instance of R is

independently selected from the group consisting of (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl, and heteroaryl; a chiral base, a carbonate base; or a combination thereof. 27. The process according to claim 26, wherein the base is a chiral base. 28. The process according to claim 26, wherein the base is a combination of the tertiary amine and a chiral base. 29. The process according to any one of claims 19– 25, wherein regioselectivity for Formula (III) to Formula (III’) or Formula (III’) to Formula (III) ranges from about 100 mol% to about 50 mol%. 30. A process for synthesizing a compound according to Formula (IV)

comprising the steps of (A) contacting a compound according to Formula (I):

with a reagent according to the formula to yield a redox-active ester compound according to Formula V:

(B) contacting the compound according to Formula V with a metal salt that is a nickel (II) or iron (III) salt, optionally a reducing agent, and an active R7 reagent to thereby yield a compound according to Formula (IV); wherein PG is an amine protecting group; R1 is selected from the group consisting of H, (C3-C7)cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R1 can be substituted with 1 to 3 J; each instance of R2 and R3 are independently selected from the group consisting of (C1- C6)alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH2, (C1-C4)alkoxy, and (C1- C4)acyloxy, wherein any carbon atom in R2 and R3 can be substituted with J, wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R2 groups taken together, and/or two R3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J; R7 is selected from the group consisting of H, (C1-C12)alkyl, (C3-C7)cycloalkyl, (C6- C10)aryl, (C2-C12)-alkenyl, wherein any alkyl, alkenyl, cycloalkyl, or aryl can be substituted with 1 to 3 J; RA1, RA2, and RA3 are independently selected from the group consisting of H, (C1- C6)alkyl, (C3-C7)cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J; B is CO2H, CH2CO2H, C(=O)NHCH2C(=O)H, CH2C(=O)H, C(=O)NHCH2B(ORB)2 or C(=O)NHCH2P(=O)(ORB)2, wherein RB is H, (C1-C6)alkyl, or (C6-C10)aryl G1 and G2 are each independently selected from the group consisting of (C1-C6)alkyl optionally substituted with 1– 3 J, (CH2)1-pN(R')2, a glycosyl residue, and a group cleavable under physiological conditions to provide a compound of formula (I) wherein G1 or G2 respectively is H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF3, OCF3, O, S, C(O), S(O), methylenedioxy, ethylenedioxy, (CH2)0-pN(R')2, (CH2)0-pSR', (CH2)0-pS(O)R', (CH2)0-pS(O)2R', (CH2)0-pS(O)2N(R')2, (CH2)0-pSO3R', (CH2)0-pC(O)R', (CH2)0-pC(O)CH2C(O)R', (CH2)0-pC(S)R', (CH2)0-pC(O)OR', (CH2)0- pOC(O)R', (CH2)0-pC(O)N(R')2, (CH2)0-pOC(O)N(R')2, (CH2)0-pC(S)N(R')2, (CH2)0- pNH-C(O)R', (CH2)0-pN(R')N(R')C(O)R', (CH2)0-pN(R')N(R')C(O)OR', (CH2)0- pN(R')N(R')CON(R')2, (CH2)0-pN(R')SO2R', (CH2)0-pN(R')SO2N(R')2, (CH2)0- pN(R')C(O)OR', (CH2)0-pN(R')C(O)R', (CH2)0-pN(R')C(S)R', (CH2)0- pN(R')C(O)N(R')2, (CH2)0-pN(R')C(S)N(R')2, (CH2)0-pN(COR')COR', (CH2)0- pN(OR')R', (CH2)0-pC(=NH)N(R')2, (CH2)0-pC(O)N(OR')R', or (CH2)0-pC(=NOR')R'; wherein p is 4; and each instance of R' is independently selected from the group consisting of hydrogen, (C1- C12)-alkyl, (C2-C12)-alkenyl, (C2-C12)-alkynyl, (C3-C10)-cycloalkyl, (C3-C10)- cycloalkenyl, [(C3-C10)cycloalkyl or (C3-C10)-cycloalkenyl]-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], (C6-C10)-aryl, (C6-C10)-aryl-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O)2. 31. The process according to claim 30, wherein the active R7 reagent is one selected from the group consisting of (R7)2Zn, R7B(OH)2, (R7)3Si(C6-C12-aryl), R7CuBr, R7CuI, Michael acceptor that corresponds to the moiety R7, and R7ZnX (wherein X is Cl or Br). 32. The process according to claim 30 or 31, wherein the reagent according to the formula is a compound conforming to the formula:

wherein each instance of R8 is independently selected from H and Cl; Z1 is N or C(O); and Z2 is N or C. 33. The process according to claim 32, wherein Z1 is C(O) and Z2 is C. 34. The process according to any one of claims 30– 33, wherein step (B) further comprises contacting with a ligand.

35. The process according to any one of claims 30– 34, wherein the metal salt is a nickel (II) salt. 36. The process according to any one of claims 30– 35, wherein step (B) comprises contacting with a reducing agent. 37. The process according to any one of claims 30– 36, wherein the active R7 reagent is a Michael acceptor that corresponds to the moiety R7. 38. The process according to any one of claims 30– 36, wherein the active R7 reagent is (R7)2Zn. 39. The process according to any one of claims 30– 36, wherein the active R7 reagent is R7B(OH)2. 40. The process according to any one of claims 30– 36, wherein the active R7 reagent is (R7)3Si(C6-C12-aryl), and R7 is H. 41. The process according to any one of claims 30– 36, wherein the active R7 reagent is R7ZnCl.

Description:
SYNTHESIS OF THE ARYLOMYCIN MACROCYCLIC CORE STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.5R21 AI109809- 02, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention. CLAIM OF PRIORITY

This application claims the benefit of priority to U.S. Provisional Application Serial No.62/348,335, filed on June 10, 2016, which is incorporated herein by reference in its entirety. BACKGROUND

The arylomycin class of natural products was first described in the form of arylomycins A and B (J. Schimana et al., The Journal of Antibiotics 55 (6) (2002) 565). Additionally, arylomycin C was later discovered in 2004 (P. Kulanthaivel, J. Biol. Chem.279 (2004) 36250). In addition to these compounds’ discoveries, the biological activity of these compounds was investigated in a number of ways. First, the inhibitory activity of arylomycins A and B on prokaryotic, human, fungal and plant cell growth was probed yielding only a narrow antimicrobial spectrum against select Gram-positive bacteria. Later, in 2004 Paetzel disclosed the structure of a co-crystal of arylomycin A bound in the active site of the type I signal peptidase (SPase) of E. coli giving evidence for this enzyme as its biomolecular target (M. Paetzel et al., Journal of Biological Chemistry 279 (29) (2004) 30781). Various other biochemical assays corroborated this finding.

SPase itself is an attractive target for the development of antimicrobial therapeutics. It is both essential to prokaryotic cell viability as well as conserved among all bacteria, giving it the potential to be a broad spectrum antibiotic. Additionally, the target’s active site, where arylomycin is known to bind, is extracellular (N. Bilgin et al., EMBO J.9 (1990) 2717), potentially making the site more accessible to small molecule inhibitors that might otherwise have trouble passing into the cytoplasm. These aspects of SPase prompted the first total synthesis of Arylomycin A (T. Roberts et al., Journal of the American Chemical Society 129 (51) (2007) 15830).

Subsequently, the Romesberg group published a number of reports describing the antibacterial spectrum of the arylomycins (T. Roberts (2007) supra; P. Smith, et al., Chemistry & Biology 17 (11) (2010) 1223; T. Roberts et al., Journal of Medicinal Chemistry 54 (14) (2011) 4954-4963; and P. Smith et al., Antimicrobial agents and chemotherapy 55 (3) (2011) 113).

While the original report by J. Schimana et al. (2002), supra, suggested a narrow spectrum of antimicrobial activity, it was discovered that a majority of the strains (both Gram- negative and Gram-positive) that were inherently resistant also contained a resistance- conferring mutation in SPase (P. Smith et al. (2010), supra). This mutation was also found in those strains in which resistance was developed through repeated exposure to the antibiotic. This discovery suggested that resistance might be overcome by subverting the resistance mechanism through synthetic modification of the natural product, and thus provide a molecule with broad spectrum activity. Synthetic efforts resulted in modified analogs of the arylomycin class of molecules (T. Roberts (2011) supra; WO 2012/036907; and

WO 2012/166665). It was found that a number of modifications to the arylomycin molecule could be made that restored activity against previously resistant strains to various degrees, thereby positioning the analogs as broad spectrum antibiotics. Prior to clinical trials of the analogs, large scale synthesis of the analogs must be performed in order to provide required amounts of active compound.

The above-mentioned analogs from the Romesberg group results from syntheses that mirror their reported total syntheses of the natural products. To date, two separate total syntheses of both arylomycins A and B have been reported (T. Roberts et al. (2007) supra; J. Dufour et al., Chemistry - A European Journal 16 (34) (2010,) 10523; and T. Roberts et al., Journal of Natural Products 74 (5) (2011) 956) as well as one total synthesis of arylomycin C (J. Liu et al., Journal of the American Chemical Society 133 (44) (2011) 17869).

In all of these reported syntheses, the synthetic routes used are essentially identical. These routes rely upon a number of amide bond forming amino acid couplings to build the hexapeptide core of the molecule, and they employ an intramolecular Suzuki-Miyaura cross- coupling to forge the macrocycle. While straight forward, the routes suffer from a number of drawbacks that make the scheme both economically and practically undesirable for large scale production.

First, the route necessitates the use of a large number of protecting group

manipulations. Thus, the total synthesis of the arylomycins and their derivatives requires a process that entails many non-productive steps.

Second, in the approach to the amino acid building blocks with the proper

functionality and protecting groups, the use of toxic methylating reagents is required (methyl iodide and/or dimethyl sulfate). These reagents are highly undesirable for use in the large scale production of compounds.

Third, the Suzuki- Miyaura cross-coupling suffers from low overall reaction yield without the possibility of recovering the starting material due to protodeboration.

Additionally, the cross-coupling reaction used to forge the macrocycle requires the use of palladium in two separate reactions (reported at 20 mol % for both), which is both expensive to use in large quantities as well as tedious to remove in order to meet FDA requirements. These aspects of the palladium chemistry involved are economically prohibitive in the context of large scale production of the arylomycins and their derivatives.

Finally, the traditional synthetic route ends with a global deprotection using either boron tribromide or aluminum trichloride and ethane thiol. Both of these deprotection protocols subject the desired materials to rather hash conditions, and as a result the yields of these reactions are consistently modest. Because the deprotection is the last step in production of the target compounds, these yields are highly undesirable. SUMMARY OF THE INVENTION

The invention described here overcomes these drawbacks by providing a new synthetic route to the arylomycin macrocyclic core. The route circumvents a number of non- constructive and/or low-yielding steps making the synthesis of this commercially desired antibiotic scaffold significantly more cost effective.

Thus, one embodiment of the invention is a process for synthesizing a compound according to Formula (I):

comprising contacting a compound according to Formula (II):

with

(a) a copper pre-catalyst,

(b) an amine, and

(c) an oxidant,

wherein

PG is an amine protecting group;

R 1 is selected from the group consisting of H, (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R 1 can be substituted with 1 to 3 J;

each instance of R 2 and R 3 are independently selected from the group consisting of (C1- C6)alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH2, (C1-C4)alkoxy, and (C1- C 4 )acyloxy,

wherein any carbon atom in R 2 and R 3 can be substituted with J,

wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R 2 groups taken together, and/or two R 3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J;

R A1 , R A2 , and R A3 are independently selected from the group consisting of H, (C 1 - C6)alkyl, (C3-C7)cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J;

B is CO2H, CH2CO2H, C(=O)NHCH2C(=O)H, CH2C(=O)H, C(=O)NHCH2B(OR B )2 or C(=O)NHCH2P(=O)(OR B )2, wherein R B is H, (C1-C6)alkyl, or (C6-C10)aryl

G 1 and G 2 are each independently selected from the group consisting of H, (C 1 -C 6 )alkyl, (CH2)1-pN(R')2, a glycosyl residue, and a group cleavable under physiological conditions to provide a compound of formula (I) wherein G 1 or G 2 respectively is H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF3, OCF3, O, S, C(O), S(O), methylenedioxy, ethylenedioxy, (CH2)0-pN(R')2, (CH2)0-pSR', (CH2)0-pS(O)R', (CH2)0-pS(O)2R', (CH2)0-pS(O)2N(R')2, (CH2)0-pSO3R', (CH 2 ) 0-p C(O)R', (CH 2 ) 0-p C(O)CH 2 C(O)R', (CH 2 ) 0-p C(S)R', (CH 2 ) 0-p C(O)OR', (CH 2 ) 0- p OC(O)R', (CH 2 ) 0-p C(O)N(R') 2 , (CH 2 ) 0-p OC(O)N(R') 2 , (CH 2 ) 0-p C(S)N(R') 2 , (CH 2 ) 0- pNH-C(O)R', (CH2)0-pN(R')N(R')C(O)R', (CH2)0-pN(R')N(R')C(O)OR', (CH2)0- p N(R')N(R')CON(R') 2 , (CH 2 ) 0-p N(R')SO 2 R', (CH 2 ) 0-p N(R')SO 2 N(R') 2 , (CH 2 ) 0- pN(R')C(O)OR', (CH2)0-pN(R')C(O)R', (CH2)0-pN(R')C(S)R', (CH2)0- pN(R')C(O)N(R') 2 , (CH 2 ) 0-p N(R')C(S)N(R') 2 , (CH 2 ) 0-p N(COR')COR', (CH 2 ) 0- p N(OR')R', (CH 2 ) 0-p C(=NH)N(R') 2 , (CH 2 ) 0-p C(O)N(OR')R', or (CH 2 ) 0-p C(=NOR')R'; wherein p is 4; and

each instance of R' is independently selected from the group consisting of hydrogen, (C1- C 12 )-alkyl, (C 2 -C 12 )-alkenyl, (C 2 -C 12 )-alkynyl, (C 3 -C 10 )-cycloalkyl, (C 3 -C 10 )- cycloalkenyl, [(C3-C10)cycloalkyl or (C3-C10)-cycloalkenyl]-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], (C6-C10)-aryl, (C6-C10)-aryl-[(C1-C12)-alkyl or (C2- C 12 )-alkenyl or (C 2 -C 12 )-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C 1 -C 12 )-alkyl or (C 2 -C 12 )-alkenyl or (C 2 -C 12 )-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O) 2 .

In some embodiments, the compound according to Formula (I) is a compound according to Formula (IA):

More specifically, in some embodiments each of n2 and n3 is 0; and each of G 1 and G 2 is H. In other embodiments, each of R A1 and R A2 is H; and R A3 is (C1-C6)alkyl. An example of R A3 is methyl.

One example of a compound according to Formula (IA), per another embodiment, is the following compound designated herein as compound 7:

In some of embodiments of the process described the pre-catalyst is a Cu(I) compound. Illustrative compounds of this type include [Cu(MeCN)4]PF6, Cu(OAc), CuI, CuBr, CuCl, and Cu(OS(O)2CF3).

Other embodiments of the process described herein provide an amine that is a bidentate diamine. More specifically, according to exemplary embodiments, the bidentate diamine is selected from the group consisting of (R a )2N(CH2)aN(R a )2 (wherein each R a is independently selected from H, (C 1 -C 6 )alkyl, and (C 6 -C 10 )aryl, and a is an integer selected from 1, 2, 3, and 4), 2,2’-bipyridine, and 1,10-phenanthroline.

In some embodiments, the bidentate diamine is (R a )2N(CH2)aN(R a )2. Illustrative diamines are those wherein each R a is (C1-C6)alkyl and a is 2, such as Me2NCH2CH2NMe2.

Other embodiments of the process provide for a molar ratio of the pre-catalyst to compound according to Formula (I) as 5:1 to 2:1. A specific example is the molar ratio of 2:1.

In still other embodiments, the molar ratio of nitrogen atoms in the amine to the copper pre-catalyst is 4:1 to 1:1. An exemplary ratio is 2:1.

According to one embodiment of the process, the oxidant is O2.

In another embodiment, the invention provides a process for synthesizing a compound according to Formula (III) or Formula (III’):

comprising contacting a compound according to Formula (I):

with a compound according to the formula [R 5 C(O)]2O or LG-R 6 in the presence of at least one base, wherein PG is an amine protecting group; R 1 is selected from the group consisting of H, (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R 1 can be substituted with 1 to 3 J; each instance of R 2 and R 3 are independently selected from the group consisting of (C1- C 6 )alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH 2 , (C 1 -C 4 )alkoxy, and (C 1 - C 4 )acyloxy, wherein any carbon atom in R 2 and R 3 can be substituted with J, wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R 2 groups taken together, and/or two R 3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J; R 4 is selected from (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, -C(O)(C 1 -C 6 )alkyl, -C(O)(C 3 - C7)cycloalkyl, and–C(O)aryl, wherein R 4 is optionally substituted with 1 to 3 J; when R 4 is -C(O)(C1-C6)alkyl, -C(O)(C3-C7)cycloalkyl, or–C(O)aryl, then the compound according to Formula (I) is contacted with a compound according to [R 5 C(O)] 2 O, wherein R 5 is (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, or aryl, respectively; when R 4 is (C1-C6)alkyl or (C3-C7)cycloalkyl, then the compound according to Formula (I) is contacted with a compound according to LG-R 6 , wherein R 6 is (C 1 -C 6 )alkyl or (C 3 -C 7 )cycloalkyl, respectively; and LG is a leaving group; R A1 , R A2 , and R A3 are independently selected from the group consisting of H, (C 1 - C 6 )alkyl, (C 3 -C 7 )cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C6-C10)aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J; B is CO 2 H, CH 2 CO 2 H, C(=O)NHCH 2 C(=O)H, CH 2 C(=O)H, C(=O)NHCH 2 B(OR B ) 2 or C(=O)NHCH2P(=O)(OR B )2, wherein R B is H, (C1-C6)alkyl, or (C6-C10)aryl G 1 and G 2 are each H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF3, OCF3, O, S, C(O), S(O), N3, methylenedioxy, ethylenedioxy, (CH2)0- pN(R')2, (CH2)0-pSR', (CH2)0-pS(O)R', (CH2)0-pS(O)2R', (CH2)0-pS(O)2N(R')2, (CH2)0- pSO 3 R', (CH 2 ) 0-p C(O)R', (CH 2 ) 0-p C(O)CH 2 C(O)R', (CH 2 ) 0-p C(S)R', (CH 2 ) 0-p C(O)OR', (CH2)0-pOC(O)R', (CH2)0-pC(O)N(R')2, (CH2)0-pOC(O)N(R')2, (CH2)0-pC(S)N(R')2, (CH2)0-pNH-C(O)R', (CH2)0-pN(R')N(R')C(O)R', (CH2)0-pN(R')N(R')C(O)OR', (CH2)0- pN(R')N(R')CON(R') 2 , (CH 2 ) 0-p N(R')SO 2 R', (CH 2 ) 0-p N(R')SO 2 N(R') 2 , (CH 2 ) 0- p N(R')C(O)OR', (CH 2 ) 0-p N(R')C(O)R', (CH 2 ) 0-p N(R')C(S)R', (CH 2 ) 0- pN(R')C(O)N(R')2, (CH2)0-pN(R')C(S)N(R')2, (CH2)0-pN(COR')COR', (CH2)0- pN(OR')R', (CH 2 ) 0-p C(=NH)N(R') 2 , (CH 2 ) 0-p C(O)N(OR')R', or (CH 2 ) 0-p C(=NOR')R'; wherein p is 4; and each instance of R' is independently selected from the group consisting of hydrogen, (C1- C 12 )-alkyl, (C 2 -C 12 )-alkenyl, (C 2 -C 12 )-alkynyl, (C 3 -C 10 )-cycloalkyl, (C 3 -C 10 )- cycloalkenyl, [(C 3 -C 10 )cycloalkyl or (C 3 -C 10 )-cycloalkenyl]-[(C 1 -C 12 )-alkyl or (C 2 - C12)-alkenyl or (C2-C12)-alkynyl], (C6-C10)-aryl, (C6-C10)-aryl-[(C1-C12)-alkyl or (C2- C12)-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C 1 -C 12 )-alkyl or (C 2 -C 12 )-alkenyl or (C2-C12)-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C2-C12)-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two

R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O)2. Another embodiment is a process for synthesizing a compound according to Formula (IV)

comprising the steps of (A) contacting a compound according to Formula (I):

(B) contacting the compound according to Formula V with a metal salt that is a nickel (II) or iron (III) salt, optionally a reducing agent, and an active R 7 reagent to thereby yield a compound according to Formula (IV); wherein PG is an amine protecting group; R 1 is selected from the group consisting of H, (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, 5- to 7- membered heteroaryl, 5- to 7-membered heterocyclyl, and (C 6 -C 10 )aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl in R 1 can be substituted with 1 to 3 J; each instance of R 2 and R 3 are independently selected from the group consisting of (C 1 - C6)alkyl nitro, halo, cyano, hydroxy, glycosyloxy, -NH2, (C1-C4)alkoxy, and (C1- C4)acyloxy, wherein any carbon atom in R 2 and R 3 can be substituted with J, wherein n2 and n3 are independently integers selected from 0, 1, 2, and 3; or wherein two R 2 groups taken together, and/or two R 3 groups taken together with the carbon atoms to which the groups are bound, can comprise fused cycloalkyl, aryl, heterocyclyl, or heteroaryl ring or rings, any of which is substituted with 1 to 3 J; R 7 is selected from the group consisting of H, (C1-C12)alkyl, (C3-C7)cycloalkyl, (C6- C 10 )aryl, (C 2 -C 12 )-alkenyl, wherein any alkyl, alkenyl, cycloalkyl, or aryl can be substituted with 1 to 3 J; R A1 , R A2 , and R A3 are independently selected from the group consisting of H, (C1- C 6 )alkyl, (C 3 -C 7 )cycloalkyl, 5- to 7-membered heteroaryl, 5- to 7-membered heterocyclyl, and (C 6 -C 10 )aryl, wherein any alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be substituted with 1 to 3 J; B is CO2H, CH2CO2H, C(=O)NHCH2C(=O)H, CH2C(=O)H, C(=O)NHCH2B(OR B )2 or C(=O)NHCH 2 P(=O)(OR B ) 2 , wherein R B is H, (C 1 -C 6 )alkyl, or (C 6 -C 10 )aryl G 1 and G 2 are each independently selected from the group consisting of (C 1 -C 6 )alkyl optionally substituted with 1– 3 J, (CH2)1-pN(R')2, a glycosyl residue, and a group cleavable under physiological conditions to provide a compound of formula (I) wherein G 1 or G 2 respectively is H; each instance of J is selected independently from the group consisting of halogen, R', OR', CN, CF 3 , OCF 3 , O, S, C(O), S(O), methylenedioxy, ethylenedioxy, (CH 2 ) 0-p N(R') 2 , (CH 2 ) 0-p SR', (CH 2 ) 0-p S(O)R', (CH 2 ) 0-p S(O) 2 R', (CH 2 ) 0-p S(O) 2 N(R') 2 , (CH 2 ) 0-p SO 3 R', (CH2)0-pC(O)R', (CH2)0-pC(O)CH2C(O)R', (CH2)0-pC(S)R', (CH2)0-pC(O)OR', (CH2)0- pOC(O)R', (CH2)0-pC(O)N(R')2, (CH2)0-pOC(O)N(R')2, (CH2)0-pC(S)N(R')2, (CH2)0- pNH-C(O)R', (CH 2 ) 0-p N(R')N(R')C(O)R', (CH 2 ) 0-p N(R')N(R')C(O)OR', (CH 2 ) 0- pN(R')N(R')CON(R')2, (CH2)0-pN(R')SO2R', (CH2)0-pN(R')SO2N(R')2, (CH2)0- pN(R')C(O)OR', (CH2)0-pN(R')C(O)R', (CH2)0-pN(R')C(S)R', (CH2)0- pN(R')C(O)N(R') 2 , (CH 2 ) 0-p N(R')C(S)N(R') 2 , (CH 2 ) 0-p N(COR')COR', (CH 2 ) 0- pN(OR')R', (CH2)0-pC(=NH)N(R')2, (CH2)0-pC(O)N(OR')R', or (CH2)0-pC(=NOR')R'; wherein p is 4; and each instance of R' is independently selected from the group consisting of hydrogen, (C 1 - C 12 )-alkyl, (C 2 -C 12 )-alkenyl, (C 2 -C 12 )-alkynyl, (C 3 -C 10 )-cycloalkyl, (C 3 -C 10 )- cycloalkenyl, [(C3-C10)cycloalkyl or (C3-C10)-cycloalkenyl]-[(C1-C12)-alkyl or (C2- C 12 )-alkenyl or (C 2 -C 12 )-alkynyl], (C 6 -C 10 )-aryl, (C 6 -C 10 )-aryl-[(C 1 -C 12 )-alkyl or (C 2 - C 12 )-alkenyl or (C 2 -C 12 )-alkynyl], mono- or bicyclic 3-10 membered heterocyclyl, mono- or bicyclic 3-10 membered heterocyclyl-[(C1-C12)-alkyl or (C2-C12)-alkenyl or (C 2 -C 12 )-alkynyl], mono- or bicyclic 5-10 membered heteroaryl, or mono- or bicyclic 5-10 membered heteroaryl-[(C 1 -C 12 )-alkyl or (C 2 -C 12 )-alkenyl or (C 2 -C 12 )-alkynyl]; or, when two R' are bound to a nitrogen atom or to two adjacent nitrogen atoms, the two R' groups together with the nitrogen atom or atoms to which they are bound can form a 3- to 8-membered monocyclic heterocyclic ring, or an 8- to 20-membered, bicyclic or tricyclic, heterocyclic ring system, wherein any ring or ring system can further contain 1-3 additional heteroatoms selected from the group consisting of N, NR’, O, S, S(O) and S(O) 2 . DETAILED DESCRIPTION

The process described herein provides a direct, oxidative biaryl coupling of the unprotected phenolic amino acid side chains in arylomycin scaffold precursors, thereby replacing the low-yielding Suzuki-Miyaura coupling described above. This approach to forging the biaryl bond in the arylomycin class of compounds, such as Formula I, highly simplifies the overall synthetic route with respect to previously reported synthetic routes. In this regard, the inventive process provides a number of advantages.

First, because the oxidative biaryl coupling is a dual C-H functionalization, the process eliminates the need for pre-functionalization of the ortho positions of the phenols. This feature alone shortens the traditional route by three steps.

Second, the inventive process removes the need for the use of expensive palladium in coupling and borylation steps in the traditional process.

Third, protection of phenolic oxygens is no longer necessary, thereby precluding the need for the use of toxic methylating agents or other phenol protection steps. Accordingly, the inventive process alleviates the need to perform a low yielding global deprotection under the harsh conditions required to remove them.

Fourth, the inventive process reduces the cost of reagents needed for the

macrocyclization by replacing two palladium-mediated reactions with one copper-mediated reaction. While the copper pre-catalyst is used in two equivalents according to some embodiments, the much lower cost of copper greatly offsets the higher pre-catalyst loading. Similarly, potentially expensive phosphine ligands needed for the optimized palladium- catalyzed reactions of the prior art are replaced in the inventive process with the very inexpensive diamines, according to some embodiments, such as TMEDA.

Fifth, the inventive process further reduces cost because the process shortens the total synthetic sequence by up to seven steps, relative to prior art processes, thereby improving the overall yield.

As mentioned above, certain analogs of the arylomycins possessed a broader spectrum of antibacterial activity, making them attractive leads for development into novel antibiotics. Many of these possess varied functional groups at the phenolic oxygens and the C-terminus. This makes novel methods to derivatize these positions of particular interest as their optimization and production efforts continue.

The previous efforts to selectively functionalize the arylomycin core at the phenolic oxygens utilized mono-protection with a Boc group. This was an unselective process and required separation of the regioisomers followed by functionalization of the remaining free phenolic oxygen and finally Boc-deprotection. Apart from being an unselective process, the separated mono-protected compounds could not be stored for an extended period of time as interconversion of the regioisomers was observed. The inventive processes for

functionalizations of the same positions with the same functional groups (alkyl amines) provides a route to the desired products with good to great selectivity without the use of protecting groups. This removes two steps and difficult one separation from this process. It also is able to selectively other functionalities selectively, namely acyl groups.

In regard to the C-terminal derivatizations, to date, very few methods have been used to modify that position on the arylomycin scaffold. Ardnt-Eistert homologation, amide bond formation, and reductions have been the only reported procedures utilized for derivatization of the native carboxylate, thus providing only a limited chemical diversity at this position of the molecule. This is unfortunate as the native carboxylate interacts with the catalytic residues of the enzyme that the arylomycins inhibit (SPase), allowing the reasoning that modification at this position could prove to be beneficial in developing more potent analogs. Thus, the decarboxylative cross-coupling methods described here are a broad expansion to the methods which have been utilized providing the potential much greater chemical diversity. Additionally, some of this diversity would not be possible to achieve with other methods. Definitions

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example, within 10%, or within 5% of a stated value or of a stated limit of a range.

All percent compositions are given as weight-percentages, unless otherwise stated. All average molecular weights of polymers are weight-average molecular weights, unless otherwise specified.

"Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is

"substantially pure" is there are only negligible traces of impurities present. The term "chemically feasible" means a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim. The structures disclosed herein, in all of their embodiments are intended to include only

"chemically feasible" structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.

When a substituent is specified to be an atom or atoms of specified identity, "or a bond", a configuration is referred to when the substituent is "a bond" that the groups that are immediately adjacent to the specified substituent are directly connected to each other in a chemically feasible bonding configuration.

All chiral, diastereomeric, racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated. Compounds used in the present invention can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.

The inclusion of an isotopic form of one or more atoms in a molecule that is different from the naturally occurring isotopic distribution of the atom in nature is referred to as an "isotopically labeled form" of the molecule. All isotopic forms of atoms are included as options in the composition of any molecule, unless a specific isotopic form of an atom is indicated. For example, any hydrogen atom or set thereof in a molecule can be any of the isotopic forms of hydrogen, i.e., protium ( 1 H), deuterium ( 2 H), or tritium ( 3 H) in any combination. Similarly, any carbon atom or set thereof in a molecule can be any of the isotopic form of carbons, such as 11 C, 12 C, 13 C, or 14 C, or any nitrogen atom or set thereof in a molecule can be any of the isotopic forms of nitrogen, such as 13 N, 14 N, or 15 N. A molecule can include any combination of isotopic forms in the component atoms making up the molecule, the isotopic form of every atom forming the molecule being independently selected. In a multi-molecular sample of a compound, not every individual molecule necessarily has the same isotopic composition. For example, a sample of a compound can include molecules containing various different isotopic compositions, such as in a tritium or 14 C radiolabeled sample where only some fraction of the set of molecules making up the macroscopic sample contains a radioactive atom. It is also understood that many elements that are not artificially isotopically enriched themselves are mixtures of naturally occurring isotopic forms, such as 14 N and 15 N, 32 S and 34 S, and so forth. A molecule as recited herein is defined as including isotopic forms of all its constituent elements at each position in the molecule. As is well known in the art, isotopically labeled compounds can be prepared by the usual methods of chemical synthesis, except substituting an isotopically labeled precursor molecule. The isotopes, radiolabeled or stable, can be obtained by any method known in the art, such as generation by neutron absorption of a precursor nuclide in a nuclear reactor, by cyclotron reactions, or by isotopic separation such as by mass spectrometry. The isotopic forms are incorporated into precursors as required for use in any particular synthetic route. For example, 14 C and 3 H can be prepared using neutrons generated in a nuclear reactor.

Following nuclear transformation, 14 C and 3 H are incorporated into precursor molecules, followed by further elaboration as needed.

The term "amino protecting group" or "N-protected" as used herein refers to those groups intended to protect an amino group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used amino protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999).

Amino protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, onitrophenoxyacetyl, Į-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,

4nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; alkoxy- or aryloxy-carbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, pmethoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, pbromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3,5dimethoxybenzyloxycarbonyl, 2,4- dimethoxybenzyloxycarbonyl, 4methoxybenzyloxycarbonyl, 2-nitro-4,5- dimethoxybenzyloxycarbonyl, 3,4,5trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1- methylethoxycarbonyl, Į,Įdimethyl3,5-dimethoxybenzyloxycarbonyl,

benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,

isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2- trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Amine protecting groups also include cyclic amino protecting groups such as phthaloyl and dithiosuccinimidyl, which incorporate the amino nitrogen into a heterocycle. Typically, amino protecting groups include formyl, acetyl, benzoyl, pivaloyl, t- butylacetyl, phenylsulfonyl, Alloc, Teoc, benzyl, Fmoc, Boc and Cbz. It is well within the skill of the ordinary artisan to select and use the appropriate amino protecting group for the synthetic task at hand.

The term "hydroxyl protecting group" or "O-protected" as used herein refers to those groups intended to protect an OH group against undesirable reactions during synthetic procedures and which can later be removed to reveal the amine. Commonly used hydroxyl protecting groups are disclosed in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999). Hydroxyl protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t- butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,

onitrophenoxyacetyl, Į-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,

4nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; acyloxy groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, pmethoxybenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, pbromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3,5dimethoxybenzyloxycarbonyl, 2,4- dimethoxybenzyloxycarbonyl, 4methoxybenzyloxycarbonyl, 2-nitro-4,5- dimethoxybenzyloxycarbonyl, 3,4,5trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1- methylethoxycarbonyl, Į,Įdimethyl3,5-dimethoxybenzyloxycarbonyl,

benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2- trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4- nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. It is well within the skill of the ordinary artisan to select and use the appropriate hydroxyl protecting group for the synthetic task at hand.

In general,“substituted”^refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non-hydrogen atom such as, but not limited to, a halogen (i.e., F, Cl, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups.

Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR', OC(O)N(R')2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R', O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R')2, SR', SOR', SO 2 R', SO 2 N(R') 2 , SO 3 R', C(O)R', C(O)C(O)R', C(O)CH 2 C(O)R', C(S)R', C(O)OR',

OC(O)R', C(O)N(R')2, OC(O)N(R')2, C(S)N(R')2, (CH2)0-2N(R')C(O)R', (CH2)0-2N(R')N(R')2, N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')CON(R')2, N(R')SO2R', N(R')SO2N(R')2, N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R', N(R')C(O)N(R') 2 , N(R')C(S)N(R') 2 ,

N(COR')COR', N(OR')R', C(=NH)N(R')2, C(O)N(OR')R', or C(=NOR')R' wherein R’ can be hydrogen or a carbon-based moiety, and wherein the carbon-based moiety can itself be further substituted.

When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. When a substituent is more than monovalent, such as O, which is divalent, it can be bonded to the atom it is substituting by more than one bond, i.e., a divalent substituent is bonded by a double bond; for example, a C substituted with O forms a carbonyl group, C=O, which can also be written as "CO", "C(O)", or "C(=O)", wherein the C and the O are double bonded. When a carbon atom is substituted with a double-bonded oxygen (=O) group, the oxygen substituent is termed an "oxo" group. When a divalent substituent such as NR is double-bonded to a carbon atom, the resulting C(=NR) group is termed an "imino" group. When a divalent substituent such as S is double-bonded to a carbon atom, the results C(=S) group is termed a "thiocarbonyl" group.

Alternatively, a divalent substituent such as O, S, C(O), S(O), or S(O)2 can be connected by two single bonds to two different carbon atoms. For example, O, a divalent substituent, can be bonded to each of two adjacent carbon atoms to provide an epoxide group, or the O can form a bridging ether group, termed an "oxy" group, between adjacent or non- adjacent carbon atoms, for example bridging the 1,4-carbons of a cyclohexyl group to form a [2.2.1]-oxabicyclo system. Further, any substituent can be bonded to a carbon or other atom by a linker, such as (CH 2 ) n or (CR' 2 ) n wherein n is 1, 2, 3, or more, and each R' is

independently selected. C(O) and S(O) 2 groups can be bound to one or two heteroatoms, such as nitrogen, rather than to a carbon atom. For example, when a C(O) group is bound to one carbon and one nitrogen atom, the resulting group is called an“amide” or“carboxamide.” When a C(O) group is bound to two nitrogen atoms, the functional group is termed a urea. When a S(O) 2 group is bound to one carbon and one nitrogen atom, the resulting unit is termed a “sulfonamide.” When a S(O)2 group is bound to two nitrogen atoms, the resulting unit is termed a“sulfamate.”

Substituted alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups as well as other substituted groups also include groups in which one or more bonds to a hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a carbon atom, or to a heteroatom such as, but not limited to, oxygen in carbonyl (oxo), carboxyl, ester, amide, imide, urethane, and urea groups; and nitrogen in imines, hydroxyimines, oximes, hydrazones, amidines, guanidines, and nitriles.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and fused ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups can also be substituted with alkyl, alkenyl, and alkynyl groups as defined herein.

By a "ring system" as the term is used herein is meant a moiety comprising one, two, three or more rings, which can be substituted with non-ring groups or with other ring systems, or both, which can be fully saturated, partially unsaturated, fully unsaturated, or aromatic, and when the ring system includes more than a single ring, the rings can be fused, bridging, or spirocyclic. By "spirocyclic" is meant the class of structures wherein two rings are fused at a single tetrahedral carbon atom, as is well known in the art.

As to any of the groups described herein, which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non–feasible. In addition, the compounds of this disclosed subject matter include all stereochemical isomers arising from the substitution of these compounds.

Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 or 1 to 6 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n- pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term“alkyl”^encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed above, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. A description herein that a group is alkyl chain“optionally comprising within the chain or at a chain terminus”^a moiety, the term signifies that the moiety can be disposed between two subunits of the alkyl chain, or can be disposed at an unsubstituted end of the chain, or can be disposed between the chain and a point of attachment of the chain, for example to a carbonyl, NR, or O group. For example, an alkylbenzoyl group is an alkyl chain with a phenyl group disposed between the alkyl and a carbonyl, fitting the above description; an N- alkylphenylcarboxamido is an alkyl chain with a phenyl group displosed between the alkyl and the aminocarbonyl group, filling within the above description.

Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some

embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term

"cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

The terms "carbocyclic," "carbocyclyl," and "carbocycle" denote a ring structure wherein the atoms of the ring are carbon, such as a cycloalkyl group or an aryl group. In some embodiments, the carbocycle has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms is 4, 5, 6, or 7. Unless specifically indicated to the contrary, the carbocyclic ring can be substituted with as many as N-1 substituents wherein N is the size of the carbocyclic ring with, for example, alkyl, alkenyl, alkynyl, amino, aryl, hydroxy, cyano, carboxy, heteroaryl, heterocyclyl, nitro, thio, alkoxy, and halogen groups, or other groups as are listed above. A carbocyclyl ring can be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring. A carbocyclyl can be monocyclic or polycyclic, and if polycyclic each ring can be independently be a cycloalkyl ring, a cycloalkenyl ring, or an aryl ring.

(Cycloalkyl)alkyl groups, also denoted cycloalkylalkyl, are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkyl group as defined above.

Alkenyl groups include straight and branched chain and cyclic alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, CH=CH(CH 3 ), CH=C(CH 3 ) 2 , C(CH 3 )=CH 2 , C(CH 3 )=CH(CH 3 ), C(CH 2 CH 3 )=CH 2 , cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

Cycloalkenyl groups include cycloalkyl groups having at least one double bond between 2 carbons. Thus for example, cycloalkenyl groups include but are not limited to cyclohexenyl, cyclopentenyl, and cyclohexadienyl groups. Cycloalkenyl groups can have from 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like, provided they include at least one double bond within a ring. Cycloalkenyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above.

(Cycloalkenyl)alkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above.

Alkynyl groups include straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to about 20 carbon atoms, and typically from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to–CŁCH, -CŁC(CH 3 ), - CŁC(CH2CH3), CH2CŁCH, CH2CŁC(CH3), and CH2CŁC(CH2CH3) among others.

The term“heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include OCH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 OH, CH 2 CH 2 NHCH 3 , CH2SCH2CH3, CH2CH2S(=O)CH3, and -CH2CH2-O-CH2CH2-O-CH3. Up to two

heteroatoms may be consecutive, such as, for example, CH2NHOCH3, or–CH2CH2SSCH3.

A "cycloheteroalkyl" ring is a cycloalkyl ring containing at least one heteroatom. A cycloheteroalkyl ring can also be termed a "heterocyclyl," described below.

The term“heteroalkenyl”^by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain monounsaturated or diunsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Up to two heteroatoms may be placed consecutively. Examples include CH=CHOCH3, CH=CHCH2OH, CH2CH=NOCH3, CH=CHN(CH3)CH3,

CH 2 CH=CHCH 2 SH, and and -CH=CH-O-CH 2 CH 2 -O-CH 3 .

Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be

unsubstituted or substituted, as defined above. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6- substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl group are alkenyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.

Heterocyclyl groups or the term "heterocyclyl" includes aromatic and non-aromatic ring compounds containing 3 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Thus a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C 2 -heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group.

The term“heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a

benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl.

Heterocyclyl groups can be unsubstituted, or can be substituted as discussed above. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with groups such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure.

A heteroaryl group designated as a C 2 -heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl,

benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed above. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed above.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, Nhydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4- pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5(2,3dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4benzo[b]thiophenyl, 5benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro- benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2indolyl, 3indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3indazolyl, 4indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1benzimidazolyl,

2benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7benzimidazolyl, 8benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6benzothiazolyl, 7benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4carbazolyl),

5Hdibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl,

5Hdibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl,

10,11dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group as defined above is replaced with a bond to a heterocyclyl group as defined above. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

Heteroarylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above.

The term“alkoxy" refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to about 12-20 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structures are substituted therewith.

The terms“halo” or“halogen” or "halide" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A“haloalkyl”^group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like. A "haloalkoxy" group includes mono-halo alkoxy groups, poly-halo alkoxy groups wherein all halo atoms can be the same or different, and per-halo alkoxy groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkoxy include trifluoromethoxy, 1,1-dichloroethoxy, 1,2-dichloroethoxy, 1,3-dibromo-3,3- difluoropropoxy, perfluorobutoxy, and the like.

The term“(CxCy)perfluoroalkyl,” wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is (C1C6)perfluoroalkyl, more preferred is

(C1C3)perfluoroalkyl, most preferred is–CF3.

The term“(C x C y )perfluoroalkylene,” wherein x < y, means an alkyl group with a minimum of x carbon atoms and a maximum of y carbon atoms, wherein all hydrogen atoms are replaced by fluorine atoms. Preferred is (C1C6)perfluoroalkylene, more preferred is (C 1 C 3 )perfluoroalkylene, most preferred is–CF 2 –.

The terms "aryloxy" and“arylalkoxy”^refer to, respectively, an aryl group bonded to an oxygen atom and an aralkyl group bonded to the oxygen atom at the alkyl moiety.

Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy.

An“acyl”^group as the term is used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl”^group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-20 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3-carbonyl) group is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl”^group. An example is a trifluoroacetyl group.

The term“amine”^includes primary, secondary, and tertiary amines having, e.g., the formula N(group) 3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to RNH 2 , for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R 3 N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

An "amino" group is a substituent of the form -NH2, -NHR, -NR2, -NR3 + , wherein each R is independently selected, and protonated forms of each, except for -NR3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary or quaternary amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

An "ammonium" ion includes the unsubstituted ammonium ion NH4 + , but unless otherwise specified, it also includes any protonated or quaternized forms of amines. Thus, trimethylammonium hydrochloride and tetramethylammonium chloride are both ammonium ions, and amines, within the meaning herein.

The term“amide”^(or“amido”) includes C- and N-amide groups, i.e., C(O)NR2, and– NRC(O)R groups, respectively. Amide groups therefore include but are not limited to primary carboxamide groups (-C(O)NH 2 ) and formamide groups (-NHC(O)H). A

"carboxamido" group is a group of the formula C(O)NR2, wherein R can be H, alkyl, aryl, etc.

The term "azido" refers to an N 3 group. An "azide" can be an organic azide or can be a salt of the azide (N3-) anion. The term "nitro" refers to an NO2 group bonded to an organic moiety. The term "nitroso" refers to an NO group bonded to an organic moiety. The term nitrate refers to an ONO 2 group bonded to an organic moiety or to a salt of the nitrate (NO 3 -) anion.

The term“urethane”^(“carbamoyl”^or“carbamyl”) includes N- and O-urethane groups, i.e., NRC(O)OR and–OC(O)NR 2 groups, respectively.

The term“sulfonamide”^(or“sulfonamido”) includes S- and N-sulfonamide groups, i.e., SO2NR2 and–NRSO2R groups, respectively. Sulfonamide groups therefore include but are not limited to sulfamoyl groups (-SO 2 NH 2 ). An organosulfur structure represented by the formula–S(O)(NR)–^is understood to refer to a sulfoximine, wherein both the oxygen and the nitrogen atoms are bonded to the sulfur atom, which is also bonded to two carbon atoms.

The term“amidine”^or“amidino”^includes groups of the formula C(NR)NR 2 .

Typically, an amidino group is–C(NH)NH 2 . The term“guanidine”^or“guanidino”^includes groups of the formula NRC(NR)NR 2 . Typically, a guanidino group is–NHC(NH)NH2.

A "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion. For example, acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NH4 + or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium, or other cations such as trimethylsulfonium, and the like. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt such as can be formed in a molecule that has at least two ionizable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form. A

"zwitterion" is a salt within the meaning herein. The compounds of the present invention may take the form of salts. The term“salts”^embraces addition salts of free acids or free bases which are compounds of the invention. Salts can be "pharmaceutically-acceptable salts." The term“pharmaceutically acceptable salt”^refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds of the invention.

Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,

benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2hydroxyethanesulfonic,

ptoluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ȕhydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates. Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (Nmethylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. Although pharmaceutically unacceptable salts are not generally useful as medicaments, such salts may be useful, for example as intermediates in the synthesis of Formula (I) compounds, for example in their purification by recrystallization. All of these salts may be prepared by conventional means from the corresponding compound according to Formula (I) by reacting, for example, the appropriate acid or base with the compound according to Formula (I). The term "pharmaceutically acceptable salts" refers to nontoxic inorganic or organic acid and/or base addition salts, see, for example, Lit et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217, incorporated by reference herein.

A "hydrate" is a compound that exists in a composition with water molecules. The composition can include water in stoichiometic quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate" refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.

A "solvate" is a similar composition except that a solvent other that water replaces the water. For example, methanol or ethanol can form an "alcoholate", which can again be stoichiometic or non-stoichiometric. As the term is used herein a "solvate" refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.

A "prodrug" as is well known in the art is a substance that can be administered to a patient where the substance is converted in vivo by the action of biochemicals within the patient's body, such as enzymes, to the active pharmaceutical ingredient. Examples of prodrugs include esters of carboxylic acid groups, which can be hydrolyzed by endogenous esterases as are found in the bloodstream of humans and other mammals. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in“Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Moreover, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups. Thus, for example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, and Y is described as selected from the group consisting of methyl, ethyl, and propyl, claims for X being bromine and Y being methyl are fully described.

If a value of a variable that is necessarily an integer, e.g., the number of carbon atoms in an alkyl group or the number of substituents on a ring, is described as a range, e.g., 0-4, what is meant is that the value can be any integer between 0 and 4 inclusive, i.e., 0, 1, 2, 3, or 4.

In various embodiments, the compound or set of compounds, such as are used in the inventive methods, can be any one of any of the combinations and/or sub-combinations of the above-listed embodiments.

Provisos may apply to any of the disclosed categories or embodiments wherein any one or more of the other above disclosed embodiments or species may be excluded from such categories or embodiments.

The present invention further embraces isolated compounds according to formula (I). The expression“isolated compound”^refers to a preparation of a compound of formula (I), or a mixture of compounds according to formula (I), wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to compound in a form in which it can be used therapeutically. For example, an“isolated compound”^refers to a preparation of a compound of formula (I) or a mixture of compounds according to formula (I), which contains the named compound or mixture of compounds according to formula (I) in an amount of at least 10 percent by weight of the total weight. Typically, the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation. The compounds of the invention and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC. Isomerism and Tautomerism in Compounds of the Invention

Tautomerism

Within the present invention it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the invention encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein. For example, tautomerism may be exhibited by a pyrazolyl group bonded as indicated by the wavy line. While both substituents would be termed a 4-pyrazolyl group, it is evident that a different nitrogen atom bears the hydrogen atom in each structure.

Such tautomerism can also occur with substituted pyrazoles such as 3-methyl, 5- methyl, or 3,5-dimethylpyrazoles, and the like. Another example of tautomerism is amido- imido (lactam-lactim when cyclic) tautomerism, such as is seen in heterocyclic compounds bearing a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:

is an example of tautomerism. Accordingly, a structure depicted herein as one tautomer is intended to also include the other tautomer.

Optical Isomerism

It will be understood that when compounds of the present invention contain one or more chiral centers, the compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present invention therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the invention.

The isomers resulting from the presence of a chiral center comprise a pair of nonsuperimposable isomers that are called“enantiomers.”^^Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the CahnIngoldPrelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). In the example below, the CahnIngoldPrelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

The present invention encompasses diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof.

Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.

“Isolated optical isomer” means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula. Preferably, the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight. Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of

DAICEL ® CHIRALPAK ® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer’s instructions. Rotational Isomerism

It is understood that due to chemical properties (i.e., resonance lending some double bond character to the C-N bond) of restricted rotation about the amide bond linkage (as illustrated below) it is possible to observe separate rotamer species and even, under some circumstances, to isolate such species (see below). It is further understood that certain structural elements, including steric bulk or substituents on the amide nitrogen, may enhance the stability of a rotamer to the extent that a compound may be isolated as, and exist indefinitely, as a single stable rotamer. The present invention therefore includes any possible stable rotamers of formula (I) which are biologically active in the treatment of cancer or other proliferative disease states.

Regioisomerism

Some compounds of the present invention have a particular spatial arrangement of substituents on the aromatic rings, which is related to the structure activity relationship demonstrated by the compound class. Often such substitution arrangement is denoted by a numbering system; however, numbering systems are often not consistent between different ring systems. In six-membered aromatic systems, the spatial arrangements are specified by the common nomenclature“para” for 1,4substitution,“meta” for 1,3substitution and“ortho” for 1,2substitution as shown below.

In various embodiments, the compound or set of compounds, such as are among the inventive compounds or are used in the inventive methods, can be any one of any of the combinations and/or sub-combinations of the above-listed embodiments. Copper Pre-catalyst

The inventive process is mediated by a copper catalytic species formed in situ, as illustrated by the appended examples. Suitable sources of copper thus are characterized as copper pre-catalysts, and they include a wide variety of commercially available Cu(I) and Cu(II) compounds. Typical embodiments of the invention employ any Cu(I) compound. Examples include but are not limited to [Cu(MeCN) 4 ]PF 6 , Cu(OAc), CuI, CuBr, CuCl, Cu(OS(O)2CF3), and Cu(BF4). Amine

The inventive process employs an amine that, together with the copper pre-catalyst, forms the active copper catalytic species that mediates the macrocyclization reaction yielding Formula (I) compounds. Any amine as described herein is suitable for this purpose.

In some embodiments, the amine is a diamine that is capable of serving as a bidentate ligand on the copper catalytic species. Many diamines are well-known to the skilled person. Illustrative diamines in this context include aliphatic diamines of the general formula (R a ) 2 N(CH 2 ) a N(R a ) 2 , wherein each R a is independently selected from H, (C 1 -C 6 )alkyl, and (C6-C10)aryl, and a is an integer selected from 1, 2, 3, and 4. A typical example illustrated below is N,N,N’N’-tetramethylethylene diamine (TMEDA), which is economical and commercially available. The amount of copper pre-catalyst suitable for use in the inventive process can vary, relative to the amount of a compound of Formula (II). Typical molar ratios of pre-catalyst to Formula (II) compound range from 5:1 to 2:1. As described generally above, an optimized ratio illustrative of the process is 2:1. In this context, ratios of copper to substrate, i.e., a compound of Formula (II), are stoichiometric and, hence, the active copper species can be understood to be a sacrificial catalyst.

Alternatively, according to some embodiments, the amount of copper is present in catalytic amounts. For example, the mole percent of copper pre-catalyst can range from about 0.5% to about 30%, from about 1% to about 25%, from about 3% to about 20%, and from about 5% to about 15%. Exemplary catalyst loadings (in mole percent) include 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30%.

The amount of amine relative to copper pre-catalyst also can vary. Without wishing to limit the invention, the inventors believe that an active copper catalyst species features two amine ligands per copper ion. In the case of diamines, according to some embodiments, just one diamine per copper would suffice. Hence, according to some embodiments, optimized ratios of the molar amount of nitrogen atoms per copper can range from 4:1 to 1:1, which range accounts for mono- and multi-dentate amines. An illustrative ratio is 2:1, as shown in the appended examples. Oxidant

The biaryl coupling reaction of the inventive process also prescribes the use of an oxidant. Obviously, a convenient and ready source of oxidant is molecular oxygen. Thus, air is one source of oxidant. The process also proceeds in the presence of pure oxygen, as illustrated in the examples below. In general, the inventive process can employ any terminal oxidant, which in the context of a catalytic process means a reagent that is consumed in the process. Other examples of suitable oxidants include a wide variety of inorganic and organic peroxides, such as hydrogen peroxide; chromates; hypervalent iodine; hypochlorites;

perchlorates; peroxy acids and related salts; and permanganate compounds, such as potassium permanganate. In efforts toward synthesizing derivatives of the arylomycin class of antibiotics, we have sought to be able to derivatize the macrocyclic core of the molecule. This has been done in two major ways, alkylation or acylation of the free phenolic oxygens and utilizing decarboxylative cross-coupling to derivatize the C-terminal carboxylate of the macrocycle.

The functionalization of the phenolic oxygens is divided into acylation and alkylation. In one embodiment, the alkylations are performed by treating the macrocyclic core with N,N- diisopropylethylamine and one equivalent of an alkylating agent in a dry polar solvent at room temperature. This yields only one regioisomer of the mono-alkylated product, alkylated at the phenolic oxygen. In another embodiment, the acylation is performed by treating the macrocyclic core with one equivalent of isobutyric anhydride, 20 mol. % of levamisole, and one equivalent of N,N-diisopropylethylamine in toluene at room temperature. This produces a 5:1 ratio of regioisomers of the mono-acylated product, acylated at the phenolic oxygen. In each of these embodiments, minor products do include bis-functionalized products, which are separable using silica gel chromatography.

The derivatizations of the C-terminal carboxylate all utilize the decarboxylative cross- coupling chemistry disclosed by the Baran lab. 1-6 This includes arylation, alkylation, Michael addition, reduction, borylation, and vinylation. In various embodiments, a macrocycle derivative (containing a functional group at both phenolic oxygens) with a free carboxylate in dichloromethane is treated with diisopropylcarbodiimide and N-hydroxyphthalimide or tetrachloro-N-hydroxyphthalimide, or is treated with HATU. After complete consumption of the starting material, the solvent is removed under vacuum. The crude compound is then subject to the conditions specific to the type of cross-coupling being performed. These conditions can be found in their respective literature references. In some cases, greater amounts of the reagents used for the cross-couplings are used than in the original reports. The reactions are worked up in concordance with literature reports and are purified by either silica gel chromatography or preparatory HPLC. Process of Synthesizing Formula III and Formula IIIಬ Compounds

Either of the phenolic oxygens in Formula I compounds is derivatized by selective alkylation or acylation by reaction of a Formula I compound with a compound according to the formula [R 5 C(O)] 2 O or LG-R 6 in the presence of at least one base, as generally described hereinabove. In some embodiments of the process, each of n2 and n3 is 0; each of R A1 and R A2 is H; and R A3 is (C1-C6)alkyl.

Acylation reactions are realized wherein R 4 is -C(O)(C 1 -C 6 )alkyl, -C(O)(C 3 - C 7 )cycloalkyl, or -C(O)aryl, and the compound according to Formula (I) is contacted with a compound according to [R 5 C(O)] 2 O, wherein R 5 is (C 1 -C 6 )alkyl, (C 3 -C 7 )cycloalkyl, or aryl, respectively. In some embodiments, R 4 is -C(O)(C1-C6)alkyl, such as -C(O)isopropyl.

Alkylation reactions are realized wherein R 4 is (C1-C6)alkyl or (C3-C7)cycloalkyl, and the compound according to Formula (I) is contacted with a compound according to LG-R 6 , wherein R 6 is (C 1 -C 6 )alkyl or (C 3 -C 7 )cycloalkyl, respectively, and LG is a leaving group. In some embodiments, R 4 is (C1-C6)alkyl. Suitable leaving groups LG, which are known to those who are skilled in the art, include bromide, iodide, tosylate, and mesylate.

In various embodiments, the base is selected from a tertiary amine according to the formula NR3, wherein each instance of R is independently selected from the group consisting of (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl, and heteroaryl; a chiral base, a carbonate base; or a combination thereof. For instance, the base is a chiral base, such as levamisole.

Alternatively, the base is a tertiary amine, such as 4-dimethylaminopyridine, di- isopropylethyl amine. In some embodiments, mixtures of chiral and tertiary amines are useful. Alternatively, or in addition to these embodiments, the base is a carbonate base, such as potassium or sodium carbonate. The amount of base can range from about 1 to about 30 mol%, such as about 10-20 mol%.

Selection of bases, or mixtures thereof, tend to direct formation of primarily Formula III or Formula IIIಬ compounds. Regioselectivity of one formula over the other ranges from about 100 mol% to about 50 mol%. For instance, regioselectivity is about 50, 60, 70, 80, 90, 95, 98, 99, or 100 mol%.

In some embodiments, the process occurs in a non-polar solvent, such as toluene. Process for Synthesizing Formula IV Compounds

In other embodiments, Formula I compounds:

undergo decarboxylative derivatization in two general steps. A Formula I compound is reacted with a reagent according to the formula to yield a redox-active ester compound according to Formula V, as described hereinabove. In various embodiments, the reagent according to the formula is a compound conforming to the formula:

Wherein each instance of R 8 is independently selected from H and Cl; Z 1 is N or C(O); and Z 2 is N or C. For example, Z 1 is C(O) and Z 2 is C. Illustrative reagents include N- hydroxyphthalamide (NHPI), tetrachloro-N-hydroxyphthalamide (TCNHPI, wherein R 8 is present in four instances as Cl), N-hyroxybenzotriazole (HOBT), and 1-Hydroxy-7- azabenzotriazole (HOAt). To facilitate formation of Formula V compounds, the step optionally includes use of a typical peptide coupling agent that is known in the art. Examples include carbodiimides, such as dicyclohexylcarbodiimide (DCC), ethyl-(Nl,N’- dimethylamino)propylcarbodiimide hydrochloride (EDC), and diisopropylcarbodiimide (DIC). Additional examples include benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy)tripyrrolidinophosphonium

hexafluorophosphate, (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium

hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate , O- (Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and O- (Benzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), O-(7- Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), O-(7- Azabenzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TATU), and O-(6- Chlorobenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HCTU). A Formula V compound is then contacted with a metal salt that is that is a nickel (II) or iron (III) salt, optionally a reducing agent, and an active R 7 reagent to thereby yield a compound according to Formula (IV). Suitable nickel salts include NiCl 2 and Ni(acac) 2 . Suitable iron salts include Fe(acac)3. To facilitate some embodiments of the reaction, as illustrated in the appended examples, a reducing agent is used. A convenient and economical reducing agent is zinc, which can be used in powder form. The active R 7 reagent is one that donates or supplies an R 7 moiety that formally replaces the carboxyl moiety in a Formula I compound. Illustrative active R 7 reagents include (R 7 )2Zn, R 7 B(OH)2, (R 7 )3Si(C6-C12-aryl), R 7 CuBr, R 7 CuI, a Michael acceptor that corresponds to the moiety R 7 , and R 7 ZnX (wherein X is Cl or Br). For example, in some embodiments, the active R 7 reagent is a Michael acceptor that corresponds to the moiety R 7 . Illustrative examples include methyl acrylate (R 7 = 2-carboxyethyl), 2-vinylpyridine (R 7 = 2- pyridylethyl), and acrylonitrile (R 7 = 2-cyanoethyl). In other embodiments, the active R 7 reagent is (R 7 ) 2 Zn. Examples include dialkyl zinc reagents and diaryl zinc reagents. Alternatively, the active R 7 reagent is R 7 ZnCl or R 7 B(OH) 2 . In other embodiments, a silane of the formula (R 7 ) 3 Si(C 6 -C 12 -aryl) can be used. For instance, PhSiH 3 is useful when R 7 is H. In some embodiments, step (B) of the process entails the use of a ligand. In some embodiments, the ligand is acetylacetonate (acac). Thus, in instances where a nickel (II) or iron (III) salt is obtained in the form of corresponding acetylacetonates, no additional ligand is needed. In other embodiments, the ligand is selected from bidentate nitrogen- or phosphorus-containing ligands. A family of bidentate nitrogen-containing ligands useful for this purpose is based on bipyridine (bipy) and its various derivatives, such as 4,4'-di-tert- butyl-2,2'-bipyridine. Examples of bidentate phosphorus-containing ligands include arylated phosphines, such as1,2-Bis(diphenylphosphino)benzene.

References:

1. Edwards, J.T.; Merchant, R.R.; Kyle S. McClymont, K.S.; Knouse, K.W.; Qin, T.; Malins, L.R.; Vokits,B.; Shaw, S.A.; Bao, D.; Wei, F.; Zhou, T.; Eastgate, M.D.; Baran, P.S., Nature, 2017 (ASAP).

2. Li, C.; Wang, J.; Barton, L.M.; Yu, S.; Tian, M.; Peters, D.S.; Kumar, M.; Yu, A.W.; Johnson, K.A.; Chatterjee, A.K.; Yan, M.; Baran, P.S., Science 2017 (ASAP).

3. Qin T.; Malins, L.R.; Edwards, J.T.; Merchant, R.R.; Novak, A.J.E.; Zhong, J.Z.; Mills, R.B.; Yan, M. Yuan, C.; Eastgate, M.D.; Baran, P.S., Angew. Chem.2017, 129, 266.

4. Wang, J.; Qin, T.; Chen, T.-G.; Wimmer, L.; Edwards,J.T.; Cornella, J.; Vokits, B.; Shaw, S.A.; Baran, P.S., Angew. Chem. Int. Ed.2016, 55, 9676.

5. Qin, T.; Cornella, J.; Li, C.; Malins, L.R.; Edwards, J.T.; Kawamura, S.; Maxwell, B.D.; Eastgate, M.D.; Baran, P.S., Science 2016, 352, 801.

6. Cornella, J.; Edwards, J.T.; Qin,T.; Kawamura, S.; Wang, J.; Pan, C.-M.; Gianatassio, R.; Schmidt, M; Eastgate, M.D.; Baran, P.S., Journal of the American Chemical Society 2015, 138, 2174. Further embodiments of the invention are illustrated in the following non-limiting examples. EXAMPLES General Methods

1H and 13 C NMR spectra were recorded on Bruker AMX 400, Bruker DRX 500, or Bruker DRX 600 spectrometers. Chemical shifts are reported relative to either chloroform (δ 7.26), methanol (δ 3.31), or dimethylsulfoxide (DMSO) (δ 2.50) for 1 H NMR and either chloroform (δ 77.16), methanol (δ 49.00), or DMSO (δ 39.52) for 13C NMR. IR

measurements were taken using a Nicolet 6700 ATR FT-IR. High resolution mass spectra were measured at the Scripps Center for Mass Spectrometry. Optical rotations were measured on a Perkin Elmer model 341 polarimeter.

Yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated. Reactions were magnetically stirred, and monitored by thin layer chromatography (TLC) with 0.25 mm Whatman pre-coated silica gel (with fluorescence indicator) plates. Flash chromatography was performed with silica gel (particle size 40-63 ^m, EMD chemicals).

Acetone was dried over anhydrous potassium carbonate, and all other dry solvents were purchased from Acros or Sigma. EDC, HOBT and 4-hydroxyphenylglycine were all purchased from Chem-Impex. All other chemicals were purchased from Acros or Sigma. Tetrakis(acetonitrile)copper(I) hexafluorophosphate was prepared as described in Kubas, G., Inorganic Syntheses 1990 (28), 68-70.

Abbreviations: THF, tetrahydrofuran; EtOH, ethanol; MeOH, methanol; AcOH, acetic acid; DCM, dichloromethane; DMF, N,N-dimethylformamide; EDC, 1- (3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc, ethyl acetate; Ar, argon; DBU, 1,8- diazabicyclo[5.4.0]undec-7-ene; TFA, trifluoroacetic acid.

Example 1: Synthesis of Boc-NH-Hpg-OH (1)

The title compound was prepared from p-hydroxyphenylglycine (Hpg) as described in a previous literature procedure reported by T. Roberts et al., J. Am. Chem. Soc.129 (2007) 15830-15838. All spectral data were consistent with those previously reported. Example 2: Synthesis of Boc-Hpg-oxazolidinone 2)

The title compound was prepared from compound 1 as described in a previous literature procedure reported by J. Dufour et al., Chemistry 16 (34) (2010) 10523-10534. All spectral data were consistent with those previously reported. Example 3: Synthesis of Boc-NMe-Hpg-OH (3)

Compound 2 (0.451 g, 1.61 mmol) was suspended in dichloromethane (2.25 mL), and the mixture was cooled to 0ºC. The reaction flask was sealed and placed under a nitrogen atmosphere. Triethylsilane (1.0 mL, 0.747 g, 6.44 mmol, 4.0 eq.) was added to mixture via syringe while stirring. Then trifluoroacetic acid (2.25 mL) was added drop-wise via syringe. The resulting solution was allowed to warm to room temperature while stirring. After 12 hours, the solution was concentrated under reduced pressure at ~35ºC. The resulting residue was suspended in water (4.5 mL) and cooled to 0ºC. Solid NaHCO3 was added to this solution until a pH of 7 was reached. To this solution was added 1N NaOH (aq.) solution (3.2 mL), then ditertbutydicarbonate (0.527 g, 2.42 mmol, 1.5 eq.) dissolved in THF (4.5 mL). The resulting solution was allowed to warm to room temperature while stirring. After stirring for 12 hours, THF was removed under reduced pressure. The remaining aqueous layer was washed twice with hexanes. The aqueous layer was then acidified to pH 2-3 with aqueous 6N HCl, then extracted two times with dichloromethane. The organic layers were combined and rinsed with brine, dried over Na2SO4, and concentrated under reduced pressure to yield an off-white solid (0.285 g, 63%) which was used without further purification. Example 4: Synthesis of Boc-NH-Ala-Tyr-OMe (4)

Boc-L-Ala-OH (4.730 g, 25.0 mmol) and L-Tyr-OMeڄHCl (5.7920 g, 25.0 mmol, 1 eq.) were dissolved in 80 mL of DMF. To this solution was added hydroxybenzotriazole (HOBT) (3.378 g, 25.0 mmol, 1 eq.) and Et 3 N (11.51 mL, 8.348 g, 82.5 mmol, 3.3 eq.). After stirring the resultant solution for 5 minutes, it was cooled to 0ºC, and 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimideÂHCl (EDC) (7.189 g, 37.5 mmol, 1.5 eq.) was added at once. The reaction mixture was allowed to warm to room temperature while stirring. The reaction was stirred for 18 hours. The resulting mixture was partitioned between EtOAc and water and extracted three times with EtOAc. The organic layers were combined and sequentially washed with sat. NaHCO3 (aq.), sat. NH4Cl (aq.), and brine. The organic layer was dried over Na 2 SO 4 , and concentrated under reduced pressure to yield a white foamy solid. The resulting off-white solid was purified by flash column chromatography (SiO2; 3:2 EtOAc/hexanes) yielding a white solid (8.885 g, 97% yield). 1 H NMR (600 MHz, CDCl3) δ (ppm) 6.92 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.1 Hz, 3H), 5.08 (s, 1H), 4.16 (s, 1H), 3.72 (s, 3H), 3.07 (dd, J = 14.0, 5.1 Hz, 1H), 2.99 (dd, J = 14.0, 5.9 Hz, 1H), 1.44 (s, 9H), 1.30 (d, J = 6.5 Hz, 3H).13C NMR (151 MHz, CDCl3) δ (ppm) 172.50, 171.87, 155.61, 155.39, 130.40, 127.03, 115.58, 80.44, 53.42, 52.43, 50.12, 37.16, 37.14, 28.35, 18.31. Example 5: Synthesis of HClڄNH2-Ala-Tyr-OMe (5)

Compound 4 (1.896 g, 5.17 mmol), was placed in an oven dried flask fixed with a magnetic stir bar, and was sealed. The flask was evacuated under reduced pressure and backfilled with dry N2 three times. To this flask, was added dry MeOH (45 mL) via cannula. The resulting solution was cooled to 0ºC, and acetyl chloride (1.10 mL, 1.218g, 15.52 mmol, 5 eq.) was added drop-wise via syringe. The reaction was allowed to warm to room temperature while stirring. It was allowed to react for 18 hours, after which it was concentrated under reduced pressure yielding a white foamy solid (quantitative yield). 1 H NMR (600 MHz, Methanol-d 4 ) δ (ppm) 7.04 (d, J = 8.5 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 4.63 (dd, J = 9.0, 5.4 Hz, 1H), 3.90 (q, J = 7.1 Hz, 1H), 3.70 (s, 3H), 3.35 (s, 1H), 3.10 (dd, J = 14.1, 5.4 Hz, 1H), 2.91 (dd, J = 14.1, 9.1 Hz, 1H), 1.49 (d, J = 7.1 Hz, 3H). 13 C NMR (151 MHz, Methanol-d 4 ) δ (ppm) 173.33, 171.20, 157.69, 131.33, 128.74, 116.48, 55.97, 52.94, 50.20, 37.50, 17.79. Example 6: Boc-NMe-Hpg-Ala-Tyr-OMe (6)

Compound 3 (1.652 g, 5.78 mmol) and compound 5 (1.778 g, 5.87 mmol, 1 eq.) were dissolved in 82 mL of MeCN/DMF (4:1). To this solution was added HOBT (0.793 g, 5.87 mmol, 1 eq.) and Et3N (2.7 mL, 1.960g, 19.4 mmol, 3.3 eq.). After stirring the resultant solution for 5 minutes, it was cooled to 0ºC, and EDC (1.688 g, 8.81 mmol, 1.5 eq.) was added at once. The reaction mixture was allowed to warm to room temperature while stirring. The reaction was stirred for 18 hours. The resulting mixture was partitioned between EtOAc and water and extracted three times with EtOAc. The organic layers were combined and sequentially washed with sat. NaHCO 3 (aq.), sat. NH 4 Cl (aq.), and brine. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The resulting off-white solid was purified by flash column chromatography (SiO 2 ; 7:3

EtOAc/hexanes) yielding a white solid (2.237g, 72%). 1 H NMR (600 MHz, CDCl3) δ (ppm) 8.02 (s, 1H), 7.70 (s, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 5.53 (s, 1H), 4.80– 4.74 (m, 1H), 4.49 (s, 1H), 3.69 (s, 3H), 3.08 (dd, J = 14.0, 4.6 Hz, 1H), 2.89 (dd, J = 14.1, 7.8 Hz, 1H), 2.71 (s, 3H), 2.01 (s, 2H), 1.46 (s, 9H), 1.21 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 172.47, 171.93, 170.77, 156.72, 156.69, 155.45, 130.50, 130.26, 127.09, 125.63, 115.98, 115.64, 81.06, 63.45, 53.85, 52.55, 49.09, 36.93, 28.41, 17.87, 1.0 Example 7: Synthesis of Macrocycle (7)

Compound 6 (10 mg, 0.0189 mmol) was added to an oven dried flask fixed with a stir bar. This flask was then sealed, evacuated and backfilled with oxygen and kept under this atmosphere. To this was added dry acetonitrile (1.5 mL) via syringe, completely dissolving 6. In a separate oven dried vial [Cu(MeCN) 4 ][PF 6 ] (0.0756 mmol) (freshly recrystallized from 1:1 MeCN/Et 2 O and dried under reduced pressure) was placed with a stir bar. The vial was sealed then evacuated and backfilled with nitrogen three times. To this was added dry acetonitrile (1.0 mL), then tetramethylethylenediamine (TMEDA) (0.0756 mmol). This was stirred for 5 minutes, then 0.5 mL was added to the reaction flask containing dissolved compound 6 (delivering 2 eq. of [Cu(MeCN)4][PF6], 2 eq. of TMEDA, and bringing the final reaction volume to 2.0 mL). This reaction mixture was stirred at room temperature under oxygen for 18 hours. The resulting solution was partitioned between EtOAc and water and extracted two times with EtOAc. The organic layers were combined, rinsed first with 5% NH4Cl (aq.), then with brine, dried over Na2SO4, and concentrated under reduced pressure. The resulting residue was purified preparative thin layer chromatography (SiO 2 ; 7:1

Et2O/benzene) to yield a white solid (7.4 mg, 75%) as a mixture of isomers. Alternative Procedure Compound 6 was added to an oven dried microwave vial and sealed. It was immediately evacuated and backfilled with nitrogen three times. To this was added dry acetonitrile to give a concentration of 0.0189 mmol/mL. In a separate oven dried microwave vial,

[Cu(MeCN) 4 ][PF 6 ] was added with a stir bar and sealed. This was immediately evacuated and backfilled three times. To this was added dry acetonitrile via syringe to give a concentration of 0.0378 mmol/mL. To the resulting clear solution was then added an equimolar amount of TMEDA via syringe. This was stirred for 1 minute under nitrogen, then it was purged with oxygen by bubbling it through the solution while stirring for 15 minutes. This produced a deep blue solution. Another separate oven dried vial fixed with a stir bar (reaction vessel) was sealed and placed under nitrogen atmosphere. To this was added 1 mL of the [Cu(MeCN)4][PF6] / TMEDA solution. While stirring under nitrogen, 1 mL of the solution of compound 6 was added to the reaction vessel over the course of 2 hours using a syringe pump. The reaction was allowed to stir for 4 days. 69% yield (HPLC). Example 7: Procedure for Selective Alkylation

Macrocycle 7 was placed in a vial fixed with a stir bar and placed under nitrogen. This was dissolved by addition of dry DMF (0.1 M). To this was added 5 eq. N,N- diisopropylethylamine (DIPEA). Finally, 1 equivalent of 2-tosyl-azidoethane was added via syringe. The resulting solution was allowed to stir at 25 ºC for 2 days, after which the reaction mixture was partitioned between water and EtOAc. It was extracted twice with EtOAc. The combined organic layers were then washed sequentially with 10% LiCl solution, sat. NH 4 Cl, and brine. The organic layer was collected, dried over Na 2 SO 4 , and solvent removed under reduced pressure. The crude product was analyzed by LCMS showing a single regioisomer (confirmed by comparison to chromatogram of 1:1 mixture of

regioisomers 8 and 9 prepared through a non-selective alkylation method). The crude material was then purified by preparatory thin layer chromatography (PTLC) providing a single regioisomer 8 or 9 in 70% yield. MS (ESI): 597.2 (M+H) + Example 8: Procedure for Selective Acylation

Macrocycle (7) was placed in a vial fixed with a stir bar and placed under nitrogen. To this was added dry toluene (0.01 M) followed by 1 equivalent DIPEA, and 20 mol % of levamisole (stock solution at 10 mg/mL in toluene). Finally, 1 equivalent of isobutyric anhydride was added and the resulting suspension was allowed to stir for 24 hours. This was partitioned between water and EtOAc and extracted with EtOAc twice. The combined organic layers were rinsed with brine, dried over Na 2 SO 4 , and the solvent was removed under reduced pressure. The crude product was analyzed by LCMS showing a 5:1 ratio of regioisomers 10 and 11 (confirmed by comparison to chromatogram of 1:1 mixture of regioisomers 10 and 11 prepared through a non-selective alkylation method).. The crude material was then purified by PTLC, separating the two isomers. The major isomer was provided in 66% yield, and the minor isomer in 14% yield. MS (ESI): 598.3 (M+H) + .

Example 9: General Giese Addition Procedure:

Carboxylic acid substrate 12 and N-hydroxyphthalimide (1.1 eq) were placed in an oven dried culture tube fixed with a stir bar. This was evacuated and backfilled with argon 3 times. To this was added dichloromethane (0.07 M) via syringe, creating a suspension. While stirring, N,Nಬ-diisopropylcarbodiimide (1.1 eq.) was added via syringe. After consumption of starting material by TLC (~1 h), the solvent was blown off under a stream of argon, and placed on high vacuum for 2 hours. After this, Zn powder (20 eq.), Ni(acac)2 (1 eq.), and lithium chloride (20 eq.) quickly added to the tube and resealed. Before stirring, the Michael acceptor (20 eq.) was added via syringe. The resulting mixture was then stirred vigorously for 18 hours at room temperature. It was quenched with 2 mL of 1:1 water/saturated aqueous NH4Cl. This was extracted twice with EtOAc. The combined organic layers were rinsed with brine and dried over Na 2 SO 4 . Solvent was removed under reduced pressure. The crude material was purified by PTLC to enrich the product (does not separate well from similar impurities). This enriched product (confirmed by LCMS) was then subject an appropriate the deprotection sequence, followed by purification by preparatory HLPC and lyophilization.

Compound 12 (40.0 mg, 0.035 mmol) and N-hydroxyphthalimide (6.4 mg, 0.039 mmol, 1.1 eq) were placed in an oven dried culture tube fixed with a stir bar. This was evacuated and backfilled with argon 3 times. To this was added dicholormethane (0.5 mL) via syringe, creating a suspension. While stirring, N,Nಬ-diisopropylcarbodiimide (6.1 PL, 4.9 mg, 0.039 mmol, 1.1 eq.) was added via syringe. After consumption of starting material by TLC (~1 h), the solvent was blown off under a stream of argon, and placed on high vacuum for 2 hours. After this, Zn powder (5.8 mg, 0.088 mmol, 2.5 eq.), NiCl 2 (H 2 O) 6 (4.21 mg, 0.018 mmol, 0.5 eq.), and 4,4'-di-tert-butyl-2,2'-bipyridine (9.5 mg, 0.035 mmol, 1.0 eq.) were quickly added to the tube and resealed. It was evacuated and backfilled with argon 3 times. Before stirring, DMF (0.4 mL) and phenylsilane (32 PL, 29 mg, 0.27 mmol, 7.5 eq.) were added to the vial. The resulting mixture was then stirred vigorously for 3 hours at room temperature. It was quenched with 2 mL of 1:1 water/saturated aqueous NH 4 Cl. This was extracted twice with EtOAc. The combined organic layers were rinsed with brine and dried over Na2SO4. Solvent was removed under reduced pressure. The crude material was purified by PTLC to enrich the product (does not separate well from similar impurities). This enriched product (mass confirmed by LCMS) was then suspended in 0.6 mL dichloromethane under nitrogen, cooled to 0 |C, and 0.1 mL of trifluoroacetic acid was added. This was stirred for 1 hour, and the solvent was then removed under reduced pressure. The resulting material was then purified by preparatory HPLC, yielding compound 16 (5.1 mg) as a white foam after lyophilization. 1H NMR (600 MHz, Methanol-d4): 0 MHz, Methanol- lyophilization. J = 8.6, 2.3 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 7.167.20 (d, tion), 6.94 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H), 6.36 (s, 1H), 5.15 (dd, J = 8.0, 5.6 Hz, 1H), 4.73 (q, J = 6.8 Hz, 1H), 4.27 (m, 5H), 4.08 (m, 1H), 3.30 (m), 3.23 erial was then purified by preparatory J = 7.7 Hz, 2H), 2.44 (s, 3H), 2.29 (m, 1H), 2.19 (m), 3.23 erial was then purified by 1.27 (m, 15H), 0.92 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz, Methanol-d4): 51 MHz, Methanol- (s, 3H), 2.29 (m, 1H), 2.19 (m), 3.23 erial was then purified by 1.27 (m, 15H), 0.92 (t, ngethane under nitrogen, cooled to 0 nched with 2 mL of 1:1 water/saturated aqueous NHs. Before stirring, DMF (0.4 mL) and phenylsilane (32 , NiCl76, 34.76, 31.77, 31.15, 30.64, 30.23, 28.68, 28.54, 28.40, 21.84, 18.14, 17.44, 12.55.

HRMS (m/z): calc’d for C44H64N7O6 [M+H]+ 786.4913, found 786.4911.

Carboxylic acid 12 substrate and HATU (1.1 eq) were placed in an oven dried culture tube fixed with a stir bar. This was evacuated and backfilled with argon 3 times. To this was added THF (0.1 M) via syringe. While stirring, triethylamine (1.1 eq.) was added via syringe. This was allowed to stir for 1 hour. In a separate vial, a THF solution of Fe(acac) 3 and 1,2- bis(diphenylphosphanyl)benzene were mixed in a 1:1.2 ratio (0.1 mL contains 1 equiv. with respect to carboxylic acid).0.1 mL of this Fe/ligand mixture was transferred to the reaction vial containing the carboxylic acid following its activation with HATU. Immediately after this, bis(4-methoxyphenyl)zinc (0.33 M in THF, 20 eq.) was added to the reaction vial via syringe. The resulting mixture was then stirred for 5 hours at room temperature. It was quenched with 2 mL of 1:1 water/saturated aqueous NH 4 Cl. This was extracted twice with EtOAc. The combined organic layers were rinsed with brine and dried over Na2SO4. Solvent was removed under reduced pressure. The crude material was purified by PTLC to give compound 17. MS (ESI): 1192.7 (M+H) +

Compound 18 (2.500 g, 10.07 mmol) and (J-Boc)-diaminobutyric acid-OMe (2.338 g, 10.07 mmol) were dissolved in DMF. To this solution was added HOBT (1.541 g, 10.07 mmol) and Et3N (4.63 mL, 3.36 g, 33.2 mmol). After stirring the resultant solution for 5 minutes, it was cooled to 0 ºC, and EDC (2.344 g, 15.10 mmol) was added at once. The reaction mixture was allowed to warm to room temperature while stirring. The reaction as allowed to stir for 12 hours. The resulting mixture was partitioned between EtOAc and water and extracted three times with EtOAc. The organic layers were combined and sequentially washed with sat. NaHCO3 (aq.), sat. NH4Cl (aq.), and brine. The organic layer was dried over Na2SO4, and concentrated under reduced pressure. The crude material was purified The resulting oil was purified by flash column chromatography (SiO 2 ; 2:1 EtOAc/Hexanes) yielding a white solid. This was then dissolved in THF (85 mL) and cooled to 0 qC. To this was added 2.0 N aq. LiOH (58 mL, 116 mmol). The solution was allowed to warm to room temperature and stirred vigorously for 2 hours. The solution was then acidified to a pH of 3 with 6.0 M HCl and partitioned with EtOAc. This was extracted 3 times with EtOAc. The combined organic layers were rinsed with brine, dried over Na 2 SO 4 , and dried under reduced pressure to yield a colorless, analytically pure viscous oil 19 (3.70 g, 82.3% yield).

Physical State: extremely viscous colorless oil;

1H NMR (600 MHz, Methanol-d 4 ): δ 7.40– 7.36 (m, 1H), 7.08– 7.04 (m, 2H), 4.59 (dd, J = 9.6, 4.4 Hz, 1H), 3.25 (dt, J = 13.0, 6.7 Hz, 1H), 3.17 (dt, J = 14.0, 7.3 Hz, 1H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.12 (m, 1H), 1.91 (m, 1H), 1.65– 1.58 (m, 2H), 1.44 (s, 9H), 1.36 – 1.25 (m, 11H), 0.90 (t, J = 7.1 Hz, 3H);

1 3 C NMR (151 MHz, Methanol-d4): δ 175.14, 173.45, 158.45, 146.22, 137.16, 134.84, 131.86, 128.47, 126.70, 80.18, 51.77, 38.23, 36.66, 33.04, 32.54, 30.58, 30.43, 30.27, 28.81, 23.74, 19.90, 14.47;

HRMS (m/z): calc’d for C25H41N2O5 [M+H] + 449.3010, found 499.3012. Example 13

Compound 7 was placed in a flame dried culture tube fixed with a stir bar, then evacuated and backfilled with nitrogen three times. To this was added methanol (0.1 M) and stirred until dissolved. This was cooled to 0 ºC, and acetyl chloride (5 eq.) was added dropwise. The solution was allowed to warm to room temperature and stirred until completion by TLC (~ 5 h). After this, the solvent was removed on a rotary evaporator, and placed under high vacuum for at least 5 hours. The resulting off white solid was then re-suspended in DMF (0.2 M) and DIPEA (2 eq.) was added to this solution and stirred. In a separate flame dried culture tube fixed with a stir bar was placed 4 (2 eq.) and (7-Azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate) (PyAOP) (2 eq.), which was then sealed, and evacuated and backfilled with nitrogen three times. To this was added DMF (0.2M) and stirred until all solids dissolved. DIPEA (2 eq.) was then added at once and the resulting solution was stirred for 15 minutes at room temperature. At this time, this solution was added transferred to the vial containing crude deprotected compound 7. This new solution was then placed in an oil bath preheated to 50 ºC and stirred for 2 days. After this time, the solution was cooled to room temperature, partitioned between EtOAc and water, and extracted 2 times with EtOAc. The combined organic layers were rinsed sequentially with aq. LiCl (10% w/w) and sat. aq. NH 4 Cl, and brine. The organic layer was then dried over Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified flash column chromatography (SiO 2 ; 100% CH 2 Cl 2 to 5% MeOH in CH 2 Cl 2 ) yielding product 20 as an off-white solid (50-84% yield).

Physical State: white solid;

TLC: Rf = 0.31 (5% MeOH in dichloromethane);

1H NMR (600 MHz, Methanol-d 4 ): Mixture of isomers (see spectrum below);

1 3 C NMR (151 MHz, Methanol-d4): Mixture of isomers (see spectrum below);

HRMS (m/z): calc’d for [M+H] + 880.4467, found 880.4462.

Compound 12 (25 mg, 0.028 mmol) was placed in a vial fixed with a stirbar and dissolved in THF (0.2 mL). The solution was cooled to 0 |C, and aq. LiOH (2M) (0.2 mL) was added slowly. The solution was allowed to warm to room temperature and stirred vigorously for 2 hours. The solution was then acidified to a pH of 3 with 1.0 M HCl and partitioned with EtOAc. This was extracted 3 times with EtOAc. The combined organic layers were rinsed with brine, dried over Na 2 SO 4 , and dried under reduced pressure to yield a white solid, which was used without further purification. The crude solid was placed in a vial fixed with a stirbar and placed under nitrogen. CH2Cl2 (0.4 mL) was added via syringe resulting in a suspension that was cooled to 0 |C. To this was added trifluoroacetic acid (0.07 mL) dropwise. The solution was allowed to warm to room temperature and stirred for 2 hours. The solvent was then removed on a rotary evaporator and the resulting oil was placed under high vacuum for at least 5 hours. The crude material was taken up in EtOH and purified by reverse phase preparatory HPLC (C18; gradient of H2O to MeCN each containing 0.1% TFA), and dried via lyophilization to yield 9 mg of compound 21.

Physical State: white foam;

HRMS (m/z): calc’d for C41H54N5O8 [M+H] + 744.3967, found 744.3968.

Example 15

Compound 22 (60.0 mg, 0.068 mmol) and K2CO3 (94.0 mg, 0.68 mmol, 10 eq.) were added quickly to a flame dried culture tube fixed with a stirbar and sealed, then evacuated and backfilled with nitrogen three times. DMF (0.7 mL) was added via syringe and stirred for 5 minutes. N-Boc-2-bromoethanamine (45.0 mg, 0.34 mmol, 5 eq.) was added via syringe and the resulting solution was placed in an oil bath preheated to 50 ºC and stirred for 2-3 days (monitored for complete conversion to the bis-alkylated product by LCMS as mono- and bis- alkylated products are inseparable by PTLC or flash column chromatography). After completion, the solution was cooled to room temperature and filtered over a bed of celite and rinsed with EtOAc. The filtrate was then partitioned between water and EtOAc and extracted twice. The combined organic layers were rinsed sequentially with aq. LiCl (10% w/w) and brine, dried over Na2SO4, and solvent removed under reduced pressure. The crude solid was purified by preparatory thin layer chromatography (SiO 2 ; 5% MeOH in CH 2 Cl 2 ) yielding compound 22 as a light yellow solid (61.9 mg, 80% yield).

Physical State: light yellow solid;

TLC: R f = 0.32 (5% MeOH in dichloromethane);

HRMS (m/z): calc’d for [M+H] + 1144.6540, found 1144.6545.

Compound 23 was synthesized via deprotection of 22 in the same manner as compound 12. The crude material was purified by reverse phase preparatory HPLC (C18; gradient of H 2 O to MeCN each containing 0.1% TFA).

Physical State: white foam;

1H NMR: (600 MHz, Methanol-d4): δ 8.39 (d, J = 9.0 Hz, 1H), 7.34– 7.31 (m, 2H), 7.26 (dd, J = 8.6, 2.4 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.12– 7.08 (m, 4H), 6.87 (d, J = 2.5 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.33 (s, 1H), 5.13 (dd, J = 7.9, 5.6 Hz, 1H), 4.91 (m, 1H), 4.81 (m, 1H), 4.32– 4.21 (m, 5H), 3.49 (dd, J = 17.1, 3.3 Hz, 1H), 3.30-3.25 (m, 3H) 3.17– 3.08 (m, 4H), 2.92 (s, 3H), 2.62 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.27 (m, 1H), 2.13 (m, 1H), 1.62 (m, 3H), 1.37– 1.27 (m, 15H), 0.90 (t, J = 7.0 Hz, 3H).

1 3 C NMR: (151 MHz, Methanol-d4): δ 174.51, 174.22, 173.32, 173.09, 172.34, 172.25, 156.60, 155.27, 146.87, 137.25, 136.62, 133.97, 133.33, 132.18, 132.13, 131.81, 131.00, 130.65, 129.91, 129.76, 128.38, 126.91, 115.77, 115.47, 66.84, 62.09, 52.24, 50.33, 50.23, 49.57, 49.43, 49.28, 49.14, 49.00, 48.86, 48.72, 48.58, 40.34, 40.21, 37.64, 36.63, 34.49, 33.65, 33.02, 32.51, 30.57, 30.56, 30.41, 30.27, 23.72, 20.02, 19.28, 14.42.

HRMS (m/z): calc’d for C 45 H 64 N 7 O 8 [M+H] + 830.4811, found 830.4812.

Experimental: Compound 22 ( g, mmol) was placed in a vial fixed with a stirbar and dissolved in THF ( mL). The solution was cooled to 0 ºC, and aq. LiOH (2M) ( mL) was added slowly. The solution was allowed to warm to room temperature and stirred vigorously for 2 hours. The solution was then acidified to a pH of 3 with 1.0 M HCl and partitioned with EtOAc. This was extracted 3 times with EtOAc. The combined organic layers were rinsed with brine, dried over Na 2 SO 4 , and dried under reduced pressure to yield a white solid compound 24, which was used without further purification. Example 18

Synthesized through general Giese addition procedure presented above.

1H NMR: (600 MHz, Methanol-d 4 ): δ 7.35 - 7.33 (m, 2H), 7.25 (dd, J = 8.6, 2.4 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.14 - 7.09 (m, 4H), 6.88 (d, J = 2.4 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 6.35 (s, 1H), 5.15 (dd, J = 8.0, 5.6 Hz, 1H), 4.72 (q, J = 6.8 Hz, 1H), 4.49 - 4.43 (m, 1H), 4.33 - 4.22 (m, 5H), 3.29 (q, J = 6.2, 5.5 Hz, 5H), 3.17 - 3.09 (m, 4H), 2.93 (s, 3H), 2.64 (t, J = 7.7 Hz, 3H), 2.52 (d, J = 7.2 Hz, 2H), 2.43 (s, 3H), 2.33 - 2.25 (m, 1H), 2.17 - 2.10 (m, 1H), 2.06 - 1.99 (m, 1H), 1.89 - 1.81 (m, 1H), 1.66 - 1.59 (m, 3H), 1.40 (d, J = 6.8 Hz, 3H), 1.37 - 1.28 (m, 16H), 0.92 (d, J = 6.8 Hz, 4H). 13 C NMR: (151 MHz, Methanol-d4): δ 174.63, 173.29, 173.08, 172.26, 162.99, 162.75, 156.59, 155.08, 146.87, 137.25, 136.61, 133.97, 132.99, 132.91, 132.17, 131.83, 131.13, 130.67, 129.92, 129.81, 128.38, 126.91, 120.95, 115.75, 115.47, 66.88, 66.84, 62.12, 50.52, 49.57, 49.00, 48.15, 40.35, 40.20, 37.64, 37.21, 36.63, 33.61, 33.20, 33.02, 32.51, 30.58, 30.56, 30.41, 30.27, 23.72, 20.02, 19.98, 14.42, 14.36. Example 19: Antibiotic Activity Procedures:

Antibiotic susceptibilities were determined using the Clinical and Laboratory Standards Institute broth microdilution method. 1 Briefly, antibiotics were prepared as 2-fold dilutions in 96-well plates containing cation-adjusted Mueller-Hinton broth containing (0.01% v/v TWEEN-80). Stock solutions of antibiotics were made in dimethyl sulfoxide (DMSO). Wells were inoculated from a fresh plate scrape diluted to a final concentration of 5 ^ 10 5 ^CFU/ml and incubated at 37rC. Growth observed visually at 20 h. All MICs are an average of at least three independent determinations. 1 Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial

susceptibility tests for bacteria that grow aerobically; Approved Standard, 9th ed.;^CLSI document M07-A9;^Clinical and Laboratory Standards

Institute: Wayne, PA, 2012.

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.