US008232268B2

(12) United States Patent (10) Patent No.: US 8,232,268 B2 Choi et al. (45) Date of Patent: *Jul. 31, 2012

(54) CARBAPENEMANTIBACTERIALS WITH OTHER PUBLICATIONS GRAM-NEGATIVE ACTIVITY AND Aldridge, K., “Ertapenem (MK-0826), a New Carbapenem: Com PROCESSES FOR THEIR PREPARATION parative In Vitro Activity Against Clinically Significant Anaerobes.” (75) Inventors: Woo-Baeg Choi, Atlanta, GA (US); Ewa Diagn. Microbiol. Infect. Dis., 2002, pp. 181-186, 44(2). Arnould et al., “New Applications of the Mitsonobu Reaction in the Kowalik, Atlanta, GA (US) Synthesis of C-2 N-Methy Carbapenems”. Tetrahedron Letters, 1992, vol. 33, No. 47, pp. 7133-7136. (73) Assignee: FOB Synthesis, Inc., Kennesaw, GA Bouffard et al., “Thienamycin Total Synthesis. 1. Synthesis of (US) AZetidinone Precursors of (+)-Thienamycin and Its Stereoisomers.” (*) Notice: Subject to any disclaimer, the term of this J. Org. Chem., 1980, pp. 1130-1142, 45. Cunha, B., "Ertapenem: A Review of Its Microbiologic, patent is extended or adjusted under 35 Pharmocokinetic and Clinical Aspects.” Drugs of Today, 2002, pp. U.S.C. 154(b) by 0 days. 195-213,38 (3). This patent is Subject to a terminal dis Edwards et al., “In Vitro Antibacterial Activity of SM-7338, a claimer. Carbapenem Antibiotic with Stability to Dehydropeptidase I.” Antimicrob. Agents Chemother, 1989, pp. 215-222, 33 (2). Hazumi et al., “Mechanism of Enhanced Antipseudomonal Activity (21) Appl. No.: 12/705,716 of BO-2727, a New Injectable 1-?3-Methyl Carbapenem.” (22) Filed: Feb. 15, 2010 Antimicrob. Agents Chemother, 1995, pp. 702-706, 39 (3). Johnston et al., “Total Synthesis of (+)-Thienamycin”. J. Am. Chem. (65) Prior Publication Data Soc., 1978, pp. 313-315, 100. Kahanet al., “Thienamycin, a New B-Lactam Antibiotic I. Discovery, US 2010/O16O284A1 Jun. 24, 2010 Taxonomy, Isolation and Physical Properties,” J. Antibiot., 1979, pp. 1-12, 32. Related U.S. Application Data Krein et al., “A Convenient Synthesis of 2-(Alkylamino) Pyridines.” J. Org. Chem., 2002, pp. 4965-4967, 67. (63) Continuation of application No. 1 1/150,428, filed on Leanza et al., “N-Acetimidoyl-and N-Formimidoylthienamycin Jun. 10, 2005, now Pat. No. 7,683,049. Derivatives: Antipseudomonal B-Lactam Antibiotics.” J. Med. Chem., 1979, pp. 1435-1436, 22. (60) Provisional application No. 60/578,632, filed on Jun. Legua et al., “Safety and Local Tolerability of Intramuscularly 10, 2004. Administered Ertapenem Diluted in Lidocaine: A Prospective, Ran domized, Double-Blind Study Versus Intramuscular Ceftriaxone.” (51) Int. Cl. Clin. Therapeut., 2002, pp. 434-444, 24 (3). C07D 477/14 (2006.01) Majumdar et al., “Pharmacokinetics of Ertapenem in Healthy Young Volunteers.” Antimicrob. Agents Chemother, 2002, pp. 3506-351 1, A6 IK3I/4439 (2006.01) 46 (11). Martins et al., “Design, Synthesis, and Biological Activity of A6 IK3I/478 (2006.01) a Novel Non-Cisplatin-type Platinum-Acridine Pharmacophore.” J. A6 IK3I/407 (2006.01) Med. Chem., 2001, pp. 4492-4496, 44. A6IP3/04 (2006.01) Marvel et al., “The Structure of Urea-Formaldehyde Resins,” J. Am. Chem. Soc., 1946, pp. 1681-1686, 68 (9). (52) U.S. Cl...... 514/210.09: 540/302 Nakagawa et al., “In Vitro Activity of a New Carbapenem Antibiotic, (58) Field of Classification Search ...... 540/302: BO-2727, with Potent Antipseudomnal Activity.” Antimicrob. Agents 514/210.09 Chemother, 1993, pp. 2756-2759, 37 (12). See application file for complete search history. Neu et al., “In Vitro Activity and B-Lactamase Stability of a New Carpabenem, SM-7338.” Agents Chemother, 1989, pp. 1009-1018, (56) References Cited 33 (7). Saino et al., “Purification and Properties of Inducible Penicillin U.S. PATENT DOCUMENTS B-Lactamase Isolated from Pseudomonas maltophilia," Antimicrob, Agents Chemother., 1982, pp. 564-570, 22 (4). 3,950,357 A 4, 1976 Kahan et al. Salzmann et al., “A Sterocontrolled Synthesis of (+)-Thienamycin.” 4.866,171 A 9/1989 Kumagai et al. J. Am. Chem. Soc., 1980, pp. 6161-6163, 102. 4,933,333 A 6/1990 Sunagawa et al. Shah et al., “Ertapenem, the First of a New Group of Carbapenems.” 4,943,569 A 7, 1990 Sunagawa J. Antimicrob. Chemother..., 2003, pp. 538-542, 52. 4,962,103 A 10/1990 Sunagawa et al. 5,011,832 A 4, 1991 DiNinno et al. (Continued) 5,034,384 A 7, 1991 Greenlee et al. 5,064,954 A 1 1/1991 Uyeo et al. 5,102,877 A 4, 1992 Murata et al. Primary Examiner — Mark Berch 5,122,604 A 6/1992 Sunagawa et al. (74) Attorney, Agent, or Firm — King & Spalding 5,539,102 A 7, 1996 Sendo et al. 6.255,300 B1 7/2001 DiNinno et al. (57) ABSTRACT 6,310,055 B2 10/2001 Dininno et al. The present invention provides B-methyl carbapenem com 6,399,597 B1 6/2002 Cama et al. pounds and pharmaceutical compositions useful in the treat 7,683,049 B2* 3/2010 Choi et al...... 540,302 ment of bacterial infections and methods for treating Such 2006/0074070 A1 4/2006 Choi et al. infections using Such compounds and/or compositions. The FOREIGN PATENT DOCUMENTS invention includes administering an effective amount of a EP OO17992 4f1980 carbapenem compound or salt and/or prodrug thereof to a EP 0184844. A 6, 1986 host in need of Such a treatment. The present invention is also EP O292191 11, 1988 in the field of synthetic organic chemistry and is specifically EP 04811 16 A1 10, 1990 provides an improved method of synthesis of B-methyl car JP 2001.491 1, 1990 bapenems which are useful as antibacterial agents. JP 4-164091 6, 1992 JP 2000.136133 5, 2000 17 Claims, 8 Drawing Sheets US 8,232.268 B2 Page 2

OTHER PUBLICATIONS MRSA 1.8-Napthosultam Pharmacophore”, Bioorganic and Medici Shimada et al. “Overview of a New Carbapenem, Panipenem/ nal Chemistry Letters 9, 1999, pp. 673-678. Yadav et al., “Reactions on a Solid Surface. A Simple, Economical, Betamipron.” Drugs Exp Clin Res., 1994, pp. 241-245, 20 (6). and Efficient Acylation of Alcohols and Amines over Al-O. J. Org. Tiraby et al., “A Standard Numbering Scheme for the Class A Chem., 2004, pp. 577-580, 69. B-Lactamases.” Biochem. J., 1991, pp. 269-270,276 (pt. 1). Yotsujii et al., “Properties of Novel B-Lactamase Produced by Weaver et al., “Thienamycin: New Beta-Lactam Antibiotic with Bacteroides fragilis, "Antimicrob. Agents Chemother, 1983, pp. 925 Potent Broad-Spectrum Activity.” Antimicrob. Agents Chemother. 929, 24 (6). 1979, pp. 518-521, 15 (4). International Search Report with Written Opinion of the Searching Wilkening et al., “Synthesis and Activity of Authority dated Nov. 3, 2005 for PCT/US05/20518. 2(Sulfonamido)Methylcarbapenems: Discovery of a Novel, Anti * cited by examiner U.S. Patent Jul. 31, 2012 Sheet 1 of 8 US 8,232,268 B2

FIGURE 1

OH H HC H

COH NH Thienamycin

NH Imipenem

CONMe

N Meropenem

H

HC H

CH Panipenem U.S. Patent Jul. 31, 2012 Sheet 2 of 8 US 8,232,268 B2

FIGURE 1 (CONTINUED)

OH CH H HC s / S O COH N1 N y \s Biapenem LC 10627

NHCH

Lenapenem

BO 2727

L-786 392 U.S. Patent Jul. 31, 2012 Sheet 3 of 8 US 8,232,268 B2

FIGURE 1 (CoNTINUED)

Faropenem

O

OH CH NH H H s S HC S

2–s O

O O o1 Nouls t-Bu CS 834

HC

Sanfeltrinem cilexetil

GV 18819 U.S. Patent Jul. 31, 2012 Sheet 4 of 8 US 8,232,268 B2

FIGURE 2: EXAMPLES OF GRAM-NEGATIVE ACTIVECP’s

CONHSO2NH2 U.S. Patent Jul. 31, 2012 Sheet 5 of 8 US 8,232,268 B2

FIGURE 2, CONTINUED

N1NH NH

U.S. Patent Jul. 31, 2012 Sheet 6 of 8 US 8,232,268 B2

FIGURE 3: SYNTHETIC ROUTE To CPI 5. OTBDMS O O O O bno r no-o-o-Bu bio-oo: -- OA —- O A O B NH O TBDMSO CO2Bn O TBDMSO CO2Bn O Xros arra-ne- oo: He -NH o c of N, TBDiasO D OTBDMS O OTES l O O O OiBu -o- l l --alle N. O. O O-iBu - C COPNB O TBDMS E N, O F O H OTES O OTES ls. O O O-iBU l N O -> / O O-Bu O G YorO O CPI5 COPNB U.S. Patent Jul . 31, 2012 Sheet 7 of 8 US 8 232,268 B2

U.S. Patent US 8,232,268 B2

CIRIINILNOO“IGITAVL

US 8,232,268 B2 1. 2 CARBAPENEMANTIBACTERIALS WITH Ed.), Merck & Co., Inc. (Rahway, N.J.), 1989, University of GRAM-NEGATIVE ACTIVITY AND Wisconsin Antimicrobial Use Guide, http://www.med PROCESSES FOR THEIR PREPARATION Sch.wisc.edu/clinsci/5amcgfamcg.html; Introduction on the Use of the Antibiotics Guideline, of Specific Antibiotic CROSS REFERENCE TO RELATED Classes, Thomas Jefferson University, http://jeffiine.tu.edu/ APPLICATIONS CWIS/OAC/antibiotics guide/intro.html; and references cited therein. This application is a continuation of U.S. patent applica The first carbapenem to be isolated was thienamycin, tion Ser. No. 1 1/150,428 filed Jun. 10, 2005 now U.S. Pat. No. shown below, which was isolated from Streptomyces cattleya 7,683,049 and claims priority to U.S. Provisional Patent 10 (U.S. Pat. No. 3.950.357) and was shown to have strong Application No. 60/578,632, filed Jun. 10, 2004. The entire antibacterial activity, including potency against Pseudomo contents of each of the above-identified patent applications nas spp. and B-lactamase stability (Kahan, J. S., et al., J. are hereby incorporated by reference. Antibiot., 32, pp. 1-12 (1979); Bodey, G.P., et al., Antimicrob, Agents Chemother, 15, pp. 518-521 (1979). The racemic FIELD OF THE INVENTION 15 synthesis of thienamycin was reported shortly thereafter by Merck (Johnston, D. B. R., et al., J. Am. Chem. Soc., 100, pp. This application provides novel carbapenem compounds 313-315 (1978); Bouffard, F. A., et al., J. Org. Chem., 45, and their salts and prodrugs, methods of treatment of gram 1130-1142 (1980)), as well as an asymmetric total synthesis negative bacterial infections with an effective amount of the (Salzmann, T.N., et al., J. Am. Chem. Soc. 102, pp. 6161-6163 compounds and pharmaceutical compositions including the (1980)). The nucleus and amino-containing side chain of this compounds. molecule, DESCRIPTION OF RELATED ART

The worldwide exploitation of antibiotics to treat infec 25 tious diseases has grown dramatically over the last forty years. In 1954, two million pounds of antibiotics were pro duced in the United States. Today, the figure exceeds 50 million pounds. According to the Centers Disease Control (CDC), humans consume 235 million doses of antibiotics 30 CO2H annually. Widespread misuse or overuse of antibiotics has fostered however, contributed to its chemical instability. In addition to the spread of antibiotic resistance and has contributed to the its potential to be hydrolyzed by the zinc-activated B-lacta development of a serious public health problem. Antibiotic mase that is present in Bacillus species, Xanthomonas, resistance occurs when bacteria that cause infection are not 35 Pseudomonas, and Bacteroides species (Saino.Y., et al., Anti killed by the antibiotics taken to stop the infection. The bac microb. Agents Chemother, 22, pp. 564-570 (1982); Yotsujii, teria Survive and continue to multiply, causing more harm. A., et al., Antimicrob. Agents Chemother, 24, pp. 925-929 For example, the bacterium Staphylococcus aureus is a major (1983)), chemical stability issues associated with the inter cause of hospital acquired infections that, historically, molecular aminolysis of the azetidinone (B-lactam) ring of responded satisfactorily to the antibiotic Vancomycin. 40 one molecule of thienamycin by the primary amine in the Recently, however, many strains of S. aureus have been found cysteamine side chain of another thienamycin molecule, to be resistant to Vancomycin. Moreover, the death rate for resulted in the use of thienamycin as a drug candidate to be Some communicable diseases such as tuberculosis have abandoned. started to rise again, in part because of increases in bacterial As a result of the problems associated with thienamycin, resistance to antibiotics. 45 N-formimidoyl thienamycin, known as imipenem, was syn Antibiotics are used therapeutically to treat bacterial infec thesized (Leanza, W.J., et al., J. Med. Chem., 22, pp. 1435 tions. Several types of antibiotics, classified according to their 1436 (1979)). This compound bears a more basic amidine mechanism of action, are currently employed. The known functionality on the 2 side chain, which is protonated at types of antibiotics include, e.g. cell wall synthesis inhibitors, physiological pH, preventing the compound from initiating a cell membrane inhibitors, protein synthesis inhibitors and 50 nucleophilic attack on another imipenem molecule. inhibitors that bind to or affect the synthesis of DNA or RNA. Cell wall synthesis inhibitors, such as beta lactam antibi

otics, generally inhibit some step in the synthesis of bacterial peptidoglycan. Penicillin is generally effective against non resistant Streptococcus, gonococcus and staphylococcus. 55 Amoxycillin and Ampicillin have broadened spectra against Gram-negative bacteria. Cephalosporins are generally used as penicillin Substitutes, against Gram-negative bacteria and \ , NH in Surgical prophylaxis. Monobactams are generally useful CO2H iN-4 for the treatment of allergic individuals. 60 Numerous antibiotic agents, suitable for use in the treat H ment of bacteria-related diseases and disorders, are known and disclosed, e.g. in The Physician's Desk Reference (PDR), However, poor urinary tract recovery from test subjects Medical Economics Company (Montvale, N.J.), (53" Ed.), revealed an instability of this compound to the mammalian 1999; Mayo Medical Center Formulary, Unabridged Version, 65 B-lactamase renal dehydropeptidase-I (DHP-I) (Shimada, J., Mayo Clinic (Rochester, Minn.), January 1998, Merck Index. et al., Drugs Exp Clin Res., 20, pp. 241-245 (1994)). Conse An Encyclopedia of Chemicals, Drugs and Biologicals, (11" quently, the compound cilastatin was developed for use in US 8,232,268 B2 3 4 co-administration in order to prevent hydrolysis and degre No. 4,962, 103: U.S. Pat. No. 4,933,333; U.S. Pat. No. 4,943, dation by DHP-I; this combination therapy is currently pre 569; U.S. Pat. No. 5,122,604; U.S. Pat. No. 5,034,384 and scribed under the name Primaxin R (Merck Frosst Std). U.S. Pat. No. 5,011,832. In response to the problem of carbapenems to destruction U.S. Pat. No. 6,255,300 to Merck & Co. describes certain by renal dehydropeptidase-1, the carbapenem antibiotic 5 carbapenem antibacterial agents in which the carbapenem meropenem (SM7338) (shown below), was developed (see, nucleus is substituted with an iodo-phenyl linked through a Edwards, J. R., et al., Antimicrob. Agents Chemother, 33, pp. methyl-oxygen lineage. The patent states that these com 215-222 (1989); Neu, H. C., et al., Antimicrob. Agents pounds are useful against gram positive bacterial infections. Chemother, 33, pp. 1009-1018 (1989)). Similarly, U.S. Pat. No. 6,310,055 provides carbapenem 10 compounds with aromatic side chains that are halogen Sub stituted, linked through an alkoxy unsaturated group. European Publication No. 0 292 191 to Merck & Co. describes certain 2-(substituted methyl)-1-alkylcarbapenem 15 compounds useful as antibiotic agents. U.S. Pat. No. 6,399,597, also to Merck & Co. describes NH certain naphthoSultam compounds that are allegedly useful in the treatment of certain drug resistant bacterial infections. CO2H Because of the difficulty in developing effective carbap enem compounds due to hydrolysis of the B-lactam ring and This compound was shown to be active against a large num low recovery, compounds with Superioranti-bacterial activity ber of Gram-negative bacteria. The drug is currently pre have not been developed. scribed for intravenous use (MerremR IV: AstraZeneca) in Therefore, it is one object of the present invention to pro the treatment of intra-abdominal infections and bacterial vide novel B-methyl compounds carbapenems that are effec meningitis. 25 tive antimicrobial agents. The carbapenem ertapenem (formerly MK-0826; Cunha, It is another object of the present invention to provide B.A., Drugs of Today, 38, pp. 195-213 (2002)) was the first of methods for the treatment of gram-negative bacteria, that is a group of carbapenems with potential against methicillin optionally can be drug-resistant and/or multi-drug resistant. resistant staphylococci (MRS) shown to be useful as a long acting, parenteral carbapenem (Shah, P. M., et al., J. Antimi 30 SUMMARY OF THE INVENTION crob. Chemother, 52, pp. 538-542 (2003); Aldridge, K. E., Diagn. Microbiol. Infect. Dis., 44(2), pp. 181-6 (2002)). It is In one embodiment of the present invention, carbapenems Suitable for administration both as a single-agent (e.g., co of the general formula (I) administration with a compound Such as cilastatin is not required), or by the intravenous or intramuscular route (Le 35 gua, P. et al., Clin. Therapeut., 24, pp. 434-444 (2002); (I) Majumdar, A. K., et al., Antimicrob. Agents Chemother, 46. pp. 3506-3511 (2002)). Eratapenem has received regulatory approval in both the United States (November, 2001) and the European Union (April, 2002). 40 One carbapenem having a fused pyrazole ring system (L-627; Biapenem) was developed by Lederle Ltd. (Japan), COM and introduced a methyl radical at the 1-?3 position of the carbapenem skeleton (see, U.S. Pat. No. 4,866,171). This 45 or a pharmaceutically acceptable salt and/or prodrug thereof structural modification reportedly gave biapenem Stability are described, wherein against hydrolysis by kidney dehydropeptidase, making co R is H or alkyl, typically lower alkyl such as CH: administration of a dehydropeptidase inhibitor unnecessary. R" is Horalkyl, typically lower alkyl such as CH: More recently, a new, injectable 1-?3-methyl carbapenem M is H or a group such that COM represents a carboxylic antibiotic having an (R)-1-hydroxymethyl-methylaminopro 50 acid, a carboxylate anion, a pharmaceutically acceptable pyl group exhibiting both broad spectrum, potent antibacte ester group, or a carboxylic acid protected by a protecting rial activity (BO-2727) and having antipseudomonal activity group; has been reported (Nakagawa, S., et al., Antimicrob. Agents P is selected from the group consisting of hydrogen, Chemother, 37, pp. 2756-2759 (1993); Hazumi, N., et al., hydroxyl, halogen (such as F), or hydroxyl protected by a Antimicrob. Agents Chemother, 39, pp. 702-706 (1995). 55 hydroxyl protecting group; and Since the discovery of thienamycin having a potential anti each Y and Y is independently selected from the group microbial activity against Gram-negative and Gram-positive consisting of hydrogen; halo. —CN; NO; NR'R'': bacteria, studies on the syntheses of carbapenem derivatives OR; SR: C(O)NR'R'': C(O)OR: S(O)R; which are analogous to thienamycin have been widely devel SOR; SONR'R'': NRSOR: C(O)R’; OC oped. As a result, it was found that carbapenem derivatives 60 (O)R’; OC(O)NR'R'': NRaC(O)NR'R'': NRCOR; having, as their 2-side chain, a Substituent derived from 4-hy —OCO.R"; NRC(O)R’; C straight- or branched-chain droxy-proline exhibit a potential antimicrobial activity and alkyl that are substituted or unsubstituted with one to four are useful as medicines or as intermediates for compounds R" groups: C, straight- or branched-chain alkyl that are possessing antimicrobial activity. substituted or unsubstituted one to four R groups and with 1-3-methyl carbapenem antibiotics, are particularly well 65 or without saturation (double or triple bonds); -A-(CH2)- known for treating a broad spectrum of gram-negative and Q and C-7 cycloalkyl, Substituted or unsubstituted with gram-positive bacterial infections. See for example U.S. Pat. one to four R groups: US 8,232,268 B2 5 6 wherein A is selected from the group consisting of O, S, NH, each R is independently selected from hydrogen; halo: NCH, NR, or -CH ; phenyl: CN: NO; NR'R'': OR"; - SR"; n is 0, 1, 2 or 3: CONR'R'': COOR"; SOR"; SOR"; each R", RandR is independently selected from hydrogen, SONR'R'': NR'SOR: COR"; NR"COR; —C straight- or branched-chain alkyl, unsubstituted or OCOR'; OCONR'R'; NR'COR'; NR" substituted with one to four R groups, or -C-7 CONR'R'': —OCOR'; —C straight- or branched cycloalkyl, unsubstituted or substituted with one to four R' chain alkyl, unsubstituted or substituted with one to four groups; R groups; or R and R” taken together with any intervening atoms rep each R" and R is independently hydrogen or - C resent a 4-6 membered Saturated ring optionally inter 10 straight- or branched-chain alkyl, unsubstituted or Sub rupted by one or more of O, S, NR, with R as defined stituted with one to four R groups; above, or—C(O)—, said ring being unsubstituted or Sub or R'' and R together with any intervening atoms represent stituted with one to four R groups; a 4-6 membered Saturated ring optionally interrupted by or R and R taken together with any intervening atoms rep one or more of O, S, NRW or —C(O)—, said ring being resent a 4-6 membered Saturated ring optionally inter 15 unsubstituted or substituted with one to four R groups; rupted by one to three of O, S, NR", with R" as defined each R" independently represents hydrogen; —C. above, or—C(O)—, said ring being unsubstituted or Sub straight- or branched-chain alkyl, unsubstituted or Sub stituted with one to four R groups; stituted with one to four R groups; C. cycloalkyl each R" is independently selected from the group consisting optionally substituted with one to four R groups; phenyl of halogen; –CN; NO; NR'R''. -OR: —SR; optionally substituted with one to four R groups, or CONRR?: COORS: SORS: - SORS: heteroaryl optionally substituted with 1-4 R groups; or SONR'R'; NRSOR: COR: NRCOR; R" and R” taken together with any intervening atoms OCOR: OCONRR1 NRCONR/R3; NRCOR; represent a 5-6 membered Saturated ring, optionally OCOR: C(NR)NR'R8; NRC(NH)NR'Rs or interrupted by one or two of O, S, SO. NH or NCH: NRC(NR)Rs: 25 each R' independently represents hydrogen or a Cs each R, R and R3 independently represents hydrogen; —R; straight- or branched chain alkyl, optionally interrupted —C straight- or branched-chain alkyl unsubstituted or by one or two of O, S, SO, SO, NR", NR'R'', or substituted with one to four R groups: —C(O)—, said chain being unsubstituted or Substituted or R and R taken together with any intervening atoms rep with one to four of halo, CN, NO, OR", SR", SOR", resent a 4-6 membered Saturated ring optionally inter 30 SOR", NR'R'', N*(R)-R", C(O) R", C(O) rupted by one to three of O, S, C(O) or NR3 with R3 as NR'R'', SONRR", COR", OC(O)R", OC(O) defined above, said ring being unsubstituted or substituted NR'R", NRC(O)R", NRC(O)NR'R", or a phenyl or with one to four R groups; heteroaryl group which is in turn optionally substituted each R independently represents halo; CN; NO; phe with from one to four R groups or with one to two C nyl; NHSOR: OR', SR': N(R'): Nt(R)3; 35 straight- or branched-chain alkyl groups, said alkyl —C(O)N(R'); SON(R'): heteroaryl; heteroarylium; groups being unsubstituted or Substituted with one to COR': C(O)R’; OCOR': NHCOR": guanidinyl: four R groups; carbamimidoyl or ureido; each R" and R is independently selected from hydrogen; each R" independently represents hydrogen, a -C, a straight phenyl; —C straight or branched chain alkyl, unsub or branched-chain alkyl group, a C-C cycloalkyl group 40 stituted or substituted with one to four R groups, and or phenyl, or when two R' groups are present, said R' optionally interrupted by O, S, NR", N'R"R" or groups may be taken in combination and represent a 4-6 —C(O)—: membered saturated ring, optionally interrupted by one or or R and R” together with any intervening atoms represent two of O, S, SO = C(O) , NH and NCH: a 4-6 membered Saturated ring optionally interrupted by each Q is selected from the group consisting of: 45 O, S, SO, NR", NR'R' or C(O)—, unsubstituted or substituted with 1-4 R groups, and when R and R together represent a 4-6 membered ring as defined above, R is as defined above or R rep resents an additional saturated 4-6 membered ring fused to the ring represented by R and R” taken together, l C. optionally interrupted by O, S, NR" or —C(O)—, said rings being unsubstituted or substituted with one to four WRN R groups. - S-6 69N-R and - Sierr The present invention also provides a pharmaceutical com e Y.7 55 position including a compound of the invention, or a pharma L (CH2) ceutically acceptable salt and/or prodrug thereof, optionally with a pharmaceutically acceptable carrier or diluent. wherein. In one embodiment, the invention provides a pharmaceu a and b are 1, 2 or 3: tical composition comprising a compound of the invention, or L is a pharmaceutically acceptable counterion; 60 a pharmaceutically acceptable salt and/or prodrug therein, in C. is selected from O, S or NR; combination with one or more other antimicrobial agents, each f3, 8, , u, and O is independently selected from CR, optionally with a pharmaceutically acceptable carrier or dilu Nor NR, provided that no more than one of B, 8, , u, ent. and O is NR; In another embodiment, the invention provides a method of each R is independently selected from hydrogen; phenyl 65 preventing or treating a bacterial infection in a host, typically or C. Straight- or branched-chain alkyl, unsubstituted an animal, and most typically a human, including adminis or substituted with one to four R groups; tering to the host a therapeutic amount of a compound of the US 8,232,268 B2 7 8 present invention, or a pharmaceutically acceptable salt and/ nents or steps, the compounds, compositions and methods or prodrug therein, optionally in a pharmaceutically accept can also “consist essentially of or “consist of the various able carrier or diluent. components and steps. In a separate embodiment, the invention provides a method The term “alkyl, as used herein, unless otherwise speci of preventing or treating a gram-negative bacterial infection fied, includes a saturated Straight, branched, or cyclic, pri in a host that includes administering a therapeutic amount of mary, secondary, or tertiary hydrocarbon of C to Co. The a compound of the present invention, or a pharmaceutically term includes both substituted and unsubstituted alkyl acceptable salt and/or prodrug therein, in combination or groups. Moieties with which the alkyl group can be substi alternation with one or more other antimicrobial agents, tuted are selected from the group consisting of hydroxyl, halo 10 (F, Cl, Br, I), amino, alkylamino, arylamino, alkoxy, aryloxy, optionally in a pharmaceutically acceptable carrier or diluent. nitro, cyano, Sulfonic acid, Sulfate, phosphonic acid, phos In one principal embodiment, the bacterial infection is due phate, or phosphonate, either unprotected, or protected as to a gram-negative bacteria. In another embodiment, the bac necessary, as known to those skilled in the art, for example, as terial infection is from a drug resistant and/or multiple-drug taught in Greene, et al., Protective Groups in Organic Syn resistant gram-negative bacteria. 15 thesis, John Wiley and Sons, Second Edition, 1991, hereby The invention also provides a compound of the present incorporated by reference. When the alkyl group is said to be invention for use in medical therapy, and the use in the prepa Substituted with an alkyl group, this is used interchangeably ration of a medicament for the treatment of bacterial infec with “branched alkyl group'. Specific examples of alkyls tions, particularly gram negative bacterial infections, alone or and/or substituted alkyls includes, but are not limited to, in combination with another agent. methyl, trifluoromethyl, ethyl, propyl, isopropyl, cyclopro pyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, DESCRIPTION OF THE FIGURES neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpenty1, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. FIG. 1 shows a nonlimiting illustrative example of known The term “lower alkyl, as used herein, and unless other carbapenems. 25 wise specified, refers to a C to Ca Saturated Straight, FIG. 2 shows a nonlimiting illustrative example of the branched, or if appropriate, a cyclic (for example, cyclopro structure of carbapenem analogs of the present invention pyl) alkyl group, including both Substituted and unsubstituted possessing gram-negative biological activity. forms. Unless otherwise specifically stated in this applica FIG. 3 shows the synthetic process of preparing carbap tion, when alkyl is a suitable moiety, lower alkyl is typical. enem Intermediate 5. 30 Similarly, when alkyl or lower alkyl is a suitable moiety, FIG. 4 is a table showing gram-negative MIC (In Vitro unsubstituted alkyl or lower alkyl is typical. Susceptibility) Data for selected compounds against selected Cycloalkyl is a species of alkyl containing from 3 to 15 organism. carbonatoms, without alternating or resonating double bonds between carbonatoms. It may contain from 1 to 4 rings which DETAILED DESCRIPTION OF THE INVENTION 35 are fused. The term “alkenyl' includes a hydrocarbon radical The invention provides carbapenem compounds or their straight, branched or cyclic containing from 2 to 10 carbon pharmaceutically acceptable salts or prodrugs, pharmaceuti atoms and at least one carbon to carbon double bond. cal compositions containing these compounds and methods Examples of alkenyl groups include ethenyl, propenyl, bute of their use in the treatment or prevention of gram-negative 40 nyl and cyclohexenyl. bacterial infections. The term “alkynyl refers to a hydrocarbon radical straight or branched, containing from 2 to 10 carbonatoms and at least Definitions one carbon to carbon triple bond. Examples of alkynyl groups include ethynyl, propynyl and butynyl. The numbering system for the carbapenem compounds 45 “Alkoxy” includes C-C alkyl-O , with the alkyl group used in this specification is set out below, wherein the num optionally substituted as described herein. bering of the carbapenem nucleus is in accordance with stan The term “alkylamino” or “arylamino” refers to an amino dards in the art (see, Tiraby, G., et al., Biochem J. 276 (pt. 1), group that has one or two alkyl or aryl Substituents, respec pp. 269-270 (1991)). tively. 50 "Aryl refers to aromatic rings e.g., phenyl, Substituted

phenyl, biphenyl, and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 55 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. The typical aryl groups are phenyl, naphthyl and phenanthrenyl. COM The term includes both substituted and unsubstituted moi eties. The aryl group can be substituted with one or more Whenever a range is presented herein it should be under 60 moieties selected from the group consisting of bromo, chloro, stood to include each element of the range. For example, the fluoro, iodo, hydroxyl, amino, alkylamino, arylamino, range “C to Calkyl independently includes C, C, C and alkoxy, aryloxy, nitro, cyano, Sulfonic acid, Sulfate, phospho C alkyl groups. When such a range is stated, each element nic acid, phosphate, or phosphonate, either unprotected, or has been contemplated and the range is used merely for con protected as necessary, as known to those skilled in the art, for Venience. 65 example, as taught in Greene, et al., Protective Groups in Generally, while the compounds, compositions and meth Organic Synthesis, John Wiley and Sons, Second Edition, ods are described in terms of "comprising various compo 1991. Typical substituted aryls include phenyl and naphthyl. US 8,232,268 B2 10 The term “alkaryl' or “alkylaryl” refers to an alkyl group with an aryl substituent. The term “aralkyl or “arylalkyl N e e e refers to an aryl group with an alkyl Substituent. N-CH S O The term "heteroaryl' or "heteroaromatic’, as used herein, s2N -NS/ -NS -N/ refers to an aromatic group that includes at least one Sulfur, oxygen, nitrogen or phosphorus in the aromatic ring. Het eroaryl or heteroaromatic compounds include monocyclic N-CH, o N-CH aromatic hydrocarbon group having 5 or 6 ring atoms, or a s N/ NS/ bicyclic aromatic group having 8 to 10 atoms, containing at 10 least one heteroatom, O.S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one, two or three additional carbon atoms are optionally replaced by a heteroa tom selected from oxygen, Sulfur or nitrogen heteroatom. Examples of this type are pyrrole, pyridine, oxazole, thiazole 15 and oxazine. Additional nitrogen atoms may be present together with the first nitrogen and oxygen or Sulfur, giving, e.g., thiadiazole. Examples include the following.

e e e NH NH S S. NN/ NS/ pyrrole (pyrrolyl) imidazole (imidazolyl) thiazole (thiazolyl) 25 When a charge is shown on a particular nitrogen atom in a e e ring which contains one or more additional nitrogenatoms, it O S is understood that the charge may reside on a different nitro O S. S. genatom in the ring by virtue of charge resonance that occurs. oxazole (oxazolyl) furan (furyl) thiophene (thienyl) e e 30 NH NH O N / S / YN2\ YN-\ NS/ N N N-CH --> N'-CH and NS NS triazole (triazolyl) pyrazole (pryazolyl) isoxazole (isoxazolyl) e e 35 N-CH --> N-CH er S O s -NS/ -N N/ N2 ND isothiazole (isothiazolyl) pyridine (pyridinyl) pyrazine (pyrazinyl) The term "heterocycloalkyl refers to a cycloalkyl group 40 (nonaromatic) in which one of the carbon atoms in the ring is replaced by a heteroatom selected from O, S or N, and in which up to three additional carbonatoms may be replaced by r r heteroatoms. 2N e The terms “quaternary nitrogen' and “positive charge' pyridazine (pyridazinyl) pyrimidine (pyrimidinyl) 45 refer to tetravalent, positively charged nitrogenatoms includ 1s ing, e.g., the positively charged nitrogen in a tetraalkylam monium group (e.g. tetramethylammonium), heteroarylium, le (e.g., N-methyl-pyridinium), basic nitrogens which are pro N tonated at physiological pH, and the like. Cationic groups triazine (triazinyl) 50 thus encompass positively charged nitrogen-containing groups, as well as basic nitrogens which are protonated at physiologic pH. The heteroaryl or heteroaromatic group can be optionally substituted with one or more substituent selected from halo The term "heteroatom” refers to oxygen, sulfur, nitrogen, gen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives, 55 phosphorus, and selenium, selected on an independent basis. Halogen and "halo', as used herein, includes bromine, amido, amino, alkylamino, dialkylamino. Functional oxygen chlorine, fluorine and iodine. and nitrogen groups on the heterocyclic or heteroaryl group The term acyl refers to a carboxylic acid ester in which the can be protected as necessary or desired. Suitable protecting non-carbonyl moiety of the ester group is selected from groups are well known to those skilled in the art, and include 60 straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and including methoxymethyl, aralkyl including benzyl, aryloxy t-butyl-diphenylsilyl, trity1 or substituted trityl, alkyl groups, alkyl Such as phenoxymethyl, aryl including phenyl option acyl groups such as acetyl and propionyl, methanesulfonyl, ally substituted with halogen, C to C alkyl or C to Ca and p-toluenylsulfonyl. alkoxy, Sulfonate esters such as alkyl or aralkyl Sulphonyl “Heteroarylium” refers to heteroaryl groups bearing a qua 65 including methanesulfonyl, the mono, di or triphosphate ternary nitrogen atom and thus a positive charge. Examples ester, trity1 or monomethoxytrityl, substituted benzyl, tri include the following. alkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. US 8,232,268 B2 11 12 Aryl groups in the esters typically include a phenyl group. "Pharmaceutically acceptable salts' include salts that The term “lower acyl refers to an acyl group in which the retain the desired biological activity of the parent compound non-carbonyl moiety is lower alkyl. and do not impart undesired toxicological effects. These salts “Carboxylate anion” refers to a negatively charged group can take the form —COOM, where M is a negative charge, COO. which is balanced by a counterion. These include salts formed “Guanidinyl refers to the group: HNC(NH)NH-. with cations such as sodium, potassium, NH, magnesium, “Carbamimidoyl refers to the group: HNC(NH)—. Zinc, ammonium, or alkylammonium cations such as tetram “Ureido” refers to the group: HNC(O)NH-. ethylammonium, tetrabutylammonium, choline, triethylhy When a group is “optionally interrupted, this includes one droammonium, meglumine, triethanolhydroammonium, cal 10 cium, and calcium polyamines Such as spermine and or more of the interrupting moieties in combination, as well as spermidine. These can also include Salts formed from said moieties located at either or both ends of the chain. Thus, elemental anions such as chloride, bromide, and iodide. They it includes terminating the group as well. can also include acid addition salts, for example, salts derived When a group is termed “substituted, unless otherwise from inorganic or organic acids. Included among such salts indicated, this means that the group contains from 1 to 4 15 are the following: acetate, adipate, alginate, ascorbic acid, substituents thereon. With respect to R. R. R. and R, the aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, Substituents available on alkyl groups are selected from the citrate, camphorate, camphorsulfonate, cyclopentanepropi values of R". Many of the variable groups are optionally onate, digluconate, dodecylsulfate, ethanesulfonate, fuma substituted with up to four R groups. With respect to R. R. rate, glucoheptanoate, gluconic acid, glycerophosphate, and R, when these variables represent substituted alkyl, the hemisulfate, heptanoate, hexanoate, hydrochloride, hydro substituents available thereon are selected from the values of bromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, R. maleate, methanesulfonate, 2-naphthalenesulfonate, nicoti When a functional group is termed “protected, this means nate, nitric acid, oxalate, palmitic acid, pamoate, pectinate, that the group is in modified form to preclude undesired side persulfate, 3-phenylpropionate, phosphoric acid, picrate, piv reactions at the protected site, and unless otherwise defined 25 alate, polygalacturonic acid; polyglutamic acid, propionate, refers to a group that is added to an oxygen, nitrogen, or p-toluenesulfonic acid, Succinate, Sulfuric acid, tannic acid, phosphorus atom to prevent its further reaction or for other tartrate, thiocyanate, tosylate and undecanoate. purposes. In some of the carbapenem compounds of the The term “prodrug includes a compound that, when present invention, M is a readily removable carboxyl protect administered to an animal, is converted under physiological ing group, and/or P represents a hydroxyl which is protected 30 conditions to a compound of the invention, for example a by a hydroxyl protecting group. Such protecting groups are pharmaceutically acceptable ester. used to protectively block the hydroxyl or carboxyl group The pharmaceutically acceptable esters are such as would during the synthesis procedures and are readily removable by be readily apparent to a medicinal chemist, and include, for procedures that will not cause cleavage or other disruption of example, those described in detail in U.S. Pat. No. 4,309.438. the remaining portions of the molecule. Such procedures 35 Included within Such pharmaceutically acceptable esters are include chemical and enzymatic hydrolysis, treatment with those which are hydrolyzed under physiological conditions, chemical reducing or oxidizing agents under mild conditions, Such as pivaloyloxymethyl, acetoxymethyl, phthalidyl, inda treatment with a transition metal catalyst and a nucleophile nyl and methoxymethyl. These are also referred to as “biola and catalytic hydrogenation. bile esters', which are biologically hydrolysable. Examples A wide variety of oxygen and nitrogen protecting groups 40 of biolabile esters include compounds in which M represents are known to those skilled in the art of organic synthesis. an alkoxyalkyl, alkylcarbonyloxyalkyl, alkoxycarbonyloxy Suitable protecting groups for the compounds of the present alkyl, cycloalkoxyalkyl, alkenyloxyalkyl, aryloxyalkyl, invention will be recognized from the present application alkoxyaryl, alkylthioalkyl, cycloalkylthioalkyl, alkenylthio taking into account the level of skill in the art, and with alkyl, arylthioalkyl or alkylthioaryl group. These groups can reference to standard textbooks, such as Greene, T. W. and 45 be substituted in the alkyl or aryl portions thereof with acyl or Wuts, P. M., Protective Groups in Organic Synthesis, 3" Ed., halo groups. The following M species are examples of biola Wiley, New York (1991). Examples of carboxyl protecting bile ester forming moieties: acetoxymethyl, 1-acetoxyethyl, groups include allyl, benzhydryl, 2-naphthylmethyl, benzyl 1-acetoxypropyl, pivaloyloxymethyl, lisopropyloxycarbony (Bn), silyl such as t-butyldimethylsilyl (TBDMS), phenacyl, loxyethyl, 1-cyclohexyloxycarbonyloxyethyl, phthalidyland p-methoxybenzyl, o-nitrobenzyl, p-methoxyphenyl, p-ni 50 (2-oxo methyl-1,3-dioxolenyl)methyl. trobenzyl, 4-pyridylmethyl and t-butyl. Examples of suitable The term "host', as used herein, refers to a unicellular or C-6 hydroxyethyl protecting groups include triethylsilyl multicellular organism in which the bacteria can replicate, (TES), t-butyldimethylsilyl (TBDMS), o-nitrobenzyloxycar including cell lines and animals. Alternatively, the host can be bonyl (ONB), p-nitrobenzyloxycarbonyl (PNB), benzyloxy carrying a part of the bacterial particles, whose replication carbonyl (CBZ), allyloxycarbonyl (Alloc), t-butyloxycarbo 55 and/or function can be altered by the compounds of the nyl (Boc), 2.2.2-trichloroethyloxycarbonyl (Troc), and the present invention. The term host refers to infected cells, cells like. transfected with all or part of the bacteria and animals, such The phrase “pharmaceutically acceptable ester, salt or as, primates (including chimpanzees) and, in one embodi hydrate.” refers to those salts, esters and hydrated forms of the ment, the host is a human. Veterinary applications are also compounds of the present invention which would be apparent 60 encompassed by the present invention. to the pharmaceutical chemist. i.e., those which are Substan The term “treatment as used herein, includes an approach tially non-toxic and which may favorably affect the pharma for obtaining beneficial or desired results including clinical cokinetic properties of said compounds, such as palatability, results, including alleviation of symptoms, diminishment of absorption, distribution, metabolism and excretion. Other extent of disease, stabilization (i.e., not worsening) state of factors that are also important in the selection are cost of the 65 disease, preventing spread of disease, preventing or reducing raw materials, ease of crystallization, yield, stability, Solubil occurrence or recurrence of disease, delay or slowing of dis ity, hygroscopicity and flowability of the resulting bulk drug. ease progression, and reduction of incidence of disease or US 8,232,268 B2 13 14 symptoms. As used herein, the phrase “anti-bacterially effec rupted by one to three of O, S, C(O)—or NR with R as tive amount’ means an amount effective for treating the bac defined above, said ring being unsubstituted or substituted terial infection. with one to four R groups; each R' independently represents halo. —CN; NO phe Compounds of the Invention nyl; NHSOR: OR', SR: N(R): N*(R)3; —C(O)N(R'); SON(R'): heteroaryl; heteroarylium; In one embodiment of the present invention, the carbap COR: C(O)R’; OCOR': NHCOR": guanidinyl: enem of the formula I, carbamimidoyl or ureido; each R" independently represents hydrogen, a -C, a straight 10 or branched-chain alkyl group, a C-C cycloalkyl group (I) or phenyl, or when two R' groups are present, said R' groups may be taken in combination and represent a 4-6 membered Saturated ring, optionally interrupted by one or two of O, S, SO = C(O) , NH and NCH: 15 Q is selected from the group consisting of

No. 8-N- s Q/ e/5, I or pharmaceutically acceptable salts and/or prodrugs thereof \,.25, Y- 3, is provided, wherein R is H or alkyl, such as lower alkyl such as CH: R" is Horalkyl, such as lower alkyl such as CH: M is H or a group such that COM represents a carboxylic acid, a carboxylate anion, a pharmaceutically acceptable ester 25 group, or a carboxylic acid protected by a protecting group; GE) P is hydrogen, hydroxyl, halogen such as F, or hydroxyl NRRR2 protected by a hydroxyl protecting group; and Y' and Y are each independently selected from the follow 30 wherein. ing: hydrogen; halo; CN; NO; NR'R''. —OR: a and b are 1, 2 or 3: –SR: C(O)NR'R'': C(O)OR: S(O)R; SOR: L is a pharmaceutically acceptable counterion; SONR'R'': NRSOR: C(O)R’; OC(O)R’; C. represents O, S or NR; OC(O)NR'R'': NRaC(O)NR'R'': NRCOR; f3, 8, , u, and O represent CR, N or NR, provided that no —OCOR'; NRC(O)R’: C, straight- or branched-chain 35 more than one of B, 8, , u, and O is NR; alkyl that are substituted or unsubstituted with one to four R' each R independently represents hydrogen; phenyl or C. groups and with or without saturation (double or triple straight- or branched-chain alkyl, unsubstituted or Substi bonds); alkylaryl, -A-(CH2)-Q and C-7 cycloalkyl, Substi tuted with one to four R groups; tuted or unsubstituted with one to four R groups. each R independently represents hydrogen; halo; phenyl: wherein A represents O, S, NH, NCH, NR, or —CH2—, 40 CN; NO; NR'R'': OR', SR'; CONR'R'': in represents an integer 0, 1, 2 or 3: COOR"; SOR"; SOR"; SONR'R'': each R, R and R independently represents hydrogen, NR'SOR: COR"; NR'COR'; OCOR'; —C straight- or branched-chain alkyl, unsubstituted or OCONR'R'; NR'COR'; NR''CONR'R'"; substituted with one to four R groups, or -C-7 —OCOR'; —C straight- or branched-chain alkyl, cycloalkyl, unsubstituted or substituted with one to four R' 45 unsubstituted or substituted with one to four R groups; groups; RandR represent hydrogen or—C straight- or branched or R and R” taken together with any intervening atoms rep chain alkyl, unsubstituted or substituted with one to four R' resent a 4-6 membered Saturated ring optionally inter groups; rupted by one or more of O, S, NR, with R as defined or R'' and R together with any intervening atoms represent a above, or—C(O)—, said ring being unsubstituted or Sub 50 4-6 membered Saturated ring optionally interrupted by one stituted with one to four R groups; or more of O, S, NRW or —C(O)—, said ring being unsub or Rand R taken together with any intervening atoms rep stituted or substituted with one to four R groups; resent a 4-6 membered Saturated ring optionally inter each R" independently represents hydrogen; —C straight rupted by one to three of O, S, NR", with R" as defined or branched-chain alkyl, unsubstituted or substituted with above, or—C(O)—, said ring being unsubstituted or Sub 55 one to four R groups; C. cycloalkyl optionally substi stituted with one to four R groups; tuted with one to four R groups; phenyl optionally substi each R" independently represents halo; CN; NO; tuted with one to four R groups, or heteroaryl optionally NRR: OR8; SR3; CONR'R' COORS: substituted with 1-4 R groups; or R' and R” taken together SOR8; SOR8; SONR'R''. NRSOR: with any intervening atoms representa 5-6 membered satu COR; NRCOR: OCOR: OCONRR1 NR 60 rated ring, optionally interrupted by one or two of O, S, CONR/R3; NRCOR: OCOR: C(NR)NR'Rs: SO, NH or NCH, NRC(NH)NR'R's or NRC(NR)Rs: R represents hydrogen or a Cs straight- or branched chain each R, RandR independently represents hydrogen; R"; alkyl, optionally interrupted by one or two of O, S, SO, —C straight- or branched-chain alkyl unsubstituted or SO, NR", N'R"R", or—C(O)—, said chain being unsub substituted with one to four R groups: 65 stituted or substituted with one to four of halo, CN, NO, or R and R taken together with any intervening atoms rep OR', SR", SOR", SOR", NR'R'', N“(R).R", C(O) resent a 4-6 membered Saturated ring optionally inter R", C(O)NR'R'', SONR'R'', COR", OC(O)R", OC(O) US 8,232,268 B2 15 16 NR'R'', NRC(O)R", NRC(O)NR'R'', or a phenyl or membered Saturated ring, optionally interrupted by one or heteroaryl group which is in turn optionally substituted two of O, S, SO,-C(O) , NH and NCH: with from one to four R groups or with one to two C Q is selected from the group consisting of: straight- or branched-chain alkyl groups, said alkyl groups being unsubstituted or substituted with one to four R' groups; N. N R'' and R represent hydrogen; phenyl; —C straight or GM GA branched chain alkyl, unsubstituted or substituted with one - St.\ 25 O - S R\-is to four R groups, and optionally interrupted by O, S, NR", l C. NR'R' or C(O) ; 10 or R and R together with any intervening atoms represent a wherein. 4-6 membered saturated ring optionally interrupted by O. a and b are 1, 2 or 3: S, SO, NR", NR'R' or C(O)—, unsubstituted or sub L is a pharmaceutically acceptable counterion; stituted with 1-4 R groups, 15 C. represents O, S or NR; and when R and R together represent a 4-6 membered ring B, 8, w, u, and O represent CR, N or NR, provided that no as defined above, R is as defined above or R represents an more than one of B, 8, , u, and O is NR; additional Saturated 4-6 membered ring fused to the ring each R independently represents hydrogen; phenyl or C. represented by R and R” taken together, optionally inter straight- or branched-chain alkyl, unsubstituted or Substi rupted by O, S, NR" or —C(O)—, said rings being unsub tuted with one to four R groups; stituted or substituted with one to four R groups. each R independently represents hydrogen; halo; phenyl: In one sub-embodiment, of Formula I, CN; NO; NR'R'; OR"; - SR"; CONR'R'': R is lower alkyl: COOR"; SOR"; SOR"; SONR'R'': R" is lower alkyl: NR'SOR: COR"; NR"COR'; OCOR'; 25 OCONR'R'': NRCOR'; NR''CONR'R'': M is H or a group such that COM represents a carboxylic —OCOR'; —C straight- or branched-chain alkyl, acid protected by a protecting group; unsubstituted or substituted with one to four R groups; P is hydroxyl or hydroxyl protected by a hydroxyl protecting group; R" and R' represent hydrogen or—C, straight- or branched 30 chain alkyl, unsubstituted or substituted with one to four R' Y' is C straight- or branched-chain alkyl that are substi groups; tuted or unsubstituted with one to four R groups and with each R" independently represents hydrogen; —C straight or without saturation (double or triple bonds); and or branched-chain alkyl, unsubstituted or substituted with Y is independently selected from CN:-NR'R'':-C(O) one to four R groups. NR'R'': C(O)OR"; NRSOR: C(O)R and -A- 35 In another embodiment, Q is selected from (CH2)-Q; wherein A represents —CH2—, in represents an integer 0, 1, 2 or 3: (CH2). each R" and R' independently represent hydrogen, —C. 40 straight- or branched-chain alkyl, unsubstituted or Substi Le (CH2) tuted with one to four R groups, or -C-7 cycloalkyl, unsubstituted or substituted with one to four R groups; or R" and R” taken together with any intervening atoms rep whereina, b, L. R. RandR areas originally defined. resent a 4-6 membered Saturated ring optionally inter 45 In one particular embodiment of the present invention, the rupted by one or more of O, S, NR, with R as defined carbapenem of the formula II, above, or—C(O)—, said ring being unsubstituted or Sub stituted with one to four R groups;

each R" independently represents halo; CN; NO; (II) NRR?: OR8; SR3; CONRR?: COOR8; 50 SOR8; SOR8; SONR'R''. NRSOR: COR; NRCOR?, OCOR: OCONRR1 NR CONR R5; NRCOR: OCOR'; C(NR)NR'R8; NRC(NH)NR'R8 or NRC(NR)R8; each R, RandR independently represents hydrogen; R"; 55 —C., straight- or branched-chain alkyl unsubstituted or substituted with one to four R groups: each R' independently represents halo. —CN; NO; phe or pharmaceutically acceptable salts and/or prodrugs thereof nyl; NHSOR: OR', SR; N(R): N*(R)3; is provided, wherein —C(O)N(R'); SON(R'): heteroaryl; heteroarylium; 60 R" is hydrogen or methyl, COR: C(O)R’; OCOR'; NHCOR": guanidinyl: P is hydrogen or hydroxyl, and carbamimidoyl or ureido; Y' is H or R, wherein R is H or C-C alkyl and Y is each R" independently represents hydrogen, a -C, a straight —(CH2)-A, wherein n=0-4 and A is CN, OR, SR, or branched-chain alkyl group, a C-C cycloalkyl group 65 N(R), COR, CO) N(R), CO) NRSON(R), or phenyl, or when two R' groups are present, said R' NRSON(R), NH C(=NR) N(R), S C groups may be taken in combination and represent a 4-6 (—NR) N(R), wherein each R is H or C-C alkyl. US 8,232,268 B2 17 18 In another particular embodiment of the present invention, R" is hydrogen or methyl, the carbapenem of the formula III, P is hydrogen or hydroxyl, Z is —O, S, N(R), and, each Y and Y independently represents hydrogen or (III) 5 —(CH2)-A, wherein n=0-4 and A= N(R), —CO.R. CONGR) –CO NRSON(R), NRSON(R), NH C(-NR) N(R), - S C(=NR) N(R), wherein each R is H or C-C alkyl, independently. In one subembodiment of Formula (V), P is hydroxyl, R' is methyl, Z is NH, and, each Y and Y independently repre 10 sents hydrogen or —(CH)-A, wherein n=0-2 and A= N COM (R), CONCR), CO. NHSONH, NHSONH, wherein R-hydrogen or methyl. In certain Subembodiments, the compounds of the inven or pharmaceutically acceptable salts and/or prodrugs thereof tion are those compounds shown in FIG. 2. is provided, wherein 15 In certain Subembodiments, the compound is compound 9: R" is hydrogen or methyl, P is hydrogen or hydroxyl, and Y is —(CH)-A, wherein n=0-4 and A is CN, OR, –SR, N(R), COR, C(=O) N(R), C(=O)– NRSON(R), NRSON(R), NH C(-NR) N(R), —S C(=NR) N(R), wherein each R is H or C-C, alkyl. In another particular embodiment of the present invention, CO2H the carbapenem of the formula IV. 25 or a pharmaceutically acceptable salt or prodrug thereof. In certain Subembodiments, the compound is compound 12:

(IV)

12 30 (CH2)-B

COM 35 or pharmaceutically acceptable salts and/or prodrugs thereof CO2H is provided, wherein R" is H or methyl, or a pharmaceutically acceptable salt or prodrug thereof. 40 In certain Subembodiments, the compound is compound P is hydrogen or hydroxyl, 15: n=0-4, and B is OR, SR, N(R), COR, CO. N(R), CO 15 NRSON(R), NRSON(R), NH C(-NR) N(R), —S C(=NR) N(R), wherein each R is H or C-C, 45 alkyl, independently. O In one subembodiment of Formula (IV), P is hydroxyl, R' is methyl, n=0, 1, and B is CONH, -CONHSONH, NHSONH, NH C(-NH) NH, S C(-NH) 50 NH. CO2H In another particular embodiment of the present invention, the carbapenem of the formula V. or a pharmaceutically acceptable salt or prodrug thereof. In certain Subembodiments, the compound is compound 55 18: (V)

18

60

65 or pharmaceutically acceptable salts and/or prodrugs thereof is provided, wherein or a pharmaceutically acceptable salt or prodrug thereof. US 8,232,268 B2 19 20 In certain Subembodiments, the compound is compound In certain Subembodiments, the compound is compound 21: 36:

21

36 N N1 NHSoNH,

CO2H 10 or a pharmaceutically acceptable salt or prodrug thereof. NHSONH In certain Subembodiments, the compound is compound 25:

15 25 or a pharmaceutically acceptable salt or prodrug thereof.

In certain Subembodiments, the compound is compound 39:

CO2H

39 or a pharmaceutically acceptable salt or prodrug thereof. 25 In certain Subembodiments, the compound is compound 28:

CONH2 28 30

or a pharmaceutically acceptable salt or prodrug thereof. 35 CO2H Process of Making Compounds or a pharmaceutically acceptable salt or prodrug thereof. The compounds of the present invention can be prepared In certain Subembodiments, the compound is compound 40 32: using the general process outlined in Scheme 1, below. Such as from carbapenem intermediate 5 illustrated in FIG. 3.

32 Reagents and Substrates used herein can be either purchased,

45 or synthesized according to known procedures. The present invention also is directed to an efficient syn thetic route to 3-methyl carbapenems from available precur CONHSONH sors with the option of introducing functionality as needed. 50 CO2H The process of synthesis is applicable to a wide range of oxygen and nitrogen linkers, as well as other heteroatom or a pharmaceutically acceptable salt or prodrug thereof. linkers, such as Sulfur and phosphorus. The carbapenems In certain Subembodiments, the compound is compound made according to the present invention can also be used as 34: 55 synthetic intermediates in the preparation of a variety of other B-methyl carbapenem analogs, as well as additional deriva 34 tives obtained by Subsequent functional group manipulations.

The invention also provides intermediates disclosed herein 60 that are useful in the preparation of compounds of the present invention as well as Synthetic methods for preparing the com NHSONH2 pounds of the invention. CO2H 65 In one embodiment of the invention, the carbapenem inter mediate is synthesized using Scheme 1, which is shown in or a pharmaceutically acceptable salt or prodrug thereof. greater detail in FIG. 3. US 8,232,268 B2 21 22

OP

OR

This carbapenem intermediate, containing an activated 25 b) coupling the compound of structure (A) with a moiety with —O(CO) R', wherein R' is an alkyl, such as —O(CO)2(i-Bu), a free hydroxyl. Such as an aromatic alcohol or a heteroaro to achieve coupling of the carbapenem to the heteroaromatic matic alcohol, or a mono- or di-substituted amine, Such as side chain, including a oxygen or nitrogen moiety, to produce a aromatic amine or heteroaromatic amine, to obtain an a 3-methyl carbapenem. B-methyl carbapenem; and then In one embodiment, a process for synthesizing a compound 30 c) optionally deprotecting the B-methyl carbapenem, if nec represented by formula I. essary. In one illustrative embodiment, the carbapenem interme diate (A), is the following compound (A*).

(I) 35

40 or a pharmaceutically acceptable salt thereof is provided. COP The present invention also is directed to methods of effi cient synthesis off-methyl carbapenems from available pre 45 The selection of reaction conditions should take into account cursors with the option of introducing functionality as the ease of substitution of the —O(CO)R in the carbapenem needed. Therefore, in one embodiment, the carbapenems are intermediate to form the desired carbapenem. synthesized using the process of the present invention. The process of synthesis is applicable to a wide range of In one embodiment, the method for preparing B-methyl oxygen and nitrogen linkers, as well as other heteroatom carbapenems includes: 50 linkers, such as Sulfur and phosphorus. The carbapenems a) preparing or obtaining a carbapenem intermediate of the made according to the present invention can also be used as structure (A), for example using the process of Scheme 1 synthetic intermediates in the preparation of a variety of other B-methyl carbapenem analogs, as well as additional deriva tives obtained by Subsequent functional group manipulations.

(A) 55 The invention also provides intermediates disclosed herein that are useful in the preparation of compounds of the present invention as well as Synthetic methods for preparing the com pounds of the invention. Suitable solvents for carrying out the processes of the 60 present disclosure are inert organic solvents, including but are not limited to, alcohols, aldehydes, amides, ethers, esters, OR halogenated solvents, hydrocarbons, glycols and glycol ethers, ketones, nitriles, and numerous other solvents com wherein mon in chemical processes, as well as mixtures of Such sol P. Rand R' are as defined above; 65 vents. These inert Solvents can be used alone or in combina P" is a Suitable carboxyl protecting group; and tion, and can be miscible or immiscible with each other, with R" is an alkyl or substituted alkyl; and then the proviso only that the compounds of interest are at least US 8,232,268 B2 23 24 partially soluble in the solvent or solvents used. In the cally modified to preclude undesired side reactions at the instance of using an immiscible, or 2-phase, solvent system, protected site. Suitable compounds for use with the com the process can also include the addition of a phase-transfer pounds of the present invention will be recognized from the agent. Suitable phase-transfer agents are known in the art, present application, and include those included in Such stan such as those described in Sasson, et al., Handbook of Phase dard reference texts known to those of skill in the art as Transfer Catalysis, Kluwer Academic Publishers, 1997. Greene, T. W. and Wuts, P. G. M., “Protective Groups in In one embodiment, the solvent is DMF, the reaction is Organic Synthesis, Third Edition'. Wiley Interscience, New carried out at about room temperature, using 5 moleq. of Pd York (1999). Examples of suitable protecting groups include dba*CHC1, (tris(dibenzylideneaceton)-dipalladium(0)- but are not limited to silyl protecting groups, including tri chloroform adduct), 30 mol eq. of either P(OEt), no acid or 10 C. alkylsilyl groups (e.g., trimethylsilyl and triethylsilyl), base. In certain cases, 0.5 eq. 2.6-lutidine can be added to diphenyl siloxy groups (e.g., t-butyldiphenylsilyl (TBDPS)), increase the rate of reaction. In certain embodiments, 0.5eq. C. alkylsilyloxy groups (e.g., tert-butyldimethylsilyl (TB PTSA are added. DMS)), substituted and unsubstituted benzyl groups (e.g., In one embodiment, the reaction is run with dippb (1,4-bis benzyl, benzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-ni (diphenylphosphine)butane) or triethyl phosphate. 15 trobenzyloxycarbonyl), carbonyls, such as 2.2.2-trichloroet In one embodiment, the reaction is run in the absence of a hyloxycarbonyl (Troc), allyloxycarbonyl (Alloc), and fluore base. In another embodiment, the reaction is carried out (run) nylmethyloxycarbonyl (Fmoc). in the absence of an acid. In a separate embodiment the The processes of preparing the compounds of the present reaction is carried out in 2.6-lutidine or p-toluenesulfonic invention are Suitably conducted at a temperature in a range of acid monohydrate. from about -78° C. to the boiling point of the reaction For the purpose of the present invention, the inert organic medium or solvent (e.g., from about -78°C. to about 200° Solvents suitable for use in preparing the compounds C.), and are typically conducted at a temperature in a range of described and claimed herein include but are not limited to from about -50° C. to the boiling point of the reaction aromatic solvents, such as benzene, toluene, chlorobenzene, medium or solvent. In one embodiment, the temperature is in styrene, tetraline, biphenyl, and Xylenes; ether solvents, such 25 a range of from about -20° C. to the boiling point of the as diethyl ether, n-butyl ether, methyl tert-butyl ether, tetrahy reaction medium or solvent. In another embodiment, the tem drofuran (THF), and 1,4-dioxane; halogenated solvents. Such perature is in the range of from about -10°C. to the boiling as chloroform, bromoform, carbon tetrachloride, dichlo point of the reaction medium or solvent. romethane, dichloroethane, trichloroethane, dichloroben The reactants used in the presently disclosed process can Zene, and chlorobenzene: alcohols, including C-Co 30 be added to the reaction vessel (also referred to herein as the alkanols, which can be linear, branched, or cyclic, and may be reaction “pot', or “round bottom) concurrently, either saturated or unsaturated, including methanol, ethanol, 2-pro together or separately, or they can be added sequentially in panol, butanol and hexanol; C-C hydrocarbon solvents, either order. which can be linear, branched, or cyclic, and may be saturated or unsaturated, including hexane, heptane, cyclohexane, 35 Method of Treatment cyclohexene, and pentane; ester and ketone solvents, such as acetone, ethyl acetate, isopropyl acetate, methylbutyl ketone The present invention also provides a method of preventing (2-hexanone), methyl ethyl ketone (MEK), methylisobutyl or treating a bacterial infection, in a host, for example an ketone (MIBK), methyl n-butyl ketone (MBK), methyl iso animal, and typically a human, including administering a propyl ketone, and cyclohexanone; and nitrogen-containing 40 therapeutic amount of a compound of the present invention, Solvents, including acetonitrile, nitromethane, N,N-dimeth or a pharmaceutically acceptable salt and/or prodrug therein, ylformamide (DMF), dimethylacetamide (DMA), hexameth optionally in a pharmaceutically acceptable carrier or diluent ylphosphoramide (HMPA), N-methylpyrrolidinone (NMP), where the bacterial infection is due to a gram-negative bac N,N'-dimethylpropylene urea (DMPU), 1,3-dimethyl-2-oxo teria. In one embodiment, the bacterial infection is a drug hexahydropyrimidine, and N-ethylpyrrolidinone. 45 resistant and/or multiple-drug resistant bacterial infection. Bases Suitable for use in conducting certain of the synthetic The invention also provides a compound of the present transformations described and claimed herein include but are invention for use in medical therapy. not limited to carbonates, including alkali metal carbonates The present invention also provides a use of a therapeutic and bicarbonates, such as Sodium carbonate, Sodium bicar amount of a compound of the present invention, or a pharma bonate, potassium carbonate, rubidium carbonate, and 50 ceutically acceptable salt and/or prodrug therein, optionally cesium carbonate; alkaline earth metal carbonates, such as in a pharmaceutically acceptable carrier or diluent, for pre magnesium carbonate, calcium carbonate, and strontium car venting or treating a gram-negative bacterial infection, in a bonate; hydroxides. Such as Sodium hydroxide and potassium host, such as an animal, and typically a human. hydroxide; and transition metal bases, such as Zinc hydrox The distinctive feature of gram-negative bacteria is the ide. Also suitable for use as bases in the transformations 55 presence of a double membrane Surrounding each bacterial described herein are organic bases, including but not limited cell. Although all bacteria have an inner cell membrane, to triethylamine (TEA); diethylamine; diisopropylamine; gram-negative bacteria have a unique outer membrane. This N,N-diisopropylethylamine (DIPEA or DIEA, also known as outer membrane excludes certain drugs and antibiotics from Hunig's base); dimethylamine; benzylamine, 4-dimethy penetrating the cell, partially accounting for why gram-nega laminopyridine (DMAP); ureas, such as tetramethylurea 60 tive bacteria are generally more resistant to antibiotics than (TMU); pyridine; 2.6-lutidine; imidazole; pyrrole; dipheny are gram-positive bacteria. The pathogenic capability of lamine; tri-n-propylamine; cyclohexylamine; tripheny gram-negative bacteria is usually associated with certain lamine: pyrrolidine: ureas, such as tetramethylurea (TMU); components of their cell walls, particularly the lipopolysac and piperidine. charide (endotoxin) layer. The outer membrane of gram As defined above, when a functional group is termed to be 65 negative bacteria is rich in lipopolysaccharide. If gram-nega “protected with a “protecting group’ (herein represented by tive bacteria enter the bloodstream, lipopolysaccharide can the letter designation, P), this means that the group is chemi trigger a cascade of events, including high fever and a drop in US 8,232,268 B2 25 26 blood pressure. Unlike Gram-positive bacteria, which evaluated using standard analytical techniques of protein bio assume a violet color in Gram staining, Gram negative bac chemistry, such as, for example, using an activity assay for a teria incorporate the counterstain rather than the primary bacterial enzyme, or using Western blotting or quantitative stain. Because the cell wall of Gram(-) bacteria is high in gel electrophoresis for a bacterial protein. Bacterial nucleic lipid content and low in peptidoglycan content, the primary acid levels in a cell can be evaluated using standard analytical crystal-violet escapes from the cell when the decolorizer is techniques such as Northern blotting and Southern Blotting added. Most enteric (bowel related) illnesses can also be or quantitation by polymerase chain reaction (PCR). attributed to this group of bacteria. Examples of gram-negative bacteria include Aeromonas Combination and Alternation Therapies sp., Acinetobacter sp. Such as Acinetobacter baumannii (or A. 10 calcoaceticus), Actinobacillus actinomycetemcomitans, In one embodiment of the invention, one or more therapeu Bacteroides sp. Such as Bacteroides fragilis, Bartonella, tic agents, including particularly antimicrobial agents such as Bdellovibrio spp., Bordetella pertussis, Brucella sp., antibiotic agents that are effective against gram negative bac Burkholderia cepacia, Burkholderia, pseudomalilei, Campy teria, can be used in combination and/or alternation with the lobacter sp., Capnocytophaga sp., Cardiobacterium hominis, 15 Chlamydia trachomatis, Citrobacter sp., Eikenella cor compound/composition of the present invention to achieve a rodens, Enterobacter sp., Escherichia coli, Francisella tulla additive and/or synergistic therapeutic effect. rensis, Flavobacterium sp., Fusobacterium sp., Helicobacter The active compounds can be administered in combina pylori, Haemophilus influenzae, Haemophilus ducreyi, Kleb tion, alternation or sequential steps with another anti-bacte siella spp. Such as Klebsiella pneumoniae, Kingella kingae, rial agent. In combination therapy, effective dosages of two or Legionella spp. Such as Legionella pneumophila, Moraxella more agents are administered together, whereas in alternation catarrhalis, Morganella, Neisseria gonorrhoeae, Neisseria or sequential-step therapy, an effective dosage of each agent meningitidis, Pasteurella pestis, Pasteurella multocida, Ple is administered serially or sequentially. The dosages given Siomonas Shigelloides, PrevOtella sp., Proteus spp., Provi will depend on absorption, inactivation and excretion rates of dencia, Pseudomonas spp. Such as Pseudomonas aeruginosa, 25 the drug as well as other factors known to those of skill in the Salmonella spp. Such as Salmonella enteriditis and Salmo art. It is to be noted that dosage values will also vary with the nella typhi, Serratia marcescens, Shigella spp., Vibrio chol severity of the condition to be alleviated. It is to be further erae, Vibrio parahaemolyticus, Vibrio vulnificus, Veillonella understood that for any particular Subject, specific dosage sp., Xanthomonas maltophilia or Stenotrophomonas malto regimens and schedules should be adjusted over time accord phila, Yersinia pestis, Yersinia enterocolitica. Additionally, 30 ing to the individual need and the professional judgment of Some organisms simply tend not to be well differentiated by the person administering or Supervising the administration of gram staining, despite any known phylogenetic affiliation the compositions. In some embodiments, an anti-bacterial with the gram-negatives or gram-positives. Rickettsia agent that exhibits an ECs of 10-15 uM or less, or typically prowazekii, Rickettsia rickettsii and Treponema pallidum. less than 1-5 uM. Chlamydias are Small, gram-negative, peptidoglycan-less 35 In one particular embodiment, the combination includes a cocci that are obligate intracellular parasites of animals. Spi B-lactamase inhibitor, such as clavulanic acid, which has rochetes are chemoheterotrophic bacteria whose cells are been used as in the delivery of prophylactic amounts of anti tightly coiled or resemble a stretched spring with gram-nega biotics in patients. Although Clavulanic acid does have some tive-like cell envelopes. Spirochetes include Spirillum minus, degree of bacterial activity, its principal role is as a beta Borrelia burgdorferi (Lyme disease), Leptospira spp. (lep 40 lactamase inhibitor. Clavulanic acid has a similar structure to to spirosis) and Treponema pallidum (syphilis). Rickettsias the beta-lactam antibiotics but binds irreversibly to the beta and actinomycetes are also gram-negative pleomorphic lactamase enzymes. Used in combination with the beta-lac bacilli and coccobacilli that are obligate intracellular para tam antibiotics, it has become one of the most prescribed sites of eucaryotes transmitted generally by insects and ticks. antibiotics in the western world prolonging the effective life The present invention also provides a use of a therapeutic 45 of antibiotics such as Ampicillin (as in GSK's AugmentinR). amount of a compound of the present invention, or a pharma It is possible that drug-resistant variants of bacteria can ceutically acceptable salt and/or prodrug therein, optionally emerge after prolonged treatment with an anti-bacterial in a pharmaceutically acceptable carrier or diluent, in the agent. The efficacy of a drug against the bacterial infection manufacture of a medicament for preventing or treating a can be prolonged, augmented, or restored by administering gram-negative bacterial infection, in a host, such as an ani 50 the compound in combination or alternation with a second, mal, and typically a human. and perhaps third, anti-bacterial agent, for example with a The invention also includes methods of inhibiting bacterial different site of activity than the principle drug. Alternatively, infection in a host. Inhibition of bacterial replication or treat the pharmacokinetics, biodistribution or other parameter of ment of an infection in a cell can be measured by showing a the drug can be altered by Such combination or alternation reduction in bacterial replication in a cell to a levellower than 55 therapy. the level in an otherwise identical cell, which was not admin Suitable antibiotic agents are disclosed, e.g. in Physicians istered the compound of the invention. The reduction can be Desk 30 Reference (PDR), Medical Economics Company by about 80%, 85%, 90%. 95%, about 99.9% or more. The (Montvale, N.J.), (53rd Ed.), 1999; Mayo Medical Center level of bacterial replication in a cell can be assessed by any Formulary, Unabridged Version, Mayo Clinic (Rochester, known methods. For example, the level of bacterial replica 60 Minn.), January 1998; Merck Index An Encyclopedia of tion in a cell can be assessed by evaluating the number of Chemicals, Drugs and Biologicals, (11th Ed.), Merck & Co., bacterial particles or amount of a bacterial component. Such Inc. (Rahway, N.J.), 1989; University of Wisconsin Antimi as a bacterial protein, a bacterial enzyme, or bacterial nucleic crobial Use Guide, http://www.medsch.wisc.edu/clinsci/ acid, in the cell or in fluid or debris associated with the cell. 5amcgfamcg.html; Introduction on the Use of the Antibiotics The number of infectious bacteria in a cell can be evaluated, 65 Guideline, of Specific Antibiotic Classes, Thomas Jefferson for example, in a plaque assay. The level of a bacterial com University. http://jeffine.tu.edu/CWIS/OAC/antibiotics ponent such as a bacterial protein or enzyme in a cell can be guide/intro.html; and references cited therein. US 8,232,268 B2 27 28 Nonlimiting examples of agents that can be used in com ytetracycline HCl); Achromycin V capsules5 (tetracycline bination or alternation with the compounds of the invention HCl); Linco-mycins; and Cleotin HCl (clindamycin HCl); include: aminoglycosides, B-lactam antibiotics, cephalospo Abelcet (amphotericin B lipid complex); AmBisome (am rius, macrollides, miscellaneous antibiotics, penicillins, tetra photericin B); Amphotec (amphotericin B cholesterol sulfate cyclines, antifungals, antimalarial agents, antituberculosis complex); Ancobon (flucytosine); Diflucan (fluconazole); agents, antibacterials, leprostatics, miscellaneous anti-infec Fulvicin P/Gamma (ultramicrosize griseofulvin); Fulvicin tives, quinolones, Sulfonamides, urinary anti-infectives, nasal P/G 165 and 330 (ultramicrosize griseofulvin); Grifulvin V antibiotics, ophthalmic antibiotics, ophthalmic antibacteri (griseofulvin); Gals-PEG (gxiseofulvin ultramicrosize): als, ophthalmic quinalones, ophthalmic sulfonamides, skin Lamisil (terbinafine hydrochloride); Nizoral (ketoconazole); and mucous membrane antibiotics, skin and mucous mem 10 Amphotericin B; Lotrimin (clotrimazole); Dapsone tablets brane antifungals, skin and mucous membrane antibacterials, (dapsone); Diflucan (fluconazole); Monistat-Derm cream skin and mucous membrane miscellaneous anti-infectives, (miconazole); Mycostalin Crc.am (nyStatin); and Sporanox skin and mucous membranes cabicides and pedulicides, skin (itraconazole); Aralen hydrochloride (chloroquine HCl); and mucous membrane antineoplasts, nitrofurans and oxazo Aralen phosphate (chloroquine phosphate); Dataprim (py lidinones. 15 rimethamine); Ladam (mefloquine HCl); and Plaquenil (hy Specific compounds include, for example, Amikacin (ami droxychloroqnine Sulfate); Capastat Sulfate (capreomycin kacin Sulfate); Craramyein (gentamicin Sulfate); Nebcin (to sulfate); Myambutol (ethambutol hydrochloride); Mycobutin bramycin sulfate); Netromycin (netilmicin sulfate); Strepto (rifabutin capsules); Nydrazid (isoniazid injection); Paser mycin Sulfate; and TOBI (tobramycin), AZactam (aminosalicylic acid); Prifiin (rifapentine); Pyrazinamide tab (aztreonam): Cefotan (cefotetan); Lorabid (loracarbef); lets (pyrazinamide); Rifadin (rifampin capsules); Rifadin IV Mefoxin (cefoxitin); Merrem (meropenem); and Primaxin (rifampin for injection); Rifamate (rifampin and isoniazid); (imipenem and cilastatin for injectable Suspension); Ancef Rifater (rifampin, isoniazid and pyrazinamide); Seromycin (cefazolin); Ceclor (cefaclor); Cedax (ceffibuten); Cefizox (cycloserine capsules); Streptomycin-Sulfate: Tice BCG (ceffizoxime sodium); Cefobid (cefoperaZone sodium); Cef (BCG vaccine); Cycloserine (seromycin capsules); Urised tin (cefuroxime axetil); Cefail (cefprozil); Ceptaz (ceftazi 25 (Methenamine); and Trecator-SC (ethionamide tablets); Alf dime); Claforan (cefotaxime); Duricef (cefadroxil monohy eron N (interferon alfa-n3); Crixivan (indinavir sulfate); drate); Fortaz (ceftazidime); Keflex (cephalexin); Keftab Cytovene (ganciclovir); Cytovene-IV (ganciclovir Sodium); (cephalexin HCl); Kefurox (cefuroxime); Kefaol (cefazolin); Epivir (lamivudine); Famvir (famciclovir); Flumadine Mandol (cefamandole nafate); Maxipime (cefepime HCl); (rimantadine HCl); Foscavir (foscarnet sodium); Hivid (zal Monocid (cefonicid sodium); Omnicef (cefdinir); Rocephin 30 citabine); Intron A (interferon alfa-2b); Invirase (saquinavir (ceftriaxone); Suprax (cefixime); Tazicef (ceftazidime); Tazi mesylate); Norvir (ritonavir); Rebetron combination therapy, dime (ceftazidime); Vantin (cefpodoxime proxetil); and which contains Rebetrol (ribavirin) and Intron A (inteferon ZinacefS (cefuroxime); Biaxin (clarithromycin); Dynabac alfa-2b); Rescriptor (delavirdine mesylate); Retrovir (Zidu (dirithromycin); E.E.S. 200 (Erythromycin Ethylsuccinate); Vudine); Retrovir IV (Ziduvudine); Symmetrel (amantadine E.E.S. 400 (Erythromycin Ethylsuccinate); Ery-Ped 200 35 HCl); Synagis (palivizumab); Valtrex (valacyclovir HCl); (Erythromycin Ethylsuccinate); EryPed 400 (Erythromycin Videx (didanosine); Viracept (nelfinavir mesylate); Viramune Ethylsuccinate); Ery-Tab (Erythromycin delayed-release tab (nevirapine); Virazole (ribavirin); Vistide (cidofovir); Zerit lets): Erythrocin Stearate (Erythromycin stearate); Ilosone (stavudine (d4T)): Symmetrel Syrup (amantadine HCl); (erythromycinestolate); PCE Dispertab (erythromycin par Combivir Tablets (lamiduvine); and Zovirax (acyclovir); ticles in tablets); Pediazole (erythromycin ethylsuccinate and 40 Dapsone Tablets (dapsone); Daraprim (pyrimethamine); Sulfisoxazole acetyl for oral Suspension); Tao (troleandomy Flagyl 375 (metronidazole); Flagyl ER Tablets (metronida cin), Zithromax (azithromycin); and Erythromycin; Cleocin zole); Flagyl I.V. (metronidazole); Furoxone (furazolidone); HCl (clindamycin hydrochloride); Cleotin Phosphate (elin Mepron (atovaquone); and Neutrexin (trimetrexate glucur damycin phosphate); Coly-Mycin M (colistimethate onate); Cipro (ciprofloxacin HCl); Floxin (ofloxacin); Leva sodium); and Vancocin HCl (vancomycin hydrochloride); 45 quin (levofloxacin); Mazaquin (lomefioxacin HCl); Noroxin Amoxil (amoxicillin); Augmentin (amoxicillin/clavulanate (norfloxacin); Penetrex (enoxacin); Raxar (grepafloxacin potassium); Bicillin C-R 900/300 (Penicillin G benzathine HCl); Trovan (trovafloxacin mesylate); and Zagam (spar and Penicillin G procaine suspension); Bicillin C-R (Penicil floxacin); Bactrim. (trimethoprim and Sulfamethoxazole); lin G benzathine and Penicillin G procaine suspension); Bactrim DS (Irimethoprim and sulfamethoxazole double Bicillin L-A (Penicillin G benzathine suspension); Geoeillin 50 strength); Pediazole (erythromycin ethylsuccinate and (carbencillin indanyl sodium); Mezlin (sterile mezlocillinso Sulfisoxazole acetyl); Septra (trimethoprimand Sulfamethox dium); Omnipen (ampicillin); Pen-Vee K (penicillin V potas azole); Septra DS (trimethoprim and sulfamethoxazole); Co sium); Pfizerpen (penicillin G potassium); Pipracil (piperacil Trimoxazole, Sulfadiazine, Battrim I.V. Infusion (sul lin sodium); Speetrobid (bacampicillin-HCl); Ticar famethoxazole); Sulfapyridine and Pediazole (erythromycin (tiearcillin disodium); Timentin (ticarcillin disodium and cla 55 ethylsuccinate and Sulfisoxazole acetyl); Furadantin (nitro Vulanate potassium); Unasyn (ampicillin sodium/sulbactam furantoin); Macrobid (nitrofurantoin monohydrate macroc Sodium); Zosyn (piperacillin Sodium and taZobactam rystals); Macrodantin (nitrofurantoin macrocrystals); sodium); and Dicloxacillin Sodium; Achromycin V (tetracy Monurol Sachet (fosfomycin tromethamine); NegGram cline HCl); Declomycin (demeclo-cycline HCl); Dynacin Caplets (nalidixic acid); Septra (trimethoprim and Sul (minocylcine HCl); Minocin (minocycline hydrochloride); 60 famethoxazole); Septra DS (trimethoprim and sulfamethox Monodox (Doxycycline monohydrate capsules); Terramycin azole); Urised (a combination of the antisepticsmethenamine, (oxytetracyline); Vectrin (minocycline hydrochloride); methylene blue, phenyl salicylate, benzoic acid and parasym Vibramycin Calcium (doxycycline sodium); Vibramycin patholytics (atropine Sulfate) hyoscyamine); (oxytetracycline Hyclate (doxycycline hyclate); Vibramycin Monohydrate HCl, sulfamethizole and phenazopyridine HCl); (meth (doxycycline monohydrate); Vibra-Tabs (doxycycline-hy 65 enamine mandelate); Bactroban (mupirocin); Chloromycetin drate); Declomycin (demeclocycline HCl); Vibramycin ophthalmic (chloramphenical); Cortisporin (neomycin and (doxycycline); Dynacin (Minocyline HCl); Terramycin (ox polymyxin 3 Sulfates and hydrocortisone acetate cream): US 8,232,268 B2 29 30 Ilotycin (erythromycin ophthalmic ointment); NeoDecadron maceutically acceptable salts and prodrugs can be calculated (neomycin Sulfate-dexamethasone sodium phosphate); Poly based on the weight of the parent nucleoside to be delivered. trim (trimethoprim and polythyxin 3 sulfate ophthalmic If the salt or prodrug exhibits activity in itself, the effective solution); Terra-Cortril (oxytetracycline HCl and hydrocorti dosage can be estimated as above using the weight of the salt Sone acetate); Terramycin (oxytetracycline); and Tobral Dex or prodrug, or by other means known to those skilled in the (tobramycin and dexamethasone ophthalmic suspension and art. ointment); Vita-A ophthalmic ointment, (Vidatabine); (nor Optionally, the active ingredient should be administered to floxacin ophthalmic solution; Ciloxan ophthalmic Solution achieve peak plasma concentrations of the active compound and ointment (Ciprofloxacin HCl); and Ocuflox ophthalmic of from about 0.2 to 70 M, e.g., about 1.0 to 10 uM. This may Solution (ofloxacin), Blephamide ophthalmic ointment (Sul 10 beachieved, for example, by the intravenous injection of a 0.1 facetamide Sodium and prednisolone acetate); and Blepha to 5% solution of the active ingredient, optionally in saline, or mide ophthalmic Suspension (sulfacetamide Sodium and pre administered as a bolus of the active ingredient. The concen drdsolone acetate); A/T/S (erythromycin); Bactroban tration of active compound in the drug composition will (mupirocin); BenZamycin (erythromycin-benzoyl peroxide depend on absorption, inactivation and excretion rates of the topical gel); Betadine (povidone-odine); Cleotin T (clindamy 15 drug as well as other factors known to those of skill in the art. cinphosphate topical Solution); Clindets (clindamycin phos It is to be further understood that for any particular subject, phate pledgets); Cortispo?in (neomycin, polymyxin B Sul specific dosage regimens should be adjusted according to the fates and hydrocortisone acetate cream); Emgel (erythromy individual need and the professional judgment of the person cin); Erycette (erythromycin topical Solution); Garamycin administering or Supervising the administration of the com (gentamicin Sulfate); Klaron (sodium sulfacetamide lotion); positions, and that the concentration ranges set forth herein Mycostatin (nyStatin cream); Theramycin Z (erythromycin are exemplary only and are not intended to limit the scope or topical solution); T-Stat (erythromycin); Chloromycetin practice of the claimed composition. The active ingredient (chloramphenicol ophthalmic ointment); Cortisporin (neo may be administered at once, or may be divided into a number mycin and polymyxin B sulfates, bacitracin Zinc and hydro of Smaller doses to be administered at varying intervals of cortisone ophthalmic ointment); Ilotycin (erythromycin); 25 time. NeoDeeadron (neomycin Sulfate-dexamethasone sodium The compound is conveniently administered in unit any phosphate); Polytrim (trimethoprim and polymyxin B sul Suitable dosage form, including but not limited to one con fate); Terra-Cortril (oxytetracycline HCl and hydrocortisone taining 7 to 3000 mg. or 70 to 1400 mg of active ingredient per acetate); Terramycin (Oxytetracycline); Exelderm (Sulcona unit dosage form. A dosage of 50-1000 mg is optional. Zole nitrate); Fungizone (amphotericin B oral Suspension); 30 The active compound can be administered in a pharmaceu Lamisil (terbinafine hydrochloride cream); Loprox (ciclo tically acceptable carrier available in the art, and can be piroxolamine); Lotrimin (clotrimazole); Lotrisone (clotrima administered by a chosen route of administration. Pharma Zole and betamethasone diproprionate); Mentax (butenafine ceutical compositions can be prepared, packaged, or sold in a HCl); Monistat-Denn (miconazole nitrate); Mycelex (clotri variety of formulations which can be suitable for one or more mazole); Mycostatin (nystatin): Naffin (natti fine HCl); 35 routes of administration Such as, for example, oral, intrave Nizoral Ocetoconazole); Nystop (nystatin); Oxistat (oxicona nous, intramuscular, topical, Subcutaneous, rectal, vaginal, Zole nitrate); Selsun RX (2.5% selenium sulfide lotion); and parenteral, pulmonary, intranasal, buccal, ophthalmic, or Spectazole (econazole nitrate); Denavir (penciclovir cream): another route of administration. The active materials can be and Zovirax (acyclovir); Benzashave Coenzoyl peroxide); administered in liquid or solid form. Other contemplated Betadine (povidone-iodine); Betasept (chlorhexidine glucon 40 formulations include projected nanoparticles, liposomal ate); Cetaphil (soap substitute); Clorpactin WCS-90 (sodium preparations, resealed erythrocytes containing the active oxychlorosene); Dapsone Tablets (dapsone): Desquam-E ingredient, and immunologically-based formulations. Coenzoyl peroxide); Desquam-X (benzoyl peroxide); Hibi The active compound may be administered intravenously clens (chlorhexidine gluconate); Hibistat (chlorhexidine glu or intraperitoneally by infusion or injection. Solutions of the conate); Impregon (tetrachlorosalicylanilide 2%); Metro 45 active compound or its salts may be prepared in water or Cream (metronidazole); MetroGel (metronidazole); Noritate saline, optionally mixed with a non-toxic Surfactant. Disper (metronidazole); pH isoHex (hexachlorophene detergent sions may be prepared in glycerol, liquid polyethylene gly cleanser); Sulfacet-R (sodium sulfacetamide 10% and sulfur cols, triacetin, mixtures thereof, and in oils. Under ordinary 5%); Sulfamylon (materfide acetate); Tfiaz Coenzoyl perox conditions of storage and use, these preparations contain a ide); and Vanoxide-HC Coenzoyl peroxide hydrocortisone); 50 preservative to prevent growth of microorganisms. Acticin (permethrin); Elimite (permethrin); Eurax (crotami Pharmaceutical dosage forms suitable for injection or influ ton); Efudex (fluoro-uracil); Fluoroplex. sion may include sterile aqueous Solutions or dispersions or sterile powders comprising the active ingredient which are Pharmaceutical Compositions adapted for the extemporaneous preparation of sterile inject 55 able or infusible solutions or dispersions, optionally encap Hosts, including humans can be treated by administering to Sulated in liposomes. The ultimate dosage form is optionally the patient an effective amount of the active compound or a sterile, fluid, and stable under conditions of manufacture and pharmaceutically acceptable prodrug or salt thereof in the storage. The liquid carrier or vehicle may be a solvent or presence of a pharmaceutically acceptable carrier or diluent. liquid dispersion medium comprising, for example, water, The active materials can be administered by any appropriate 60 ethanol, a polyol (for example, glycerol, propylene glycol, route, for example, orally, parenterally, intravenously, intra liquid polyethylene glycols, and the like), vegetable oils, dermally, Subcutaneously, or topically, in liquid or Solid form. nontoxic glyceryl esters, and Suitable mixtures thereof. An optional dose of the compound for treatment of a bac For oral therapeutic administration, the active compound terial (Such as a gram negative bacteria) infection is about 1 to can be combined with one or more excipients and used in the 50 mg/kg, or 1 to 20 mg/kg, of body weight per day, more 65 form of ingestible tablets, buccal tablets, troches, capsules, generally 0.1 to about 100 mg per kilogram body weight of elixirs, Suspensions, syrups, wafers, and the like. Such com the recipient per day. The effective dosage range of the phar positions and preparations can contain at least 0.1% (W/w) of US 8,232,268 B2 31 32 active compound. The percentage of the compositions and number have a diameter less than 6 nanometers. The active preparations can, of course, be varied, for example from about ingredient can also be in the form of droplets of a solution or 0.1% to nearly 100% of the weight of a given unit dosage Suspension, for example those that have an average diameter form. The amount of active compound in Such therapeutically in the range from about 0.1 to about 200 nanometers. useful compositions is such that an effective dosage level will The formulations described herein as being useful for pull be obtained upon administration. monary delivery are also useful for intranasal delivery of a The tablets, troches, pills, capsules, and the like may also pharmaceutical composition of the invention. Anotherformu contain one or more of the following: binders, such as micro lation Suitable for intranasal administration is a coarse pow crystalline cellulose, gum tragacanth, acacia, corn starch, or der comprising the active ingredient and having an average gelatin; excipients, such as dicalcium phosphate, starch or 10 lactose; a disintegrating agent, such as corn starch, potato particle from about 0.2 to 500 micrometers. starch, alginic acid, primogel, and the like; a lubricant, such as A pharmaceutical composition of the invention may be magnesium Stearate or Sterotes; a glidant, such as colloidal prepared, packaged, or sold in a formulation Suitable for silicon dioxide; a Sweetening agent, Such as Sucrose, fructose, ophthalmic administration. For topical administration, the lactose, saccharin, or aspartame; a flavoring agent Such as 15 present compounds can be applied in pure form, i.e., as a peppermint, methylsalicylate, oil of wintergreen, or cherry liquid. However, typically, the compounds are administered flavoring; and a peptide antibacterial agent, such as envu to the skin as compositions or formulations, in combination virtide (FuzeonTM). When the unit dosage form is a capsule, it with a dermatologically acceptable carrier. Useful solid car can contain, in addition to materials of the above type, a liquid riers include finely divided Solids such as talc, clay, microc carrier, such as a vegetable oil or a polyethylene glycol. rystalline cellulose, silica, alumina, and the like. Useful liquid Various other materials may be present as coatings or to carriers include water, alcohols, glycols, and blends of two or otherwise modify the physical form of the solid unit dosage more of these, in which the present compounds can be dis form. solved or dispersed at effective levels, optionally with the aid In one embodiment, the active compounds are prepared of non-toxic Surfactants. Adjuvants such as fragrances and with carriers that will protect the compound against rapid 25 additional antimicrobial agents can be added to optimize elimination from the body, such as a controlled release for properties for a given use. The resulting liquid compositions mulation, including implants and microencapsulated delivery can be applied using absorbent pads, used to impregnate systems. Biodegradable, biocompatible polymers may be bandages or other dressings, or sprayed onto the affected area used. Such as ethylene vinyl acetate, polyanhydrides, polyg using pump-type or aerosol sprayers. lycolic acid, collagen, polyorthoesters, and polylacetic acid. 30 The compounds/compositions of the present invention are Methods for preparation of such formulations will be appar optionally administered in a controlled release formulation, ent to those skilled in the art. The materials may also be which can be a degradable or nondegradable polymer, hydro obtained commercially from Alza Corporation. gel organogel or other physical construct that modifies the Other formulations can also be developed. For example, bioabsorption, half life or biodegradation of the active the compounds can be administered in liposomal Suspensions 35 agent(s). The controlled release formulation can be a material (including liposomes targeted to infected cells with mono that is painted or otherwise applied onto the afflicted site, clonal antibodies to bacterial antigens). These may be pre either internally or externally. In one embodiment, the inven pared according to methods known to those skilled in the art, tion provides a biodegradable bolus or implant. The con for example, as described in U.S. Pat. No. 4.522,811. For trolled release formulation with appropriated selected imag example, liposome formulations may be prepared in a variety 40 ing agent can be used to coat a transplanted organ or tissue to of lipid(s) (such as Stearoyl phosphatidyl ethanolamine, prevent rejection. It can alternatively be implanted or other Stearoyl phosphatidylcholine, arachadoyl phosphatidylcho wise applied near the site of potential infection. line, and cholesterol). Thickeners such as Synthetic polymers, fatty acids, fatty A pharmaceutical composition of the invention may be acid salts and esters, fatty alcohols, modified celluloses, or prepared, packaged, or sold in a formulation Suitable for 45 modified mineral materials can also be employed with liquid rectal administration. Such a composition may be in the form carriers to form spreadable pastes, gels, ointments, soaps, and of for example, a Suppository, a retention enema preparation, the like, for application directly to the skin of the user. and a solution for rectal or colonic irrigation. A pharmaceu The compound or a pharmaceutically acceptable prodrug tical composition of the invention may also be prepared, or salts thereof can also be mixed with other active materials packaged, or sold in a formulation Suitable for vaginal admin 50 that do not impair the desired action, or with materials that istration. Such a composition may be in the form of, for Supplement the desired action, such as antibiotics, antifun example, a Suppository, an impregnated or coated vaginally gals, anti-inflammatories, or other antibacterials, including insertable material Such as a tampon, a douche preparation, or other nucleoside compounds. Solutions or Suspensions used a solution for vaginal irrigation. for parenteral, intradermal, Subcutaneous, or topical applica A pharmaceutical composition of the invention may be 55 tion can include the following components: a sterile diluent prepared, packaged, or sold in a formulation Suitable for Such as water for injection, Saline Solution, fixed oils, poly pulmonary administration via the buccal cavity. Such a for ethylene glycols, glycerine, propylene glycol or other syn mulation may comprise dry particles which comprise the thetic Solvents; antibacterial agents such as benzyl alcohol or active ingredient and which have a diameter in the range from methyl parabens; antioxidants such as ascorbic acid or about 0.5 to about 7 nanometers, or from about 1 to about 6 60 Sodium bisulfite; chelating agents such as ethylenediamine nanometers. Such compositions are conveniently in the form tetraacetic acid; buffers such as acetates, citrates or phos of dry powders for administration, which can include par phates and agents for the adjustment of tonicity Such as ticles wherein at least 98% of the particles by weight have a Sodium chloride or dextrose. The parental preparation can be diameter greater than 0.5 nanometers and at least 95% of the enclosed in ampoules, disposale Syringes or multiple dose particles by number have a diameter less than 7 nanometers. 65 vials made of glass or plastic. If administered intravenously, Typically least 95% of the particles by weight have a diameter useful carriers are physiological saline orphosphate buffered greater than 1 nanometer and at least 90% of the particles by saline (PS). US 8,232,268 B2 33 34 In one embodiment, the active compounds are prepared (doublet), t (triplet), q (quartet), bs or brs (broad singlet), dd with carriers that will protect the compound against rapid (doublet of doublet), and m (multiplet). UV spectra were elimination from the body, such as a controlled release for obtained on a Beckman DU 650 spectrophotometer. Mass mulation, including implants and microencapsulated delivery spectra were measured on a Micromass Inc. Autospec High systems. Biodegradable, biocompatible polymers can be Resolution double focussing sector (EBE) MS spectrometers. used. Such as ethylene vinyl acetate, polyanhydrides, polyg Infrared spectra were obtained on a Nicolet 510 FT-IR spec trometer. All reactions were monitored using thin layer chro lycolic acid, collagen, polyorthoesters and polylactic acid. matography on Analtech, 200 mm silica gel GF plates. Dry Methods for preparation of such formulations will be appar 1,2-dichloroethane, dichloromethane, acetonitrile, N,N-dim ent to those skilled in the art. The materials can also be ethylformamide, and THF were obtained by drying over 4A obtained commercially from Alza Corporation. 10 The concentration of the compound(s) in a liquid compo molecular sieves. sition, such as a lotion, will, for example, range from about ABBREVIATIONS 0.1% to about 95% by weight, or from about 0.5% to about 25% by weight. The concentration in a semi-solid or solid ACN: acetonitrile composition Such as a gel or a powder will, for example, range 15 DCE: 1,2-dichloroethane from about 0.1% to 100% by weight, or about 0.5% to about DCM; dichloromethane 5% by weight. Single doses for intravenous injection, Subcu DDQ: dichlorodicyano quinone taneous, intramuscular or topical administration, infusion, DIEA: diisopropylethylamine ingestion or suppository will generally be from about 0.001 to DIHO: deionized water about 5000 mg, and be administered from about 1 to about 3 DMAP: 4-dimethylamino pyridine times daily, to yield levels of about 0.01 to about 500 mg/kg, DMF: N,N-dimethyl formamide for adults. DPPB: 1,4-bis(diphenylphosphine)butane The invention also includes one or more compounds dis LAH: lithiumaluminum hydride closed herein, or any combination thereof, or salt thereof, in LHMDS: lithium hexamethyldisilazide an amount effective to inhibit bacterial (such as a gram nega 25 Pd/C: palladium on carbon tive bacteria) replication in a host. The compound can be PNB: p-nitrobenzyl useful for inhibiting bacterial replication in a cell or neutral PTSA: p-toluenesulfonic acid monohydrate ization (i.e. inactivation) of extracellular bacteria. TBAF: tetrabutylammonium fluoride As used herein, to inhibit bacterial replication in a host TBDMS: t-butyldimethylsilyl means to reduce the bacterial load in a host to a level which is 30 TEA: triethylamine lower than the level of the bacterial load in an otherwise TES: triethylsilyl identical host which was not administered the compound. TFA: trifluoroacetic acid Bacterial load in a mammal can be reduced by about 1 to 12 THF: tetrahydrofuran logo or more relative to an otherwise identical mammal TLC: thin layer chromatography which was not administered the compound. Bacterial load in 35 TBDPS: t-butyldiphenylsilyl a mammal can be assessed by a number of methods known in the art such as, for example, obtaining a tissue or fluid sample Preparation of the Carbapenem Intermediate (CPI) 5 from the mammal and assessing the amount of bacterial com ponents in the mammal contained therein using technology Carbapenem Intermediate (CPI) 5 was prepared according which is either immunological, biochemical or molecular 40 to the synthetic scheme shown in FIG. 3. In the first step of the biological in nature and which is well known to the skilled process, benzylpropionate is reacted with isobutoxycarbony artisan and which are described elsewhere herein. Inhibition loxyacetic acid methyl ester in a solvent at low temperature in of bacterial replication in a cell is assessed using similar or the presence of LDA to form ketoester A. The ketoester A is identical assays as those used to assess bacterial load in a then contacted with the acetoxyazetidinone B (prepared by mammal. 45 any number of known, synthetic routes) in a solvent, and The invention also includes a kit for administering a com Sodium carbonate is added. The reaction ages for a period of pound of the invention, a pharmaceutically acceptable salt time at a temperature Such that the reaction goes substantially thereof, or a pharmaceutical composition, to a host for treat to completion, generating the target lactam C. ment of a bacterial (such as a gram negative bacteria) infec The lactam C is dissolved in a solvent, such as DMF, to tion. Typically, the host is a human. The kit comprises one or 50 which a suitable base (such as DIEA) and TBSOTfare added, more compounds of the invention, or a combination thereof, and the mixture allowed to age for a period of time at a and optionally an instructional material, which describes temperature. Following workup, the bis-TBS-ketoester D is adventitially administering the composition to the mammal isolated. by any of the routes of administration described herein. In The crude ketoester D is dissolved in ethyl acetate in an another embodiment, this kit comprises a (typically sterile) 55 appropriate reaction vessel. Formic acid and a catalyst, Such Solvent Suitable for dissolving or Suspending the composition as Pd/C, are added to the reaction vessel, and the entire of the invention prior to administering the compound to the mixture is hydrogenated at an appropriate hydrogen pressure mammal. (40-50 psi) for a period of time such that the decarboxylation reaction proceeds to completion. The reaction mixture is fil EXAMPLES 60 tered over a pad of Celite(R), and the solvent is removed under vacuum. Product E is isolated following purification by col Melting points were determined on a Mel-temp II labora umn chromatography. tory device and are uncorrected. Nuclear magnetic resonance The bis-TBDMS ketolactam E is then de-silylated using 2 spectra were obtained on a GE 300 Plus (300 MHz), a Varian N HCl in ACN and the product is isolated after a standard INOVA 400 (400 MHz), and a Varian INOVA 600 (600 MHz) 65 aqueous workup. The crude product is dissolved in a solvent, spectrometer, chemical shifts (Ö) are reported in parts per such as DCM, and allowed to react with triethylsilyl chloride million (ppm), and the signals are described as S (singlet), d and imidazole for several hours (monitored by TLC) at rt. US 8,232,268 B2 35 36 Following aqueous workup, O-TES ketolactam F was iso -continued lated and purified on silica gel. OH N-PNB, O-TES ketolactam G is produced by reacting ketolactam F with p-nitrobenzyl oxalylchloride in a suitable solvent (DCM, for example) in the presence of a base (DIEA, 5 for example). The mixture is allowed to age for a period of time (and at an appropriate temperature) to effect a Substan tially complete reaction as monitored by an appropriate means (e.g., TLC or HPLC). Following workup in a usual manner, intermediate G was isolated. 10 To a solution of compound G is a suitable solvent was added triethylphosphite, and the mixture heated to reflux until Step 1: General Procedure for the Palladium complete by TLC. Following workup and purification in the Coupling Reaction appropriate manner, CPI 5 was isolated. 15 The Amine Series: Preparation of Gram-Negative Active Car Method A bapenems To a dry, round-bottom flask, anhydrous DMF (40 mL) was The amine series of 1-3-methylcarbapenem analogs pos added and the solvent was stirred and degassed at room tem sessing Gram-negative activity were synthesized using Syn perature with two cycles of nitrogen/vacuum. Pd(dba) thetic methods described above and as illustrated in Scheme CHCl (51.8 mg 0.05 mmol) and DPPB (64.0 mg, 0.15 2 below, unless otherwise noted. In general, a series of sec mmol) were then added. The solution was degassed with two ondary amines (2) were coupled to CPI 5 (FIG. 3) in DMF nitrogen/vacuum cycles and aged for 20 minutes. The appro using a combination of Pd(dba)-CHCl with DPPB (Method priate secondary amine (2) (1.1 mmol) was added and the A) or triethylphosphite (Method B) at it to produce the mixture was againdegassed under reduced pressure for 5 min. coupled intermediate 3. In some cases, 2.6-lutidine (Method 25 CPI 5 (590.7 mg, 1.0 mmol) was then added, the resulting C) or PTSA (Method D) were added to drive the reaction to mixture was again degassed with two nitrogen/vacuum completion. The secondary amines were either purchased cycles, and the resulting mixture was aged for 12-36 h (moni from commercial sources or prepared by alkylation of tored by TLC) at rt. The solvent was then removed under N-Boc-protected primary amines with various alkyl halides reduced pressure and the resulting residue was purified by followed by cleavage of the Boc protecting group with TFA/ 30 flash chromatography on silica gel to produce coupled inter water in DCM. mediate 3 in 50-98% yield. Removal of the TES protecting group in the series of inter Method B mediate 3 was accomplished with aqueous triflic acid solu tion in IPA/water (pH 2.4, Method E) or with 0.06N HCl in To a dry, round-bottom flask, anhydrous DMF (40 mL) was IPA/THF (Method F) at 0° C. to rt. 35 added and the solvent was stirred and degassed at room tem Lastly, the PNB group(s) in intermediate 4 were removed perature with two cycles of nitrogen/vacuum. Pd(dba) by hydrogenation of the corresponding PNB esters using CHCl (51.8 mg 0.05 mmol) and P(OEt) (24.9 mg, 0.15 standard conditions (atmospheric H. pressure, 5%Pt/C, THF/ mmol). were then added. The solution was degassed with two i-PrCH/0.5 M potassium phosphate buffer solution (pH 7.0) nitrogen/vacuum cycles and aged for 20 minutes. The appro at 0°C.) and the final products 6 were isolated after purifica 40 priate secondary amine (2) (1.1 mmol) was added and the tion on SP-207 resin. mixture was againdegassed under reduced pressure for 5 min. CPI 5 (590.7 mg, 1.0 mmol) was then added, the resulting mixture was again degassed with two nitrogen/vacuum SCHEME 2 cycles, and the resulting mixture was aged for 12-36 h (moni 45 tored by TLC) at rt. The solvent was then removed under R1 M reduced pressure and the resulting residue was purified by CPI + NH Hess flash chromatography on silica gel to produce coupled inter V mediate 3 in 50-98% yield. 5 R2 2 Method C 50 Used a similar procedure as was described in Method B except that 2.6-lutidine (0.5 eq) was added in conjunction with reagent 2. 55 Method D

Used a similar procedure as was described in Method B except that PTSA (0.5 eq) and 4 A molecular sieves were added in conjunction with the palladium catalyst. 60 Step 2: General Procedure for the Removal of the TES Protecting Group Method E 65 Preparation of the pH 2.4 aqueous/IPA triflic acid stock solution: 620 uL of triflic acid was added to a stirred solution US 8,232,268 B2 37 38 of DIHO (100 mL) and IPA (500 mL). Additional triflic acid (d. J=14.1 Hz, 2H), 5.21 (d. J=14.1 Hz, 2H), 4.29-4.20 (m, was added dropwise to adjust the pH to 2.4. TES-Intermediate 2H), 3.93 (d. J=15.0 Hz, 1H), 3.47-3.36 (m, 1H), 3.25 (dd, 3 (100 mg) was then added to 3 mL of the aqueous triflic J–4.8; 2.7 Hz, 1H), 3.14 (d. J=14.7 Hz, 1H), 2.78-2.69 (m, acid:IPA stock solution and the resulting mixture was stirred 1H), 2.64-2.56 (m. 1H), 2.51-2.47 (m, 2H), 2.25 (s.3H), 1.24 at rt. The reaction pH was then adjusted to 2.4 by the incre 5 (d. J=7.2 Hz, 3H), 1.18 (d. J–7.5 Hz, 3H), 0.93 (t, J=7.8 Hz, mental addition of triflic acid stock solution, if necessary. The 9H), 0.59 (q, J–7.5 Hz, 6H). reaction mixture was stirred at rt until complete by TLC (-2 h), neutralized with 0.5M potassium phosphate buffer solu PNB-Protected Cyanoethylamine CP Intermediate 8 tion (pH 7.0, 5-10 mL), and the resulting mixture was stirred for 10 min and then extracted with ethyl acetate. The organic 10 layer was washed with brine, dried over anhydrous sodium

Sulfate, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel using as eluent hexane/ethyl acetate with gradient or ethyl acetate/methanol with gradient to produce alcohol 4 in 15 67-92% yield. Method F COPNB Alcohol 4 (100 mg) was dissolved in THF/IPA (2 mL/2 mL) and cooled to 0°C. 0.06N HCl solution was then added Method E: Percent yield: 75%; H NMR (CDC1, 300 (1-3 mL. pH adjusted to 2.4) and the resulting mixture was MHz): 88.16 (d. J=9.3 Hz, 2H), 7.60 (d. J=8.4 Hz, 2H), 5.43 aged at 0°C. for 2-18h (monitored by TLC). The reaction was (d. 14.4 Hz, 2H), 5.15 (d. J=14.4 Hz, 2H), 4.24-4.18 (m, 2H), neutralized with 0.5M potassium phosphate buffer solution 3.90 (d. J=14.7 Hz, 1H), 3.50-3.40 (m, H), 3.26 (dd, J=6.6;3.0 (pH 7.0, 5-10 mL), and the resulting mixture was carried on to 25 HZ, 1H), 3.11 (d. J=14.7 Hz, 1H), 2.86 (brs, 1H), 2.75-2.66 Step 3 without further isolation or purification. (m. 1H), 2.60-2.52 (m, 1H), 2.50-2.45 (m, 2H), 2.23 (s.3H), 1.28 (d. J=6.9 Hz, 3H), 1.15 (d. J–7.5 Hz, 3H). Step 3: General Procedure for the Removal of the PNB Protecting Group Cyanoethylamine CP Analog 9 30 To a round-bottom flask equipped with a side arm were added alcohol intermediate 4 (200mg) and THF/IPA (3 mL/3 mL), and the resulting mixture was stirred at rt. Aqueous 0.5 M potassium phosphate buffer solution (pH 7.0, 6 mL), was then added and the resulting mixture was cooled to 0°C.5% 35 Pt/C (40 mg) was added and the reaction flask was fitted with a hydrogen balloon, degassed under reduced pressure, and charged with hydrogen. The resulting mixture was aged for 4-14 hrs. (monitored by TLC) at 0°C., diluted with chilled CO2H ethyl acetate (20 mL) and DI HO (10 mL), and filtered over 40 a pad of Celite. The celite was washed with chilled DI HO (30 mL) and ethyl acetate (30 mL), the layers separated, and Percent yield: 50%; H NMR (DO, 400 MHz): 8 4.22 the aqueous layer was extracted with cold ethyl acetate (30 4.15 (m, 2H), 3.75 (d. J=14.4 Hz, 1H), 3.42-3.39 (m, 2H), mL). The aqueous fraction was then concentrated under 3.24-3.16 (m. 1H), 3.05-2.98 (m, 1H), 2.83-2.72 (m, 3H), reduced pressure to remove any organics and lyophilized. The 45 2.38 (s.3H), 1.23 (d. J=6.4 Hz, 3H), 1.08 (d. J–7.6 Hz, 3H). crude material was purified on SP-207 resin with IPA/DIHO and the column fractions concentrated under reduced pres Example 2 sure at 10°C. to remove i-PrCH and then lyophilized to give the desired, final product 6 as fluffy solids. TES-Protected N-Methyl Acetamide CP 50 Intermediate 10 Example 1 TES-Protected Cyanoethylamine CP Intermediate 7 10

55

60 COPNB Method A: Percent yield: 98%; H NMR (CDC1, 300 COPNB MHz): 8 8.20 (d. J=8.1 Hz, 2H), 7.65 (d. J=8.4 Hz, 2H), 6.87 65 (brs, 1H), 6.32 (brs, 1H), 5.43 (d. J=14.1 Hz, 1H), 5.21 (d. Method A: Percent yield: 98%; H NMR (CDC1, 300 J=14.1 Hz, 1H), 4.29-4.19 (m, 2H), 4.03 (d. J=14.7 Hz, 1H), MHz): 8 8.20 (d. J=9.0 Hz, 2H), 7.66 (d. J=8.4 Hz, 2H), 5.44 3.33-3.23 (m, 2H), 3.14 (14.1 Hz, 1H), 3.07 (d. J=17.1 Hz,

US 8,232,268 B2 55 56 Step I: General Procedure for Mesylate Formation 2H), 4.06 (d. J=14.7 Hz, 1H), 3.26-3.11 (m, 5H), 2.82-2.73 (m. 2H), 2.26 (s.3H), 1.22 (d. J=6.3 Hz, 3H), 1.13 (d. J–7.5 Methanesulfonyl chloride (1.3 mmol) and triethylamine Hz, 3H), 0.91 (t, J–7.5 Hz, 9H),057 (q, J=7.5 Hz, 6H). (1.5 mmol) were added at 0°C. under nitrogen atmosphere to a solution of alcohol intermediate 55 or 58 (1.0 mmol) in dry PNB-Protected N-Methyl Thioguanidine CP DCM (15 mL). The reaction mixture was stirred at 0°C. for Intermediate 61 1 hand then at 5°C. for 15 minutes (monitoring by TLC). The reaction mixture was then washed with aq. NaHCO, water, brine, and dried over NaSO. Evaporation of the solvent 10 61 gave crude meSylate, which was immediately used without purification. NH Step II: General Procedure for Mesylate Displacement by Amine Sidechains NH2 15 To a solution of freshly prepared mesylate (1.0 mmol) in dry DMF (15 mL) at 0°C. under Natmosphere was added a COPNB solution of thiourea (1.5 mmol) in dry DMF (3 mL) and the resulting mixture was stirred at 0°C. for 1 h and then allowed Method E; Percent yield:90%; H NMR (acetone-d, 300 to warm to rt and stirred overnight (monitored by TLC). The MHz): 89.60 (brs, 1H), 8.61 (brs, 2H), 8.22 (d. J=8.4 Hz, reaction mixture was then evaporated under reduced pressure 2H), 7.79 (d. J=8.4 Hz, 2H), 5.52 (d. J=14.1 Hz, 1H), 5.30 (d. and residue was purified by flash chromatography. The last J=14.1 Hz, 1H), 4.29 (dd, J=10.5:3.0 Hz, 1H), 4.16-4.08 (m, two deprotection steps were performed as previously 1H), 4.03 (d. J=14.7 Hz, 1H), 3.48-3.31 (m, 5H), 2.94-2.87 described. (m. 2H), 2.33 (s.3H), 1.23 (d. J–7.2 Hz, 3H), 1.19 (d. J–7.2 25 Hz, 3H). Example 19 N-Methyl Thioguanidine CP Analog 62 TES-Protected N-Methyl Hydroxyethyl CP Intermediate 55 30 62

55

35 NH2

CO2H

COPNB 40 Percent yield: 20%; H NMR (D.O, 300 MHz): & 4.20 4.13 (m, 2H), 3.80 (d. J=13.8 Hz, 1H), 3.51 (d. J=13.8 Hz, Method A: Percent yield: 74%; H NMR (CDC1, 300 1H), 3.40-3.39 (m. 1H), 3.32-3.30 (m, 2H), 3.18-3.12 (m, MHz): 88.19 (d. J=8.4 Hz, 2H), 7.65 (d. J=8.4 Hz, 2H), 5.44 1H), 3.09-2.92 (m, 2H), 2.47 (s.3H), 1.22 (d. J=6.9 Hz, 3H), (d. J=14.1 Hz, 1H), 5.20 (d. J=14.1 Hz, 1H), 4.25-4.18 (m, 1.07 (d. J–7.2 Hz, 3H). 2H), 3.93 (d. J=14.7 Hz, 1H), 3.63-3.56 (m, 2H), 3.36-3.30 45 (m. 1H), 3.22 (dd, J=5.7; 3.3 Hz, 1H), 3.14 (d. J=14.7 Hz, Example 20 1H), 2.70-2.56 (m, 2H), 2.49-2.41 (m. 1H), 2.21 (s.3H), 1.22 (d. J=6.3 Hz, 3H), 1.14 (d. J–7.5 Hz, 3H), 0.91 (t, J=7.5 Hz, TES-Protected Prolinol CP Intermediate 58 9H), 0.57 (q, J–7.5 Hz, 6H). 50 TES-Protected N-Methyl Thioguanidine CP 58 Intermediate 57

55 57

NH OH COPNB NH2 60 Method D: Percent yield: 66%; H NMR (CDC1, 300 MHz): 8 8.20 (d. J=9.3 Hz, 2H), 7.66 (d. J=8.4 Hz, 2H), 5.45 COPNB (d. J=14.1 Hz, 1H), 5.22 (d. J=14.1 Hz, 1H), 4.27-4.19 (m, 1H), 4.21 (dd, J=11.4; 3.9 HZ, 1H), 3.96 (d. J=15.0 Hz, 1H), Percent yield: 54%; H NMR (CDC1, 300 MHz): 8 9.21 65 3.65 (dd, J=11.1; 4.2 Hz, 1H), 3.47-3.41 (m, 1H), 3.39-3.32 (brs, 3H), 8.18 (d. J–7.8 Hz, 2H), 7.65 (d.J=8.4 Hz, 2H), 5.42 (m. 2H), 3.24 (dd, J=4.8; 2.7 Hz, 1H), 3.00-2.94 (m, 1H), (d. J=14.1 Hz, 1H), 5.21 (d. J=14.1 Hz, 1H), 428-4.23 (m, 2.70-2.65 (m, 1H), 2.54 (brs, 1H), 2.27-2.18 (m, 1H), 1.98 US 8,232,268 B2 57 58 1.63 (m, 4H), 1.25 (d. J=6.0 Hz, 3H), 1.15 (d. J=7.2 Hz, 3H), added a solution of methylamine in THF (2.0M in THF, 115 0.93 (t, J=7.2 Hz, 9H), 0.59 (q, J=7.5 Hz, 6H). mL, 230 mmol), so as to maintain an internal temperature below 5°C. The reaction mixture was aged at 0-5°C. for 3 h TES-Protected Thioguanidine CP Intermediate 60 (monitored by TLC) and then allowed to warm to rt and evaporated under reduced pressure. The crude hydrobromide salt was dissolved in methanol and the pH adjusted to 8-9 with NaOH solution in methanol. The solvent was removed under 60 reduced pressure and crude material was dried under high vacuum, and then treated with ACN and stirred for several 10 minutes. The insoluble material was filtered off and washed with ACN (2x10 mL). The combined filtrate was concen trated under reduced pressure, and the residue was purified by flash chromatography on silica gel (eluent-acetonitrile fol COPNB lowed by acetonitrile/water with gradient) to give the desired 15 product 64 in 75% yield. "H NMR (DMSO-d,300 MHz): 87.28 (brs, 1H), 7.05 (br Percent yield: 43%; H NMR (CDC1, 300 MHz): 8 9.33 s, 1H), 3.00 (s. 2H), 2.22 (s.3H). (brs, 2H), 8.20 (d. J=9.3 Hz, 2H), 7.67 (d.J=8.7 Hz, 2H), 5.43 (d. J=14.1 Hz, 2H), 5.25 (d. J=14.1 Hz, 2H), 4.30-4.19 (m, 2-(N-Methylamino)-1-Carbonyl Ethylsulfamide 65 3H), 3.50-3.34 (m, 2H), 3.27-3.25 (m, 1H), 3.20-3.07 (m, 3H), 2.95-2.90 (m, 1H), 2.45-2.35 (m, 1H), 2.10-1.98 (m, N-Boc-protected sarcosine was activated as its N-hydrox 1H), 1.85-1.70 (m,3H), 1.21 (d. J=6.9 Hz,3H), 1.17 (d. J=7.8 y succinimido ester and then allowed to react with sulfamide Hz, 3H), 0.92 (t, J–8.4 Hz, 9H), 0.58 (q, J=8.4 Hz, 6H). to give, after deprotection, the desired product 65. Thioguanidine CP Analog 63 25 s O 1. Nulls.OH -- 63 s O O 30 1. NullsO-N Her NH

O 35 CO2H s O 1 Null NHSONH -> Method F. Percent yield: 15% (from 60); H NMR (D.O. O 400MHz): 84.24-4.20 (m, 1H), 4.18 (dd, J=9.6; 2.8 Hz, 1H), 3.83 (d. J=13.2 Hz, 1H), 3.46 (d. J=13.2 Hz, 1H), 3.41 (dd. 40 J=6.4; 2.8 Hz, 1H), 3.33-3.26 (m, 2H), 3.23–3.16 (m, 1H), 1. Null NHSONH 3.10-3.05 (m. 1H), 3.04-2.98 (m, 1H), 2.37-2.32 (m, 1H), 65 2.13-2.08 (m. 1H), 1.86-1.68 (m,3H), 1.28 (d. J=6.4 Hz, 3H), 1.11 (d. J–7.2 Hz, 3H). 45 Step 1 To a stirred solution of N BOC amino acid (10 mmol) in Synthesis of the Amine Sidechains dry DCM (30 mL) at 0°C. were added N-hydroxysuccinim ide (15 mmol) and 1-3-(dimethylamino)propyl-3-ethylcar Preparation of Sarcosinamide 64 bodiimide hydrochloride (EDC, 12 mmol). The resulting 50 mixture was allowed to warm to room temperature, stirred for ~20 h (monitoring by TLC) and concentrated under reduced 64 pressure. The residue was then dissolved in ethyl acetate (50 NH2 mL), washed with water (4x10 mL) and brine (1 omL), dried YNH, i. n- Her over NaSO, concentrated, and dried under high vacuum to O 55 give the hydroxysuccinimido ester in quantitative yield. NH2 "H NMR (CDC1,300 MHz): 84.24 (s. 2H), 3.00 (s.3H), 2.86 (s, 4H), 1.47 (s, 9H). non-n Step 2 O To a solution of the N-Boc-N-hydroxysuccinimido ester of 60 sarcosine (2.86 g, 10.0 mmol) in dry DMF (20 mL) was added N-Methylglycinamide was prepared from methylamine sulfamide (1.92g, 20.0 mmol) and the resulting mixture was and C-bromoacetamide as previously described in literature stirred under nitrogen at 90°-95°C. for 8 h (monitoring by for sarcosinamide hydrochloride, (Marvel, C. S.; Elliott, J. R.: TLC). The mixture was then filtered, and the filtrate was Boettner, F. E.; Yuska, H., J. Am. Chem. Soc. 1946, 68, 1681 evaporated under reduced pressure. The residue was dried 1686). 65 under high vacuum and then dissolved in ethyl acetate (20 To a stirred solution of bromoacetamide (3.73 g, 27 mmol) mL). The resulting solution was extensively washed with in anhydrous THF (40 mL) at 0°C. under N atmosphere was water (monitoring by TLC) and the combined organics were US 8,232,268 B2 59 60 dried over NaSO and concentrated under reduced pressure temperature, and stirred overnight (monitored by TLC). The to give crude material. After drying under high vacuum, the reaction mixture was then washed with 1M aq. NaHCO material was triturated from ethyl acetate and hexanes and the Solution, water, and brine. The organic layer was dried over insoluble material removed by filtration. The filtrate was Na2SO, concentrated, and the remaining residue was puri evaporated under reduced pressure and the remaining Solid fied by flash chromatography to give the desired N-Boc-N'- was washed with ethyl ether, dissolved in DCM, and the PNB protected intermediate in 73% yield. product precipitated by adding hexanes. "H NMR (CDC1, 300 MHz): 8 8.21 (d. J=8.4 Hz, 2H), Percent yield: 70%; H NMR (CDC1,300 MHz): 8 6.60 7.50 (d. J=9.0 Hz, 2H), 5.19 (s. 2H), 3.38 (brs, 4H), 2.89 (s. 6.30 (m, 2H), 3.88 (s. 2H), 2.94 (s.9H). 3H), 1.45 (s, 9H). Step 3 10 To a stirred solution of Boc-protected amine (1.0 mmol) in Step 4 DCM (2-5 mL) at 0°C. was added trifluoroacetic acid (95% Sidechain 66 was prepared using standard TFA deprotec aqueous solution, 0.5 mL) and the mixture was stirred at 0°C. tion reaction conditions and purified by flash column chro matography on silica gel. for 30 minutes, and then allowed to warm to rt and stirred 15 overnight. The volatiles were evaporated under reduced pres Percent yield; quantitative; H NMR (acetone-de, 300 sure and the residue was treated two times with ethyl ether and evaporated yielding the corresponding amine TFA salt. MHz): 8 8.20 (d. J=8.7 Hz, 2H), 7.62 (d. J=8.1 Hz, 2H), 7.41 The salt was dissolved in methanol and treated with NaOH (brs, 1H), 5.20 (s. 2H), 3.55 (brs, 2H), 3.18 (brs, 2H), 2.74 solution in methanol (pH adjusted to 8-9) and concentrated. (s, 3H). The remaining solid was partitioned between ethyl acetate and water, the layers separated, and the organic layer was N-Methyl-N'-(Aminosulfonyl)Ethylenediamine 67 dried and concentrated to produce the desired product in quantitative yield. "H NMR (DMSO-d 300 MHz): 8 7.25 (s, 1H), 7.03 (s, 1H), 3.04 (s. 2H), 2.22 (s.3H). 25 N -e- N-Methyl-N'-PNB Ethylenediamine 66 1. N1)NH, Boc 30 N He H N He 1N1 NHSoNH, 1 N1\N, N Boc 1N1 NHsoNH, 1N1N NHcocF, - - 35 67 Boc This sidechain was prepared by treating N-Boc-N-methyl N 1. n1n NH2 He ethylenediamine with an excess of Sulfamide in refluxing Boc 40 dioxane followed by N-Boc deprotection. Step 1 1N1N NHcoPNB - - A solution of N-Boc-N-methylethylenediamine (1 g, 5.74 mmol) and Sulfamide (1.1 g, 11.48 mmol) in dioxane (20 mL) N was stirred at reflux under N atmosphere (monitored by 1. N1 NHcopNB 45 TLC). The mixture was then filtered to remove the insoluble 66 material and the filtrate was concentrated under reduced pres sure and treated with ethyl acetate. The insoluble material was again removed by filtration and the filtrate concentrated under The synthesis involved the selective protection of the two reduced pressure to give the desired product in 96% yield. amine groups in N-methylethylenediamine, first with ethyl 50 trifluoroacetate and, second, with di-tert-butyl dicarbonate, 'HNMR (acetone-d 300 MHz): 85.91 (brs, 2H), 5.77 (br followed by removal of the trifluoroacetyl group and reaction s, 1H), 3.38 (t, J=6.3 Hz, 2H), 3.20 (t, J=6.3 Hz, 2H), 2.85 (s, of this amine with p-nitrobenzylchloroformate. The synthesis 3H), 1.40 (s.9H). ends with the removal of the Boc-protecting group. Step 2 Steps 1 and 2 55 Percent yield: 91%; H NMR (acetone-d 300 MHz) 8: The procedure used to make N-Boc-N-methyl ethylenedi 6.62 (brs, 2H), 6.34 (brs, 1H), 3.48 (t, J=4.8 Hz, 2H), 3.32 (t, amine was similar to that previously described in literature J–4.8 Hz, 2H)2.80 (s.3H). (Martins, E. T.; Baruah, H.; Kramarczyk, J.; Saluta, G.; Day, C. S. Kucera, G. L.; Bierbach, U.J. Med. Chem. 2001, 44, 4492-4496). 60 1-2-(N-Methylamino)Ethyl Imidazole 68 Step 3 To a stirred solution of N-Boc-N-methyl ethylenediamine (1.39 g, 8.0 mmol) and DIEA (1.05 g, 8.1 mmol) in dry DCM (50 mL) at 0°C. was added a solution of p-nitrobenzylchlo roformate (1.75 g, 8.1 mmol) in dry DCM (10 mL) dropwise 65 H over 5-10 minutes. The reaction mixture was stirred at this 1N1 So 1N1)-on temperature for 30 minutes and then allowed to warm to room US 8,232,268 B2 61 62 -continued This sidechain was prepared by displacement of pyrazole from the bis-PNB-protected 1H-pyrazole-carboxamidine (shown above), followed by removal of the Boc protecting group. Step 1: N-Boc-N-Methylaminoethyl-N',N'-Bis-PNB 68 Guanidine 1-2-(N-methylamino)ethyl)imidazole was synthesized To a solution of N-Boc-N-methylethylenediamine (627 from the mesylate of 2-(N-Boc-N-methylamono)ethanol, 10 mg, 3.6 mmol) in dry DMF (10 mL) was added bis-PNB which was allowed to react with imidazole in the presence of protected pyrazole-1-carboxamidine (750 mg, 1.6 mmol) and LiHMDS. The starting material was prepared by selective the resulting mixture was stirred at rt for 3 days (monitoring protection of amino group with Boc-anhydride in the pres by TLC). The solvent was then removed under reduced pres ence of Al-O according to the procedure described in litera Sure and purified by flash chromatography to produce the ture (Yadar, V. K. Ganesh Babu, K. J. Org. Chem. 2004, 69. 15 desired Boc-protected amine in 50% yield. 577-580). 'HNMR (CDC1,300 MHz)8: 11.70 (s.1H),8.47 and 8.35 Preparation of (s+s, 1H), 8.14 (d. J=8.1 Hz, 2H), 8.11 (d. J–8.1 Hz, 2H), 7.48 1-2-(N-Boc-N-Methylamino)Ethyl Imidazole (d. J=8.1 Hz, 2H), 7.46 (d. J–8.1 Hz, 2H), 5.21 (s. 2H), 5.15 To a stirred solution of imidazole (1.02g, 15 mmol) at -30° (s. 2H), 3.56-3.51 (m, 2H), 3.41 (t, J=5.4 Hz, 2H), 2.81 (s, C. under N atmosphere in dry DMF (20 mL) was added 3H), 1.34 (s.9H). LHMDS (1 M solution in hexanes, 16.0 mL, 16.0 mmol), Step 2: Amine 69 dropwise, by Syringe. The reaction mixture was stirred at -30°C. for 40 minutes. To this mixture was added a solution 25 Percent yield: 89%; H NMR (CDC1,300 MHz) 8: 11.63 of the mesylate from 2-(N-Boc-N-methylamino)ethanol (s, 1H), 9.80 (s, 1H),8.65 (t, J=6.0 Hz, 1H), 8.19 (d. J–4.5 Hz, (freshly prepared from 2-(N-Boc-N-methylamino)ethanol, 2H), 8.16 (d. J=4.5 Hz, 2H), 7.51 (d. J=5.7 Hz, 2H), 7.48 (d. mesyl chloride, and DIEA, (1.75 g, 10 mmol)) in DMF (6 mL) J=5.1 Hz, 2H), 5.23 (s. 2H), 5.20 (s. 2H), 3.76 (brs, 2H), 2.70 (s, 3H) via syringe. The resulting mixture was stirred at -30°C. for 30 30 minutes and then at rt overnight (monitored by TLC). The 2-(N-Methylaminomethyl)Thiazole 70 mixture was then evaporated under reduced pressure and purified by flash chromatography on silica gel to produce the desired Boc-protected amine in 54% yield. "H NMR (CDC1, 300 MHz) 8: 7.09 (s, 1H), 6.69 (s, 1H), 35 70 6.60 and 6.57 (s+s, 1H), 3.76 (t, J=6.0Hz, 2H), 3.16(brs, 2H), 2.40 and 2.28 (s+s, 3H), 1.10 and 1.05 (s+s, 9H). Step 2: Preparation of Imidazole 68 40 (r. - (r Percent yield: 83%; H NMR (CDC1,300 MHz) 8: 7.51 (s, 1H), 7.07 (s, 1H), 6.96 (s, 1H), 4.07 (t, J=6.0 Hz, 2H), 2.93 (t, J=6.0 Hz, 2H), 2.43 (s, 3H), 1.13 (brs, 1H). To a flask containing dry DCM (60 mL) and 4A molecular N-Methylaminoethyl-N',N'-bis-PNB-Oxycarbonyl sieves were added methylamine (2.0M solution in THF, 4.0 Guanidine 69 mL, 8.0 mmol), NaSO (1.42g, 10.0 mmol), and 2-thiazole 45 carboxaldehyde (0.452 g, 4.0 mmol). The reaction mixture was stirred at room temperature and monitored by "H NMR. Upon completion, the reaction mixture filtered to remove the NaSO and molecular sieves and the filtrate was concen trated under reduced pressure. The crude imine was dissolved 50 in absolute EtOH (40 mL), treated with NaBH (0.227g, 6.0 mmol) and aged for several hours at rt (monitored by TLC). The reaction mixture was then quenched with water (8 mL) and concentrated under reduced pressure. The residue was treated with brine, extracted with DCM (3x50 mL), and the NHCOPNB He 55 combined organic layers were dried, and evaporated under reduced pressure to give the desired product in 69% yield. NCOPNB H NMR (CDC1, 300 MHz) 8: 7.69 (d. J=3.6 Hz, 1H), Boc NCOPNB 7.24 (d. J=3.6 Hz, 1H0, 4.06 (s. 2H), 2.49 (s.3H), 1.80 (brs, 1H). N ls 60 1. n-n NHCOPNB 2-(Aminosulfonyl)Aminomethylpiperazine 71 N - 1NCOPNB 1. n-n NHCOPNB 65 69 US 8,232,268 B2 63 64 -continued Step 4: 2-(Aminosulfonyl)Aminomethylpiperazine

r NBZ He A mixture of 1,4-dibenzyl-piperazine-2-methylsulfamide NH2 (1.82 g, 4.87 mmol) in MeOH (20 mL) and 10% Pd/C (180 mg) was treated with hydrogen gas (65 psi) for 27 hat rt. The is O mixture was then filtered through a pad of Celite and the celite washed well with methanol. The filtrate was concentrated under reduced pressure to give the desired product 71 in 96% r Her NH2 yield. os--- 10 H NMR (DMSO-d 300 MHz) 8: 6.48 (brs, 1H), 6.42(br s, 1H), 3.20-3.13 (m, 2H), 2.77-2.62 (m, 3H), 2.57-2.50 (m, r NBZ He 2H), 2.47-2.39 (m with DMSO-d peak, 1H), 2.11 (dd. BZN NHSONH J=11.4; 9.6 Hz, 1H). 15 (2S)-2-(Aminosulfonyl)aminocarbonylpyrrolidine is--r NHSONH2 72 71

Cbz O The synthesis involved preparation of 1,4-dibenzyl-pipera Zine-2-carboxylic acid amide, reduction of the amide to the N amine with LAH, conversion of the amine to the sulfonamide, OH --> and deprotection of the bis-benzyl protecting groups. 25 Cbz O O

Step 1: 1,4-Dibenzy piperazine-2-Carboxylic Acid N Amide O-N He

This compound was prepared in 75% yield according to the 30 procedure described in literature (Butts, C. P.; Jazdzyk, M. O Chem. Commu. 2003, (13) 1530-1531) Cbz O N Step 2: 1,4-Dibenzyl-2-aminomethyl-piperazine NHSONH -> 35 To a stirred suspension of LAH (0.9 g, 25.9 mmol) in dry O H THF (150 mL) was added 1,4-dibenzyl-piperazine-2-car N boxylic acid amide (4.0 g, 12.93 mmol) and the mixture was NHSONH2 stirred under reflux for 20 h (monitored by TLC). The stirred mixture was cooled with an ice-bath and quenched with 40 Rochelle’s salt (6 mL), warmed to room temperature, and the 72 Suspension was poured over a pad of silica gel (20 mm). The celite was washed with 15% MeOH in CHC1 with 1% NH-OH (3x50 mL) and eluted through pad of silica gel. The (2S)-1-Cbz-2-(Aminosulfonyl)AminocarbonylPyr eluent was concentrated under reduced pressure to give the 45 rolidine desired product in 99% yield. "H NMR (CDC1, 300 MHz): 8 7.32-7.31 (m, 10H), 4.05 Percent yield: 42%; H NMR (CDC1, 300 MHz): 8 7.34 (d. J–13.8 Hz, 1H), 3.52 and 3.47 (AB, J–12.9 Hz, 2H), 3.27 7.26 (m, 5H), 6.76 (s, 1H), 6.42-6.26 (m, 2H), 5.11 (s. 2H), (d. J=13.8 Hz, 1H), 3.06 (dd, J=13.2; 5.7 Hz, 1H), 2.82-2.69 4.31 (brs, 1H), 3.52-3.40 (m, 2H), 2.20-2.11 (m, 2H), 1.95 (m,3H), 2.63-2.58 (m, 1H), 2.50-2.43 (m, 1H), 2.36-2.16 (m, 50 1.86 (m, 3H). 3H). (2S)-2-(Aminosulfonyl)AminocarbonylPyrrolidine Step 3: 1,4-Dibenzyl-2-(Aminosulfonyl)Aminom 72 ethylpiperazine 55 Percent yield: 95%; H NMR (CDC1, 300 MHz) 8: 7.40 (brs, 1H), 6.16 (brs, 1H), 3.69 (dd, J=9.0; 5.1 Hz), 3.06-2.85 To a solution of 1,4-dibenzyl-2-aminomethyl-piperazine (m. 2H), 2.35 (brs, 1H), 2.17-2.05 (m. 1H), 1.94-1.83 (m, (2.5g, 8.46 mmol) in dioxane (50 mL), was added sulfamide 1H), 1.80-1.59 (m, 2H). (1.63 g, 16.9 mmol) and the resulting mixture was stirred at 95°C. under N. atmosphere for several hours (monitored by 60 Synthesis of Secondary Amines by Alkylation TLC). The solvent was removed under reduced pressure and the crude material was purified by flash chromatography on The synthesis of a series of 2-, 3- or 4-(alkylamino)alky silica gel using acetonitrile/water with gradient as eluent. lpyridines was achieved by protection of 2-, 3- or 4-ami Percent yield: 58%; H NMR (CDC1, 300 MHz) 8: 737 noalkylpyridines with Boc anhydride in t-butyl alcohol fol 7.29 (m. 10H), 5.64 (brs, 1H), 4.24 (brs, 2H), 3.94 (d. J=12.9 65 lowed by treatment with sodium hydride in DMF and HZ, 1H), 3.59-3.46 (m, 4H), 3.13 (d. J=12.9 HZ, 1H), 2.96 alkylation with various alkyl halides. Lastly, removal of the 2.91 (m. 1H), 2.81-2.76 (m, 1H). Boc protecting group was accomplished with 95% TFA in US 8,232,268 B2 65 66 DCM. This synthetic strategy was first reported in literature -continued for preparation of 2-(alkylamino)pyridines (Krein, D. M.: 30 Lowary, T. L.J. Org. Chem. 2002, 67,4965-4967). Examples of Prepared Amines 5 2-(Methylaminomethyl)-5-methylpyrazine—66% overall yield; H NMR (CDC1, 300 MHz) 8: 8.42 (s, 1H), 8.35 (s, 1H), 3.83 (s. 2H), 2.50 (s.3H), 2.43 (s.3H). 2-(methylaminomethyl)pyridine 55% overall yield; H 10 NMR (CDC1,300 MHz) 8: 8.58 (s, 1H), 7.71 (t, J=8.4 Hz, 1H), 7.31-7.24 (m, 2H), 4.01 (s. 2H), 2.54 (s.3H). 3-(4-thiazolomethylamino)methylpyridine 42% over all yield; H NMR (CDC1, 300 MHz) 8: 8.79 (d. J=1.2 Hz, 42 1H), 8.57 (d. J–4.2 Hz, 1H), 8.08 (dt, J=8.1; 2.4 Hz, 7.35 (d. 15 J=6.9 HZ, 1H), 7.24 (brs, 1H), 7.19-7.15 (m. 1H), 4.06 (s, 2H), 3.99 (s. 2H), 2.80 (s, 1H). Dilution Antimicrobial Susceptibility Tests 2O The agar dilution method for determining the antimicrobial Susceptibility was carried out using an agar dilution method with Mueller-Hinton agar (see, M7-A5, Vol. 20 (2), 2000). A final inoculum of 104 CFU was applied with a replicating 45 device. Broth dilution tests were performed with 5x105 CFU as in tubes containing 1 mL of broth. Incubation of test tubes containing agar and broth was done at 35 C for 18 h. The susceptibilities of streptococci were determined by Mueller Hinton agar supplemented with 5% sheep blood, and the Susceptibility of anaerobic species was determined with bru- 30 cella agar Supplemented with 5% sheep blood, hemin, and Vitamin K. Incubation of anaerobic cultures was done for 48 48 h in jars. The susceptibilities of methicillin-resistant staphy lococci were determined on Mueller-Hinton agar or in broth supplemented with 3% NaCl. All assays were run with the 35 indicated control strains, available from the American Type Culture Collection, Rockville, Md.). Results of the antimi crobial susceptibility tests of compounds 9-63 against Gram negative organisms are shown in Table I. The compositions, methods and/or processes disclosed and 40 51 claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be appar ent to those of skill in the art that variations may be applied to 45 the compositions, methods and/or processes and in the steps or in the sequence of steps of the methods described herein without departing from the concept and scope of the inven 54 tion. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may so be substituted for the agents described herein while the same or similar results would be achieved. All such similar substi tutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention. The invention claimed is: 55 1. A compound selected from the group consisting of 62

NH2, and US 8,232,268 B2 67 68 -continued 6. The compound of claim 1, wherein the compound is: 63

48 1.NH 5 CO2H

10 CO2H or a pharmaceutically acceptable salt or prodrug thereof. 2. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt or prodrug thereof. 7. The compound of claim 1, wherein the compound is: 15 23

51 r in N 2

CO2H CO2H or a pharmaceutically acceptable salt or prodrug thereof. 25 or a pharmaceutically acceptable salt or prodrug thereof. 3. The compound of claim 1, wherein the compound is: 8. The compound of claim 1, wherein the compound is:

30

OH 30 54

35

CO2H CO2H N or a pharmaceutically acceptable salt or prodrug thereof. 40 4. The compound of claim 1, wherein the compound is: or a pharmaceutically acceptable salt or prodrug thereof. 9. The compound of claim 1, wherein the compound is:

42

45 62

NH2 21 50 CO2H CO2H or a pharmaceutically acceptable salt or prodrug thereof. 5. The compound of claim 1, wherein the compound is: 5s or10. a The pharmaceutically compound of claimacceptable 1, wherein salt or the prodrug compound thereof. is:

45 63

60 NH

CO2H 65 or a pharmaceutically acceptable salt or prodrug thereof. or a pharmaceutically acceptable salt or prodrug thereof. US 8,232,268 B2 69 70 11. A pharmaceutical composition comprising a com 15. The method of claim 12, wherein the infection is by a pound of claim 1 and a pharmaceutically acceptable carrier. multiple-drug resistant strain. 12. A method of preventing or treating an infection by a 16. The method of claim 12, wherein the compound is gram negative bacteria in a host comprising administering to administered in combination or alternation with at least one the host a therapeutic amount of a compound of claim 1, other antimicrobial agent. optionally in a pharmaceutically acceptable carrier. 17. The method of claim 12, wherein the compound is administered in combination with a B-lactamase inhibiting 13. The method of claim 12, wherein the host is a human. agent. 14. The method of claim 12, wherein the infection is by a drug resistant bacterial strain.