Heterocyclic Compounds And Their Use in Preventing or Treating Bacterial Infections

Abstract

The present invention relates to compounds of formula (I) and their use for treating or preventing a bacterial infection or as an antibacterial agent and/or as a β-lactamase inhibitor.

##STR00001##

Claims

1-19. (canceled)

20. A compound of formula (I), in which: ##STR00040## wherein: R.sup.1 is chosen in the group consisting of H, CN, C(═O)NR.sup.2R.sup.3, C(═O)NR.sup.4NR.sup.2R.sup.3, C(═O)NR.sup.2OR.sup.3, (CH.sub.2).sub.nOR.sup.2, (CH.sub.2).sub.nNR.sup.2R.sup.3, (CH.sub.2).sub.nNR.sup.4C(═NR.sup.4)N(R.sup.4).sub.2, C(═NOZ.sup.4)NZ.sup.1Z.sup.2, (CH.sub.2).sub.n-(5 to 6-membered)heteroaryl comprising 1 or 4 heteroatoms independently chosen in the group consisting of N, O or S; n is an integer comprised between 1 and 6; R.sup.2 and R.sup.3, identical or different, are chosen in the group consisting of H, linear or branched (C1-C6)alkyl, (C3-C11)cycloalkyl, (4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S, (5 to 10-membered)heteroaryl comprising from 1 to 4 heteroatom independently chosen in the group consisting of N, O or S, C(═O)(C1-C6)alkyl, C(═O)(4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S, or form together with the nitrogen atom to which they are linked a (4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatom independently chosen in the group consisting in N, O or S, wherein the alkyl, cycloalkyl, aryl, heterocyclyl and heteroaryl are optionally substituted by one or more R.sup.5; R.sup.4, each identical or different, is independently chosen in the group consisting of H and linear or branched (C1-C6)alkyl optionally substituted by one or more R.sup.5; R.sup.5, each identical or different, is chosen in the group consisting of OH, O(C1-C6)alkyl, NH.sub.2, NH(C1-C6)alkyl, N[(C1-C6)alkyl].sub.2, C(═O)NH.sub.2, C(═O)NH(C1-C6)Alkyl, C(═O)N[(C1-C6)Alkyl].sub.2; Y.sup.1 is chosen in the group consisting of SO.sub.3H, CHFC(═O)Y.sup.2 and CF.sub.2C(═O)Y.sup.2, SO.sub.3(C1-C6)alkyl-C(═O)O(C1-C6)alkyl; Y.sup.2 is chosen in the group consisting of OH, O(C1-C6)alkyl linear or branched, O(C3-C11)cycloalkyl, O-(4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatom independently chosen in the group consisting of N, O and S; NY.sup.3Y.sup.4, wherein the alkyl, cycloalkyl and heterocyclyl are optionally substituted by one or more Y.sup.5; Y.sup.3 and Y.sup.4, each identical or different, is chosen in the group consisting of linear or branched (C1-C6)alkyl, linear or branched O(C1-C6)alkyl, (C3-C11)cycloalkyl, (4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S, or form together with the nitrogen atom to which they are linked a (4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S; wherein the alkyl, cycloalkyl and heterocyclyl is optionally substituted by one or more Y.sup.5; Y.sup.5, each identical or different, is chosen in the group consisting of linear or branched (C1-C6)alkyl, (C3-C6)cycloalkyl, linear or branched O(C1-C6)alkyl, linear or branched O(C1-C6)alkyl-O(C1-C6)alkyl, linear or branched (C1-C6)alkyl-O(C1-C6)alkyl; and O(C3-C6)cycloalkyl; X.sup.1═X.sup.2 is chosen in the group consisting of N═CX.sup.4, CX.sup.3=CA.sup.1 and CA.sup.1=CX.sup.4; A.sup.1 is chosen in the group consisting of H, (CH.sub.2).sub.m—C(═O)NA.sup.2A.sup.3, (CH.sub.2).sub.n—NA.sup.2A.sup.3; A.sup.2 and A.sup.3, each identical or different is chosen in the group consisting of H, linear or branched (C1-C6)alkyl, linear or branched O—(C1-C6)alkyl; X.sup.3 and X.sup.4 is chosen in the group consisting of (CH.sub.2).sub.m—C(═O)NX.sup.6X.sup.7, (CH.sub.2).sub.m—C(═NX.sup.6)NHX.sup.7, (CH.sub.2).sub.n—NX.sup.6X.sup.7, (CH.sub.2).sub.n—NX.sup.6C(═O)X.sup.7, (CH.sub.2).sub.n—NHC(═NX.sup.6)NHX.sup.7, (CH.sub.2).sub.n—NHC(═NX.sup.6)X.sup.7, (CH.sub.2).sub.m-(5- to 6-membered heteroaryl comprising from 1 to 4 heteroatom independently chosen in the group consisting of N, O, S), (CH.sub.2).sub.m-(4- to 6-membered heterocyclyl comprising from 1 to 2 heteroatom independently chosen in the group consisting of N,O,S), wherein the heteroaryl is substituted at least by one or more Z.sup.3 and the heterocyclyl is optionally substituted by Z.sup.3; m is an integer comprised between 0 and 6; X.sup.6, X.sup.7 and X.sup.8, each identical or different, are chosen in the group consisting of H, linear or branched (C1-C6)alkyl-Z.sup.3, or form together with the nitrogen atom to which they are linked a 4 to 6-membered heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S, wherein the heterocyclyl are optionally substituted by Z.sup.3; Z.sup.1 and Z.sup.2, each identical or different are chosen in the group consisting of H, linear or branched (C1-C6)alkyl; Z.sup.3, each identical or different, is chosen in the group consisting of (CH.sub.2).sub.m—NZ.sup.1Z.sup.2, (CH.sub.2).sub.m—NHC(═NH)Z.sup.1, (CH.sub.2).sub.m—NHC(═NH)NHZ.sup.1; any sulphur atom present within a heterocycle can be oxidized to form a S═O group or a S(O).sub.2 group; any nitrogen atom present within a heterocycle or present within group wherein it is trisubstituted thus forming a tertiary amino group, can be further quaternized by a methyl group; Z.sup.4 represents H or linear or branched (C1-C6)alkyl; or a racemate, an enantiomer, a diastereoisomer, a geometric isomer or a pharmaceutically acceptable salt thereof, with the exception that if one of X.sup.3 or X.sup.4 represent (CH.sub.2).sub.m—C(═O)NX.sup.6X.sup.7, or (CH.sub.2).sub.n—NX.sup.6X.sup.7 then at least one of X.sup.6 or X.sup.7 is different from H.

21. The compound of claim 20, wherein: R.sup.1 is chosen in the group consisting of H, CN, C(═O)NR.sup.2R.sup.3, C(═O)NR.sup.4NR.sup.2R.sup.3, C(═O)NR.sup.2OR.sup.3, (CH.sub.2).sub.nOR.sup.2, (CH.sub.2).sub.nNR.sup.2R.sup.3, (CH.sub.2).sub.nNR.sup.4C(═NR.sup.4)N(R.sup.4).sub.2, (CH.sub.2).sub.n-(5 to 6-membered)heteroaryl comprising 1 or 4 heteroatoms independently chosen in the group consisting of N, O or S; and/or Y.sup.1 is chosen in the group consisting of SO.sub.3H, CHFC(═O)Y.sup.2 and CF.sub.2C(═O)Y.sup.2.

22. The compound of claim 20, wherein: R.sup.1 is chosen in the group consisting of H, CN, C(═O)NR.sup.2R.sup.3, C(═O)NHNHR.sup.2, C(═O)NHOR.sup.2, CH.sub.2OR.sup.2, CH.sub.2NHR.sup.2, CH.sub.2NR.sup.4C(═NR.sup.4)N(R.sup.4).sub.2, C(═NOZ.sup.4)NZ.sup.1Z.sup.2, CH.sub.2-(5 to 6-membered)heteroaryl comprising 1 or 4 heteroatoms independently chosen in the group consisting of N, O or S; R.sup.2 and R.sup.3, each identical or different, are chosen in the group consisting of H, linear or branched (C1-C6)alkyl, (4 to 6-membered)heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S, C(═O)(4 to 6-membered heterocyclyl comprising 1 or 2 heteroatoms independently chosen in the group consisting of N, O or S); R.sup.4, each identical or different, is independently chosen in the group consisting of H, linear or branched (C1-C6)alkyl, wherein the alkyl is optionally substituted by one or more R.sup.5; R.sup.5, each identical or different, is chosen in the group consisting of OH, O(linear or branched-C1-C6)alkyl, NH.sub.2, NH(linear or branched C1-C6)alkyl, N[(linear or branched C1-C6)Alkyl].sub.2, C(═O)NH.sub.2, C(═O)NH(linear or branched C1-C6)alkyl, C(═O)N[linear or branched (C1-C6)alkyl].sub.2; Y.sup.2 is chosen in the group consisting of OH, O(C1-C6)alkyl linear or branched, O-(4 to 6-membered)heterocyclyl comprising 1 or 2 independently heteroatom chosen in the group consisting of N, O and S; wherein the alkyl, heterocyclyl are optionally substituted by one or more Y.sup.5; Y.sup.5, each identical or different, is chosen in the group consisting of linear or branched (C1-C6)alkyl, (C3-C6)cycloalkyl, linear or branched O(C1-C6)alkyl, linear or branched O(C1-C6)alkyl-O(C1-C6)alkyl, linear or branched (C1-C6)alkyl-O(C1-C6)alkyl; and O(C3-C6)cycloalkyl; and X.sup.1═X.sup.2 is chosen in the group consisting of N═CX.sup.4, CX.sup.3=CA.sup.1 and CA.sup.1=CX.sup.4.

23. The compound of claim 22, wherein R.sup.1 is chosen in the group consisting of H, CN, C(═O)NR.sup.2R.sup.3, C(═O)NHNHR.sup.2, C(═O)NHOR.sup.2, CH.sub.2OR.sup.2, CH.sub.2NHR.sup.2, CH.sub.2NR.sup.4C(═NR.sup.4)N(R.sup.4).sub.2CH.sub.2-(5 to 6-membered)heteroaryl comprising 1 or 4 heteroatoms independently chosen in the group consisting of N, O or S.

24. The compound of claim 20 having a structure corresponding to formula (IA): ##STR00041##

25. The compound of claim 20 having a structure corresponding to formula (IB): ##STR00042##

26. The compound of claim 20 having a structure corresponding to formula (IC): ##STR00043##

27. The compound of claim 20 have a structure corresponding to formula (I*), formula (IA*), formula (IB*), or formula (IC*): ##STR00044##

28. The compound of claim 20 selected from the group consisting of: [2-(2-aminoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid; [2-(guanidinomethyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid; [2-(2-guanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid; [trans-4-(dimethylcarbamoyl)-2-(2-guanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] hydrogen sulfate; [N-[[trans-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-7-sulfooxy-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methyl]carbamimidoyl]ammonium 2,2,2-trifluoroacetate; [(trans-4-(dimethylcarbamoyl)-3-(2-guanidinoethyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-7-yl] hydrogen sulfate.

29. A pharmaceutical composition comprising a compound according to claim 20 and a pharmaceutically acceptable excipient.

30. The pharmaceutical composition according to claim 29 further comprising an antibacterial compound.

31. The pharmaceutical composition according to claim 30 wherein the antibacterial compound is selected from aminoglycosides, β-lactams, glycylcyclines, tetracyclines, quinolones, fluoroquinolones, glycopeptides, lipopeptides, macrolides, ketolides, lincosamides, streptogramins, oxazolidinones, polymyxins and mixtures thereof.

32. The pharmaceutical composition according to claim 29 further comprising a β-lactam compound.

33. The pharmaceutical composition according to claim 32, the β-lactam compound is selected from the group consisting of penicillin, cephalosporins, penems, carbapenems, monobactam, and combinations thereof.

34. A pharmaceutical composition comprising a compound according to claim 20 and ceftazidime.

35. A kit comprising: a first pharmaceutical composition that comprises a first pharmaceutically active compound a first pharmaceutically acceptable excipient; and a second pharmaceutical composition that comprises a second pharmaceutically active compound and second pharmaceutically acceptable excipient; wherein the first and second pharmaceutically active compounds are different compounds according to claim 20.

36. A kit comprising: a first pharmaceutical composition comprising the compound of claim 20; and a second pharmaceutical composition comprising ceftazidime.

37. A method for treating or preventing a bacterial infection, the method comprising the administration to a person in need thereof of a compound according to claim 20.

38. The method according to claim 37, wherein the bacterial infection is caused by bacteria producing one or more β-lactamase.

39. The method according to claim 38, wherein the bacteria is a gram-positive bacteria or gram-negative bacteria.

40. A method for treating or preventing a bacterial infection, the method comprising the simultaneous, separate or sequential administration to a patient in need thereof of a kit according to claim 35.

Description

EXAMPLES

[0099] The following examples are provided for the purpose of illustrating the present invention and by no means should be interpreted to limit the scope of the present invention.

[0100] The first part represents the preparation of the compounds (intermediates and final compounds) whereas the second part describes the evaluation of antibacterial activity of compounds according to the invention.

[0101] Preparation of the compounds and biological activity: Abbreviations or symbols used herein include: [0102] ACHN: 1,1′-azobis(cyclohexanecarbonitrile) [0103] ACN: acetonitrile [0104] AcOH: acetic acid [0105] Bn: benzyl [0106] Boc: tert-butoxycarbonyl [0107] Boc.sub.2O: tert-butoxycarbonyl anhydride [0108] BocON: [2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile] [0109] bs: broad singlet [0110] Burgess reagent: methyl N-(triethylammoniosulfonyl)carbamate [0111] CFU: colony-forming units [0112] CLSI: clinical laboratory standards institute [0113] d: doublet [0114] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene [0115] DCM: dichloromethane [0116] dd: double doublet [0117] ddd: double double doublet [0118] ddt: double double triplet [0119] dq: double quartet [0120] dt: double triplet [0121] DTAD: di-tert-butylazodicarboxylate [0122] DEAD: diethyl azodicarboxylate [0123] Dess-Martin periodinane: 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one [0124] DHP 3,4-dihydro-2H-pyran [0125] DIAD: diisopropyl azodicarboxylate [0126] DIPEA: N,N-diisopropylethylamine [0127] DMAP: 4-dimethylaminopyridine [0128] DMF: N,N-dimethylformamide [0129] DMSO: dimethylsulfoxide [0130] EtOAc: ethyl acetate [0131] Et.sub.2O: diethyl ether [0132] h: hours [0133] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate [0134] m: multiplet [0135] min: minutes [0136] MeOH: methanol [0137] MeONa: sodium methoxide [0138] MIC: minimum inhibitory concentration [0139] MS: mass spectrometry [0140] MsCI: methanesulfonyl chloride [0141] NBS: N-bromosuccinimide [0142] NMR: nuclear magnetic resonance spectroscopy [0143] Ns: nosyl, nitrobenzenesulfonyl [0144] Pd(Ph.sub.3).sub.4: tetrakis(triphenylphosphine)palladium(0) [0145] PG: protective group [0146] PhSH: thiophenol [0147] PMe.sub.3: trimethylphosphine [0148] PPh.sub.3: triphenylphosphine [0149] Ppm: parts per million [0150] q: quartet [0151] rt: room temperature [0152] s: singlet [0153] SEM: [2-(trimethylsilyl)ethoxy]methyl [0154] t: triplet [0155] TBAF: tetra-n-butylammonium fluoride [0156] TBDMSCl: tert-butyldimethylsilyl chloride [0157] TBDMSOTf: trifluoromethanesulfonic acid tert-butyldimethylsilyl ester [0158] TBSOTf: trimethylsilyl trifluoromethanesulfonate [0159] tBuOK: potassium tert-butoxide [0160] TEA: triethylamine [0161] TFA: trifluoroacetic acid [0162] THF: tetrahydrofuran [0163] THP: tetrahydropyranyl [0164] TLC: thin layer chromatography [0165] TMSI: Iodotrimethylsilane

Example 1: Synthesis of [2-(2-aminoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid

[0166] ##STR00020## ##STR00021##

Step 1: Preparation of Intermediate tert-butyl 7-[tert-butyl(dimethyl)silyl]oxy-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (1b)

[0167] Intermediate (1a) (WO2017109025, 4.86 g, 19 mmol) was dissolved in DCM (38 mL). TEA (5.87 mL, 42 mmol), TBDMSCl (3.15 g, 21 mmol) and DMAP (catalytic amount) were added. The mixture was stirred at rt overnight. As starting material was not totally consumed, TBDMSCl (1.14 g, 7.5 mmol) was added and mixture was stirred at rt for the night. The mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (1b) (5.45 g, 14.8 mmol, 78%). MS m/z ([M+H].sup.+) 371. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.18 (s, 3H), 0.20 (s, 3H), 0.93 (s, 9H), 1.49 (s, 9H), 3.05-3.35 (m, 1H), 4.00-4.35 (m, 1H), 4.45 (d, J=16.8 Hz, 1H), 4.80-5.05 (m, 2H), 8.70 (s, 1H).

Step 2: Preparation of Intermediate lithium 5-tert-butoxycarbonyl-7-[tert-butyl(dimethyl)silyl]oxy-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-2-carboxylate (1c)

[0168] At −78° C., a solution of nBuLi 1.6M in hexane (16.5 mL, 26 mmol) was added to a solution of Intermediate (1b) (4.9 g, 13 mmol) in THF (100 mL). After 30 min, CO.sub.2 gas was bubbled in the mixture for 5 min at −78° C. The mixture was warmed rt for 45 min and then concentrated to give a crude which was used in next step without purification. MS m/z ([M+H].sup.+) 415.

Step 3: Preparation of Intermediate O5-tert-butyl O2-methyl 7-[tert-butyl(dimethyl)silyl]oxy-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-2,5-dicarboxylate (1d)

[0169] At rt, a solution of intermediate (1c) (13 mmol), K.sub.2CO.sub.3 (2.76 g, 20 mmol) and Me.sub.2SO.sub.4 (1.88 mL, 20 mmol) in acetone (85 mL) was stirred for 18 h. As the conversion was not complete, Me.sub.2SO.sub.4 (1.88 mL, 20 mmol) was added and mixture was stirred at rt for 18 h more. The mixture was quenched by addition of TEA (5.8 mL, 42 mmol) and then stirred at rt for 30 min. The mixture was diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (1d) (3.75 g, 8.7 mmol, 68% on 2 steps). MS m/z ([M+H].sup.+) 429. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.19 (s, 3H), 0.22 (s, 3H), 0.94 (s, 9H), 1.49 (s, 9H), 3.00-3.20 (m, 1H), 4.00 (s, 3H), 4.11-4.41 (m, 1H), 4.40 (dd, J=1.8, 17.1 Hz, 1H), 4.85-5.05 (m, 2H).

Step 4: Preparation of Intermediate O5-tert-butyl O2-methyl 7-hydroxy-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-2,5-dicarboxylate (1e)

[0170] At rt, a solution of TBAF 1M in THF (8.7 mL, 8.7 mmol) was added to a solution of intermediate (1d) (3.75 g, 8.7 mmol) in THF (44 mL). After 30 min, the mixture was diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (DCM/Acetone: 10/0 to 5/5) to give intermediate (1e) (2.2 g, 7.0 mmol, 80%). MS m/z ([M+H].sup.+) 315. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.48 (s, 9H), 3.74-3.90 (m, 2H), 4.01 (s, 3H), 4.59 (dd, J=1.3, 17.2 Hz, 1H), 4.78-4.88 (m, 1H), 4.96-5.05 (m, 1H).

Step 5: Preparation of Intermediate O5-tert-butyl O2-methyl 7-[allyloxy-(2-nitrophenyl)sulfonyl-amino]-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-2,5-dicarboxylate (1f)

[0171] At 0° C., DTAD (2.09 g, 9.1 mmol) was added portion wise to a solution of intermediate (1e) (2.2 g, 7.0 mmol), N-allyloxy-2-nitro-benzenesulfonamide (2.35 g, 9.1 mmol) and PPh.sub.3 (2.38 g, 9.1 mmol) in toluene (70 mL). The mixture was stirred at rt for 2 h 30 and then concentrated. The residue was roughly purified by flash chromatography on silica gel (DCM/Acetone: 10/0 to 7/3) to give intermediate (1f) which was used in next step without further purification. MS m/z ([M+H].sup.+) 555.

Step 6: Preparation of Intermediate O5-tert-butyl O2-methyl 7-(allyloxyamino)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-2,5-dicarboxylate (1a)

[0172] At 0° C., K.sub.2CO.sub.3 (7.25 g, 52.5 mmol) and thiophenol (3.59 mL, 35.0 mmol) were added to a solution of intermediate (1f) (7.0 mmol) in ACN (70 mL). After 3 h 30 at rt, the mixture was filtered on celite which was washed with ACN and DCM. The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (DCM/Acetone: 10/0 to 7/3) to provide intermediate (1g) (1.62 g, 4.4 mmol, 62% on 2 steps). MS m/z ([M+H].sup.+) 370. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.48 (s, 9H), 3.57-3.79 (m, 1H), 4.00 (s, 3H), 4.00-4.05 (m, 1H), 4.31 (d, J=6.0 Hz, 2H), 4.39-4.45 (m, 1H), 4.51-4.64 (m, 1H), 4.73-4.92 (m, 1H), 5.24 (dd, J=1.5, 10.5 Hz, 1H), 5.33 (dd, J=1.5 17.3 Hz, 1H), 5.93 (ddt, J=5.9, 10.3, 17.3 Hz, 1H).

Step 7: Preparation of Intermediate methyl 7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carboxylate (1h)

[0173] At 0° C., diphosgene (0.37 mL, 3.0 mmol) was added to a solution of intermediate (1g) (1.62 g, 4.4 mmol) and TEA (0.92 mL, 6.6 mmol) in DCM (44 mL). The mixture was stirred at 0° C. for 45 min then a solution of MeSO.sub.3H (4.27 mL, 65.8 mmol) in DCM (22 mL) was added. After 1 h at 0° C., TEA (12.23 mL, 87.8 mmol) was added and mixture was stirred at rt for 10 min. After partial concentration under azote, the mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (1h) (777 mg, 2.6 mmol, 60%). MS m/z ([M+H].sup.+) 296. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.26 (d, J=11.3 Hz, 1H), 3.78 (dd, J=2.9, 11.3 Hz, 1H), 4.00 (s, 3H), 4.37-4.53 (m, 3H), 4.67 (d, J=9.4 Hz, 1H), 4.69 (d, J=5.0 Hz, 1H), 5.29-5.40 (m, 2H), 6.00 (dddd, J=6.0, 6.7, 10.3, 17.1 Hz, 1H).

Step 8: Preparation of Intermediate 7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carboxylic acid (1i)

[0174] At 0° C., LiOH 1N (2.6 mL, 2.6 mmol) was added to a solution of intermediate (1h) (775 mg, 2.6 mmol) in a mixture of THF (26 mL) and water (13 mL). The mixture was stirred at 0° C. After 40 min, starting material remained and supplementary LiOH 1N (0.26 mL, 0.26 mmol) was added. The mixture was stirred at 0° C. for 15 min more until complete consumption of intermediate (1h). At 0° C., the mixture was quenched by addition of HCl 1N (2.88 mL, 2.88 mmol), diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give intermediate (1i) (639 mg, 2.27 mmol, 87%) which was used in next step without further purification. MS m/z ([M+H].sup.+) 282.

Step 9: Preparation of Intermediate tert-butyl N-[2-[(7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carbonyl)amino]ethyl]carbamate (1i)

[0175] DIPEA (0.23 mL, 1.33 mmol) and HATU (243 mg, 0.64 mmol) were added to a solution of intermediate (1i) (150 mg, 0.53 mmol) in DCM (5 mL). After 10 min, tert-butyl N-(2-aminoethyl)carbamate (128 mg, 0.80 mmol) was added and mixture was stirred at rt for 18 h. The mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified twice by column chromatography on silica gel (DCM/Acetone: 10/0 to 7/3) to give intermediate (1j) (52 mg, 0.123 mmol). MS m/z ([M−H].sup.−) 422.

Step 10: Preparation of Intermediate triphenyl-propenylphosphonium [2-[2-(tert-butoxycarbonylamino)ethylcarbamoyl]-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] sulfate (1k)

[0176] AcOH (14 μL, 0.24 mmol) and Pd(PPh.sub.3).sub.4 (71 mg, 0.06 mmol) were successively added to a solution of intermediate (1j) (52 mg, 0.12 mmol) in anhydrous DCM (1.2 mL). The mixture was stirred at rt for 2 h. Pyridine (1.2 mL) and sulfur trioxide pyridine complex (97 mg, 0.61 mmol) were then added and the mixture was stirred at rt for the night. The heterogeneous mixture was diluted with DCM and solids were filtered off. The filtrate was concentrated and the crude was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (1k) (22 mg, 0.028 mmol, 24%). MS m/z ([M−H].sup.−) 462.

Step 11: preparation of [2-(2-aminoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid, Example 1

[0177] At 0° C., TFA (0.1 mL) was added to a solution of intermediate (1k) (22 mg, 0.028 mmol) in DCM (0.13 mL). The mixture was stirred at 0° C. for 30 min. At 0° C., Et.sub.2O was added to give a precipitate and supernatant was eliminated (operation done 3 times). The residue was triturated twice with ACN and supernatant was eliminated each time. The precipitate was filtered on PTFE membrane and dried under vacuum. The crude was purified by column chromatography on C18 (Water/ACN 99/1 to 70/30). The fractions containing the desired compound were combined, partially concentrated under flux of nitrogen to remove ACN, frozen and lyophilized to provide Example 1 (6 mg, 0.016 mmol, 60%). MS m/z ([M+H].sup.+) 364. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 3.30 (t, J=5.7 Hz, 2H), 3.61 (d, J=11.7 Hz, 1H), 3.68-3.84 (m, 2H), 3.97 (dd, J=3.0, 11.7 Hz, 1H), 4.58 (d, J=16.8 Hz, 1H), 4.65 (d, J=16.8 Hz, 1H), 5.30 (d, J=2.8 Hz, 1H). .sup.19F NMR (377 MHz, D.sub.2O): no fluorine.

Example 2: Synthesis of [2-(guanidinomethyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid

[0178] ##STR00022## ##STR00023##

Step 1: Preparation of Intermediate tert-butyl 7-[tert-butyl(dimethyl)silyl]oxy-2-formyl-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (2a)

[0179] At −78° C. a solution of nBuLi 1.6M in hexanes (2.16 mL, 5.4 mmol) was added to a solution of intermediate (1b) (2.0 g, 5.4 mmol) in THF (27 mL). The mixture was stirred at −78° C. for 30 min before a solution of DMF (0.42 mL, 5.4 mmol) in THF (3 mL) was added. The mixture was stirred for 10 min at −78° C. The temperature was allowed to rise up at 0° C. for 1 h. The reaction was quenched by addition of NH.sub.4Cl solution, diluted with AcOEt, washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was used in next step without purification. MS m/z ([M+H].sup.+) 399. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.20 (s, 3H), 0.22 (s, 3H), 0.94 (s, 9H), 1.50 (s, 9H), 2.95-3.20 (m, 1H), 4.15-4.43 (m, 1H), 4.41 (d, J=16.8 Hz, 1H), 4.87-5.08 (m, 2H), 9.91 (s, 1H).

Step 2: Preparation of Intermediate tert-butyl 7-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (2b)

[0180] At 0° C., NaBH.sub.4 (225 mg, 5.94 mmol) was added to a solution of intermediate (2a) (5.4 mmol) in a mixture of THF (5 mL)/MeOH (15 mL). The mixture was stirred at rt for 30 min. Reaction was quenched by addition of acetone and water. After partial concentration, the residue was diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 5/5) to give intermediate (2b) (1.38 g, 3.5 mmol, 64% on 2 steps). MS m/z ([M+H].sup.+) 401. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.18 (s, 3H), 0.20 (s, 3H), 0.93 (s, 9H), 1.48 (s, 9H), 2.74 (bs, 1H), 3.04-3.30 (m, 1H), 3.99-4.32 (m, 1H), 4.39 (dd, J=1.7, 17.0 Hz, 1H), 4.73-4.96 (m, 2H), 4.93 (s, 2H).

Step 3: Preparation of Intermediate tert-butyl 7-[tert-butyl(dimethyl)silyl]oxy-2-(tetrahydropyran-2-yloxymethyl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (2c)

[0181] DHP (0.17 mL, 1.87 mmol) and APTS (catalytic amount) were added to a solution of intermediate (2b) (500 mg, 1.25 mmol) in DCM (12 mL). The mixture was stirred at rt for 3 h, diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (DCM/Acetone: 10/0 to 7/3) to give intermediate (2c) (570 mg, 1.18 mmol, 95%). MS m/z ([M+H].sup.+) 485. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.18 (s, 3H), 0.20 (s, 3H), 0.93 (s, 9H), 1.48 (s, 9H), 1.52-1.92 (m, 6H), 3.08-3.32 (m, 1H), 3.53-3.60 (m, 1H), 3.89 (ddt, J=2.8, 8.8, 11.4 Hz, 1H), 3.97-430 (m, 1H), 4.39 (d, J=16.8 Hz, 1H), 4.76 (d, J=13.7 Hz, 1H), 4.77-4.81 (m, 2H), 4.86-4.98 (m, 1H), 4.94 (d, J=13.7 Hz, 1H).

Step 4: Preparation of Intermediate tert-butyl 7-hydroxy-2-(tetrahydropyran-2-yloxymethyl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (2d)

[0182] At 0° C., TBAF 1M in THF (1.18 mL, 1.18 mmol) was added to a solution of intermediate (2c) (570 mg, 1.18 mmol) in THF (6 mL). The mixture was stirred at 0° C. for 1 h, diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 4/6) to give intermediate (2d) (413 mg, 1.12 mmol, 95%). MS m/z ([M+H].sup.+) 371. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.49 (s, 9H), 1.51-1.90 (m, 6H), 3.57 (ddt, J=2.9, 5.6, 9.8 Hz, 1H), 3.60-3.70 (m, 1H), 3.83-3.92 (m, 1H), 3.98 (dd, J=4.2, 13.7 Hz, 1H), 4.42 (d, J=17.0 Hz, 1H), 4.74-4.86 (m, 3H), 4.89-4.96 (m, 1H), 4.95 (d, J=14.0 Hz, 1H).

Step 5: Preparation of Intermediate tert-butyl 7-(allyloxyamino)-2-(tetrahydropyran-2-yloxymethyl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (2e)

[0183] At −78° C., a solution of Ms.sub.2O (580 mg, 3.33 mmol) in DCM (2.5 mL) was added to a solution of TEA (0.61 mL, 4.44 mmol) and intermediate (2d) (410 mg, 1.11 mmol) in DCM (11 mL). After 45 min at −78° C., a solution of NH.sub.2OAlI (566 mg, 7.76 mmol) in DCM (2.5 mL) was added. The mixture was stirred 15 min at −78° C. Temperature was allowed to rise up at rt for 1 h. The mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/AcOEt: 10/0 to 2/8) to give intermediate (2e) (375 mg, 0.88 mmol, 80%). MS m/z ([M+H].sup.+) 426. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.49 (s, 9H), 1.51-1.92 (m, 6H), 3.45-3.65 (m, 2H), 3.88 (ddd, J=3.1, 8.9, 11.4 Hz, 1H), 4.10-4.53 (m, 5H), 4.73-4.86 (m, 3H), 4.98 (dd, J=6.2, 14.6 Hz, 1H), 5.21-5.26 (m, 1H), 5.29-5.36 (m, 1H), 5.94 (ddt, J=5.9, 10.3, 17.3 Hz, 1H).

Step 6: Preparation of Intermediate 7-allyloxy-2-(hydroxymethyl)-5,8-methano-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-6-one (2f)

[0184] At 0° C., diphosgene (74 μL, 0.61 mmol) was added to a solution of intermediate (2e) (370 mg, 0.87 mmol) and TEA (0.18 mL, 1.3 mmol) in DCM (9 mL). The mixture was stirred at 0° C. for 45 min then a solution of MeSO.sub.3H (0.85 mL, 13.05 mmol) in DCM (1 mL) was added. After 1 h at 0° C., TEA (2.4 mL, 17.4 mmol) was added and the mixture was stirred at rt for 1 h. The mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 2/8) to give intermediate (2f) (163 mg, 0.61 mmol, 71%). MS m/z ([M+H].sup.+) 268. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.17 (d, J=11.0 Hz, 1H), 3.66 (dd, J=2.9, 11.1 Hz, 1H), 4.29 (d, J=16.8 Hz, 1H), 4.30-4.43 (m, 2H), 4.49 (d, J=16.8 Hz, 1H), 4.55 (d, J=2.3 Hz, 1H), 4.78 (s, 2H), 5.22-5.27 (m, 1H), 5.30 (dq, J=1.4, 17.2 Hz, 1H), 5.88-5.99 (m, 1H).

Step 7: Preparation of Intermediate tert-butyl N-[(7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-2-yl)methyl]-N—(N-tert-butoxycarbonylcarbamimidoyl)carbamate (2g)

[0185] Under argon atmosphere and at 0° C., DTAD (48 mg, 0.21 mmol) was added portionwise to a solution of intermediate (2f) (50 mg, 0.17 mmol), tert-butyl N—(N-tert-butoxycarbonylcarbamimidoyl)carbamate (54 mg, 0.21 mmol) and PPh.sub.3 (55 mg, 0.21 mmol) in toluene (1.2 mL). The mixture was stirred at rt for 2 h and then concentrated. The residue was purified by column chromatography on silica gel (DCM/AcOEt: 10/0 to 4/6) to provide intermediate (2g) (40 mg, 0.08 mmol, 47%). MS m/z ([M+H].sup.+) 509. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.39 (s, 9H), 1.49 (s, 9H), 3.21 (d, J=10.9 Hz, 1H), 3.70 (dd, J=2.9, 11.0 Hz, 1H), 4.32 (d, J=16.8 Hz, 1H), 4.35-4.48 (m, 2H), 4.52 (d, J=16.8 Hz, 1H), 4.56 (d, J=2.7 Hz, 1H), 5.26-5.38 (m, 3H), 5.49 (d, J=16.0 Hz, 1H), 5.92-6.04 (m, 1H), 9.40-9.43 (m, 2H).

Step 8: Preparation of Intermediate triphenyl-propenylphosphonium [2-[[tert-butoxycarbonyl-(N-tert-butoxycarbonylcarbamimidoyl)amino]methyl]-5,8-dimethyl-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] sulfate (2h)

[0186] AcOH (9 μL, 0.16 mmol) and Pd(PPh.sub.3).sub.4 (45 mg, 0.04 mmol) were successively added to a solution of intermediate (2g) (40 mg, 0.08 mmol) in anhydrous DCM (0.8 mL). After stirring for 1 h 30 at rt, pyridine (0.8 mL) and sulfur trioxide pyridine complex (62 mg, 0.39 mmol) were added and the mixture was stirred at rt overnight. The heterogeneous mixture was diluted with DCM and the solids were filtered off. The filtrate was concentrated and purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (2h) (38 mg, 0.05 mmol, 58%) with traces of POPh.sub.3. MS m/z ([M+H].sup.+) 549 and 303.

Step 9: preparation of [2-(quanidinomethyl)-5,8-dimethyl-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid, Example 2

[0187] At 0° C., TFA (0.15 mL) was added to a solution of intermediate (2h) (38 mg, 0.05 mmol) in DCM (0.15 mL). The mixture was stirred at 0° C. for 3 h 30. As reaction was not completed, TFA (0.15 mL) was added and mixture was stirred at 0° C. for 1 h 30 more (operation repeated twice). At 0° C., a precipitate was obtained by addition of Et.sub.2O and the supernatant was eliminated (operation repeated 3 times). The residue was triturated with ACN and supernatant was eliminated (operation repeated twice). The precipitate was filtered, dried under vacuum and purified by column chromatography on C18 (Water/ACN 99/1 to 50/50). The fractions containing the desired compound were combined, partially concentrated under flux of nitrogen to remove ACN, frozen and lyophilized to provide Example 2 as zwitterion (2.1 mg, 0.006 mmol, 14%). MS m/z ([M−H].sup.−) 347. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 3.59 (d, J=11.6 Hz, 1H), 3.92 (dd, J=3.0, 11.6 Hz, 1H), 4.50 (d, J=16.8 Hz, 1H), 4.59 (d, J=16.8 Hz, 1H), 4.78 (s, 2H), 5.20 (d, J=2.8 Hz, 1H). .sup.19F NMR (377 MHz, D.sub.2O): no fluorine.

Example 3: Synthesis of [2-(2-guanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid

[0188] ##STR00024##

Step 1: Preparation of Intermediate tert-butyl N-[2-[(7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carbonyl)amino]ethyl]-N—(N-tert-butoxycarbonylcarbamimidoyl)carbamate (3a)

[0189] DIPEA (0.23 mL, 1.33 mmol) and HATU (243 mg, 0.64 mmol) were added to a solution of intermediate (1i) (150 mg, 0.53 mmol) in DCM (3.5 mL). After 10 min, tert-butyl N—[N′-(2-aminoethyl)-N-tert-butoxycarbonyl-carbamimidoyl]carbamate (193 mg, 0.64 mmol) was added and mixture was stirred at rt for 4 h. As starting material was not totally consumed, HATU (60 mg, 0.16 mmol) was added and mixture was stirred for 40 min more. The mixture was diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified twice by column chromatography on silica gel (DCM/AcOEt: 10/0 to 7/3) and by preparative TLC (eluting DCM/AcOEt 9/1) to give intermediate (3a) (105 mg, 0.160 mmol). MS m/z ([M+H].sup.+) 566.

Step 2: Preparation of Intermediate triphenyl-propenylphosphonium [2-[2-[tert-butoxycarbonyl-(N-tert-butoxycarbonylcarbamimidoyl)amino]ethylcarbamoyl]-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] sulfate (3b)

[0190] AcOH (19 μL, 0.32 mmol) and Pd(PPh.sub.3).sub.4 (92 mg, 0.06 mmol) were successively added to a solution of intermediate (3a) (105 mg, 0.16 mmol) in anhydrous DCM (1.6 mL). The mixture was stirred at rt for 2 h. Pyridine (1.6 mL) and sulfur trioxide pyridine complex (127 mg, 0.80 mmol) were then added and the mixture was stirred at rt overnight. The heterogeneous mixture was diluted with DCM and solids were filtered off. The filtrate was concentrated and the crude was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (3b) (38 mg, 0.041 mmol, 26%). MS m/z ([M+H].sup.+) 606 and 303.

Step 3: preparation of [2-(2-quanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfonic acid. Example 3

[0191] At 0° C., TFA (0.4 mL) was added to a solution of intermediate (3b) (38 mg, 0.041 mmol) in DCM (0.4 mL). The mixture was stirred at 0° C. for 7 h and at −20° C. for 18 h. At 0° C., Et.sub.2O was added to give a precipitate and supernatant was eliminated (operation repeated 3 times). The residue was triturated twice with ACN and supernatant was eliminated each time. After trituration, the obtained solid was filtered on PTFE membrane and dried under vacuum. The crude was purified by column chromatography on C18 (Water/ACN 99/1 to 80/20). The fractions containing the desired compound were combined, partially concentrated under flux of nitrogen to remove ACN, frozen and lyophilized to provide Example 3 as zwitterion (2 mg, 0.005 mmol, 13%). MS m/z ([M−H].sup.−) 404. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 3.45-3.51 (m, 2H), 3.54-3.64 (m, 2H), 3.65-3.73 (m, 1H), 3.96 (dd, J=3.0, 11.7 Hz, 1H), 4.58 (d, J=16.9 Hz, 1H), 4.65 (d, J=16.9 Hz, 1H), 5.29 (d, J=2.8 Hz, 1H). .sup.19F NMR (377 MHz, D.sub.2O): no fluorine.

Example 4 (comparative): synthesis of [2-(aminomethyl)-5,8-dimethyl-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]hydrogen sulfate

[0192] ##STR00025##

Step 1: Preparation of Intermediate (7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-2-yl)methyl methanesulfonate (4a)

[0193] Under argon atmosphere and at 0° C., TEA (0.15 mL, 1.12 mmol) and Ms.sub.2O (130 mg, 0.75 mmol) were added to a solution of intermediate (2f) (100 mg, 0.37 mmol) in DCM (1.9 mL). The mixture was stirred at 0° C. for 1 h 30, diluted with DCM and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to provide intermediate (4a) (118 mg) which was used in next step without purification. MS m/z ([M+H].sup.+) 346.

Step 2: Preparation of Intermediate 7-allyloxy-2-(azidomethyl)-5,8-methano-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-6-one (4b)

[0194] Under argon atmosphere, NaN.sub.3 (111 mg, 1.71 mmol) was added to a solution of intermediate (4a) (118 mg) in DMF (1.1 mL). The mixture was stirred at 60° C. for 1 h, diluted with AcOEt and washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to provide intermediate (4b) (68 mg, 0.23 mmol, 63% on 2 steps) which was used in next step without purification. MS m/z ([M+H].sup.+) 293.

Step 3: Preparation of Intermediate tert-butyl N-[(7-allyloxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-2-yl)methyl]carbamate (4c)

[0195] Under argon atmosphere and at 0° C., PMe.sub.3 1M in THF (0.35 mL, 0.35 mmol)) was added to a solution of intermediate (4b) (68 mg) in THF (2.4 mL). The mixture was stirred at 0° C. for 45 min then BOC-ON (86 mg, 0.35 mmol) in solution in THF (0.5 mL) was added. The mixture was stirred at 0° C. for 1 h and then concentrated. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 4/6) to provide intermediate (4c) (51 mg, 57%). MS m/z ([M+H].sup.+) 367. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.46 (s, 9H), 3.21 (d, J=11.0 Hz, 1H), 3.70 (dd, J=2.9, 11.0 Hz, 1H), 4.33 (d, J=16.8 Hz, 1H), 4.33-4.50 (m, 3H), 4.53 (d, J=16.8 Hz, 1H), 4.57 (d, J=16.8 Hz, 1H), 5.25-5.38 (m, 3H), 5.93-6.04 (m, 1H).

Step 4: Preparation of Intermediate tert-butyl N-[(7-hydroxy-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-2-yl)methyl]carbamate (4d)

[0196] AcOH (16 μL, 0.27 mmol) and Pd(PPh.sub.3).sub.4 (79 mg, 0.07 mmol) were successively added to a solution of intermediate (4c) (50 mg, 0.13 mmol) in anhydrous DCM (1.4 mL). After stirring for 1 h 30 at rt, pyridine (1.4 mL) and sulfur trioxide pyridine complex (108 mg, 0.68 mmol) were added and the mixture was stirred at rt overnight. The mixture was diluted with DCM and the solids were filtered off. The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (4d) (75 mg, 0.10 mmol, 78%). MS m/z ([M−H].sup.−) 405.

Step 5: preparation of [2-(aminomethyl)-5,8-dimethyl-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] hydrogen sulfate. Example 4

[0197] At 0° C., TFA (0.3 mL) was added to a solution of intermediate (4d) (75 mg, 0.10 mmol) in DCM (0.1 mL). After 30 min at 0° C., conversion was not complete. TFA was so added at 0° C. until complete conversion. At 0° C., a precipitate was obtained by addition of Et.sub.2O and the supernatant was eliminated (operation repeated 3 times). The residue was triturated with ACN and supernatant was eliminated (operation repeated twice). The solid was filtered, dried under vacuum and purified twice by column chromatography on C18 (Water/ACN 99/1 to 50/50). The fractions containing the desired compound were combined, partially concentrated under flux of nitrogen to remove ACN, frozen and lyophilized to provide Example 4 as zwitterion (3.8 mg, 0.012 mmol, 12%). MS m/z ([M−H].sup.−) 306. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 3.60 (d, J=11.6 Hz, 1H), 3.94 (dd, J=3.0, 11.6 Hz, 1H), 4.54 (d, J=16.9 Hz, 1H), 4.57 (s, 2H), 4.62 (d, J=16.9 Hz, 1H), 5.23 (d, J=2.9 Hz, 1H). .sup.19F NMR (377 MHz, D.sub.2O): no fluorine.

Example 9: Synthesis of [trans-4-(dimethylcarbamoyl)-2-(2-guanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]hydrogen sulfate

[0198] ##STR00026## ##STR00027## ##STR00028## ##STR00029##

Step 1: Preparation of Intermediate tert-butyl 7-[allyloxy-(2-nitrophenyl)sulfonyl-amino]-2-(tetrahydropyran-2-yloxymethyl)-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-5-carboxylate (9a)

[0199] Under inert atmosphere, DIAD (10.47 g, 51.82 mmol) was added portionwise to a solution of intermediate (2d) (16 g, 43.18 mmol), N-allyloxy-2-nitro-benzenesulfonamide (13.38 g, 51.82 mmol) and PPh.sub.3 (13.59 g, 51.8 mmol) in toluene (308 mL). The mixture was stirred at rt for 16 h and then concentrated. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (9a) (20.1 g, 31.27 mmol, 72%). MS m/z ([M+H].sup.+) 611. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.45 (s, 9H), 1.53-1.94 (m, 6H), 3.34-3.63 (m, 2H), 3.87 (t, J=10.2 Hz, 1H), 4.18 (s, 2H), 4.33 (d, J=17.2 Hz, 2H), 4.67-4.87 (m, 2H), 4.87-5.01 (m, 2H), 5.08 (d, J=10.2 Hz, 2H), 5.32 (s, 1H), 5.57 (s, 1H), 7.67 (d, J=7.8 Hz, 1H), 7.75-7.85 (m, 2H), 8.16 (dd, J=7.9, 1.5 Hz, 1H).

Step 2: Preparation of Intermediate N-allyloxy-N-[2-(hydroxymethyl)-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridin-7-yl]-2-nitro-benzenesulfonamide hydrochloride (9b)

[0200] Under inert atmosphere intermediate (9a) (20 g, 32.88 mmol) was diluted in HCl 4N in dioxane (312 mL). The reaction mixture was stirred at rt for 1 h 20. The precipitate was filtered. The solid was successively washed with dioxane and Et.sub.2O to give intermediate (9b) (15.1 g, 31.27 mmol, 95%). MS m/z ([M+H].sup.+) 427. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 3.64-3.73 (m, 1H), 3.81 (dd, J=3.5, 14.0 Hz, 1H), 3.90-3.99 (m, 1H), 4.05 (dd, J=6.3, 11.9 Hz, 1H), 4.30 (dd, J=1.5, 16.0 Hz, 1H), 4.44 (d, J=15.9 Hz, 1H), 4.82 (d, J=1.1 Hz, 2H), 4.89-5.02 (m, 2H), 5.37 (ddd, J=5.3, 10.3, 17.0 Hz, 1H), 5.67 (s, 1H), 7.88 (ddd, J=1.6, 7.3, 8.0 Hz, 1H), 7.92-8.03 (m, 2H), 8.20 (dd, J=1.4, 8.0 Hz, 1H).

Step 3: Preparation of Intermediate N-allyloxy-N-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridin-7-yl]-2-nitro-benzenesulfonamide (9c)

[0201] Under inert atmosphere, intermediate (9b) (15.1 g, 32.6 mmol) was diluted in anhydrous DCM (65.2 mL). At 0° C., TBDMSCl (7.4 g, 48.9 mmol), TEA (13.6 mL, 97.8 mmol) and DMAP (398 mg, 3.3 mmol) were added. The mixture was stirred at rt for 16 h. The mixture was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 6/4) to give intermediate (9c) (15.7 g, 28.46 mmol, 87%). MS m/z ([M+H].sup.+) 541. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.00 (s, 6H), 0.82 (s, 9H), 2.88 (dd, J=4.7, 14.6 Hz, 1H), 3.02 (s, 1H), 3.67 (d, J=17.0 Hz, 1H), 3.86 (d, J=17.0 Hz, 1H), 3.97 (s, 1H), 4.31 (dd, J=6.2, 11.3 Hz, 1H), 4.78 (s, 2H), 4.92-5.04 (m, 3H), 5.40-5.53 (m, 1H), 7.51 (dd, J=1.3, 7.9 Hz, 1H), 7.62 (td, J=1.4, 7.7 Hz, 1H), 7.66-7.72 (m, 1H), 8.01 (dd, J=1.5, 7.9 Hz, 1H).

Step 4: Preparation of Intermediate N-allyloxy-N-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-6,7-dihydrothiazolo[4,5-c]pyridin-7-yl]-2-nitro-benzenesulfonamide (9d)

[0202] Under inert atmosphere at 0° C. intermediate (9c) (4.94 g, 9.1 mmol) was diluted in DCM (55.4 mL). A solution of NCS (1.6 g, 11.9 mmol) in DCM (55.4 mL) was added dropwise. After stirring for 2 h at 0° C., DIPEA (5.9 mL, 33.8 mmol) was added dropwise and the reaction mixture was stirred at rt for 16 h. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 6/4) to give intermediate (9d) (4.44 g, 8.24 mmol, 83%). MS m/z ([M+H].sup.+) 539. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.14 (s, 6H), 0.96 (s, 9H), 3.76-3.91 (m, 1H), 4.02 (bs, 2H), 4.37 (dd, J=6.3, 11.2 Hz, 1H), 4.92 (d, J=5.4 Hz, 2H), 5.04-5.15 (m, 2H), 5.55 (m, 2H), 7.66 (dd, J=1.3, 7.9 Hz, 1H), 7.73 (td, J=1.3, 7.7 Hz, 1H), 7.82 (td, J=1.4, 7.7 Hz, 1H), 8.07 (d, J=7.9 Hz, 1H), 8.40 (s, 1H).

Step 5: Preparation of Intermediate N-allyloxy-N-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-cyano-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridin-7-yl]-2-nitro-benzenesulfonamide (9e)

[0203] Under inert atmosphere at 0° C., intermediate (9d) (4.44 g, 8.24 mmol) was diluted in anhydrous DCM (57 mL). TMSCN (10.3 mL, 82.4 mmol) was added. The reaction mixture was stirred at rt for 46 h. The mixture was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (9e) (4.45 g, 7.9 mmol, 87%). MS m/z ([M+H].sup.+) 566. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.00 (s, 6H), 0.79 (s, 9H), 2.11 (s, 1H), 3.21 (d, J=13.9 Hz, 1H), 3.95 (s, 1H), 4.28 (dd, J=6.2, 11.3 Hz, 1H), 4.69-4.80 (m, 2H), 4.85 (s, 1H), 4.86-4.97 (m, 2H), 4.99 (dd, J=1.9, 3.5 Hz, 1H), 5.32-5.46 (m, 1H), 7.46-7.53 (m, 1H), 7.60 (td, J=1.3, 7.7 Hz, 1H), 7.68 (td, J=1.5, 7.7 Hz, 1H), 7.96 (dd, J=1.4, 7.9 Hz, 1H).

Step 6: Preparation of Intermediate methyl 7-[allyloxy-(2-nitrophenyl)sulfonyl-amino]-2-(hydroxymethyl)-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine-4-carboxylate (9f)

[0204] Under inert atmosphere at 0° C., acetyl chloride (7.8 mL, 110.2 mmol) was diluted in anhydrous MeOH (20 mL). After stirring for 2 h at rt, a solution of intermediate (9e) (4.45 g, 7.9 mmol) in anhydrous MeOH (9.8 mL) was added. The reaction mixture was heated at 50° C. for 16 h 30. The mixture was concentrated to dryness under reduced pressure. The residue was diluted with DCM and sat NaHCO.sub.3. Aqueous layer was extracted with DCM. The combined organic layers were dried over Na.sub.2SO.sub.4, evaporated to dryness to give intermediate (9f) (3.55 g, 7.34 mmol, 83%). MS m/z ([M+H].sup.+) 485.

Step 7: Preparation of Intermediate methyl 7-[allyloxy-(2-nitrophenyl)sulfonyl-amino]-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine-4-carboxylate (9q)

[0205] Using the procedure described in example 9 (step 3), intermediate (9f) (3.55 g, 7.34 mmol) was converted into intermediate (9g) (3.31 g, 5.5 mmol, 71%) after purification by flash chromatography on silica gel (DCM/acetone: 100/0 to 60/40). MS m/z ([M+H].sup.+) 599.

Step 8: Preparation of Intermediate O5-tert-butyl O4-methyl 7-[allyloxy-(2-nitrophenyl)sulfonyl-amino]-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-4,5-dicarboxylate (9h)

[0206] Under inert atmosphere intermediate (9g) (3.31 g, 5.5 mmol) was diluted in anhydrous DCM (55 mL). DIPEA (1.25 mL, 7.19 mmol) and Boc.sub.2O (1.57 g, 7.2 mmol) were successively added. After stirring for 65 h at rt, the mixture was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (cyclohexane/EtOAc: 10/0 to 0/10) to give intermediate (9h) (3.52 g, 5.04 mmol, 87%). MS m/z ([M+H].sup.+) 699. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.00 (d, J=3.7 Hz, 6H), 0.83 (s, 9H), 1.30 (d, J=9.2 Hz, 9H), 3.38-3.73 (m, 4H), 4.00-4.48 (m, 3H), 4.80 (s, 2H), 4.85-5.08 (m, 2H), 5.09-5.31 (m, 1H), 5.31-5.76 (m, 2H), 7.54 (dd, J=8.3, 11.5 Hz, 1H), 7.60-7.76 (m, 2H), 7.95-8.10 (m, 1H).

Step 9: Preparation of Intermediate O5-tert-butyl O4-methyl 7-(allyloxyamino)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-6,7-dihydro-4H-thiazolo[4,5-c]pyridine-4,5-dicarboxylate (9i)

[0207] Under inert atmosphere, intermediate (9h) (3.52 g, 5.04 mmol) was diluted in anhydrous ACN (34 mL). Thiophenol (2.6 g, 25.2 mmol) and K.sub.2CO.sub.3 (5.22 g, 37.8 mmol) were successively added. The reaction mixture was stirred at rt for 2 h. The mixture was filtered over a pad of Célite® which was washed with ACN. The filtrate was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (cyclohexane/EtOAc: 10/0 to 7/3) to give intermediate (9i) (2.59 g, 5.04 mmol, 95%). MS m/z ([M+H].sup.+) 514. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.00 (d, J=1.4 Hz, 6H), 0.82 (s, 9H), 1.24-1.43 (m, 9H), 3.32-3.43 (m, 1H), 3.66 (d, J=1.5 Hz, 3H), 4.07-4.68 (m, 4H), 4.80 (m, 2H), 5.00-5.33 (m, 2H), 5.36-5.70 (m, 1H), 5.82 (m, 1H).

Step 10: Preparation of Intermediate methyl trans-7-allyloxy-2-(hydroxymethyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-4-carboxylate (9i)

[0208] Under inert atmosphere at 0° C., intermediate (9i) (2.59 g, 5.05 mmol) was diluted in anhydrous DCM (51 mL). TEA (1.06 mL, 7.58 mmol) was added. Diphosgene (427 μL, 3.54 mmol) was added dropwise. After stirring for 45 min at 0° C., a solution of MeSO.sub.3H (4.92 mL, 75.77 mmol) in DCM (5.6 mL) was added dropwise. After stirring for 1 h at 0° C., TEA (14.08 mL, 101.03 mmol) was added dropwise. The reaction mixture was stirred at rt for 1 h. The reaction mixture was diluted with DCM and the solution was washed with H.sub.2O and brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 5/5) to give intermediate (9j) (1.195 g, 3.67 mmol, 73%). MS m/z ([M+H].sup.+) 326. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.64 (dd, J=2.9, 11.5 Hz, 1H), 3.77 (dd, J=0.8, 11.6 Hz, 1H), 3.88 (s, 3H), 4.46 (qdt, J=1.2, 6.7, 12.3 Hz, 2H), 4.65 (dd, J=0.7, 2.9 Hz, 1H), 4.91 (s, 2H), 5.28 (s, 1H), 5.30-5.47 (m, 2H), 6.00-6.05 (m, 1H).

Step 11: Preparation of Intermediate trans-7-allyloxy-4-methoxycarbonyl-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carboxylic acid (9k)

[0209] Under inert atmosphere, periodic acid (1.38 g, 6.06 mmol) was diluted in ACN (16 mL) and water (1.2 mL). Chromium(VI)oxide (14.3 mg, 0.14 mmol) was added. Under inert atmosphere at 0° C. of intermediate (9j) (464 mg, 1.43 mmol) was diluted in ACN (16 mL). The previous solution was added dropwise over 30 min. The reaction mixture was stirred at rt for 1 h. The reaction mixture was diluted with DCM and the solution was washed with aq. citric acid 10%, brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give intermediate (9k) (305 mg, 0.89 mmol, 60%) which was used in next step without further purification. MS m/z ([M+H].sup.+) 340.

Step 12: Preparation of Intermediate (methyl trans-7-allyloxy-2-[2-(tert-butoxycarbonylamino)ethylcarbamoyl]-5,8-dimethyl-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-4-carboxylate (9l)

[0210] Under inert atmosphere at 0° C., intermediate (9k) (305 mg, 0.89 mmol) was diluted in anhydrous DMF (3.9 mL). N-Boc-ethylenediamine (171 μL, 1.08 mmol), EDC (248 mg, 1.29 mmol), HOBt (166 mg, 1.08 mmol) and DIPEA (470 μL, 2.7 mmol), were successively added. After stirring for 19 h at 50° C., the mixture was diluted with EtOAc and the solution was washed with brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 8/2) to give intermediate (9l) (183 mg, 0.32 mmol, 35%). MS m/z ([M+H].sup.+) 482. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.44 (s, 9H), 3.38 (s, 2H), 3.56 (qd, J=7.2, 13.8 Hz, 2H), 3.66 (dd, J=2.9, 11.7 Hz, 1H), 3.79 (dd, J=0.8, 11.8 Hz, 1H), 3.89 (s, 3H), 4.46 (qdt, J=1.2, 6.6, 12.2 Hz, 2H), 4.62-4.75 (m, 1H), 4.89 (s, 1H), 5.30-5.47 (m, 3H), 5.98-6.03 (m, 1H), 7.49 (s, 1H).

Step 13: Preparation of Intermediate trans-7-allyloxy-2-[2-(tert-butoxycarbonylamino)ethylcarbamoyl]-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-4-carboxylic acid (9m)

[0211] Under inert atmosphere at 0° C. intermediate (9l) (183 mg, 0.32 mmol) was diluted in a mixture of THF (1.7 mL) and water (1.1 mL). A solution LiOH 1N (319 μL, 0.32 mmol) was added dropwise. After 1 h at rt, further LiOH 1N (62 μL, 0.06 mmol) was added at 0° C. After 2 h 20, further LiOH 1N (62 μL, 0.06 mmol) was added at 0° C. The mixture was stirred at rt for 1 h more until complete consumption of intermediate (9l). At 0° C., the mixture was quenched by addition of HCl 1N to pH=2. The mixture was extracted with DCM. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give intermediate (9m) (149 mg, 0.32 mmol, 100%) which was used in next step without further purification. MS m/z ([M+H].sup.+) 468.

Step 14: Preparation of Intermediate tert-butyl N-[2-[[trans-7-allyloxy-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carbonyl]amino]ethyl]carbamate (9n)

[0212] Under inert atmosphere at 0° C. intermediate (9m) (199 mg, 0.43 mmol) was diluted in anhydrous DMF (1.8 mL). Dimethylamine hydrochloride (69 mg, 0.85 mmol), DIPEA (222 μL, 1.28 mmol) and HATU (178 mg, 0.47 mmol) were successively added. After stirring for 2 h at rt, the reaction mixture was diluted with EtOAc and the solution was washed with brine. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 5/5) to give intermediate (9n) (274 mg, 0.40 mmol, 94%). MS m/z ([M+H].sup.+) 495. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.34-1.49 (m, 9H), 3.04 (d, J=1.5 Hz, 3H), 3.30 (s, 2H), 3.34 (d, J=1.5 Hz, 3H), 3.44-3.55 (m, 3H), 3.70 (d, J=11.3 Hz, 1H), 4.34-4.50 (m, 2H), 4.67 (t, J=2.0 Hz, 1H), 5.30-5.44 (m, 3H), 5.95-6.03 (m, 1H).

Step 15: Preparation of Intermediate tert-butyl N-[2-[[trans-7-allyloxy-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepine-2-carbonyl]amino]ethyl]-N—(N-tert-butoxycarbonylcarbamimidoyl)carbamate (9o)

[0213] At 0° C., TFA (1.73 mL) was added to a solution of intermediate (9n) (210 mg, 0.425 mmol) in DCM (4.2 mL). After stirring for 1 h at 0° C., the mixture was concentrated to dryness under reduced pressure. The residue was diluted in anhydrous THF (4.25 mL). TEA (180 μL, 1.27 mmol) and 2-(trifluoromethylsulfonyl)guanidine (332 mg, 0.85 mmol) were successively added. After stirring for 1 h 30 at rt, the mixture was concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 7/3) to give intermediate (9o) (152 mg, 0.23 mmol, 53%). MS m/z ([M+H].sup.+) 637. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.43 (m, 18H), 2.98 (s, 3H), 3.28 (s, 3H), 3.45 (m, 2H), 3.53-3.69 (m, 3H), 3.73 (d, J=11.4 Hz, 1H), 4.37 (qdt, J=1.2, 6.6, 12.4 Hz, 2H), 4.60 (d, J=2.6 Hz, 1H), 5.21-5.38 (m, 3H), 5.86-5.99 (m, 1H), 7.23 (s, 1H), 8.48 (s, 1H).

Step 16: Preparation of Intermediate allyl(triphenyl)phosphonium [trans-2-[2-[tert-butoxycarbonyl-(N-tert-butoxycarbonylcarbamimidoyl)amino]ethylcarbamoyl]-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl]sulfate (9p)

[0214] AcOH (27 μL, 0.48 mmol) and Pd(PPh.sub.3).sub.4 (138 mg, 0.12 mmol) were successively added to a solution of intermediate (9o) (152 mg, 0.24 mmol) in anhydrous DCM (4.9 mL). After 2 h 20 at rt, further AcOH (1.5 μL, 0.026 mmol) and Pd(PPh.sub.3).sub.4 (70 mg, 0.06 mmol) were added. After 3 h 40 at rt, further AcOH (7 μL, 0.122 mmol) was added. After 4 h 30 at rt, conversion was not complete and phenylsilane (10.3 μL, 0.084 mmol) was added. The mixture was stirred at rt for 1 h 30 more until complete consumption of intermediate (9o). Pyridine (2.4 mL) and sulfur trioxide pyridine complex (190 mg, 1.19 mmol) were then added and the mixture was stirred at rt for the night. The heterogeneous mixture was diluted with DCM and solids were filtered off. The filtrate was concentrated and the crude was purified by column chromatography on silica gel (DCM/Acetone: 10/0 to 0/10) to give intermediate (9p) (100 mg, 0.10 mmol, 39%) which was used in next step without further purification. MS m/z ([M+H].sup.+) 677.

Step 17: Synthesis of [trans-4-(dimethylcarbamoyl)-2-(2-guanidinoethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothiazolo[4,5-e][1,3]diazepin-7-yl] hydrogen sulfate. Example 9

[0215] At 0° C., TFA (0.455 mL) was added to a solution of intermediate (9p) (100 mg, 0.102 mmol) in DCM (1.02 mL). At 0° C., further TFA was added four times every 2 h per portion of 150 μL. The mixture was stirred for further 5 h 30 until complete conversion on intermediate (9p). The mixture was diluted in Et.sub.2O. The precipitate was filtered and triturated in Et.sub.2O and AON. The crude was purified by column chromatography on C18 (Water/AON 99/1 to 90/10). Fractions of interest were combined, partially concentrated in vacuo, frozen and lyophilized to provide Example 9 (27 mg, 0.057 mmol, 42%). MS m/z ([M+H].sup.+) 477. .sup.1H NMR (400 MHz, D.sub.2O): δ (ppm) 2.99 (s, 3H), 3.33 (s, 3H), 3.39 (dd, J=5.1, 6.4 Hz, 2H), 3.52-3.60 (m, 3H), 3.74 (dd, J=2.9, 12.1 Hz, 1H), 5.23 (d, J=2.5 Hz, 1H), 5.66 (s, 1H).

Example 10: Synthesis of [N-[[trans-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-7-sulfooxy-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methyl]carbamimidoyl]ammonium 2,2,2-trifluoroacetate

[0216] ##STR00030## ##STR00031## ##STR00032##

Step 1: Preparation of Intermediate 2-(4-bromo-3-thienyl)-N,N-dimethyl-2-oxo-acetamide (10a)

[0217] At −78° C. under nitrogen atmosphere, to a solution of 3,4-dibromothiophene (10 g, 41.33 mmol) in anhydrous Et20 (100 mL) was dropwise added a n-butyllithium solution 2.5 M in hexanes (18.2 mL, 45.47 mmol). The mixture was stirred at −78° C. for 20 min then added (via cannula) to a solution of ethyl N,N-dimethyloxamate (6.18 mL, 45.47 mmol) in anhydrous Et.sub.2O (100 mL) at −78° C. The mixture was stirred at −78° C. for 30 min. Water (100 mL) was added and the mixture was stirred for 5 min at rt. The layers were separated. The aqueous layer was extracted with Et.sub.2O (100 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to provide intermediate (10a) (8.72 g, 33.26 mmol, 80%) as a yellow oil which was used without further purification. MS m/z ([M+H].sup.+) 262/264. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.01 (s, 3H), 3.07 (s, 3H), 7.36 (d, J=3.5 Hz, 1H), 8.25 (d, J=3.5 Hz, 1H).

Step 2: Preparation of Intermediate 2-(4-bromo-3-thienyl)-2-hydroxy-N,N-dimethyl-acetamide (10b)

[0218] At 0° C., sodium borohydride (1.26 g, 33.26 mmol) was portionwise added to a solution of intermediate (10a) (8.72 g, 33.26 mmol) in methanol (100 mL). The mixture was stirred at 0° C. for 40 min then water (50 mL) was added. Methanol was evaporated in vacuo. The resulting solution was extracted with AcOEt (2×60 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (cyclohexane/AcOEt 50/50 to 0/100) to provide intermediate (10b) (7.51 g, 28.43 mmol, 85% mmol) as a white solid. MS m/z ([M+H].sup.+) 264/266. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 2.74 (s, 3H), 3.05 (s, 3H), 4.00 (bs, 1H), 5.35 (s, 1H), 7.18 (d, J=3.4 Hz, 1H), 7.30 (d, J=3.4 Hz, 1H).

Step 3: Preparation of Intermediate 2-(4-bromo-3-thienyl)-2-(2,2-diethoxyethylamino)-N,N-dimethyl-acetamide (10c)

[0219] At 0° C., methanesulfonic anhydride (7.43 g, 42.65 mmol) was added to a solution of intermediate (10b) (7.51 g, 28.43 mmol) in DCM (120 mL) and TEA (7.9 mL, 56.86 mmol). The mixture was stirred at 0° C. for 1 h. Aminoacetaldehyde diethyl acetal (8.47 mL, 58.28 mmol), TEA (7.9 mL, 56.86 mmol) and tetrabutylammonium iodide (2.1 g, 5.68 mmol) were added and the mixture was stirred at rt overnight. The mixture was concentrated in vacuo. The residue was dissolved in MTBE (100 mL) and a saturated solution of NaHCO.sub.3 (100 mL). Layers were separated. The aqueous layer was extracted with MTBE (100 mL). The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 100/0 to 60/40) to provide intermediate (10c) (10.08 g, 26.57 mmol, 93%). MS m/z ([M+H].sup.+) 379/381. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.10-1.27 (m, 6H), 2.59 (dd, J=5.8, 12.1 Hz, 1H), 2.80 (dd, J=5.2, 12.1 Hz, 1H), 2.89 (s, 3H), 2.97 (s, 3H), 3.43-3.59 (m, 2H), 3.60-3.73 (m, 2H), 4.60 (t, J=5.5 Hz, 1H), 4.85 (s, 1H), 7.27 (d, J=3.5 Hz, 1H), 7.28 (d, J=3.5 Hz, 1H).

Step 4: Preparation of Intermediate tert-butyl 3-bromo-4-(dimethylcarbamoyl)-7-hydroxy-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10d)

[0220] A mixture of intermediate (10c) (10.08 g, 26.57 mmol) in a hydrochloric acid solution (6M in water, 180 mL) was heated at 80° C. overnight. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in water (150 mL) and NaHCO.sub.3 (17.8 g, 212.6 mmol) was carefully added. A solution of di-tert-butyl dicarbonate (8.12 g, 37.20 mmol) in THF (150 mL) was added and the mixture was stirred at rt overnight. THF was evaporated in vacuo. The aqueous solution was extracted with MTBE (2×150 mL). The organic layers were combined, washed with aqueous HCl 0.1 M, brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/AcOEt 10/0 to 5/5) to provide intermediate (10d) as a (6.07 g, 14.97 mmol, 56%, mixture of trans and cis isomers) as a light yellow foam. MS m/z ([M+H].sup.+) 405/407. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.48 and 1.49 (s, 9H), 2.96-2.99 (m, 3H), 3.35-3.60 (m, 3.5H), 4.33 (d, J=14.4 Hz, 0.5H), 4.08 (dd, J=4.5, 12.7 Hz, 0.5H), 4.33 (d, J=14.4 Hz, 0.5H), 4.73-4.85 (m, 1H), 5.92 and 6.12 (2s, 1H), 7.19 and 7.23 (2s, 1H).

Step 5: Preparation of Intermediate tert-butyl 3-bromo-7-[tert-butyl(dimethyl)silyl]oxy-4-(dimethylcarbamoyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10e)

[0221] To a solution of intermediate (10d) (6.07 g, 14.97 mmol) in DMF (60 mL) were successively added DMAP (183 mg, 1.50 mmol), imidazole (2.04 g, 29.95 mmol) and tert-butyldimethylsilyl chloride (3.38 g, 22.46 mmol). The mixture was stirred at rt for 2 h. Water (100 mL) was added. The mixture was extracted with MTBE (2×100 mL). The organic layers were combined, washed with brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-heptane/AcOEt 10/0 to 5/5) to provide intermediate (10e) (6.61 g, 12.72 mmol, 84%, mixture of diastereoisomers) as a colorless oil. MS m/z ([M+Na].sup.+) 541/543, ([M+H].sup.+) 519/521. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.13-0.20 (m, 6H), 0.88-0.95 (m, 9H), 1.48 (s, 9H), 2.96 and 2.99 (s, 3H), 3.37-3.51 (m, 3.5H), 3.88 (dd, J=3.0, 13.9 Hz, 0.5H), 4.11 (dd, J=5.4, 12.9 Hz, 0.5H), 4.17 (d, J=14.0 Hz, 0.5H), 4.75 (dd, J=5.4, 10.1 Hz, 0.5H), 4.85 (dd, J=2.0, 2.9 Hz, 0.5H), 5.95 and 6.12 (2s, 1H), 7.13 and 7.18 (2s, 1H).

Step 6: Preparation of Intermediate tert-butyl 4-(dimethylcarbamoyl)-7-hydroxy-3-vinyl-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10f)

[0222] To a degassed solution of intermediate (10e) (1.0 g, 1.92 mmol) in dioxane (6.4 mL) were added tributyl(vinyl)tin (692 mg, 2.11 mmol) and Pd(PPh.sub.3).sub.4 (111 mg, 0.10 mmol). The flask was sealed and the mixture was heated at 100° C. for 8 h. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (Petroleum Ether/AcOEt 10/0 to 5/5) to provide intermediate (10f) (897 mg, 1.92 mmol, 99%, mixture of diastereoisomers) as a solid. MS m/z ([M+H]) 467. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 0.15-0.20 (m, 6H), 0.91-0.95 (m, 9H), 1.48-1.54 (m, 9H), 2.94-2.97 (m, 3H), 3.32-3.44 (m, 3H), 3.82-4.19 (m, 2H), 4.79-4.86 (m, 1H), 5.14-5.20 (m, 1H), 5.44-5.51 (m, 1H), 6.01-6.17 (m, 1H), 6.29-6.42 (m, 1H), 7.16-7.18 (m, 1H).

Step 7: Preparation of Intermediate tert-butyl 4-(dimethylcarbamoyl)-3-formyl-7-hydroxy-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10g)

[0223] To a solution of intermediate (10f) (787 mg, 1.69 mmol) in THF/H2O (17/17 mL) were added OsO.sub.4 (4% in H2O) (215 mL, 0.03 mmol) and NaIO.sub.4 (905 mg, 4.22 mmol). The mixture was stirred at rt for 5 h. A saturated solution of Na.sub.2S.sub.2O.sub.3 was added and the mixture was extracted twice with AcOEt. The organic layers were combined, washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/AcOEt 10/0 to 6/4) to provide intermediate (10g) (800 mg, 1.69 mmol, 99%) as a solid. MS m/z ([M+H].sup.+) 469.

Step 8: Preparation of Intermediate tert-butyl 4-(dimethylcarbamoyl)-7-hydroxy-3-(hydroxymethyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10h)

[0224] Under inert atmosphere, to a solution of intermediate (10g) (700 mg, 1.49 mmol) in MeOH (13 mL) was added NaBH.sub.4 (80 mg, 2.09 mmol) at 0° C. The mixture was stirred at 0° C. for 30 min. Water was added and MeOH was evaporated in vacuo. The mixture was extracted twice with AcOEt. The organic layers were combined, washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give crude product (10h) (560 mg, 1.19 mmol, 80%) as a white solid. MS m/z ([M+H].sup.+) 471.

Step 9: Preparation of Intermediate tert-butyl 3-(azidomethyl)-4-(dimethylcarbamoyl)-7-hydroxy-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10i)

[0225] To a solution of intermediate (10h) (415 mg, 0.88 mmol) in anhydrous DCM (8.8 mL) at −10° C. were successively added TEA (0.5 mL, 3.53 mmol) and methanesulfonic anhydride (460 mg, 2.64 mmol). The mixture was stirred at −10° C. for 30 min and at rt for 3 h. A saturated solution of NaHCO.sub.3 was added and the mixture was extracted twice with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was solubilized in anhydrous DMF (4.3 mL) and sodium azide (256 mg, 3.9 mmol) was added. The mixture was stirred at rt for 30 min before being poured in a saturated solution of NaCl. The mixture was extracted twice with AcOEt. The organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to provide intermediate (10i) (509 mg, <100%) as a crude product. MS m/z ([M+H].sup.+) 496.

Step 10: Preparation of Intermediate tert-butyl 3-(azidomethyl)-4-(dimethylcarbamoyl)-7-hydroxy-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10i)

[0226] To a solution of intermediate (10i) (430 mg, 0.87 mmol) in anhydrous THF (4.3 mL) at 0° C. under inert atmosphere was added a solution of TBAF 1M in THF (1 mL, 1.04 mmol). The mixture was stirred at rt for 20 min then water was added to the mixture. The aqueous layer was extracted twice with AcOEt. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/Acetone 10/0 to 8/2) to provide intermediate (10j) (330 mg, 0.86 mmol, 72%) as a white solid. MS m/z ([M+H].sup.+) 382.

Step 11: Preparation of Intermediate tert-butyl 7-(allyloxyamino)-3-(azidomethyl)-4-(dimethylcarbamoyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (10k)

[0227] To a solution of intermediate (10j) (236 mg, 0.62 mmol) in DCM (6.2 mL) at −78° C. were added TEA (0.30 mL, 2.16 mmol) and methanesulfonic anhydride (222 mg, 1.24 mmol). The mixture was stirred at −78° C. for 50 min. A solution of o-allylhydroxylamine 90% in DCM (350 mg, 4.33 mmol) was added and the mixture was stirred at rt for 4 h. Water was added. Layers were separated. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-heptane/AcOEt 10/0 to 6/4) to provide intermediate (10k) (158 mg, 0.36 mmol, 60%) as a mixture of diastereoisomers. MS m/z ([M+H].sup.+) 437. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.49-1.51 (m, 9H), 3.0 (s, 3H), 3.34 (s, 3H), 3.65 (d, J=14.0 Hz, 1H), 4.05-4.55 (m, 5H), 5.19-5.40 (m, 2H), 5.73 (d, J=8.0 Hz, 1H), 5.90-6.02 (m, 1H), 6.09-6.18 (m, 1H), 6.38-6.85 (m, 1H), 7.0-7.2 (m, 1H).

Step 12: Preparation of Intermediate trans-7-allyloxy-3-(2-azidomethyl)-5,8-methano-N,N-dimethyl-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepine-4-carboxamide (101)

[0228] To a solution of intermediate (10k) (155 mg, 0.36 mmol) in anhydrous DCM (2 mL) under inert atmosphere at 0° C. were added TEA (0.1 mL, 0.71 mmol) and diphosgene (34 μL, 0.278 mmol). The mixture was stirred at 0° C. for 2 h. A saturated solution of NaHCO.sub.3 was added. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was solubilized in dioxane (0.5 mL) and a solution of HCl 4N in dioxane (2 mL, 7.1 mmol) was added. The mixture was stirred at rt for 5 h and then concentrated in vacuo. The residue was dissolved in DCM (2 mL) and TEA (0.2 mL, 0.73 mmol) was added. The mixture was stirred at rt overnight. Water was added. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/AcOEt 10/0 to 5/5) to provide intermediate (101) as trans isomer (25 mg, 0.07 mmol, 19%, trans isomer) and the cis isomer (8 mg, 0.02 mmol, 5.5%) as white solids.

[0229] Trans isomer: MS m/z ([M+H].sup.+) 363. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.04 (s, 3H), 3.34 (s, 3H), 3.48 (dd, J=2.9, 11.0 Hz, 1H), 3.55 (dd, J=0.8, 11.1 Hz, 1H), 4.08 (dd, J=I. 1, 14.2 Hz, 1H), 4.25 (dd, J=0.7, 14.1 Hz, 1H), 4.30-4.54 (m, 3H), 5.27-5.41 (m, 3H), 6.03 (ddt, J=6.3, 10.3, 16.9 Hz, 1H), 7.09 (s, 1H).

[0230] Cis isomer: MS m/z ([M+H].sup.+) 363. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 3.06 (s, 3H), 3.20 (d, J=10.8 Hz, 1H), 3.33 (s, 3H), 3.76 (dd, J=2.9, 10.7 Hz, 1H), 4.38-4.42 (m, 4H), 4.44 (d, J=2.8 Hz, 1H), 5.18 (s, 1H), 5.24-5.35 (m, 2H), 6.00 (ddt, J=6.3, 10.3, 16.7 Hz, 1H), 7.05 (d, J=0.9 Hz, 1H).

Step 13: Preparation of Intermediate tert-butyl N-[[[trans-7-allyloxy-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methylamino]-(tert-butoxycarbonylamino)methylene]carbamate (10m)

[0231] To a solution of intermediate (101) (25 mg, 0.07 mmol) in anhydrous THF (0.7 mL) at 0° C. was added trimethylphosphine 1M in THF (0.1 mL, 0.1 mmol). The mixture was stirred at 0° C. for 2 h. Then, TEA (21 μL, 0.15 mmol) and 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (35 mg, 0.09 mmol) were added at rt and the mixture was stirred for 2 h. The mixture was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (n-Heptane/AcOEt 10/0 to 2/8) to provide intermediate (10m) (25 mg, 0.04 mmol, 62%) as a white solid. MS m/z ([M+H].sup.+) 579. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.49 (s, 9H), 1.51 (s, 9H), 3.00 (s, 3H), 3.31 (s, 3H), 3.45 (dd, J=2.9, 11.0 Hz, 1H), 3.58 (d, J=11.0 Hz, 1H), 3.99-4.29 (m, 2H), 4.30-4.59 (m, 4H), 5.14-5.47 (m, 2H), 6.02 (ddt, J=6.3, 10.3, 17.0 Hz, 1H), 7.10 (d, J=0.9 Hz, 1H), 8.45 (s, 1H), II. 46 (s, 1H).

Step 14: Preparation of Intermediate tert-butyl N-[(tert-butoxycarbonylamino)-[[trans-4-(dimethylcarbamoyl)-7-hydroxy-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methylamino]methylene]carbamate (10n)

[0232] To a solution of intermediate (10m) (24 mg, 0.04 mmol) in DCM (0.4 mL) were successively added Pd(PPh.sub.3).sub.4 (24 mg, 0.02 mmol) and AcOH (17 μL, 0.304 mmol). The mixture was stirred at rt for 45 min. Further Pd(PPh.sub.3).sub.4 (88 mg, 0.078 mmol) and AcOH (5 μL, 0.08 mmol) were added. The mixture was stirred for further 50 min. The mixture was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 5/5) to provide intermediate (10n) (29 mg).

Step 15: Preparation of Intermediate tert-butyl N-[(tert-butoxycarbonylamino)-[[trans-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-7-sulfooxy-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methylamino]methylene]carbamate (10o)

[0233] A solution of intermediate (10n) in pyridine (400 μL) was heated at 40° C. in the presence of sulfur trioxide pyridine complex (33 mg, 0.2 mmol) for 2 h. The mixture was concentrated in vacuo. The residue was triturated in DCM and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (10o) (22 mg, 0.036 mmol, 89.5%). MS m/z ([M+H].sup.+) 619. MS m/z ([M−H].sup.−) 617.

Step 16: preparation of [N-[[trans-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-7-sulfooxy-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]methyl]carbamimidoyl]ammonium 2,2,2-trifluoroacetate. Example 10

[0234] A mixture of intermediate (10o) (22 mg, 0.036 mmol) in DCM (0.4 mL) and TFA (3.3 mL) was stirred at 0° C. for 6 h. The mixture was diluted with Et.sub.2O and the supernatant was removed (twice). The solid was triturated with ACN and filtered. The solid was washed with ACN and dried in vacuo in the presence of P.sub.2O.sub.5 to provide Example 10 (6 mg, 0.030 mmol, 36%) as TFA salt. MS m/z ([M+H].sup.+) 419. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 2.93 (s, 3H), 3.26 (s, 3H), 3.48 (dd, J=3.0, 11.2 Hz, 1H), 3.95 (dd, J=4.2, 15.7 Hz, 1H), 4.12 (dd, J=6.4, 15.8 Hz, 1H), 4.81 (d, J=2.7 Hz, 1H), 5.35 (s, 1H), 7.28 (s, 1H), 7.74 (d, J=6.0 Hz, 1H).

Example 11: Synthesis [(trans-4-(dimethylcarbamoyl)-3-(2-guanidinoethyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-7-yl]hydrogen sulfate

[0235] ##STR00033## ##STR00034##

Step 1: Preparation of Intermediate tert-butyl 7-[tert-butyl(dimethyl)silyl]oxy-4-(dimethylcarbamoyl)-3-(2-tetrahydropyran-2-yloxyethyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (11a)

[0236] To a degassed solution of intermediate (10e) (1.35 g, 2.60 mmol) in toluene (15 mL) and water (5 mL) were added potassium 2-(tetrahydro-2H-pyran-2-yloxy)ethyltrifluoroborate (613 mg, 2.60 mmol), cesium carbonate (2.54 g, 7.79 mmol), RuPhos (121 mg, 0.26 mmol) and palladium(II) acetate (29 mg, 0.13 mmol). The flask was sealed and the mixture was heated at 95° C. for 5 h. The mixture was cooled to rt and water was added. The layers were separated. The aqueous layer was extracted with toluene. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (cyclohexane/AcOEt 10/0 to 5/5) to provide intermediate (11a) (1.21 g, 2.12 mmol, 81%) as a yellow oil. MS m/z ([M+Na].sup.+) 591.

Step 2: Preparation of Intermediate tert-butyl 4-(dimethylcarbamoyl)-7-hydroxy-3-(2-tetrahydropyran-2-yloxyethyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (11b)

[0237] To a solution of intermediate (11a) (890 mg, 1.56 mmol) in anhydrous THF at 0° C. under inert atmosphere was added a solution of TBAF 1M in THF (2.35 mL, 2.35 mmol). The mixture was stirred at 0° C. for 2.5 h then concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 7/3) to provide intermediate (11 b) (761 mg, quantitative) as a yellow oil. MS m/z ([M+H].sup.+) 455.

Step 3: Preparation of Intermediate tert-butyl 7-(allyloxyamino)-4-(dimethylcarbamoyl)-3-(2-tetrahydropyran-2-yloxyethyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (11c)

[0238] To a solution of intermediate (11b) (755 mg, 1.66 mmol) in DCM (10 mL) at −78° C. were added TEA (0.68 mL, 4.98 mmol) and methanesulfonic anhydride (434 mg, 2.49 mmol). The mixture was stirred at −78° C. for 45 min. A solution of o-allylhydroxylamine 50% in diethyl ether (1.14 g, 8.30 mmol) was added and the mixture was stirred at rt for 2.5 h. Water was added. Layers were separated. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 8/2) to provide intermediate (11c) (603 mg, 1.18 mmol, 71%) as a yellow oil. MS m/z ([M+Na].sup.+) 532, m/z ([M+H].sup.+) 510.

Step 4: Preparation of Intermediate tert-butyl 7-(allyloxyamino)-4-(dimethylcarbamoyl)-3-(2-hydroxyethyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (11d)

[0239] A mixture of intermediate (11c) (443 mg, 0.87 mmol) and PPTS (328 mg, 1.30 mmol) in methanol (12 mL) was refluxed for 1 h. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 8/2) to provide intermediate (11d) (250 mg, 0.58 mmol, 67%) as a colorless oil. MS m/z ([M+H].sup.+) 426.

Step 5: Preparation of Intermediate tert-butyl 7-(allyloxyamino)-3-(2-azidoethyl)-4-(dimethylcarbamoyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-carboxylate (11e)

[0240] To a solution of intermediate (11d) (200 mg, 0.47 mmol) in anhydrous DCM (10 mL) at 0° C. were successively added DIPEA (0.123 mL, 0.705 mmol) and methanesulfonyl chloride (44 μL, 0.564 mmol). The mixture was stirred at 0° C. for 1 h. Water was added. The layers were separated. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was dissolved in anhydrous DMF (2.5 mL) and sodium azide (61 mg, 0.94 mmol) was added. The mixture was heated at 50° C. overnight before being poured in a saturated solution of NaHCO.sub.3. The mixture was extracted twice with AcOEt. The organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 7/3) to provide intermediate (11e) (143 mg, 0.32 mmol, 67%, mixture of isomers) as a colorless oil. MS m/z ([M+Na].sup.+) 473, m/z ([M+H].sup.+) 451. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.47 and 1.48 (2s, 9H), 2.50-2.74 (m, 2H), 2.97 (s, 3H), 3.19-3.62 (m, 6H), 4.12-4.57 (m, 4H), 5.11-5.42 (m, 2H), 5.85-6.12 (m, 2H), 6.91 and 6.96 (2s, 1H).

Step 6: Preparation of Intermediate trans-7-allyloxy-3-(2-azidoethyl)-5,8-methano-N,N-dimethyl-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepine-4-carboxamide (11f)

[0241] To a solution of intermediate (11e) (179 mg, 0.397 mmol) in anhydrous DCM (5 mL) under inert atmosphere at 0° C. were added TEA (83 μL, 0.596 mmol) and diphosgene (34 μL, 0.278 mmol). The mixture was stirred at 0° C. for 40 min. A saturated solution of NaHCO.sub.3 (5 mL) was added. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. HCl 4N in dioxane (3.97 mL, 15.89 mmol) was added to the residue and the mixture was stirred at rt for 2 h before being concentrated in vacuo. The residue was dissolved in DCM (5 mL) and TEA (0.277 mL, 1.98 mmol) was added. The mixture was stirred at rt overnight. Water was added. The aqueous layer was extracted with DCM. The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (cyclohexane/AcOEt 5/5 to 0/10) to provide intermediate (11f) as trans isomer (70 mg, 0.185 mmol, 46%) and the cis isomer (40 mg, 0.106 mmol, 26%) as white solids.

[0242] Trans isomer: MS m/z ([M+Na].sup.+) 399, m/z ([M+H].sup.+) 377. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 2.51 (t, J=7.4 Hz, 2H), 3.03 (s, 3H), 3.34 (s, 3H), 3.36-3.53 (m, 3H), 3.56 (d, J=11.0 Hz, 1H), 4.34-4.49 (m, 3H), 5.23 (s, 1H), 5.25-5.41 (m, 2H), 5.93-6.08 (m, 1H), 6.93 (s, 1H).

[0243] Cis isomer: MS m/z ([M+Na].sup.+) 399, m/z ([M+H].sup.+) 377. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 2.51-2.64 (m, 1H), 2.66-2.75 (m, 1H), 3.03 (s, 3H), 3.13 (d, J=10.7 Hz, 1H), 3.32 (s, 3H), 3.35-3.44 (m, 1H), 3.51-3.61 (m, 1H), 3.73 (dd, J=2.9, 10.7 Hz, 1H), 4.39 (d, J=6.3 Hz, 2H), 4.42 (d, J=2.8 Hz, 1H), 5.12 (s, 1H), 5.20-5.37 (m, 2H), 5.89-6.07 (m, 1H), 6.92 (s, 1H).

Step 7: Preparation of Intermediate tert-butyl N-[2-[trans-7-allyloxy-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-3-yl]ethyl]-N—(N-tert-butoxycarbonylcarbamimidoyl)carbamate (11a)

[0244] To a solution of intermediate (11f) (70 mg, 0.186 mmol) in anhydrous THF (2 mL) at 0° C. was added trimethylphosphine 1M in THF (0.28 mL, 0.28 mmol). The mixture was stirred at rt for 1 h before adding further trimethylphosphine 1M in THF (0.34 mL, 0.34 mmol). The stirring was resumed for 3 h. TEA (52 μL, 0.37 mmol) and 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (95 mg, 0.242 mmol) were added and the mixture was stirred for 30 min. The mixture was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (cyclohexane/AcOEt 8/2 to 2/8) to provide intermediate (11 g) (90 mg, 0.152 mmol, 81%) as a colorless oil. MS m/z ([M+H].sup.+) 593. .sup.1H NMR (400 MHz, CDCl.sub.3): δ (ppm) 1.47 (s, 9H), 1.49 (s, 9H), 2.40-2.60 (m, 2H), 3.02 (s, 3H), 3.33 (s, 3H), 3.44 (dd, J=2.9, 11.0 Hz, 1H), 3.57 (d, J=11.0 Hz, 1H), 3.59-3.67 (m, 2H), 4.30-4.52 (m, 3H), 5.16 (s, 1H), 5.22-5.44 (m, 2H), 5.89-6.10 (m, 1H), 6.94 (s, 1H), 8.38 (s, 1H), 11.45 (s, 1H).

Step 8: Preparation of Intermediate allyl(triphenyl)phosphonium [trans-3-[2-[tert-butoxycarbonyl(ethanimidoyl)amino]ethyl]-4-(dimethylcarbamoyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-7-yl] sulfate (11h)

[0245] To a solution of intermediate (11 g) (90 mg, 0.152 mmol) in DCM (2 mL) were successively added Pd(PPh.sub.3).sub.4 (88 mg, 0.078 mmol) and AcOH (17 μL, 0.304 mmol). The mixture was stirred at rt for 45 min. Further Pd(PPh.sub.3).sub.4 (88 mg, 0.078 mmol) and AcOH (17 μL, 0.304 mmol) were added. The stirring was resumed for 45 min. Further Pd(PPh.sub.3).sub.4 (44 mg, 0.038 mmol) and AcOH (9 μL, 0.15 mmol) were added. The stirring was resumed for 1 h. The mixture was concentrated in vacuo. The residue was co-evaporated twice with toluene. The residue was heated at 40° C. in the presence of pyridine (2 mL) and sulfur trioxide pyridine complex (121 mg, 0.76 mmol) for 2 h. The mixture was concentrated in vacuo. The residue was triturated in DCM and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (DCM/acetone 10/0 to 0/10) to provide intermediate (11h) (90 mg, 0.096 mmol, 63%). MS m/z ([M−H].sup.−) 631.

Step 9: preparation of [(trans-4-(dimethylcarbamoyl)-3-(2-guanidinoethyl)-5,8-methano-6-oxo-4,8-dihydrothieno[2,3-e][1,3]diazepin-7-yl] hydrogen sulfate. Example 11

[0246] A mixture of intermediate (11h) (90 mg, 0.096 mmol), DCM (0.2 mL) and TFA (2 mL) was stirred at 0° C. for 5 h. n-Heptane was added and the mixture was concentrated in vacuo. This operation was performed twice. The residue was purified by reversed phase chromatography on C18 (Water/acetonitrile 10/0 to 0/10). Fractions containing targeted compound were combined, frozen and lyophilized. The residue was stirred in water (1.5 mL) for 10 min. The solid was filtered. The solid was rinsed with water and dried in vacuo in the presence of P.sub.2O.sub.5 to provide Example 11 (13.3 mg, 0.030 mmol, 32%) as a white solid. MS m/z ([M+H].sup.+) 433. MS m/z ([M−H].sup.−) 431. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ (ppm) 2.34-2.45 (m, 1H), 2.50-2.57 (m, 1H), 2.93 (s, 3H), 3.29 (s, 3H), 3.31-3.37 (m, 3H), 3.46 (dd, J=3.0, 11.2 Hz, 1H), 4.78 (d, J=2.7 Hz, 1H), 5.29 (s, 1H), 7.15 (s, 1H), 7.40 (t, J=5.8 Hz, 1H).

TABLE-US-00001 Example Structure 4 COMPARATIVE [00035] Covered by general formula in WO2004052891 (not described) 5 COMPARATIVE [00036] Described as example 20 in WO2002100860 6 COMPARATIVE [00037] Described as example 64 in WO2002100860 7 COMPARATIVE [00038] Described as example 65 in WO2002100860 8 COMPARATIVE [00039] Described as example 44 in WO2004052891

Biological Activity

[0247] MIC of Compounds and Synergy with Ceftazidime Against Bacterial Isolates (Table 1 and 2)

[0248] Compounds of the present invention were assessed against genotyped bacterial strains alone or in combination with the β-lactam ceftazidime. In the assays, MICs of said compounds, or of ceftazidime at fixed concentrations of said compounds were determined by the broth microdilution method according to the Clinical Laboratory Standards Institute (CLSI—M7-A7). Briefly, compounds alone according to the invention were prepared in DMSO and spotted (2 μL each) on sterile polystyrene plates (Corning, 3788). Compounds and ceftazidime dilutions were prepared in DMSO and spotted (1 μL each) on sterile polystyrene plates (Corning, 3788). Log phase bacterial suspensions were adjusted to a final density of 5×10.sup.5 cfu/mL in cation-adjusted Mueller-Hinton broth (Beckton-Dickinson) and added to each well (98 μL). Microplates were incubated for 16-20 h at 35° C. in ambient air. The MIC of the compounds was defined as the lowest concentration of said compounds that prevented bacterial growth as read by visual inspection. The MIC of ceftazidime at each compound concentration was defined as the lowest concentration of ceftazidime that prevented bacterial growth as read by visual inspection.

TABLE-US-00002 TABLE 1 Bacterial species used in MIC determination Strains Resistance mechanism E. coli ECO 190317 TEM-1, SHV-12, CTX-M-15, OXA-1 E. coli ECO UFR39 CTX-M-15, NDM-1 E. cloacae ECL P99 AmpC K. pneumoniae KPN 121206 SHV-1, NDM-1 P. aeruginosa PAE 107051 TEM-24, OXA-1 P. aeruginosa PAE UFR92 derepressed AmpC, OprD- P. aeruginosa PAE 105250 (AH) OXA-15 P. aeruginosa PAE UFR49 VIM-2

TABLE-US-00003 TABLE 2 MIC of Ceftazidime (CAZ) and compounds alone (μg/mL) MIC compounds of the invention alone (μg/mL) PAE ECO ECO ECL KPN PAE PAE 105250 PAE Compounds 190317 UFR39 P99 121206 107051 UFR92 (AH) UFR49 CAZ 128 >256 128 >256 256 32 256 64 1 0.25 0.5 2 32 8 8 8 16 2 0.063 0.125 1 >32 2 4 4 4 3 0.5 2 0.5 16 4 8 8 16 4 0.25 0.25 1 32 >32 32 32 32 COMPARATIVE 5 >32 >32 >32 >32 >32 >32 >32 >32 COMPARATIVE 6 2 4 16 >32 >32 >32 >32 >32 COMPARATIVE 7 2 4 8 >32 >32 >32 >32 >32 COMPARATIVE 8 >32 >32 >32 >32 >32 >32 >32 >32 COMPARATIVE 9 32 >32 >32 >32 8 32 8 4 10  2 32 16 32 4 8 8 8 11  4 >32 16 >32 4 8 4 4

TABLE-US-00004 TABLE 3 MIC of Ceftazidime (CAZ) alone and CAZ/compound combinations combination of CAZ and compounds of the invention at 4 μg/mL: MIC (μg/mL) PAE ECO ECO ECL KPN PAE PAE 105250 PAE Compounds 190317 UFR39 P99 121206 107051 UFR92 (AH) UFR49 CAZ 128 >256 128 >256 256 32 256 64 CAZ + 1 <0.125 <0.125 <0.125 >128 4 4 32 32 CAZ + 2 <0.125 <0.125 <0.125 >128 <0.125 <0.125 <0.125 <0.125 CAZ + 3 <0.125 <0.125 <0.125 >128 <0.125 8 <0.125 64 CAZ + 4 <0.125 <0.125 <0.125 >128 4 8 128 64 COMPARATIVE CAZ + 5 128 >128 >128 >128 >128 32 >128 64 COMPARATIVE CAZ + 6 <0.125 <0.125 1 >128 8 32 >128 64 COMPARATIVE CAZ + 7 <0.125 <0.125 <=0.125 >128 16 32 >128 64 COMPARATIVE CAZ + 8 128 >128 64 >128 >128 32 >128 64 COMPARATIVE CAZ + 9 64 >128 128 >128 16 32 64 0.5  CAZ + 10 <0.125 >128 32 >128 <0.125 32 16 8  CAZ + 11 <0.125 >128 128 >128 <0.125 16 0.125 0.25