Macrocyclic amidinourea derivatives, methods of preparation and uses thereof as chitinase inhibitors

Abstract

The present invention relates to macrocyclic amidinourea derivatives of formula 8, methods of preparation and uses thereof, pharmaceutical compositions in particular to be used as chitinase inhibitors in the treatment of a fungal infection. ##STR00001##

Claims

1. A compound having the general formula 8: ##STR00056## wherein n.sub.1 is a number from 0 to 4; n.sub.2 is a number from 1 to 7; R.sub.1 is H; linear or branched C.sub.1-C.sub.6 alkyl; propargyl, cyclopropylmethyl, but-2-en-1-yl, -methylallyl, -methylallyl, ,-dimethylallyl, benzyl, allyl, pyridin-ylmethyl; methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, or prop-2-enylcarbamoyl; R.sub.2 is H or ##STR00057## R.sub.3 is OH, nitro, NH.sub.2, NHR.sub.8, NR.sub.9R.sub.10, C.sub.1-C.sub.6 alkyl, COOH, CONH.sub.2, CONR.sub.11H, CONR.sub.12R.sub.13, cyano, F, Cl, or Br; wherein R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13, are the same or each independently C.sub.1-C.sub.6 alkyl, methylcyclopropyl or propan-2-yl; X is CH.sub.2 or C(O); Y is CH.sub.2, or XY is ##STR00058## wherein R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are the same or each independently H, OH, nitro, NH.sub.2, NHR.sub.14, NR.sub.15R.sub.16, C.sub.1-C.sub.6 alkyl, COOH, CONH.sub.2, CONR.sub.17H, CONR.sub.18R.sub.19, cyano, F, Cl, or Br; and wherein R.sub.14, R.sub.15, R.sub.16, R.sub.17, R.sub.18, and R.sub.19 are the same or each independently C.sub.1-C.sub.6 alkyl, methylcyclopropyl or propan-2-yl; and Z is CH.sub.2, ##STR00059## or pharmaceutically acceptable salts thereof.

2. The compound according to claim 1 wherein R.sub.2 is ##STR00060## XY is ##STR00061## Z is CH.sub.2; or pharmaceutically acceptable salts thereof.

3. The compound according to claim 1 wherein R.sub.2 is H; XY is ##STR00062## Z is ##STR00063## or pharmaceutically acceptable salts thereof.

4. The compound according to claim 1 having formula 8a ##STR00064## or pharmaceutically acceptable salts thereof.

5. The compound according to claim 4 wherein XY is ##STR00065## with R.sub.4, R.sub.5, R.sub.6 and R.sub.7=H or pharmaceutically acceptable salts thereof.

6. The compound according to claim 4 wherein X is C(O) and Y is CH.sub.2, or pharmaceutically acceptable salts thereof.

7. The compound according to claim 1 selected from: ##STR00066## ##STR00067## or pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition comprising at least one compound according to claim 1 and appropriate excipients or diluents.

9. The pharmaceutical composition according to claim 8 further comprising at least one therapeutic agent selected from the group consisting of: an antifungal agent, and an anti-inflammatory agent.

10. The pharmaceutical composition according to claim 9 wherein the further antifungal agent is selected from the group consisting of: amphotericin, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Imidazole, triazole, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole; Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole; Abafungi; Amorolfin, Butenafine, Naftifine, Terbinafine; Anidulafungin, Caspofungin, Micafungin; Benzoic acid; Ciclopirox; Flucytosine; Griseofulvin; Haloprogin; Tolnaftate; Undecylenic acid; Crystal violet and Balsam of Peru.

11. A process for the preparation of a compound of formula 8 ##STR00068## said process comprising the following steps: i) reacting a compound of formula 2 ##STR00069## in the presence of AllylNH.sub.2, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride [EDC], 1-hydroxybenzotriazole [HOBt], and N,N-Diisopropylethylamine [DIPEA] in N,N-dimethylformamide [DMF] to obtain a resultant compound: ##STR00070## ii) reacting the resultant compound of step (i) with diisobutylaluminium hydride [DIBAL-H] in dichloromethane [DCM] at room temperature to obtain a compound of formula 3 ##STR00071## iii) reacting the compound of formula 3 of step (ii) with a compound of formula 1 in tetrahydrofuran [THF] under reflux for 12 h to obtain a compound of formula 4 ##STR00072## iv) reacting the compound of formula 4 of step (iii) in the presence of a second generation Grubbs' catalyst in toluene or DCM at a concentration of 2-10 mM at 40-80 C. to obtain a compound of formula 5 ##STR00073## v) reacting the compound of formula 5 of step (iv) with hydrogen over palladium on carbon [Pd/C] in ethanol [EtOH] to obtain a compound of formula 6 ##STR00074## vi) reacting the compound of formula 6 of step (v) with R.sub.1NBoc(CNBoc)SMe in THF under reflux for 12 h to obtain a compound of formula 7 ##STR00075## vii) reacting the compound of formula 7 of step (vi) with TFA to obtain a compound of Formula 8, or a pharmaceutically acceptable salts thereof, wherein n.sub.1 is a number from 0 to 4; n.sub.2 s a number from 1 to 7; R.sub.1 is H; linear or branched C.sub.1-C.sub.6 alkyl; propargyl, cyclopropylmethyl, but-2-en-1-yl, -methylallyl, -methylallyl, ,-dimethylallyl, benzyl, alkyl, pyridin-ylmethyl; methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, or prop-2-enylcarbamoyl; R.sub.2 is H or ##STR00076## R.sub.3 is OH, nitro, NH.sub.2, NHR.sub.8, NR.sub.9R.sub.10, C.sub.1-C.sub.6 alkyl, COOH, CONH.sub.2, CONR.sub.11H, CONR.sub.12R.sub.13, cyano, F, Cl, or Br; wherein R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, a R.sub.13, are the same or each independently C.sub.1-C.sub.6 alkyl, methylcyclopropyl or propan-2-yl; X is CH.sub.2 or C(O); Y is CH.sub.2, or XY is ##STR00077## wherein R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are the same or each independently H, OH, nitro, NH.sub.2, NHR.sub.14, NR.sub.15R.sub.16, C.sub.1-C.sub.6 alkyl, COOH, CONH.sub.2, CONR.sub.17H, CONR.sub.18R.sub.19, cyano, F, Cl, or Br; and wherein R.sub.14, R.sub.15, R.sub.16, R.sub.17, R.sub.18, and R.sub.19 are the same or each independently C.sub.1-C.sub.6 alkyl, methylcyclopropyl or propan-2-yl; and Z is CH.sub.2, ##STR00078##

12. A method for inhibiting a chitinase in a mammal comprising administering the compound according to claim 1 in a suitable amount to the mammal in need thereof.

13. A method for treating a fungal infection in a mammal comprising administering the compound according to claim 1 in a suitable amount to the mammal in need thereof.

14. The method according to claim 13, wherein the fungal infection is of a Candida species.

15. The method according to claim 14, wherein the Candida species is selected from the group consisting of: C. albicans, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis, C. kefyr and C. glabrata.

16. The method according to claim 14 wherein the Candida species is drug resistant.

17. The method according to claim 16 wherein the Candida species is resistant to fluconazole and/or voriconazole.

18. A method for treating an IL-13 and/or Th-2-mediated disease in a mammal comprising administering the compound according to claim 1 in a suitable amount to the mammal in need thereof.

19. The method according to claim 18 wherein the IL-13 and/or Th-2-mediated disease is asthma.

20. The method according to claim 18 wherein the IL-13 and/or Th-2-mediated disease is a Th-2-mediated inflammation.

21. The method according to claim 18 wherein the IL-13 and/or Th-2-mediated disease is an allergic airway disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Chemistry

(2) The compounds having formula 8 described in the invention can be synthesized as described in scheme 1 below:

(3) ##STR00019##
Reagents and Conditions:

(4) (i) AllylNH.sub.2, EDC, HOBt, DIPEA, DMF (ii) DIBAL-H, DCM, r.t. (iii) THF, reflux, 12 h (iv) Grubbs' Cat. 2nd gen., toluene or DCM 2-10 mM, 40-80 C. (v) H.sub.2, Pd/C, EtOH (vi) R.sub.1NBoc(C=NBoc)SMe, THF, reflux, 12 h.

(5) Wherein n.sub.1, n.sub.2, R.sub.1, R.sub.2, X, Y and Z are as defined herein above.

(6) Unless otherwise indicated, commercially available reagents and solvents were used without further purification. Specifically, the following abbreviations may have been used in the descriptions of the experimental methods:

(7) min: minutes; h: hour(s); r.t.: room temperature, NMR: Nuclear Magnetic Resonance;

(8) MHz: Megahertz; .sup.1H: proton; .sup.13C: carbon 13; mg: milligrams; mmol: millimoles; mL: milliliters; L: microliters; N: Normal;

(9) NMR Nuclear Magnetic Resonance; LC-MS Liquid Chromatography Mass Spectrum; TMS: tetramethylsilane; THF: tetrahydrofuran; Tf: trifluoromethylsulfonyl; Boc: tert-butyloxycarbonyl; DCM: dichloromethane; EtOAc: ethyl acetate; EDC: N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; HOBT: 1-Hydroxybenzotriazole; DMF: N,N-dimethylformamide; DIPEA: N,N-Diisopropylethylamine; DIBAL-H: Diisobutylaluminium hydride; MeOH: methyl alcohol; MeOD: deuterated methyl alcohol; EtOH: ethyl alcohol; Et.sub.2O: diethyl ether; Et.sub.3N: triethylamine; DMSO: dimethyl sulfoxide; CbzCl: Benzyl Chloroformate; EP: Petroleum ether; DCC: N,N-dicyclohexyl methanediimine; TMSCI: Trimethylsilyl chloride; Py: pyridine.

(10) Except where indicated otherwise, all temperatures are expressed in C. (degrees centigrade) or K (Kelvin).

(11) The structure of the intermediates and of the final compounds of the synthesis were confirmed by NMR and/or LC-MS analysis.

(12) The .sup.1H-NMR spectra were acquired with a Varian Gemini 200 (200 MHz) or with a Brucker Avance DPX400 (400 MHz).

(13) The .sup.13C-NMR spectra were acquired with a Brucker Avance DPX400 (400 MHz).

(14) The chemical shifts are expressed in parts per million (ppm, units). The coupling constants are expressed in Hertz (Hz) and the splitting patterns are described as s (singlet), bs (broad signal), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet).

(15) The LC-MS analyses were carried out on a system consisted of a Varian apparatus (Varian Inc) including a vacuum solvent degassing unit, two pumps (212-LC), a Triple Quadrupole MSD (Mod. 320-LC) mass spectrometer with ES interface and Varian MS Workstation System Control Vers. 6.9 software. Chromatographic separation was obtained using a Pursuit C18 column (502.0 mm) (Varian) with 3 m particle size and gradient elution: eluent A being CH.sub.3CN and eluent B consisting of an aqueous solution of formic acid (0.1%). The analysis started with 0% of eluent A, which was linearly increased up to 50% in 10 min, then slowly increased up to 60% up to 15 min. The flow rate was 0.3 ml/min and injection volume was 5 L. The instrument operated in positive mode and parameters were: detector 1850 V, drying gas pressure 25.0 psi, desolvation temperature 300.0 C., nebulizing gas 45.0 psi, needle 5000 V and shield 600 V. Nitrogen was used as nebulizer and drying gas. Collision induced dissociation was performed using Argon as the collision gas at a pressure of 1.8 mTorr in the collision cell, the collision energy was set to 149 eV. Three transitions were recorded at 279.8 (m/z), 321.9 (m/z) and 368.0 (m/z) setting capillary voltage to 27.5 V, 23.5 V and 19.5 V respectively.

Example 16

(16) For reasons of clarity, the detailed synthesis of example 16 has been reported in scheme 2 below.

(17) The yields were calculated assuming that products were 100% pure if not stated otherwise.

(18) ##STR00020##
Reagents and Conditions:

(19) (i) BrCH.sub.2CHCH.sub.2, K.sub.2CO.sub.3, DCM, reflux (ii) NH.sub.2OH, Py, EtOH, reflux (iii) Zn, HCl, THF, reflux (iv) (BocNH).sub.2C=NTf, Et.sub.3N, DCM (v) AllylNH.sub.2, EDC, HOBt, DIPEA, DMF (vi) DIBAL-H, DCM, r.t. (vii) THF, reflux, 12 h (viii) Grubbs' Cat. 2nd gen., toluene or DCM 2-10 mM, 40-80 C. (ix) H.sub.2, Pd/C, EtOH (x) CrotylNBoc(C=NBoc)SMe, THF, reflux, 12 h

Procedure for the Preparation of 2-prop-2-enoxybenzaldehyde 10

(20) ##STR00021##

(21) 2-hydroxybenzaldehyde 9 (0.5 ml, 4.69 mmol, Sigma-Aldrich catalogue id: S356) was dissolved in CH.sub.3CN (10 ml) and K.sub.2CO.sub.3 (0.97 ml, 7.03 mmol) and 3-bromoprop-1-ene (0.45 ml, 5.16 mmol, Sigma-Aldrich catalogue id: 337528) were added. Mixture was stirred at reflux for 12 hours. Solvent was then evaporated in vacuum and the residue dissolved in EtOAc (5 mL). Water (10 ml) was added and the mixture stirred for 10 min at room temperature. Organic phase was then separated and the aqueous layer was extracted with EtOAc (210 ml). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give title compound 10.

(22) Yield 99%. .sup.1H NMR (400 MHz, CDCl.sub.3) =10.25 (1H, s), 7.54-7.52 (1H, m), 7.23-7.19 (1H, m), 6.72-6.66 (2H, m), 5.82-5.75 (1H, m), 5.20-5.15 (1H, d, J=17.6 Hz), 5.05-5.03 (1H, d, J=10.8 Hz), 4.33-4.31 (2H, m) ppm.

(23) .sup.13C NMR (CDCl.sub.3) 188.9, 160.6, 135.6, 132.2, 127.8, 124.7, 120.5, 117.5, 112.7, 68.7 ppm,

(24) Liquid chromatography Mass Spectroscopy (LCMS) m/z (ES+) m/z: 347.0 [2M+Na].sup.+, 185.0 [M+Na].sup.+, 163.0 [M+H].sup.+.

(25) Elemental analysis for C.sub.10H.sub.10O.sub.2: Calcd. C, 74.06; H, 6.21. Found C, 74.36; H, 6.52.

Procedure for the Preparation of (2-prop-2-enoxyphenyl)methanamine 11

(26) ##STR00022##

(27) 2-prop-2-enoxybenzaldehyde 10 (4.69 mmol) was dissolved in EtOH (10 ml) and pyridine (0.45 ml, 5.63 mmol) and NH.sub.2OH (490 mg, 7.03 mmol) were added. The mixture was heated at reflux for 2 h. Brine was added, the aqueous layer was extracted with EtOAc (210 ml) and the combined organic phases were dried over Na.sub.2SO.sub.4 and filtered. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (SiO.sub.2) using hexane-Et.sub.2O, 3:1 as the eluent affording a mixture of E/Z isomers of oxime.

(28) N-[(2-prop-2-enoxyphenyl)methylidene]hydroxylamine (818 mg, 4.62 mmol) was dissolved in THF (30 ml) and Zn (3.00 g, 46.23 mmol) was added. HCl 2N (12 ml) was added and mixture was stirred at reflux for 3 h. The reaction mixture was cooled to room temperature, filtered on a celite pad to remove the excess zinc and condenser under reduced pressure. pH was adjusted to >10 by addition of ammonium hydroxide. The aqueous layer was extracted with EtOAc (210 ml) and the combined organic phases were dried over Na.sub.2SO.sub.4 and filtered. The solvent was evaporated under reduced pressure to give title compound 11.

(29) Yield 99%. .sup.1H NMR (400 MHz, CDCl.sub.3) =7.06-7.01 (2H, m), 6.77-6.73 (1H, m), 6.67-6.65 (1H, m), 5.92-5.84 (1H, m), 5.28-5.24 (1H, d, J=17.2 Hz), 5.13-5.10 (1H, d, J=10.4 Hz), 4.36-4.35 (2H, m), 3.70-3.68 (2H, m), 1.65 (2H, br s) ppm.

(30) .sup.13C NMR (CDCl.sub.3) 156.1, 133.2, 131.9, 128.2, 127.7, 120.5, 116.8, 111.3, 68.3, 42.4 ppm.

(31) LCMS m/z (ES+) m/z=349.0 [2M+Na].sup.+, 202.0 [M+Na].sup.+, 163.9 [M+H].sup.+.

(32) Elemental analysis for C.sub.10H.sub.13NO: Calcd. C, 73.59; H, 8.03; N, 8.58. Found C, 73.86; H, 8.47; N, 9.01.

Procedure for the Preparation of tert-butyl N-({[(tert-butoxy)carbonyl]amino}(trifluoromethanesulfonylimino)methyl)carbamate (also named as 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine)

(33) ##STR00023##

(34) Tert-butyl N[N-[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]carbamate (7.12 g, 27.45 mmol, Sigma-Aldrich catalogue id: 496871) was dissolved in dry DCM (136 ml) and Et.sub.3N (4.2 ml, 30.19 mmol) was added. The solution was cooled at 78 C. and trifluoromethylsulphonyl trifluoromethanesulphonate (Sigma-Aldrich, catalogue ID: 91737) solution 1 M in methylene chloride (35.68 ml) was added in one portion. The mixture was then allowed to warm to r.t. and stirred for 4 h. Water was added to the mixture and organic layers were washed two times with brine while aqueous phase was extracted twice with EtOAc. Finally combined organic phases were dried over Na.sub.2SO.sub.4, filtered and evaporated in vacuum. The solid residue was recrystallized from hexane led to the title compound tert-butyl N-({[(tert-butoxy)carbonyl]amino}(trifluoromethanesulfonylimino)methyl)carbamate (also named as 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine).

(35) Yield 70%. .sup.1H NMR (400 MHz, CDCl.sub.3) =8.00 (2H, s), 1.4 (18H, m) ppm.

(36) .sup.13C NMR (CDCl.sub.3) 151.0, 148.0, 117.5, 85.9, 27.7 ppm.

(37) LCMS m/z (ES+) m/z=805.0 [2M+Na].sup.+, 414.0 [M+Na].sup.+, 391.9 [M+H].sup.+.

(38) Elemental analysis for C.sub.12H.sub.20F.sub.3N.sub.3O.sub.6S: Calcd. C, 36.83; H, 5.15; N, 10.74. Found C, 37.13; H, 5.35; N, 10.94.

Procedure for the Preparation of tert-butyl N-[[(2-methylpropan-2-yl)oxycarbonylamino]-[(2-propoxyphenyl)methylamino]methyl]carbamate 1a

(39) ##STR00024##

(40) (2-prop-2-enoxyphenyl)methanamine 11 (345 mg, 2.1 mmol), Et.sub.3N (0.58 ml, 4.2 mmol) and 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine were mixed in DCM (25 ml) and mixture stirred at room temperature overnight. The aqueous layer was separated and extracted with EtOAc (210 ml) and the combined organic phases were dried over Na.sub.2SO.sub.4 and filtered. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (hexane-Et.sub.2O, 8:2) affording guanidine title compound 1a.

(41) Yield 75%. .sup.1H NMR (400 MHz, CDCl.sub.3) =11.42 (1H, s), 8.62 (1H, s), 7.09-7.07 (1H, d, J=7.6 Hz), 7.03-6.99 (1H, m), 6.71-6.63 (2H, m), 5.92-5.83 (1H, m), 5.22-5.18 (1H, d, J=17.6 Hz), 5.06-5.04 (1H, d, J=10.4 Hz), 4.46-4.45 (2H, m), 4.37-4.35 (2H, m), 1.31 (9H, s), 1.26 (9H, s) ppm.

(42) .sup.13C NMR (CDCl.sub.3) 163.4, 156.4, 155.8, 152.9, 132.9, 129.4, 128.7, 125.5, 120.4, 117.1, 111.3, 82.3, 78.5, 68.5, 40.6, 28.0, 27.7 ppm.

(43) LCMS m/z (ES+) m/z=833.0 [2M+Na].sup.+, 428.0 [M+Na].sup.+, 406.2 [M+H].sup.+.

(44) Elemental analysis for C.sub.21H.sub.13N.sub.3O.sub.5: Calcd. C, 62.20; H, 7.71; N, 10.36. Found C, 62.56; H, 7.92; N, 10.85.

Procedure for the Preparation of 8-(benzyloxycarbonylamino)octanoic acid 2a

(45) ##STR00025##

(46) 8-aminooctanoic acid (500 mg, 3.14 mmol, Sigma-Aldrich catalogue ID: 855294) and K.sub.2CO.sub.3 (867 mg, 6.28 mmol) were dissolved in THF (15 ml) (suspension). Then CbzCl (0.67 ml, 4.71 mmol) was added and the mixture was stirred at r.t. overnight.

(47) The reaction was quenched with EtOAc and H.sub.2O. Aqueous phase was separated and pH of aqueous phase was adjusted to pH=2 by addition of HCl 4N and extracted twice with EtOAc. Combined organic phase were then dried over Na.sub.2SO.sub.4, filtered and evaporated in vacuum affording the title compound 2a.

(48) Yield 99%. .sup.1H NMR (400 MHz, CDCl.sub.3) 11.01 (1H, s), 7.28-7.19 (5H, m), 5.11-5.07 (1H, d, J=16 Hz), 5.05-5.03 (1H, d, J=8 Hz), 3.18-3.16 (2H, m), 2.44-2.40 (2H m), 1.64-1.62 (2H, m), 1.48-1.43 (2H, m), 1.32 (6H, m) ppm.

(49) .sup.13C NMR (CDCl.sub.3) 178.7, 156.4, 136.5, 128.4, 128.0, 128.0, 66.5, 41.4, 33.8, 29.7, 28.6, 26.4, 24.7 ppm.

(50) LCMS m/z (ES+) m/z=609.0 [2M+Na].sup.+, 316.0 [M+Na].sup.+, 294.1 [M+H].sup.+.

(51) Elemental analysis for C.sub.16H.sub.23NO.sub.4: Calcd. C, 65.51; H, 7.90; N, 4.77. Found C, 65.96; H, 8.35; N, 5.04.

Procedure for the Preparation of benzyl N-[8-oxo-8-(prop-2-enylamino)octyl]carbamate

(52) ##STR00026##

(53) The 8-(benzyloxycarbonylamino)octanoic acid 2a (14 mmol), HOBT (424 mg, 3.14 mmol), EDC (487 mg, 3.14 mmol), DIPEA (0.66 ml, 3.77 mmol), allylamine (0.23 ml, 3.14 mmol) were mixed in DMF (5 ml). Mixture was stirred overnight at room temperature. The reaction was quenched with NaHCO.sub.3, and then aqueous phase was separated and extracted with EtOAc (35 ml). The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (EtOAcEP, 4:1) affording benzyl N-[8-oxo-8-(prop-2-enylamino)octyl]carbamate.

(54) Yield 75%. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.28-7.19 (5H, m), 5.79-5.70 (1H, m), 5.74 (1H, br s), 5.11-5.07 (1H, d, J=17.2 Hz), 5.03-5.01 (1H, d, J=10.4 Hz), 5.00 (2H, s), 4.78 (1H, br s), 3.81-3.78 (2H, t, J=4.8 Hz), 3.10-3.08 (2H, m), 2.12-2.08 (2H, t, J=7.6 Hz), 1.55 (2H, m), 1, 40 (2H, m), 1.23-1.18 (8H, m) ppm.

(55) .sup.13C NMR (CDCl.sub.3) 172.8, 156.3, 136.5, 134.2, 128.4, 127.9, 127.6, 116.9, 66.4, 41.7, 40.8, 36.4, 29.7, 28.9, 28.7, 26.3, 25.4 ppm.

(56) LCMS m/z (ES+) m/z=687.0 [2M+Na].sup.+, 355.0 [M+Na].sup.+, 333.1 [M+H].sup.+.

(57) Elemental analysis for C.sub.19H.sub.28N.sub.2O.sub.3: Calcd. C, 68.65; H8.49; N8.43. Found C, 69.06; H, 8.95; N, 8.93.

Procedure for the Preparation benzyl N-[8-(prop-2-enylamino)octyl]carbamate 3a

(58) ##STR00027##
benzyl N-[8-oxo-8-(prop-2-enylamino)octyl]carbamate (747 mg, 2.25 mmol) was dissolved in CH.sub.2Cl.sub.2 dry (5 ml) and cooled at 78 C. DIBAL (3.37 ml, 3.37 mmol) was added and mixture was stirred 1 h at 78 C. then mixture was warmed at room temperature DIBAL (4.5 ml, 4.5 mmol) was added again and mixture was stirred for h. The reaction was quenched with EtOAc and Rochelle salt. The crude title compound 3a was used in the next step.

(59) Yield 99%. .sup.1H NMR (400 MHz, CDCl.sub.3) =7.28-7.19 (5H, m), 5.79-5.70 (1H, m), 5.11-5.07 (1H, d, J=17.2 Hz), 5.03-5.01 (1H, d, J=10.4 Hz), 5.00 (2H, s), 3.22-3.20 (2H, m), 2.09-2.05 (4H, m), 1.40-1.50 (4H, m), 1.23-1.18 (8H, m) ppm.

(60) .sup.13C NMR (CDCl.sub.3) 156.3, 141.2, 134.2, 129.0, 128.1, 127.9, 116.0, 65.0, 52.1, 49.8, 41.9, 31.0, 29.9, 29.4, 27.0, 26.8 ppm.

(61) LCMS m/z (ES+) m/z=659.0 [2 M+Na].sup.+, 341.0 [M+Na].sup.+, 319.0 [M+H].sup.+.

Procedure for the Preparation of benzyl N-[8-[[N-[(2-methylpropan-2-yl)oxycarbonyl]-N-[(2-prop-2-enoxyphenyl)methyl]carbamimidoyl]carbamoyl-prop-2-enylamino]octyl]carbamate 12

(62) ##STR00028##

(63) tert-butyl N-[[(2-methylpropan-2-yl)oxycarbonylamino]-[(2-propoxyphenyl)methylamino]methyl]carbamateBenzyl 8-(allylamino) octylcarbamate 1a (2.25 mmol) and compound 3a (1.4 mmol) were mixed in THF (10 ml), Et.sub.3N (1.4 mmol) was added and mixture was refluxed overnight. Solvent was removed and the residue was purified by flash chromatography (hexane-Et.sub.2O from 9:1 to 1:1) affording the title compound 12.

(64) Yield 80%. .sup.1H NMR (400 MHz, CDCl.sub.3) 12.27 (1H, s), 8.47 (1H, s), 7.26-7.21 (5H, m), 7.18-7.12 (2H, m), 6.83-6.76 (2H, m), 6.05-5.97 (1H, m), 5.75-5.67 (1H, m), 5.36-5.32 (1H, d, J=17.2 Hz), 5.21-5.18 (1H, d, J=10.4 Hz), 5.07-5.01 (1H, m), 5.00-4.99 (2H, m), 4.98-4.96 (1H, m), 4.52-4.47 (4H, m), 3.86-3.84 (2H, m), 3.37-3.33 (1H, m), 3.18-3.14 (1H, m), 3.09-3.06 (2H, m), 1.43 (4H, m), 1.37 (9H, m), 1.21-1.16 (8H, m) ppm.

(65) .sup.13C NMR (CDCl.sub.3) 163.6, 156.3, 153.9, 153.1, 136.7, 135.3, 134.6, 129.2, 128.8, 128.2, 128.0, 127.8, 127.0, 120.4, 117.1, 115.4, 111.4, 81.7, 68.6, 66.1, 50.3, 48.3, 40.9, 40.2, 29.8, 29.2, 28.5, 27.9, 27.8, 26.8, 26.5 ppm.

(66) LCMS m/z (ES+) m/z=672.3.0 [M+Na].sup.+, 688.2 [M+K].sup.+, 650.3 [M+H].sup.+.

(67) Elemental analysis for C.sub.36H.sub.51N.sub.5O.sub.6: Calcd. C, 66.54; H, 7.91; N, 10.78. Found C, 66.93; H, 8.11; N, 10.98.

Procedure for the Preparation of benzyl N-[8-[4-[(2-methylpropan-2-yl)oxycarbonylimino]-6-oxo-12-oxa-3, 5,7-triazabicyclo[11.4.0]heptadeca-1(13),9,14,16-tetraen-7-yl]octyl]carbamate 13

(68) ##STR00029##

(69) The next step is a ring closing metathesis and was made by two different procedures:

(70) A. The starting material compound 12 (0.43 mmol) was dissolved in DCM (solution 2 mM); second generation Grubbs catalyst (0.043 mmol, Sigma-Aldrich catalogue ID: 569747) was dissolved in 2 ml of DCM and added to previous solution via syringe pump over 4 h at 40/50 C. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (hexane-Et.sub.2O, 9:1) affording the title compound 13.
B. The starting material compound 12 (0.76 mmol) was dissolved in toluene (solution 10 mM); Grubbs catalyst (0.15 mmol) was dissolved in 2 ml of toluene and added to previous solution via syringe pump over 4 h at 100 C. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (hexane-Et.sub.2O, 9:1) affording the title compound 13.

(71) Procedure B; Yield 30%. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.33-7.16 (5H, m), 7.02-7.89 (4H, m), 5.38-5.32 (2H, m), 5.07-5.05 (2H, m), 4.65-4.45 (4H, m), 3.80-3.70 (2H, m), 3.17-3.14 (4H, m), 1.43 (9H, m), 1.26 (12H, m) ppm.

(72) .sup.13C NMR (CDCl.sub.3) 163.8, 156.3, 155.3, 153.4, 152.9, 136.6, 129.8, 129.3, 128.7, 128.4, 121.6, 113.4, 82.0, 75.0, 66.4, 48.1, 46.3, 41.0, 29.8, 29.3, 29.1, 28.6, 28.0, 26.9, 26.5, 24.6 ppm.

(73) LCMS m/z (ES+) m/z=660.3[M+Na].sup.+, 688.2 [M+K].sup.+, 622.3 [M+H].sup.+.

(74) Elemental analysis for C.sub.34H.sub.47N.sub.5O.sub.6: Calcd. C, 65.68; H, 7.62; N, 11.26; O, 15.44. Found C, 65.98; H, 7.91; N, 11.72; O, 15.83.

Procedure for the Deprotection of tert-butyl N-[7-(8-aminooctyl)-6-oxo-12-oxa-3,5,7-triazabicyclo[11.4.0]heptadeca-1(13),14,16-trien-4-ylidene]carbamate 14

(75) ##STR00030##

(76) Compound 13 (0.097 mmol) was diluted in EtOH (9 ml) and Pd/C, 10% wt. (45 mg) was added. H.sub.2 was insufflated and the mixture was stirred for 5 h. The reaction mixture was filtered through celite and then solvent was evaporated in vacuum. Crude title compound 14 was used in the next step without any further purification.

(77) Yield 99%. .sup.1H NMR (400 MHz, CDCl.sub.3) =7.27-7.08 (2H, m), 6.88-6.77 (2H, m), 4.61-4.60 (2H, m), 4.02-3.98 (2H, m), 3.30-3.26 (4H, m), 3.14-3.10 (2H, m), 1.76-1.75 (2H, m), 1.59-1.54 (6H, m), 1.43 (9H, m), 1.20-1.14 (8H, m) ppm

(78) LCMS m/z (ES+) m/z=512.3[M+Na].sup.+, 528.2 [M+K].sup.+, 490.3 [M+H].sup.+.

Procedure for the Preparation of tert-butyl N-but-2-enyl-N[N-[(2-methylpropan-2-yl)oxycarbonyl]-N-methylsulfanylcarbamimidoyl]carbamate

(79) ##STR00031##

(80) To a stirred suspension of KOH (2.8 mmol) in a solution of CH.sub.2Cl.sub.2/CH.sub.3CN (19:1, 3.5 mL), Tetrabutylammonium bromide (0.2 mmol) and Tert-butyl N[N-[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]carbamate (1 mmol, Sigma-Aldrich catalogue id: 496871) were added. After few minutes, a solution of (E)-1-bromobut-2-ene (2.4 mmol, Sigma-Aldrich catalogue ID: C86405) in CH.sub.2Cl.sub.2/CH.sub.3CN (19:1, 3.5 mL) was added dropwise and the resulting solution was stirred at r.t. for 16-18 h. The reaction mixture was then poured on ice and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (25 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude products were purified by flash column Chromatography (SiO.sub.2) using 1:9 MeOH/CH.sub.2Cl.sub.2 as the eluent to yield the title compound tert-butyl N-but-2-enyl-N[N-[(2-methylpropan-2-yl)oxycarbonyl]-N-methylsulfanylcarbamimidoyl]carbamate.

(81) Time 16 h, yield 70%. .sup.1H NMR (400 MHz, CDCl.sub.3) 5.34-5.21 (2H, m), 3.69-3.68 (2H, d, J=8 Hz), 2.02-2.01 (3H, s), 1.15 (9H, s), 1.12 (9H, m) ppm.

(82) .sup.13C NMR (CDCl.sub.3) 162.8, 157.7, 151.5, 129.6, 126.5, 82.0, 82.4, 45.4, 27.9, 17.5, 15.3 ppm.

(83) LCMS m/z (ES+) m/z=710.9 [2M+Na].sup.+, 367.0 [M+Na].sup.+, 345.1 [M+H].sup.+

(84) Elemental analysis for C.sub.16H.sub.28N.sub.2O.sub.4S: Calcd. C, 55.79; H, 8.19; N, 8.13. Found C, 55.83; H, 8.21; N, 8.34.

Procedure for the Preparation of tert-butyl N[N-[(E)-but-2-enyl]-N-[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]-N-[8-[4-[(2-methylpropan-2-yl)oxycarbonylimino]-6-oxo-12-oxa-3,5,7-triazabicyclo[11.4.0]heptadeca-1 (13), 14,16-trien-7-yl]octyl]carbamate 15

(85) ##STR00032##

(86) To a stirred solution of the compound 14 (0.097 mmol) in dry THF (5 ml) a solution of tert-butyl N-but-2-enyl-N[N-[(2-methylpropan-2-yl)oxycarbonyl]-N-methylsulfanyl carbamimidoyl]carbamate (0.10 mmol) in dry THF (4 ml) was added dropwise. Then Et.sub.3N (0.14 mmol) was added. The mixture was then stirred at reflux overnight. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography (SiO.sub.2) using 1:1 hexane-Et.sub.2O as the eluent affording title compound 15.

(87) Yield 50%. .sup.1H NMR (400 MHz, CDCl.sub.3) 11.97 (1H, s), 8.44 (1H, s), 7.28-6.74 (4H, m), 5.57-5.55 (1H, m), 5.44-5.41 (1H, m), 4.09-4.07 (2H, d, J=8 Hz), 4.02-4.00 (2H, d, J=6.4 Hz), 3.31-3.30 (2H, m), 3.17-3.10 (6H, m), 1.60-1.58 (3H, d, J=6.4 Hz), 1.42-1.36 (27H, m), 1.22-1.17 (16H, m) ppm.

(88) .sup.13C NMR (CDCl.sub.3) 163.9, 158.0, 155.5, 153.4, 153.0, 130.3, 128.3, 128.2, 127.9, 126.1, 120.4, 111.7, 81.9, 67.1, 47.4, 46.3, 43.7, 37.1, 39.5, 29.3, 28.5, 28.0, 26.9, 26.7, 24.5, 23.8, 20.9, 17.6 ppm.

(89) LCMS m/z (ES+) m/z=808.3[M+Na].sup.+, 824.0 [M+K].sup.+, 786.2[M+H].sup.+.

(90) Elemental analysis for C.sub.41H.sub.67N.sub.7O.sub.8: Calcd. C, 62.65; H, 8.59; N, 12.47. Found C, 63.08; H, 8.98; N, 12.84.

Procedure for the Preparation of Example 16: 1-[(E)-but-2-enyl]-3-[8-(4-imino-6-oxo-12-oxa-3,5,7-triazabicyclo[11.4.0]heptadeca-1(13),14,16-trien-7-yl)octyl]guanidine 16

(91) ##STR00033##

(92) Guanylated compound 15 (0.025 mmol) was dissolved in dry DCM (30 mL for 1 mmol) and treated with a 10% solution of freshly distilled TFA. The resulting solution was stirred at room temperature under argon. After 24 h the reaction mixtures were concentrated under reduced pressure affording the crude title compound 16 (brown oil) in quantitative yield.

(93) Yield quantitative. .sup.1H NMR (400 MHz, MeOD) 7.33-6.94 (4H, m), 5.68-5.64 (1H, m), 5.44-5.41 (1H, m), 4.44-4.38 (2H, m), 4.21-4.20 (2H, m), 3.68-3.67 (2H, d, J=4.8 Hz), 3.54 (2H, m), 3.30-3.28 (2H, m), 3.12-3.09 (2H, t, J=6.8 Hz), 1.65-1.64 (3H, m), 1.55-1.45 (4H, m), 1.32-1.21 (12H, m) ppm.

(94) .sup.13C NMR (MeOD) 163.5, 157.6, 156.2, 155.8, 131.0, 129.9, 128.6, 127.9, 127.1, 124.8, 121.3, 111.4, 69.0, 47.2, 43.0, 41.0, 31.5, 29.2, 28.7, 28.4, 27.2, 26.1, 24.9, 22.0, 16.2 ppm.

(95) LCMS m/z (ES+) m/z=508.3[M+Na].sup.+, 524.0 [M+K].sup.+, 486.2[M+H].sup.+.

(96) Elemental analysis for C.sub.26H.sub.43N.sub.7O.sub.2: Calcd. C, 64.30; H, 8.92; N, 20.19. Found C, 64.68; H, 9.08; N, 20.84.

Example 17

1-[(E)-but-2-enyl]-3-[8-(4-imino-6-oxo-13-oxa-3,5,7-triazabicyclo[12.4.0]octadeca-1(14),15,17-trien-7-yl)octyl]guanidine 17

(97) ##STR00034##

(98) Compound 17 was synthesized starting from the appropriate intermediate 1f, according to the procedure as for example 16.

(99) ##STR00035##
Analytical Data for Compound 17

(100) .sup.1H NMR (400 MHz MeOD) 7.23-6.88 (4H, m), 5.62-5.64 (1H, m), 5.40-5.35 (1H, m), 4.43-4.42 (2H, m), 4.06 (2H, m), 3.65 (2H, m), 3.45-3.41 (2H, m), 3.23 (2H, m), 3.08-3.00 (2H, m), 1.63-1.62 (3H, m), 1.49-1.48 (6H, m), 1.28-1.17 (12H, m) ppm.

(101) .sup.13C NMR (100 MHz MeOD) 163.5, 156.7, 156.2, 154.7, 130.0, 128.5, 127.9, 127.1, 124.8, 120.0, 111.1, 64.6, 49.2, 46.6, 42.4, 41.0, 39.6, 28.7, 28.3, 27.0, 26.0 ppm. LC-MS m/z (ES+) m/z=522.3[M+Na].sup.+, 538.0 [M+K].sup.+, 500.2[M+H].sup.+

Example 18

1-[(E)-but-2-enyl]-3-[8-(4-imino-6-oxo-14-oxa-3,5,7-triazabicyclo[13.4.0]nonadeca-1(15), 16,18-trien-7-yl)octyl]guanidine 18

(102) ##STR00036##

(103) Compound 18 was synthesized starting from the appropriate intermediate 1g, according to the procedure as for example 16.

(104) ##STR00037##
Analytical Data for Compound 18

(105) .sup.1H NMR (400 MHz MeOD) 12.31 (1H, s), 8.05 (1H, s), 7.26-7.22 (2H, m), 6.92-6.84 (2H, m), 5.66-5.65 (1H, m), 5.49-5.42 (1H, m), 4.37 (2H, m), 3.97 (2H, m), 3.68 (2H, m), 3.51 (2H, m), 3.30 (2H, m), 3.15-3.11 (4H, m), 1.63-1.62 (3H, m), 1.50-1.48 (6H, m), 1.28-1.23 (12H, m) ppm.

(106) .sup.13C NMR (100 MHz MeOD) 163.7, 157.5, 156.1, 155.8, 130.3, 128.5, 124.9, 119.8, 110.9, 67.2, 53.3, 46. 42.5, 41.0, 29.0, 28.7, 28.4, 27.1, 26.7, 26.4, 26.4, 26.3, 26.2, 26.1, 26.0 ppm.

(107) LC-MS m/z (ES+) m/z=536.3[M+Na].sup.+, 552.0 [M+K].sup.+, 514.2[M+H].sup.+

Example 19

1-[(E)-but-2-enyl]-3-[8-(6-imino-2,8-dioxo-1-oxa-5,7,9-triazacyclotetradec-9-yl)octyl]guanidine 19

(108) ##STR00038##

(109) Compound 19 was synthesized according to scheme 1 by reacting intermediate 1b with intermediate 3a, and applying a similar procedure as for example 16.

(110) The synthesis of intermediate 1 b is described in scheme 3.

(111) ##STR00039##

(112) Reagents and conditions: (i) (BocNH).sub.2C=NTf, Et.sub.3N, DCM, (ii) TMSCI, Et.sub.3N, DCM, reflux; (iii) DMAP, DCC, but-3-en-1-ol (sigma-Aldrich, catalogue ID: 496839), DCM, rt 24 h;

(113) 3-aminopropanoic acid (600 mg 6.74 mmol, Sigma-Aldrich catalogue ID: 146064) was dissolved in CH.sub.2Cl.sub.2 (10 ml) and trimethylsilyl chloride (0.86 ml, 6.74 mmol) was added dropwise at room temperature over 5 min. The reaction mixture was then refluxed for 1 h and then cooled to 0 C. Triethylamine (1.22 ml, 8.76 mmol) was then added followed by N,N-bis(tert-butoxycarbonyl)-S-methylisothiourea (977 mg, 3.37 mmol). The resulting mixture was refluxed for 1 h, then cooled down to room temperature and added with MeOH (10 ml) and stirred for 10 min. Solvent was then evaporated and the residue diluted with water and then acidified with HCl 1N to pH 2. The mixture was extracted with AcOEt (210 ml). The organic layers were collected and washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduce pressure. The residue was used in the next step without any further purification. To a solution of 3-[[N,N-bis[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]amino]propanoic acid (8.98 mmol) in dry DCM (20 ml), the but-3-en-1-ol (17.96 mmol, Sigma-Aldrich catalogue ID: 496839) and DMAP (0.89 mmol) were added. The mixture was cooled down to 0 C. and DCC (13.47 mmol) was then added. The mixture was allowed to warm to r.t. overnight while stirring under argon. The white precipitated formed during the reaction was filtered off. The solution was then washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by flash chromatography using hexane Et.sub.2O 4:6 as eluent affording desired compound 1b, but-3-enyl 3-[[N,N-bis[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]amino]propanoate.

(114) Analytical Data for Compound 19

(115) .sup.1H NMR (400 MHz MeOD) 12.31 (1H, s), 8.05 (1H, s), 5.64 (1H, m), 5.42 (1H, m), 4.13 (2H, m), 3.78 (2H, m), 3.64 (2H, m), 3.43 (2H, m), 3.08 (2H, m), 2.51 (2H, m), 1.63 (3H, m), 1.55 (4H, m), 1.49 (4H, m), 1.27 (12H, m) ppm.

(116) .sup.13C NMR (100 MHz MeOD) 171.3, 171.1, 155.1, 154.0, 137.5, 128.6, 63.9, 42.5, 41.0, 37.6, 34.1, 29.2, 28.7, 28.4, 27.6, 26.1, 21.9, 18.7 ppm.

(117) LCMS m/z (ES+) m/z=488.0[M+Na].sup.+, 466.2[M+H].sup.+.

Example 20

1-[(E)-but-2-enyl]-3-[8-(6-imino-2,8-dioxo-1-oxa-5,7,9-triazacyclopentadec-9-yl)octyl]guanidine 20

(118) ##STR00040##

(119) Compound 20 was synthesized according to scheme 1 by reacting intermediate 1h with intermediate 3a, and applying a similar procedure as described for example 16.

(120) The synthesis of intermediate 1h is described in scheme 4

(121) ##STR00041##

(122) Reagents and conditions: (i) (BocNH).sub.2C=NTf, Et.sub.3N, DCM (ii) TMSCI, Et.sub.3N, DCM, reflux; (iii) DMAP, DCC, pent-4-en-1-ol (sigma-Aldrich, catalogue ID: 111279), DCM, rt 24 h;

(123) 3-aminopropanoic acid (600 mg 6.74 mmol, Sigma-Aldrich catalogue ID: 146064) was dissolved in CH.sub.2Cl.sub.2 (10 ml) and trimethylsilyl chloride (0.86 ml, 6.74 mmol) was added dropwise at room temperature over 5 min. The reaction mixture was then refluxed for 1 h and then cooled to 0 C. Triethylamine (1.22 ml, 8.76 mmol) was then added followed by N,N-bis(tert-butoxycarbonyl)-S-methylisothiourea (977 mg, 3.37 mmol). The resulting mixture was refluxed for 1 h, then cooled down to room temperature and added with MeOH (10 ml) and stirred for 10 min. Solvent was then evaporated and the residue diluted with water and then acidified with HCl 1N to pH 2. The mixture was extracted with AcOEt (210 ml). The organic layers were collected and washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and evaporated under reduce pressure. The residue was used in the next step without any further purification. To a solution of 3-[[N,N-bis[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]amino]propanoic produced in the previous step (8.98 mmol) in dry DCM (20 ml), the pent-4-en-1-ol (17.96 mmol, Sigma-Aldrich catalogue ID: 111279) and DMAP (0.89 mmol) were added. The mixture was cooled down to 0 C. and DCC (13.47 mmol) was then added. The mixture was allowed to warm to r.t. overnight while stirring under argon. The white precipitated formed during the reaction was filtered off. The solution was then washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by flash chromatography using hexane-Et.sub.2O 4:6 as eluent affording desired compound 1b, but-3-enyl 3-[[N,N-bis[(2-methylpropan-2-yl)oxycarbonyl]carbamimidoyl]amino]propanoate.

(124) Analytical Data for Compound 20

(125) .sup.1H NMR (300 MHz MeOD) 12.31 (1H, s), 8.05 (1H, s), 5.65 (1H, m), 5.43 (1H, m), 4.06 (2H, m), 3.68 (2H, m), 3.58 (2H, m), 3.44 (2H, m), 3.30 (2H, m), 3.11-3.07 (2H, m), 2.62-2.59 (2H, m), 1.63 (3H, d, J=6.0 Hz), 1.51 (8H, m), 1.27 (12H, m) ppm.

(126) .sup.13C NMR (100 MHz MeOD) 171.3, 171.1, 155.1, 154.0, 136.1, 129.2, 76.4, 41.1, 41.0, 37.3, 36.8, 29.4, 29.2, 29.3, 29.0, 28.8, 28.4, 28.5, 26.1, 21.8, 19.0 ppm.

(127) LCMS m/z (ES+) m/z=502.0[M+Na].sup.+, 480.2[M+H].sup.+.

Example 21

1-[(E)-but-2-enyl]-3-[6-[4-imino-6-oxo-2-(1-phenyltriazol-4-yl)-14-oxa-3,5,7-triazabicyclo[13.4.0]nonadeca-1(15),16,18-trien-7-yl]hexyl]guanidine 21

(128) ##STR00042##

(129) Compound 21 was synthesized according to scheme 1 by reacting intermediate 1c with intermediate 3a and applying a similar procedure as described for example 16.

(130) The synthesis of intermediate 1c is described in scheme 5.

(131) ##STR00043## ##STR00044##
Reagents and Conditions:

(132) (i) Br(CH.sub.2).sub.3CHCH.sub.2 (1.2 eq), K.sub.2CO.sub.3 (1.2 eq), CH.sub.3CN, reflux, 12 h (ii) Ethinylmagnesium bromide (1.5 eq), THF, 15 C., 4 h (iii) N.sub.3Ph (2.5 eq), NaAscorbate (0.1 eq), CuSO.sub.4 (0.01 eq), H.sub.2O/t-BuOH, MW, 125 C., 30 (iv) MnO.sub.2 (10 eq), DCM, r.t. 18 h (v) NH.sub.2OH (2.5 eq), Py (1.2), EtOH, reflux, 4 h (vi) Zn (10.0 eq), HCl (2N, 10.0 eq), THF, reflux, 2 h (vii) (BocNH.sub.2).sub.2C=Tf (1.1 eq), Et.sub.3N (2.0), DCM, r.t. 18 h

(133) Analytical Data of Compound 21

(134) .sup.1H NMR (400 MHz, MeOD) 8.56 (1H bs), 7.87-7.85 (2H, d, J=7.2 Hz), 7.59-7.55 (2H, t, J=8.0 Hz), 7.50-7.47 (1H, t, J=7.2 Hz), 7.39-7.35 (1H, t, J=7.2 Hz), 7.30-7.29 (1H, d, J=4.0 Hz), 7.07-7.05 (1H, t, J=8.4 Hz), 6.90 (2H, bs), 6.35 (1H, s), 5.73-5.67 (1H, m), 5.47-5.43 (1H, m), 4.13 (2H, s), 3.71-3.70 (2H, d, J=5.6 Hz), 3.47 (2H, s), 3.33 (2H, s), 3.07-3.04 (2H, t, J=6.8), 1.69-1.67 (3H, d, J=6.0 Hz), 1.59-1.52 (8H, m), 1.28-1.23 (12H, m)

(135) .sup.13C NMR (400 MHz, MeOD) 163.6, 162.4, 156.3, 156.1, 153.1, 152.1, 149.0, 137.5, 137.0, 131.5, 131.2, 130.4, 129.5, 128.8, 127.6, 124.9, 120.5, 120.15, 119.4, 119.2, 115.3, 97.01, 67.6, 66.4, 50.4, 48.9, 48.3, 47.5, 45.5, 42.5, 41.0, 30.7, 30.1, 29.2, 28.8, 28.4, 26.1, 17.5

(136) LCMS m/z (ES+) m/z=329.1 [M+2H].sup.2+, 657.3 [M+H].sup.+, 695.3 [M+Na].sup.+

Example 22

1-[(2E)-but-2-en-1-yl]-3-(8-{16-imino-14-oxo-2-oxa-13,15,17-triazatricyclo[17.4.0.04,9]tricosa-1(19),4(9),5,7,20,22-hexaen-13-yl}octyl)guanidine 22

(137) ##STR00045##

(138) Compound 22 was synthesized according to according to scheme 1 by reacting intermediate 1d with intermediate 3a and applying a similar procedure as for example 16.

(139) The synthesis of intermediate is 1d reported in scheme 6.

(140) ##STR00046##
Reagents and Conditions:

(141) (i) PPh.sub.3 (1.1 eq), THF, reflux, 12 h (ii) CH.sub.2O (1.8 eq), KOH (2.0 eq), DCM, r.t., 1 h (iii) Salicylaldehyde (1.0 eq), K.sub.2CO.sub.3 (1.5 eq), CH.sub.3CN, reflux, 12 h (iv) NH.sub.2OH (2.5 eq), Py (1.2 eq), EtOH, reflux, 4 h (v) Zn (10.0 eq), HCl (2N, 10 eq), THF, reflux, 2 h (iv) (BocNH.sub.2).sub.2C=Tf (1.1 eq), Et.sub.3N (2.0 eq), DCM, r.t. 18 h.

(142) Analytical Data of Compound 22.

(143) .sup.1H NMR (400 MHz, MeOD) 7.46-7.44 (1H, d, J=6.0 Hz), 7.35 (2H, m), 7.23 (3H, m) 6.98 (1H, m), 6.87-6.88 (1H, t, J=4.5), 6.84 (1H, m), 5.75-5.69 (1H, m), 5.51-5.48 (1H, m), 5.07 (2H, s), 4.46-4.40 (2H, d, J=24 Hz), 3.74-3.72 (2H, d, J=5.6 Hz), 3.40-3.30 (4H, m), 3.17-3.13 (2H, t, J=8.8), 2.63-2.59 (2H, t, J=7.4), 1.72-1.69 (3H, d, J=6.0 Hz), 1.57-1.54 (4H, m), 1.32-1.28 (10H, m)

(144) .sup.13C NMR (400 MHz, MeOD) 163.7, 156.4, 155.9, 153.2, 136.8, 136.6, 135.3, 134.7, 133.7, 129.7, 129.6, 129.4, 128.6, 128.1, 125.7, 125.6, 124.9, 121.1, 119.5, 114.8, 111.9, 76.2, 48.4, 47.5, 42.5, 41.0, 40.4, 37.4, 29.2, 28.9, 28.8, 28.7, 28.5, 28.4, 28.1, 26.1, 16.3

(145) LCMS m/z (ES+) m/z=281.7 [M+2H].sup.2+, 562.3 [M+H].sup.+.

Example 23

1-[(2E)-but-2-en-1-yl]-3-(8-{12-imino-14-oxo-3-oxa-11,13,15-triazatricyclo[17.3.1.04,9]tricosa-1(23),4(9),5,7,19,21-hexaen-15-yl}octyl)guanidine

(146) ##STR00047##

(147) Compound 23 was synthesized according to according to scheme 1 by reacting intermediate 1e with intermediate 3a and applying a similar procedure as for example 16.

(148) The synthesis of intermediate 1e reported in scheme 7.

(149) ##STR00048## ##STR00049##
Reagents and Conditions:

(150) (i) PPh.sub.3 (1.1 eq), THF, reflux, 12 h (ii) CH.sub.2O (1.8 eq), KOH (2.0 eq), DCM, r.t., 1 h (iii) Salicylaldehyde (1.0 eq), K.sub.2CO.sub.3 (1.5 eq), CH.sub.3CN, reflux, 12 h (iv) NH.sub.2OH (2.5 eq), Py (1.2 eq), EtOH, reflux, 4 h (v) Zn (10.0 eq), HCl (2N, 10 eq), THF, reflux, 2 h (iv) (BocNH.sub.2).sub.2C=Tf (1.1 eq), Et.sub.3N (2.0 eq), DCM, r.t. 18 h.

(151) Analytical Data of Compound 23

(152) .sup.1H NMR (400 MHz, MeOD) 7.31-7.19 (7H, m), 7.07 (1H, s), 6.97-6.93 (1H, t, J=7.2), 5.75-5.70 (1H, m), 5.59-5.47 (1H, m), 5.13 (2H, s), 4.53 (2H, s), 3.74-3.72 (2H, d, J=4.8 Hz), 3.35 (2H, s), 3.17-3.13 (4H, t, J=7.2), 2.66-2.65 (2H, d, J=5.2), 2.09 (2H, s), 1.71-1.69 (3H, d, J=6.4 Hz), 1.56-1.48 (4H, m), 1.27-1.19 (10H, m)

(153) .sup.13C NMR (400 MHz, MeOD) 161.7, 157.2, 155.9, 154.1, 153.2, 147.8, 141.1, 136.6, 130.1, 130.0, 128.8, 128.2, 127.9, 127.4, 125.9, 125.4, 124.8, 122.7, 120.2, 111.4, 70.2, 69.2, 44.8, 44.4, 42.6, 42.5, 41.0, 37.4, 31.5, 29.2, 28.8, 28.7, 28.5, 27.5, 27.1, 26.1, 25.8, 16.3

(154) LCMS m/z (ES+) m/z=281.7 [M+2H].sup.2+, 562.3 [M+H].sup.+

(155) Biological Assays

(156) Antimicrobial Susceptibility Testing

(157) Sterile plastic microtitration plates containing flat-bottomed wells were used. The plates contained serial dilution of the antifungal agents with a volume of assay medium of 100 L/well. Two drug-free medium wells were used as sterility and growth controls. The trays were inoculated with 100 L/well of the final inoculum, with the exception of sterility control wells. The range of concentrations tested for each drug was 1.25-80 M. The microtitration plates were incubated at 37 C. for 24 h. The minimal inhibitory concentrations (MICs) were determined at 24 h both visually and spectrophotometrically measuring the turbidity at 595 nm with a Varian model 1475 spectrophotometer.

(158) The various strain tested are indicated in Table 1.

(159) TABLE-US-00001 TABLE 1 Strains used in the biological assay. Species Strains Source Candida ATCC60193, ATCC14053, ATCC90028, ATCC24433, ATCC (LGC Albicans ATCC200955, ATCC10231, ATCC76615, ATCC2091, Standards) ATCC96110, ATCC36232, ATCC90255, ATCC44806, ATCC44373, ATCC90257, ATCC44808, ATCC56883, ATCC48274, ATCC34133, ATCC90268, ATCC52302, ATCC38289, ATCC56880 Candida ATCC56822, ATCC22017, ATCC34134, ATCC22949, ATCC (LGC guilliermondii ATCC76759, ATCC20118, ATCC20382, ATCC6260, Standards) ATCC22948, ATCC42050 Candida ATCC32545, ATCC2340, ATCC34077, ATCC38293, ATCC (LGC krusei ATCC32672, ATCC32672, ATCC14243, ATCC201071, Standards) ATCC22985, ATCC20405, ATCC90878, ATCC2159, ATCC34135 Candida ATCC-MYA-4646, ATCC28476, ATCC22019, ATCC58789, ATCC (LGC parapsilosis ATCC20246, ATCC96140, ATCC20181, ATCC16632, Standards) ATCC20224, ATCC7333, ATCC90018, ATCC20179, ATCC96137, ATCC7330, ATCC96142, ATCC201075, ATCC201076, ATCC58791, ATCC34136, ATCC20406, ATCC96041, ATCC90875, ATCC60548 Candida ATCC15114, ATCC38292, ATCC28776, ATCC28724, ATCC (LGC tropicalis ATCC20005, ATCC22577, ATCC60557, ATCC46536, Standards) ATCC42678, ATCC36729, ATCC201380 Candida ATCC8618, ATCC66028D, ATCC8641, ATCC204093, ATCC (LGC kefyr ATCC44691, ATCC9767, ATCC2540, ATCC8654, Standards) ATCC8619, ATCC200553 Candida ATCC15545, ATCC15126, ATCC90876, ATCC32554, ATCC (LGC glabrata ATCC66032, ATCC90525, ATCC60406, ATCC64677, Standards) ATCC36909D, ATCC2001, ATCC200989, ATCC46433, ATCC58561, ATCC26512, ATCC32936, ATCC48435, ATCC200918, ATCC28226, ATCC28290, ATCC38326, ATCC90030, ATCC34139, ATCC32312, ATCC201069, ATCC MYA-275, ATCC MYA2950D-5
Results

(160) Results of biological tests are shown in Table 2, biological data are reported as MIC.sub.90 that is the minimum concentration required to inhibit the growth of at least the 90% of the strains tested for each species.

(161) TABLE-US-00002 TABLE 2 Antifungal activity of examples 16-23 Species (No. strains MIC.sub.90 (g/mL).sup.a tested) F.sup.b 16 17 18 19 20 21 22 23 C. albicans (22) 2 16 4 4 32 16 32 64 256 C. guilliermondii (10) 4 16 2 2 32 8 32 64 256 C. krusei (13) 256 32 8 4 64 32 128 256 256 C. parapsilosis (24) 0.5 8 2 2 32 8 32 64 256 C. tropicalis (11) 2 8 2 1 32 16 64 128 256 C. kefyr (10) 1 4 4 2 32 16 64 128 256 C. glabrata (26) 16 64 8 16 64 32 64 128 128 .sup.aMIC.sub.90 values were determined at 24 h both visually and spectrophotometrically. .sup.bFluconazole

(162) Compounds 16, 17 and 18 possessing an aromatic ring fused to the macrocyclic core proved to be more active when compared to compounds 19 and 20 bearing an ester moiety. It is noteworthy that compounds 18 and 20, with a macrocyclic core of 15 atoms showed one of the highest activity toward C. albicans, C. guillermondii and C. parapsilosis when compared with homologue compounds with a lover number of atoms in the macrocyclic core. In particular compounds 18 and 20 are more active than fluconazole on C. guillermondii and C. krusei.

(163) Mutant Strains

(164) Compounds 16-19 were thus assayed against mutant C. albicans and C. glabrata strains bearing mutation on ERG11, CDR1, CDR2, SNQ2 and MDR1 genes, these mutants were obtained from clinical isolates and have been reported in the scientific literature. The ERG11 gene, which encodes the cytochrome P450, confers resistance to drugs belonging to the azole class, such as voriconazole and fluconazole. CDR1 and CDR2 genes encodes for ATP binding cassette (ABC) transporters, their overexpression confers resistance to the azole class. MDR1 gene, which encodes a membrane transport protein of the major facilitator superfamily, confers resistance to a broad range of drugs and toxic compounds. The SNQ2 gene encodes for yet another ABC transporter that confers resistance to azole and non-azole drugs.

(165) Voriconazole, an azole active on fluconazole resistant strains, was chosen as reference compound. Data are reported in Table 3 as MIC value (minimum inhibitory concentration (MIC) is the lowest concentration of an agent that will inhibit the visible growth of a microorganism after overnight incubation) obtained as the mean of three measurements for each strain.

(166) TABLE-US-00003 TABLE 3 Antifungal activity of compounds 16-19 on C. albicans and C. glabrata fluconazole resistant strains. MIC (g/mL).sup.a Strains Species Resistance mechanism FLU.sup.b VOR.sup.c 16 17 18 19 DSY284 C. albicans ERG11 Mutation 256 4 16 2 8 16 DSY296 C. albicans ERG11 Mutation 128 8 16 4 16 16 DSY348 C. albicans ERG11 Mutation 32 0.25 8 2 8 8 DSY289 C. albicans ERG11 Mutation (2) 256 8 8 2 8 8 DSY291 C. albicans ERG11 Mutation (2) 2 0.03 8 4 16 8 DSY292 C. albicans ERG11 Mutation (3) 64 2 16 4 16 16 DSY775 C. albicans ERG11 Mutation 128 8 16 4 16 16 DSY732 C. albicans CDR1-CDR2 16 0.25 8 2 16 8 DSY735 C. albicans CDR1-CDR2 64 2 8 2 16 8 DSY750 C. albicans MDR1 2 0.016 16 4 16 16 DSY751 C. albicans MDR1, ERG11 256 0.25 16 4 16 16 Mutation DSY2323 C. albicans CDR2-MDR1 32 0.25 8 4 16 16 DSY530 C. glabrata CDR1 64 0.5 64 8 32 64 DSY754 C. glabrata CDR1 64 2 64 16 32 64 DSY756 C. glabrata CDR1-CDR2-SNQ2 128 4 64 8 64 128 DSY2254 C. glabrata CDR1-CDR2 128 2 64 16 64 128 DSY2271 C. glabrata CDR2 64 0.125 64 8 32 64 .sup.aMIC values were determined at 24 h both visually and spectrophotometrically. .sup.bFluconazole, .sup.cVoriconazole; numbers in parentheses represent the number of mutation on the gene.

(167) All compounds 16-19 proved to be active against C. albicans and C. glabrata fluconazole resistant strains. Compound 17 resulted more active than voriconazole itself against mutant strains DSY289, DSY296 and DSY775. Compound 18 was more active than fluconazole but in general less active than compounds 16 and 17 and voriconazole, except for DSY289 strain.

(168) Chitinase Inhibition Assays

(169) Chitinase inhibition assays were conducted against an example of the GH18 family of chitinases, i.e the chitinase enzyme extracted from Trichoderma viride. This enzyme has an high degree of identity compared to chitinase 4 of Candida albicans, and is commercially available from Sigma-Aldrich (catalogue ID: C8241). The chitinolytic enzymes from T. viride are a mixture of extracellular chitinolytic enzymes, which exhibit exo- and endochitinase activities. The major activity was found to be that of N-acetyl--glucosaminidase.

(170) The substrate used was the NP-GlcNAc (Sigma-Aldrich, catalogue ID: N9376) dissolved in MES 50 mM at pH6.0; the final concentration was 250 M. The enzyme was resuspended at 0.11 mg/ml in MES 50 mM at pH 6.0, with a final concentration for the assay of 73 nM diluting with MES 50 mM and BSA 20 g/ml at pH 6.0. A stock solution of the inhibitors was prepared at 50 mM in DMSO 100%.

(171) The mixture with the inhibitor at various concentrations and the enzyme was prepared directly into the reading plate and left to incubate at room temperature for 20 minutes or three hours. At the end of the incubation times, the substrate was added and the reading was conducted by recording the values up to a maximum time of 10 minutes. The substrate concentration was measured by reading UV absorption at 300 nm (.sub.M.sup.300: 11100), while, the product of hydrolysis were measured at 400 nm (.sub.M.sup.400: 2120). Both IC.sub.50 and K.sub.i can be calculated.

(172) The results for compound 17, one of the most active compound against candida strains, are reported in Table 4. For comparison, the inventors have also tested compounds disclosed in WO2009/113033 (reference compounds 34-39).

Compound 34: 1-(cyclopropylmethyl)-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine

(173) ##STR00050##

Compound 35: 1-[(E)-but-2-enyl]-3-[8-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)octyl]guanidine

(174) ##STR00051##

Compound 36: 1-[(E)-but-2-enyl]-3-[6-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)hexyl]guanidine

(175) ##STR00052##

Compound 37: 1-[6-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)hexyl]-3-(2-methylprop-2-enyl)guanidine

(176) ##STR00053##

Compound 38: 1-ethyl-3-[6-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)hexyl]guanidine

(177) ##STR00054##

Compound 39: 1-[6-(4-imino-2-oxo-1,3,5-triazacyclotridec-1-yl)hexyl]-3-prop-2-ynylguanidine

(178) ##STR00055##

(179) TABLE-US-00004 TABLE 4 Inhibition assay of chitinase from Thricoderma viride of compound 17 and prior art compounds 34-39. Enz.-inhib. incubation Enz.-inhib. incubation time 20 min time 3 h Inhib. Inhib. con. con. range IC.sub.50 K.sub.i range IC.sub.50 K.sub.i Inhibitor [M] [M] [M] [M] [M] [M] 17 640-20 151 48.8 1000-10 55 17.8 34 640-20 252 81.4 1000-10 130 42 35 640-40 185 59.6 1000-40 157 50.8 36 1000-10 111 35.8 37 640-40 276 81.4 1000-10 108 34.8 38 640-80 508 163.9 1000-10 140 45.3 39 640-40 170 55 1000-10 97 31.3

(180) Compound 17 (with the aromatic ring fused in the macrocyclic core) is an active inhibitor of the chitinase enzyme. This compound is more active than compounds 34-39. It is worth to note that the IC.sub.50 values decrease with time, suggesting a slow binding of the inhibitor.

REFERENCES

(181) 1. Groll, A. H.; Lumb, J., New developments in invasive fungal disease. Future Microbiol. 2012, 7 (2), 179-84. 2. Mishra, N. N.; Prasad, T.; Sharma, N.; Payasi, A.; Prasad, R.; Gupta, D. K.; Singh, R., Pathogenicity and drug resistance in Candida albicans and other yeast species. A review. Acta Microbiol. Immunol. Hung. 2007, 54 (3), 201-35. 3. Casalinuovo, I. A.; Di Francesco, P.; Garaci, E., Fluconazole resistance in Candida albicans: a review of mechanisms. Eur. Rev. Med. Pharmacol. Sci. 2004, 8 (2), 69-77. 4. Manetti, F.; Castagnolo, D.; Raffi, F.; Zizzari, A. T.; Rajamaki, S.; D'Arezzo, S.; Visca, P.; Cona, A.; Fracasso, M. E.; Doria, D.; Posteraro, B.; Sanguinetti, M.; Fadda, G.; Botta, M., Synthesis of new linear guanidines and macrocyclic amidinourea derivatives endowed with high antifungal activity against Candida spp. and Aspergillus spp. J. Med. Chem. 2009, 52 (23), 7376-9. 5. Botta, M.; Raffi, F.; Visca, P. Linear and cyclic guanidine derivatives as antifungal agents and their method of preparation. WO2009113033A2, 2009. 6. Kuranda, M. J.; Robbins, P. W., Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J. Biol. Chem. 1991, 266 (29), 19758-67. 7. Sakuda, S., Studies on the chitinase inhibitors, allosamidins. In Chitin enzymology, Muzzarelli, R. A. A., Ed. Atec Edizioni: 1996; Vol. 2, pp 203-212. 8. Takaya, N.; Yamazaki, D.; Horiuchi, H.; Ohta, A.; Takagi, M., Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. Biosci. Biotechnol. Biochem. 1998, 62 (1), 60-5. 9. Zhu, Z.; Zheng, T.; Homer, R. J.; Kim, Y.-K.; Chen, N. Y.; Cohn, L.; Hamid, Q.; Elias, J. A., Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation. Science 2004, 304 (5677), 1678-1682. 10. Boot et al., Identification of a novel acidic mammalian chitinase distinct from chitotriosidase. J. Biol. Chem. 2001, 276, 6770-8. 11. Hartl. D.; et al., Acidic Mammalian Chitinase Regulates Epithelial Cell Apoptosis via a Chitinolytic-Independent Mechanism. J-Immunolol 2009; 182:5098-5106.