LINEAR GUANIDINE DERIVATIVES, METHODS OF PREPARATION AND USES THEREOF

Abstract

The present invention relates to linear guanidine derivatives, methods of preparation, uses and pharmaceutical compositions thereof. The compounds of Formulas 1 or 2 exhibit high antimicrobial activity against Gram positive and Gram negative bacteria.

Claims

1. A compound of general formula 1: ##STR00044## or a pharmaceutical acceptable salt, hydrate or solvate thereof; wherein: R.sub.1, R.sub.3, R.sub.5, R.sub.7, R.sub.9 and R.sub.11 are the same or different groups, selected between H, methyl, ethyl, propyl, prop-2-ynyl, but-2-enyl, 2-methylprop-2-enyl, 3-methylbut-2-enyl, phenyl, benzyl, dimethylphenyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, acetyl, propanoyl, N-alkyl-carbamoyl, N-alkyl-thiocarbamoyl, N-alkyl-carbamimidoyl or saturated linear or branched C.sub.1-10 alkyl; R.sub.2, R.sub.4, R.sub.6, R.sub.8, R.sub.10 and R.sub.12 are the same or different groups, selected between H, methyl, ethyl, or saturated linear or branched C.sub.1-5 alkyl; X is O or S; L is a number from 0 to 2; n.sub.1, n.sub.2 and n.sub.3 can be the same or different and are numbers from 2 to 10; and m.sub.1, m.sub.2 and m.sub.3 can be the same or different and are numbers from 2 to 10.

2. The compound according to claim 1 wherein L=0.

3. The compound according to claim 1 wherein n.sub.1=n.sub.2=m.sub.1=m.sub.2=6.

4. The compound according to claim 1 wherein R.sub.1 and/or R.sub.5 is cyclopropylmethyl.

5. The compound according to claim 1 wherein R.sub.1 and/or R.sub.5 is ethyl, benzyl, propargyl or but-2-enyl.

6. The compound according to claim 1 being selected from the group consisting of: 1,3-bis(6-(3-(cyclopropylmethyl)guanidino)hexyl)-1,3-bis(6-guanidinohexyl)urea; 1,3-bis(8-(3-(cyclopropylmethyl)guanidino)octyl)-1,3-bis(8-guanidinooctyl)urea; 1,3-bis(8-(3-(cyclopropylmethyl)guanidino)octyl)-1-(8-guanidinooctyl)-3-(8-((3-(8-((8-(3- cyclopropylmethyl))guanidinooctyl)amino)octyl)carbamoyl)guanidino)octyl)urea; 1,3-bis(8-(3-(ethyl)guanidino)octyl)-1,3-bis(8-guanidinooctyl)urea; 1,3-bis(8-(3-(buten-2-yl)guanidino)octyl)-1,3-bis(8-guanidinooctyl)urea; 1,3-bis(8-(3-(benzyl)guanidino)octyl)-1,3-bis(8-guanidinooctyl)urea; 1,3-bis(8-(3-(propargyl)guanidino)octyl)-1,3-bis(8-guanidinooctyl)urea; 1,3-bis(9-(3-(cyclopropylmethyl)guanidino)nonyl)-1,3-bis(9-guanidinononyl)urea; 1,3-bis(9-(3-(ethyl)guanidino)nonyl)-1,3-bis(9-guanidinononyl)urea; 1,3-bis(9-(3-(buten-2-yl)guanidino)nonyl)-1,3-bis(9-guanidinononyl)urea; 1,3-bis(9-(3-(benzyl)guanidino)nonyl)-1,3-bis(9-guanidinononyl)urea; 1,3-bis(9-(3-(propargyl)guanidino)nonyl)-1,3-bis(9-guanidinononyl)urea; 1,3-bis(10-(3-(cyclopropylmethyl)guanidino)decyl)-1,3-bis(10-guanidinodecyl)urea; 1,3-bis(10-(3-(ethyl)guanidino)decyl)-1,3-bis(10-guanidinodecyl)urea; 1,3 -bis(10-(3-(buten-2-yl)guanidino)decyl)-1,3-bis(10-guanidinodecyl)urea; 1,3 -bis(10-(3-(benzyl)guanidino)decyl)-1,3-bis(10-guanidinodecyl)urea; and 1,3 -bis(10-(3-(propargyl)guanidino)decyl)-1,3-bis(10-guanidinodecyl)urea.

7. A compound having the general formula 2: ##STR00045## or a pharmaceutical acceptable salt, hydrate or solvate thereof; wherein: R.sub.1, R.sub.3, R.sub.5, and R.sub.7, are the same or different groups, selected between H, methyl, ethyl, propyl, prop-2-ynyl, but-2-enyl, 2-methylprop-2-enyl, 3-methylbut-2-enyl, phenyl, benzyl, dimethylphenyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, acetyl, propanoyl, N-alkyl-carbamoyl, N-alkyl-thiocarbamoyl, N-alkyl-carbamimidoyl or saturated linear or branched C.sub.1-10 alkyl; R.sub.2, R.sub.4, R.sub.6, and R.sub.8, are the same or different groups, selected between H, methyl, ethyl, or saturated linear or branched C.sub.1-5 alkyl L is a number from 1 to 3; n.sub.1 and n.sub.2 can be the same or different and are numbers from 2 to 10; and m.sub.1 and m.sub.2 can be the same or different and are numbers from 2 to 10.

8. The compound according to claim 7 wherein L=1.

9. The compound according to claim 7 wherein R.sub.1 and/or R.sub.5 is cyclopropylmethyl.

10. The compound according to claim 7 being selected from the group consisting of: 1-(6-carbamimidamidohexyl)-1-[6-[[N-(cyclopropylmethyl)carbamimidoyl]amino]hexyl]-3-[N-[6-[6-[[N (cyclopropylmethyl)carbamimidoyl]amino]hexylamino]hexyl]carbamimidoyl]urea; 1-(8-carbamimidamidooctyl)-1-[8-[[N-(cyclopropylmethyl)carbamimidoyl]amino]octyl]-3-[N-[8-[8-[[N-(cyclopropylmethyl)carbamimidoyl]amino]octylamino]octyl]carbamimidoyl]urea; 1-(6-carbamimidamidohexyl)-16-[[N-(cyclopropylmethyl)carbamimidoyflamino]hexyl]-3-[N-[6-[6-[[N-(cyclopropylmethyl)carbamimidoyl]amino]hexyl-[[N-[6-[6-[[N-(cyclopropylmethyl) carbamimidoyl]amino]hexylamino]hexyl]carbamimidoyl]carbamoyl]amino]hexyl]carbamimidoyl]urea; and 1-(8-carbamimidamidooctyl)-1-[8-[[N-(cyclopropylmethyl)carbamimidoyl]amino]octyl]-3-[N-[8-[8-[[N-(cyclopropylmethyl)carbamimidoyl]amino]octyl-[[N-[8-[8-[[N-(cyclopropylmethyl) carbamimidoyl]amino]octylamino]octyl]carbamimidoyl]carbamoyl]amino]octyl]carbamimidoyl] urea.

11. (canceled)

12. A method the treatment of a bacterial infection comprising administering a compound of claim 1 to a patient in need thereof.

13. The method according to claim 12 wherein the bacteria is Gram-positive or Gram-negative.

14. The method according to claim 13 wherein the bacteria is selected from the group consisting of: Enterococci, Staphylococci, Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Enterobacter spp., Achromobacter spp., Aeromonas spp., Alcaligenes spp., Burkholderia cepacia, Chryseobacterium meningosepticum, Escherichia coli, Stenotrophomonas maltophilia and Bacillus subtilis.

15. The method according to claim 12 wherein the bacteria is resistant to at least one antibiotic/antibacterial agent.

16. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutical acceptable salt or solvate thereof and acceptable carriers, excipients or diluents.

17. (canceled)

18. The pharmaceutical composition according to claim 16 further comprising at least one other therapeutic agent.

19. A process for the preparation of a compound of formula 1 as defined in claim 1 comprising: ##STR00046##

20. A process for the preparation of a compound of formula 2 as defined in claim 7 comprising: ##STR00047##

21. A process for the preparation of an intermediate of formula 7a or 7b comprising: ##STR00048##

Description

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Chemistry

[0150] All commercially available chemicals were used as purchased. DCM was dried over sodium hydride. THF were dried over Na/benzophenone prior to use. Anhydrous DMF was used as purchased. Anhydrous reactions were run under a positive pressure of dry N.sub.2 or argon.

[0151] Chromatographic separation of final products were conducted using a Polaris C18 column (150-4.6 mm, 5 pm particle size) at a flow rate of 0.8 mL min-1 with a mobile phase composed of 50% CH.sub.3CN/50% H.sub.2O-formic acid 0.1%.

Instrumentation

[0152] .sup.1H-NMR and .sup.13C-NMR were measured on a 400 mHz spectrometer and are reported in parts per million (δ scale) and internally referenced to the CDCl.sub.3 or CD.sub.3OD signal, respectively at δ 7.24 ppm and 3.31 ppm. Chemical shifts for carbon are reported in parts per million (δ scale) and referenced to the carbon resonances of the solvent (CDCl.sub.3 at δ 77.00 and CD.sub.3OD at δ 49.00 ppm). Data is presented as follows: chemical shift, multiplicity (s=singlet, d=doublet, m=multiplet and/or multiplet resonances, br s=broad singlet), coupling constant in Hertz (Hz), and integration. Mass spectra (MS) data were acquired on an Agilent 1100 LC/MSD VL system (G1946C) with a 0.4 mL/min flow rate using a binary solvent system of 95:5 methanol/water. UV detection was monitored at 254 nm. Mass spectra were acquired in positive mode scanning over the mass range.

[0153] Compounds of general formula 1, when R.sub.2, R.sub.4, R.sub.6, R.sub.8 are H, L=0 and X is O, described in this invention can be synthesized as reported in Scheme 1 starting from triamine 3. The guanylation in steps “i” and “ii” can be conducted using an appropriate guanylating agent, preferably a N-substituted N,N′-di-Boc-S-methylisothiourea or a N-substituted N.N′-di-Boc-1 H-pyrazolecarboxamidine. Linear intermediates 7a or 7b represent the key intermediates for the synthesis. Compound 7a-b can be prepared with different R groups (R.sub.1, R.sub.2, R.sub.3, R.sub.4) and with different n (n.sub.1, n.sub.2) and m (m.sub.1, m.sub.2) numbers.

##STR00010##

[0154] The present invention provides also an alternative synthesis of compound 7a or 7b reported in scheme 2. This synthetic pathway is more complex but more versatile.

##STR00011##

[0155] Compounds of general formula 2, when R.sub.2, R.sub.4, R.sub.6, R.sub.8 are H, described in this invention can be synthesized as reported in Scheme 3 starting from different or equal compounds of formula 7a-b.

##STR00012##

[0156] In scheme 3 in step (i) by varying the concentration in the range from 50 to 300 mM and the reflux time from 5 h to 36 h, products with an increasing number of L units can be synthesized and the reaction's status can be monitored through mass spectrometry.

[0157] Depending on the synthetic strategy used in the last step the resulting compounds can be isolated as salts, such as trifluoroacetate salts.

[0158] In scheme 1-3 as a result of the last step, the cleavage of the Boc protecting group,—the compounds are obtained as trifluoroacetate salts, but the counterion can be changed if appropriate purification steps are applied, such as HPLC with acidified water.

[0159] The guanylating agents used for each synthesis have been obtained through Mitsunobu reaction between the desired alcohol and the tert-butyl N-[[(2-methylpropan-2-yl)oxycarbonylamino]-pyrazol-1-ylmethylidene]carbamate (Scheme 4).[9]

##STR00013##

[0160] Compound 12 has been obtained by reacting the opportune linear dibromide with sodium azide using DMF as solvent.(Scheme 5)

##STR00014##

Example 2

Synthesis of Representative Compound 28

Synthesis of Compound 28

[0161] ##STR00015##

Synthesis of Boc-Protected Guanidine 20:

[0162] ##STR00016##

[0163] 1,8-diaminooctane 18 (6.0 g, 41.67 mmol) was dissolved in half solution of CH.sub.3CN/MeOH 9:1 (75.0 mL) and 1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea 19 (4.036 g, 13.89 mmol) in the other half (75.0 mL). The two solutions were mixed and the temperature was increased to 40-50° C. DIPEA (3.0 mL) was added to the reaction mixture and it was stirred 12 h. The reaction mixture was then concentrated and the crude product was purified by flash chromatography (CH.sub.3CN/MeOH/Et3N 8:2:1) to afford compound 20 as a pale yellow oil (yield 89%). .sup.1H NMR (CDCl.sub.3) δ (ppm):1.30 (m, 12H); 1.49 (s, 18H); 2.67 (t, 2H, J =7.0 Hz); 3.40 (m, 2H); 8.28 (bs, 1H); 11.49 (bs, 1H).LCMS m/z (ES+)=387 [M+H].sup.+

Synthesis of 1-azido-8-bromooctane 21:

##STR00017##

[0164] To a solution of dibromooctane (2.0 mL, 10.9 mmol) in DMF (2.0 mL), NaN.sub.3 (354.3 mg, 5.45 mmol) was added and the reaction mixture was stirred 12 h at 50° C. After cooling, the reaction mixture was diluted with AcOEt. The organic phase was extracted twice with H.sub.2O and then with Brine. The combined organic layers were dried over Na.sub.2SO.sub.4 and then evaporated. The crude product was purified with chromatography column in silica gel (eluent: Petroleum Ether) to afford compound 21 as a yellow oil (yield 82%). .sup.1H NMR (CDCl.sub.3) δ (ppm): 1.33 (m, 6H); 1.43 (m, 2H); 1.60 (m, 2H); 1.85 (m, 2H); 3.25 (m, 2H); 3.40 (m, 2H).

Synthesis of azide 22:

##STR00018##

[0165] DMF dry (5.0 mL) and molecular sieves (600.0 mg), previously dried in oven, were inserted into a flask with a magnetic stirrer under N.sub.2 atmosphere and stirred. CsOHxH.sub.2O (266.0 mg, 1.585 mmol) was added and the mixture was stirred for 10 minutes. Then, a solution of 20 (612.0 mg, 1.585 mmol) in DMF dry was added and the mixture was stirred for further 30 minutes. Then, compound 21(297.0 mg, 1.268 mmol) was added and the reaction mixture was stirred 12 h. The mixture was diluted with AcOEt, filtered from the solid, washed and concentrated. The residue was treated with NaOH 1N and extracted with AcOEt. The organic phase was washed with H.sub.2O, LiCl 5% and Brine. The crude product was purified with chromatography column in silica gel (eluent: DCM/MeOH 95:5; 9:1; 8:2) to afford compound 22 with a yield of 43%. .sup.1H NMR (CDCl.sub.3) δ (ppm): 1.25 (m, 24H); 1.44 (s, 18H); 2.52 (t, 4H, J =7.0 Hz); 3.19 (t, 2H, J=6.8 Hz); 3.34 (q, 2H, J=5.6 Hz); 8.23 (bs, 1H); 11.50 (bs, 1H). .sup.13C NMR (CDCl.sub.3) δ (ppm): 26.53, 26.68, 27.18, 27.95, 28.70, 28.96, 29.07, 30.01, 40.84, 49.98, 51.34, 53.31, 79.03, 82.84, 153.22, 155.98, 163.55. LCMS m/z (ES+)=540.1 [M+H].sup.+

Synthesis of Primary Amine 23

[0166] ##STR00019##

[0167] Compound 22 (341.1 mg, 0.63 mmol) was dissolved in THF (13.5 mL);

[0168] Triphenilphosphine (246.3 mg, 0.94 mmol) was added and the reaction mixture was stirred at room temperature for 30 minutes, monitoring the level of starting material. Then, H.sub.2O (170.0 μL, 9.45 mmol) was added and the mixture was stirred 12 h. H.sub.2O and AcOEt were added to the reaction mixture and the organic phase was washed with H.sub.2O and Brine. The crude product was purified with a silica gel column chromatography (DCM/MeOH 9:1, DCM/MeOH 8:2, DCM/MeOH/Et.sub.3N 8:2:1), affording compound 23 with a yield of 91%. .sup.1H NMR (MeOD) δ (ppm): 1.35 (m, 24H); 1.52 (s, 18H); 2.62 (t, 4H, J=7.6 Hz); 3.30 (m, 2H); 3.39 (m, 2H). LCMS m/z (ES+)=514.2 [M+H].sup.+

Synthesis of tert-butyl (((tert-butoxycarbonyl)imino)(1H-pyrazol-1-yl)methyl) (cyclopropylmethyl)carbamate 24

##STR00020##

[0169] N,N′-Di-Boc-1H-pyrazole-1-carboxamidine (500.0 mg, 1.61 mmol) was dissolved in THF dry (6.2 mL). Then Triphenylphosphine (631.4 mg, 2.41 mmol) and Hydroxymethyl-cyclopropane (150.5 mg, 2.09 mmol) were added. The reaction mixture was cooled at 0° C. and Diisoporpyl azodicarboxylate (0.47 mL, 2.41 mmol) was added dropwise. The temperature was increased to 70° C. and the reaction mixture was stirred at reflux 12 h. The reaction mixture was concentrated and then diluted with DCM and H.sub.2O. The aqueous phase was extracted for three times with DCM; the organic phases were collected, washed with brine twice and dried over Na.sub.2SO.sub.4. Solvent was removed in vacuum. The crude product was purified with chromatography column in silica gel (eluent: Petroleum Ether/AcOEt 9:1) to afford compound 24 as a yellow oil (yield 82%). .sup.1H NMR (CDCl.sub.3) δ (ppm): 0.45 (d, 2H, J=4.8 Hz); 0.49 (d, 2H, J=5.6 Hz); 1.27 (s, 9H); 1.49 (s, 9H); 1.54 (s, 1H); 3.60 (d, 2H, J=6.8 Hz); 6.41 (t, 1H, J=2.2 Hz); 7.69 (d, 1 H, J=1.2 Hz); 7.95 (s, 1H).LCMS m/z (ES+)=387.1 [M+Na].sup.+

Synthesis of Protected Diguanidine 25

[0170] ##STR00021##

[0171] A solution of compound 24 (262.1 mg, 0.72 mmol) in THF (5.8 mL) was added to compound 23. DIPEA (0.1 mL, 0.60 mmol) was added and the reaction mixture was stirred 12 h at room temperature. Then, the mixture was diluted with AcOEt and washed with NaHCO.sub.3, water and Brine. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified with a silica gel column chromatography (DCM/MeOH 9:1), affording compound 25 with a yield of 70%. .sup.1H NMR (CDCl.sub.3) δ (ppm): 0.24 (d, 2H, J=4.8 Hz); 0.45 (d, 2H, J=7.6 Hz); 1.04 (m, 1H); 1.31 (m, 24H); 1.49 (s, 36H); 2.58 (t, 4H; J=7.2 Hz); 3.30 (m, 2H); 3.39 (q, 2H, J=6.5 Hz); 3.53 (m, 2H); 8.28 (bs, 1H). .sup.13C NMR (CDCl.sub.3) δ (ppm): 3.44, 10.48, 26.80, 27.17, 28.16, 28.85, 29.08, 29.71, 40.85, 43.79, 49.77, 52.06, 79.05, 81.76, 82.86, 153.22, 155.98, 163.54. LCMS m/z (ES+)=810.3 [M+H].sup.+

Synthesis of Carbamoyl Chloride 26

[0172] ##STR00022##

[0173] Compound 25 (42.4 mg, 0.05 mmol) was dissolved in THF dry (1.0 mL) and stirred under nitrogen atmosphere. The reaction mixture was cooled to 0° C. and DIPEA (9.0 μL, 0.05 mmol) and Triphosgene (14.9 mg, 0.05 mmol) were added. The mixture was stirred 10 minutes at 0° C. and then at room temperature for 1 h. Then, AcOEt and NaHCO.sub.3 (s.s.) were added to the reaction mixture and it was stirred for 10 minutes. The aqueous phase was extracted twice with AcOEt and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified with a silica gel column chromatography (DCM/MeOH 98:2), affording compound 26 with a yield of 72%. .sup.1H NMR (CDCl.sub.3) δ (ppm): 0.24 (d, 2H, J=4.8 Hz); 0.44 (d, 2H, J=8.0 Hz); 0.86 (m, 2H); 1.31 (s, 16H); 1.49 (s, 36H); 1.59 (s, 8H); 3.30 (m, 4H); 3.36 (m, 4H); 3.54 (m, 2H); 8.27 (bs, 1H); 11.49 (bs, 1H). .sup.13C NMR (CDCl.sub.3) δ (ppm): 3.47, 10.50, 26.05, 26.66, 27.38, 27.99, 28.15, 28.22, 28.84, 29.04, 29.58, 40.81, 43.78, 49.82, 51.12, 79.11, 82.92, 153.11, 156.01, 162.55. LCMS m/z (ES+)=872.2 [M+H].sup.+; 436.5 [M+2H].sup.2+

Synthesis of Urea 27

[0174] ##STR00023##

[0175] Compounds 25 (42.1 mg, 0.052 mmol) and 26 (31.0 mg, 0.035 mmol) were dissolved in DCM dry (3.0 mL). DIPEA (6.0 μL, 0.035 mmol) and Nal (catalytic) were added and the reaction mixture was stirred 12 h at 35-40° C. After cooling, AcOEt, NaOH 1N and water were added to the reaction mixture and it was stirred for 10 minutes. The aqueous phase was extracted three times with AcOEt and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified with a silica gel column chromatography (EP/AcOEt 8:2), affording compound 27 with a yield of 60%. .sup.1H NMR (CDCl.sub.3) δ (ppm): 0.26 (d, 4H, J=4.8 Hz); 0.43 (d, 4H, J=7.6 Hz); 1.03 (m, 2H); 1.29 (m, 48H); 1.48 (s, 72H); 3.05 (m, 8H); 3.28 (m, 4H); 3.38 (m, 4H); 3.53 (m, 4H); 8.26 (bs, 2H); 11.49 (bs, 2H). LCMS m/z (ES+)=823.5 [M+2H].sup.2+; 549.4 [M+3H].sup.3+

Synthesis of Final Compound 28

[0176] ##STR00024##

[0177] Compound 27 (12.5 mg, 7.6×10.sup.−3 mmol) was dissolved in DCM dry (1.8 mL) and TFA 10% (0.2 mL) was added. The reaction mixture was stirred at room temperature for 7.5h. Then the solvent was evaporated and compound 28 was obtained in quantitative without any further purification as trifluoroacetate salt. .sup.1H NMR (MeOD) δ (ppm): 0.26 (d, 4H, J=4.8 Hz); 0.58 (d, 4H, J=7.2 Hz); 1.05 (m, 2H); 1.34 (m, 24H); 3.05 (d, 4H, J=6.8 Hz); 3.15 (m, 16H). .sup.13C NMR (CDCl.sub.3) δ (ppm): 2.39, 9.53, 26.17, 26.54, 27.51, 28.36, 28.45, 28.83, 28.93, 40.97, 41.09, 45.78, 46.87, 47.08, 47.30, 156.32, 158.66, 165.23. LCMS m/z (ES+)=845.0 [M+H].sup.+; 423.3 [M+2H].sup.2+; 282.5 [M+3H].sup.3+; 212.1 [M+4H].sup.4+

Example 3

Synthesis of Representative Compound 30

Synthesis of Compound 30

[0178] ##STR00025##

Synthesis of Primary Amine 29

[0179] ##STR00026##

[0180] A 100 millimolar solution in dry THF of compound 25 (54.3 mg, 0.07 mmol) was heated at reflux for 12 h. Then, the mixture was diluted with AcOEt and washed with NaHCO.sub.3, water and Brine. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified with a silica gel column chromatography (DCM/MeOH 9:1), affording compound 29 with a yield of 30%. .sup.1H NMR (CDCl.sub.3) δ (ppm): 0.22 (d, 4H, J=4.8 Hz); 0.44 (d, 4H, J=7.6 Hz); 1.02 (m, 2H); 1.27 (m, 48H); 1.44 (s, 36H); 1.46 (s, 36H); 2.83 (t, 4H; J=7.2 Hz); 3.27 (m, 8H); 3.37 (m, 4H); 3.51 (m, 4H); 7.96 (s, 1H).8.26 (s, 1H). LCMS m/z (ES+)=773.5 [M+2H].sup.2+; 516.2 [M+3H].sup.3+

Synthesis of Final Compound 30

[0181] ##STR00027##

[0182] Compound 29 (17.2 mg, 0.01 mmol) was dissolved in DCM dry (1.8 mL) and TFA 10% (0.2 mL) was added. The reaction mixture was stirred at room temperature for 10 h.

[0183] Then the solvent was evaporated and compound 30 was obtained in quantitative without any further purification as trifluoroacetate salt. .sup.1H NMR (MeOD) δ (ppm): 0.24 (d, 4H, J=5.2 Hz); 0.56 (d, 4H, J=7.6 Hz); 1.04 (m, 2H); 1.26-1.33 (m, 36H); 1.45-1.55 (m, 12H); 2.52 (t, 4H, J=7.2 Hz); 3.04 (d, 4H, J=6.8 Hz) 3.11-3.17 (m, 12H), 3.32 (s, 4H). 2.43, 9.88, 26.17, 26.54, 27.33, 28.38, 28.77, 28.93, 40.31, 41.19, 45.89, 47.28, 47.31, 156.21, 158.70, 164.77, 168.22. LCMS m/z (ES+)=845.0 [M+H].sup.+; 423.3 [M+2H].sup.2+; 282.5 [M+3H].sup.3+; 212.1 [M+4H].sup.4+

TABLE-US-00001 TABLE 1 Synthesized compounds 1 [00028] R.sub.2, R.sub.4, R.sub.6, R.sub.3, R.sub.7 (R.sub.11 if R.sub.5 (R.sub.9 if L X n.sub.(1-3) m.sub.(1-3) R.sub.1 R.sub.8, R.sub.10 R.sub.12 applicable) applicable) Compound 31 0 O 4 4 [00029] H H [00030] Compound 28 0 O 6 6 [00031] H H [00032] Compound 32 1 O 6 6 [00033] H H [00034] 2 [00035] R.sub.2, R.sub.3, R.sub.4, L n.sub.(1-2) m.sub.(1-2) R.sub.1 R.sub.6, R.sub.8, R.sub.3, R.sub.7 R.sub.5 Compound 33 1 4 4 [00036] H H [00037] Compound 30 1 6 6 [00038] H H [00039] Compound 34 2 4 4 [00040] H H [00041] Compound 35 2 6 6 [00042] H H [00043] All the compounds have been tested as trifluoroacetate salts.

Example 4

Antimicrobial Susceptibility Testing

Methods

[0184] Bacterial strains were obtained from the ATCC or CCUG culture collections or present in the authors' collection of clinical isolates [10]. Compounds were re-suspended in dimethyl sulfoxyde (DMSO) at a final concentration of 10 mg/ml and subsequently diluted in the culture medium. The minimum inhibitory concentrations (MICs) of the compounds of the invention were determined using the microdilution broth method using Mueller-Hinton broth as recommended by the Clinical Laboratory Standards Institute (CLSI [11]). Bacterial inoculum was 5×10.sup.4 CFU/well. MICs were recorded after 18-24 hours incubation at 35-37° C.

Results

[0185] Results of the antimicrobial susceptibility assays are shown in Table 2. Compound 28, belonging to formula 1, bearing a cyclopropylmethyl group as substituent R.sub.1 and R.sub.5 and zero repeating units (L is 0), seems to be the most active among the series. Although most of the compounds showed a higher antimicrobial activity against Gram-positive bacteria, compounds 28 and partially compound 32 show the broadest antimicrobial spectrum, and result active against both Gram-positive and Gram-negative organisms, especially clinically relevant Enterobacteriaceae, i.e. Escherichia coli and Klebsiella pneumonia.

TABLE-US-00002 TABLE 2 Biological results of five tested compounds Compound MIC (μg/ml) Bacterial strain 31 28 32 33 30 Achromobacter xylosoxidans AX 22 >256 8 64 >256 >256 Acinetobacter baumannii ATCC 17978 128 4 32 256 >256 Alcaligenes faecalis 424/98 >256 8 32 >256 32 Burkholderia cepacia RII >256 8 128 >256 128 Chryseobacterium meningosepticum CCUG >256 64 64 >256 >256 4310 Escherichia coli CCUG.sup.T 16 1 8 32 >256 Klebsiella pneumoniae ATCC 13833 32 1 8 64 >256 Pseudomonas aeruginosa ATCC 27853 256 4 32 >256 >256 Stenotrophomonas maltophilia 634/08 >256 16 64 >256 >256 Bacillus subtilis ATCC 6633 4 0.5 8 8 <0.125 Enterococcus faecalis ATCC 19433 32 <0.125 <0.125 32 8 Staphylococcus aureus ATCC 25923 4 2 8 16 4 Acinetobacter baumannii AC-54/97 (IMP-2) — 2 16 8 32 Enterobacter cloacae VA-417/02 (VIM-4) — 1 16 64 — Klebsiella pneumoniae 081R — 2 16 — — MIC = Minimal Inhibitory Concentration

Example 5

In Depth Biological Evaluation of Compound 28

[0186] The biological profile of compound 28 which was the most active of the series was further investigated by evaluating the bactericidal activity (Table 3) as well as the activity against selected resistant strains (Table 4).

TABLE-US-00003 TABLE 3 Bactericidal activity of compound 28 Compound 28 Bacterial strain MIC μg/ml MBC μg/ml Achromobacter xylosoxidans AX 22 8 8 Acinetobacter baumannii ATCC 17978 4 4 Alcaligenes faecalis 424/98 8 8 Burkholderia cepacia RII 8 8 Chryseobacterium meningosepticum 64 64 CCUG 4310 Escherichia coli CCUG.sup.T 1 1 Klebsiella pneumoniae ATCC 13833 1 1 Pseudomonas aeruginosa ATCC 27853 4 4 Stenotrophomonas maltophilia 634/08 16 16 Bacillus subtilis ATCC 6633 0.5 0.5 Enterococcus faecalis ATCC 19433 <0.125 <0.125 Staphylococcus aureus ATCC 25923 2 2 Acinetobacter baumannii AC-54/97 (IMP-2) 2 2 Enterobacter cloacae VA-417/02 (VIM-4) 1 1 Klebsiella pneumoniae 081R 2 2 MIC = Minimal Inhibitory Concentration; MBC = Minimal Bactericidal Concentration

[0187] The Antibacterial activity of compound 28 was also tested on a panel of Gram-negative and Gram-positive clinical isolates, showing various antimicrobial susceptibility profiles (Table 4). The class of drugs for which the isolate was resistant to is indicated in column 2 (PEN, penicillins; ES-CEPH, expanded-spectrum cephalosporins; CARB, carbapenems; AZT; aztreonam; AG, aminoglycosides; FQ, fluoroquinolones; SXT, trimethoprim/sulfamethoxazole; FOS, fosfomycin; GLY, glycopeptides; LNZ, linezolid; COL-R, colistin resistant; COL-S colistin sensitive; PDR, poly-drug resistant).

TABLE-US-00004 TABLE 4 Biological activity of compound 28 against a selected panel of resistant pathogens Comp 28 MIC Bacterial Strain Resistance Profile (μg/ml) GRAM-NEGATIVES Acinetobacter baumannii AC-54/97 PEN, ES-CEPH, 4 CARB, AZT, AG, FQ, FOS, SXT Enterobacter cloacae VA-417/02 PEN, CEPH, 2 CARB, AG, FQ Klebsiella pneumoniae 7023 PEN, ES-CEPH, 2 CARB, AG, FQ, SXT Klebsiella pneumoniae KP0787 PDR 1 Pseudomonas aeruginosa 101/1477 PEN, ES-CEPH, 8 CARB, AG Pseudomonas aeruginosa VR143/97 PEN, ES-CEPH, 8 CARB, MON, AG, FQ Pseudomonas aeruginosa 14X-34 PDR 8 Klebsiella pneumoniae BO1 COL-S 4 Klebsiella pneumoniae BO4 COL-R 8 Klebsiella pneumoniae B1 COL-S 4 Klebsiella pneumoniae B2 COL-R 4 Klebsiella pneumoniae 207-1 COL-S 4 Klebsiella pneumoniae 207-2 COL-R 8 Klebsiella pneumoniae 081R PDR 4 Klebsiella pneumoniae 167R PDR 4 GRAM-POSITIVES Staphylococcus aureus ATCC 43300 PEN 4 (MRSA) Staphylococcus aureus ATCC 700699 GLY 4 (VanA) Staphylococcus haemolyticus SI-6/2011 AG 2 Staphylococcus warneri SI-5/2011 PEN, AG 2

Example 6

ADME Properties of Compound 28

[0188] In vitro ADME properties (apparent permeability in gastrointestinal model, microsomal stability and binding to plasma proteins) for compound 28 were evaluated.

Parallel Artificial Membrane Permeability Assay

Method

[0189] Donor solution (0.5 mM) was prepared by diluting 1 mM dimethylsulfoxide (DMSO) compound stock solution using tris-HCl buffer (50 mM) at pH 7.4. Filters were coated with 5 μL of a 1% (w/v) dodecane solution of phosphatidylcholine. Donor solution (150 μL) was added to each well of the filter plate. To each well of the acceptor plate were added 300 μL of solution (50% DMSO in phosphate buffer). Compound 28 was tested in three different plates on different days. The sandwich was incubated for 5 h at room temperature under gentle shaking. After the incubation time, the plates were separated, and samples were taken from both receiver and donor sides and analyzed using LC with UV detection at 210 and 254 nm. LC analysis were performed with a Perkin-Elmer (series 200) instrument equipped with an UV detector (Perkin-Elmer 785A, UV/vis Detector). Chromatographic separation were conducted using a Polaris C18 column (150-4.6 mm, 5 μm particle size) at a flow rate of 0.8 mL min-1 with a mobile phase composed of 50% CH.sub.3CN/50% H.sub.2O-formic acid 0.1%.

[0190] Permeability (Papp) was calculated according to equation 1, obtained from Sugano [12] and Wohnsland [13] equation with some modification to obtain values in cm/s.

[00001]$\begin{array}{cc}{P}_{\mathrm{app}}=\frac{V_D.Math.V_A}{\left(V_D+V_A\right).Math.\mathrm{At}}-\ln \left(1-r\right)& \left(1\right)\end{array}$

[0191] Where V.sub.A is the volume in the acceptor well, VD is the volume in the donor well (cm.sup.3), A is the effective area of the membrane (cm.sup.2), t is the incubation time (s) and r the ratio between drug concentration in the acceptor and equilibrium concentration of the drug in the total volume (V.sub.D+V.sub.A). Drug concentration was estimated by using the peak area integration.

Results

[0192] The apparent permeability (P.sub.app) was measured by using the Parallel Artificial Membrane Permeability Assay (PAMPA), at pH 7.4. PAMPA assays on compound 28 revealed a low permeability value (1.6×10.sup.−6 cm/s) at physiological pH.

Metabolic Stability Assay

Methods

[0193] Compound 28 in DMSO solution was incubated at 37° C. for 60 min in 50 mM Tris-HCl buffer (pH 7.4), 5 pL of human liver microsomal proteins (0.2 mg mL.sup.−1), in the presence of a NADPH-generating system at a final volume of 0.5 mL (compounds' final concentration, 50 μM); DMSO did not exceed 2% (final solution). The reaction was stopped by cooling in ice and adding 1.0 mL of acetonitrile. The reaction mixtures were then centrifuged, and the parent drug and metabolites were subsequently determined by LC-UV-MS as reported for the solubility assay

Results

[0194] Metabolic stability of compound 28 was measured on human liver microsomial enzymes. Compound 28 showed a very good metabolic stability (99.9% in 1 h of incubation at 37° C.).

Binding Fluorimetric Assay

Methods

[0195] A quantitative analysis of the potential interaction was performed by fluorimetric titration: 0.2 mL. solution, containing a fixed concentration of HSA (10 μM in phosphate buffer 1 mM), was titrated with different amounts of 28, Carbamazepine and Paracetamol (2 μM to 2500 μM by stock solutions in DMSO). The solutions were mixed and after allowing 30 minutes at room temperature to reach the equilibrium conditions, the spectra were recorded. All fluorescence studies were done at room temperature, Tryptophan fluorescence emission spectra over 250-500 nm wavelength range were recorded with excitation wavelength set at 290 and the emission peaks of HAS were observed at 340 nm. 28 analyzed by the fluorimetric titration, showed decrease of intrinsic fluorescence of Tryptophan and the percentage of bound albumin at various concentrations, was calculated. The percentages obtained were plotted against the concentrations used and the KD values were calculated using GraphPad software (version 5.0).

Results

[0196] Quenching of the intrinsic Tryptophan of Human Serum Albumin (HAS) was monitored by fluorescence spectroscopy in order to determine the dissociation constant (KD) with compound 28 to HAS. Paracetamole and Carbamazepine were used for comparison. When a fixed concentration of HSA was titrated with different amounts of our compound, a scarce intrinsic fluorescence decrease was observed. [14] The experimental results suggest that 28 as similar behaviour to paracetamol. [15] On the contrary, carbamazepine shows a remarkable intrinsic fluorescence decrease. [16]

[0197] FIG. 1 shows binding curves for Carbamazepine, Paracetamol and 28. Calculated experimental Kd values are: [0198] for compound 28 >400 μM [0199] PARACETAMOL >400 μM [0200] CARBAMAZEPINE 102.4±19.7 μM.

Water Solubility

Methods

[0201] Solid compound 28 (1.01 mg) was added to 1 mL of water. The sample was shaken in a shaker bath at room temperature for 24 h. The suspension was filtered through a 0.45-μm nylon filter (Acrodisc), and the solubilised compound determined by LC-UV-MS assay. The determination was performed in triplicate. For the quantification was used an LC-UV-MS 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 (50×2.0 mm) (Varian) with 3 μm particle size and gradient elution: eluent A being CH3CN and eluent B consisting of an aqueous solution of formic acid (0.1%).

[0202] 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 0.4 ml/min and injection volume was 20 μL. 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. UV lamp was set to 210 nm. Calibration curve was obtained by analysing standard methanolic solution of compound 28 at serial dilutions. The calculation was based on the integral value of the UV peak at 14.6 min retention time.

Results

[0203] The water solubility of compound 28 was also determined by LC-UV-MS analysis, using the calibration curve method. Experimental solubility value found is 292 μg/mL.

REFERENCES

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