Antimicrobial compounds
09969758 ยท 2018-05-15
Assignee
Inventors
- Rosaleen Joy Anderson (Sunderland Tyne and Wear, GB)
- Alexandre F. Bedernjak (Morthomiers, FR)
- Marie Cellier (Montalieu-Vercieu, FR)
- Keng Tiong Ng (Kuala Lumpur, MY)
- Sylvain Orenga (Neuville-sur-Ain, FR)
- John D. Perry (Tyne and Wear, GB)
- Linda Varadi (Sajovamos, HU)
Cpc classification
C12Q1/18
CHEMISTRY; METALLURGY
C07K5/0827
CHEMISTRY; METALLURGY
C07K5/06191
CHEMISTRY; METALLURGY
C07F9/3886
CHEMISTRY; METALLURGY
C07K5/1027
CHEMISTRY; METALLURGY
A61P31/00
HUMAN NECESSITIES
C12Q1/04
CHEMISTRY; METALLURGY
International classification
C12Q1/04
CHEMISTRY; METALLURGY
C07F9/38
CHEMISTRY; METALLURGY
Abstract
An antimicrobial compound, as well as the salts, derivatives and analogs thereof, said compound being represented by the general formula (I): ##STR00001##
wherein R.sub.1 represents a peptide part P1 or a peptide part P2.
Claims
1. An antimicrobial compound of formula (I): ##STR00046## or a salt thereof, wherein R.sub.1 is: a peptide part P1 consisting of a linear sequence of one to five amino acid residues, wherein at least one of the amino acid residues is a -chloroalanine residue; or a peptide part P2 of formula (II): ##STR00047## wherein X is hydrogen or chlorine, and Y is hydrogen or a linear sequence of one to four amino acid residues; and wherein if X is hydrogen, Y is not hydrogen and an alanine residue is not in the N-terminal position of the linear amino acid sequence or bound to another amino acid residue via a peptide bond between the amino function of the alanine residue and the carboxylic acid function of another amino acid residue.
2. The antimicrobial compound of claim 1, wherein at least one of the amino acid residues of the linear sequence of one to four amino acid residues Y is selected from a -chloroalanine residue, a norvaline residue, and a methionine residue.
3. The antimicrobial compound of claim 1, wherein the -chloroalanine residue is a -chloro-L-alanine residue.
4. The antimicrobial compound of claim 2, wherein the -chloroalanine residue is a -chloro-L-alanine residue.
5. The antimicrobial compound of claim 2, wherein the norvaline residue is a L-norvaline residue.
6. The antimicrobial compound of claim 2, wherein the methionine residue is a L-methionine residue.
7. The antimicrobial compound of claim 1, wherein R.sub.1 is represented by the general formula (III): ##STR00048## wherein R.sub.2 is hydrogen or a linear sequence of one to four amino acid residues.
8. The antimicrobial compound of claim 7, wherein at least one of the amino acid residues is selected from a -chloroalanine residue, a norvaline residue, and a methionine residue.
9. The antimicrobial compound of claim 8, wherein the -chloroalanine residue is a -chloro-L-alanine residue.
10. The antimicrobial compound of claim 8, wherein the norvaline residue is a L-norvaline residue.
11. The antimicrobial compound of claim 8, wherein the methionine residue is a L-methionine residue.
12. The antimicrobial compound of claim 1, wherein R.sub.1 is represented by the general formula (IV): ##STR00049## wherein R.sub.3 is hydrogen or a linear sequence of one to three amino acid residues.
13. The antimicrobial compound of claim 12, wherein at least one of the amino acid residues is a -chloroalanine residue.
14. The antimicrobial compound of claim 13, wherein the -chloroalanine residue is a -chloro-L-alanine residue.
15. The antimicrobial compound of claim 1, wherein R.sub.1 is a linear chain of two or three amino acid residues.
16. The antimicrobial compound of claim 15, wherein R.sub.1 is a linear chain of two amino acid residues.
17. The antimicrobial compound of claim 1, wherein the amino acid residue(s) is/are selected from the group consisting of: glycine, sarcosine, -chloro-L-alanine, -chloro-D-alanine, D/L-alanine, D/L-arginine, D/L-asparagine, D/L-aspartic acid, D/L-cysteine, D/L-glutamic acid, D/L-gamma-glutamic acid, D/L-glutamine, D/L-histidine, D/L-isoleucine, D/L-leucine, D/L-lysine, D/L-methionine, D/L-norvaline, D/L-phenylalanine, D/L-proline, D/L-pyroglutamic acid, D/L-serine, D/L-threonine, D/L-tryptophan, D/L-tyrosine, and D/L-valine.
18. The antimicrobial compound of claim 17, wherein the amino acid residue(s) is/are selected from -chloro-L-alanine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-gamma-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-norvaline, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
19. The antimicrobial compound of claim 17, wherein the amino acid residue(s) is/are selected from -chloro-L-alanine, -chloro-D-alanine, D/L-alanine, D/L-methionine, D/L-norvaline, and D/L-valine.
20. The antimicrobial compound of claim 1, wherein R.sub.1 is the peptide part P2 and X is hydrogen.
21. The antimicrobial compound of claim 20, wherein Y is one amino acid residue selected from: glycine, sarcosine, -chloro-L-alanine, -chloro-D-alanine, D/L-arginine, D/L-asparagine, D/L-aspartic acid, D/L-cysteine, D/L-glutamic acid, D/L-gamma-glutamic acid, D/L-glutamine, D/L-histidine, D/L-isoleucine, D/L-leucine, D/L-lysine, D/L-methionine, D/L-norvaline, D/L-phenylalanine, D/L-proline, D/L-pyroglutamic acid, D/L-serine, D/L-threonine, D/L-tryptophan, D/L-tyrosine, and D/L-valine.
22. The antimicrobial compound of claim 21, wherein the amino acid residue is selected from a L-methionine residue and a L-norvaline residue.
23. The antimicrobial compound of claim 1, wherein the antimicrobial compound is selected from: -chloro-L-alanyl-D/L-1-aminoethylphosphonic acid; -chloro-L-alanyl--chloro-L-alanyl-D/L-1-aminoethylphosphonic acid; L-norvalinyl--chloro-L-alanyl-D/L-1-aminoethylphosphonic acid; L-methionyl--chloro-L-alanyl-D/L-1-aminoethylphosphonic acid; L-norvalinyl-L-alanyl-D/L-1-aminoethylphosphonic acid; and L-methionyl-L-alanyl-D/L-1-aminoethylphosphonic acid.
24. The antimicrobial compound of claim 1, wherein the antimicrobial compound is selected from: -chloro-L-alanyl-L-1-aminoethylphosphonic acid; -chloro-L-alanyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid; L-norvalinyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid; L-methionyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid; L-norvalinyl-L-alanyl-L-1-aminoethylphosphonic acid; and L-methionyl-L-alanyl-L-1-aminoethylphosphonic acid.
25. The antimicrobial compound of claim 1, wherein the antimicrobial compound is selected from: -chloro-L-alanyl-L-1-aminoethylphosphonic acid; and -chloro-L-alanyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid.
26. The antimicrobial compound of claim 1, wherein the amino function of the N-terminal amino acid residue of P1 or P2 is protected by a protecting group selected from: a tertiobutylocarbonyl group, a 9-fluorenylmethoxycarbonyl group and a benzyloxycarbonyl group.
27. A reaction medium comprising at least one antimicrobial compound of claim 1.
28. The reaction medium of claim 27, wherein the final concentration of the antimicrobial compound is between 0.002 mg/L and 1024.0 mg/L.
29. The reaction medium of claim 28, wherein the final concentration of the antimicrobial compound is between 0.003 mg/L and 32.0 mg/L.
30. The reaction medium of claim 29, wherein the final concentration of the antimicrobial compound is between 0.2 mg/L and 8.0 mg/L.
31. The reaction medium of claim 30, wherein the final concentration of the antimicrobial compound is between 0.2 and 2.0 mg/L.
32. The reaction medium of claim 27, wherein the reaction medium further comprises: at least one target microorganism.
33. The reaction medium of claim 32, wherein the at least one target microorganism is a microorganism from a sample of industrial or clinical origin.
34. The reaction medium of claim 33, wherein the antimicrobial compound is a selective agent, and wherein the selective agent is capable of inhibiting the survival and/or growth of non-target microorganism(s).
35. The reaction medium of claim 34, further comprising at least one growth nutrient, wherein the nutrient is a nutrient that enables the growth of the at least one target microorganism.
36. The reaction medium of claim 34, wherein the reaction medium further comprises an enzyme substrate, wherein the enzyme substrate is specific to an enzyme activity of the at least one target microorganism.
37. The reaction medium of claim 34, wherein the selective agent is selected from: -chloro-L-alanyl-L-1-aminoethylphosphonic acid; and -chloro-L-alanyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid; or a mixture thereof.
38. The reaction medium of claim 34, wherein the target microorganism is selected from: at least one Gram-negative target microorganism belonging to the genus Salmonella, the genus Acinetobacter, or the genus Pseudomonas; and at least one Gram-positive target microorganism, belonging to the genus Listeria or the genus Streptococcus.
39. The reaction medium of claim 35, wherein the reaction medium further comprises: a buffer; at least one chromogenic marker at a concentration of between 0.05 and 15.0 g/L; and agar at a concentration of between 9.0 and 28.0 g/L, wherein the at least one target microorganism belongs to the genus Salmonella, wherein the at least one nutrient agent is a peptone at a concentration of between 0.2 and 30.0 g/L, and wherein the selective agent is at a concentration of between 0.002 and 1024.0 mg/L.
40. The reaction medium of claim 34, wherein the non-target microorganism(s) belong to a genus selected from: Enterobacter; Escherichia; Klebsiella; Serratia; Yersinia; Enterococcus; and Staphylococcus.
41. The reaction medium of claim 40, wherein the non-target microorganism(s) is/are resistant to at least one conventional antibacterial.
42. A method for detecting, identifying or counting at least one target microorganism from a sample of industrial or clinical origin, the method comprising: a) seeding the reaction medium of claim 33; b) incubating the reaction medium for a period of time sufficient to detect, identify or count the at least one target microorganism; and c) detecting, identifying or counting colonies formed by the at least one target microorganism.
43. A medication for human or veterinary use comprising the antimicrobial compound of claim 1.
44. A method of treating microbial infections comprising administering the medication of claim 43 to a human or animal in need thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS AND FIGURES
(1)
(2)
(3)
(4)
(5)
(6)
DETAILED DESCRIPTION OF THE INVENTION
(7) The examples below will enable the present invention to be better understood. Nevertheless, these examples are given merely by way of illustration and must by no means be regarded as limiting the scope of said invention in any way.
Example 1: Synthesis Method of Antimicrobial Compounds C and E According to the Invention (FIG. 1)
1.1 General Points
(8) The synthesis of the prior art compounds represented by formulae A, B, D and F in
(9) The NMR spectra were obtained on a Bruker Ultrashield 300 spectrometer (at 300 MHz for the .sup.1H spectra and at 75 MHz for the .sup.13C spectra). The chemical shifts are indicated in ppm downfield from tetramethylsilane, using the residual chloroform (=7.26 in .sup.1H NMR) or the middle peak of the CDCl.sub.3 carbon triplet (=77.23 in .sup.13C NMR) as an internal standard. The melting points were obtained by means of a Reichart-Kofler heating plate microscope and are non-corrected. The infrared spectra were recorded using a PerkinElmer Spectrum BX FT-IR instrument. The low-resolution mass spectra were recorded on a Bruker Esquire 3000plus analyser using a positive ion mode electrovaporisation source. The high-resolution mass spectra were obtained on an LTQ Orbitrap XL instrument in nanospray ionisation mode. The elemental analyses were performed using an Exeter Analytical CE-440 Elemental Analyzer. All the commercially available reagents and solvents were obtained from Sigma-Aldrich, Alfa-Aesar, Fisher Scientific and Fluka, and were used without any additional purification. Thin-layer chromatography was performed on Merck silica gel plates (60E-254).
1.2 Preparation of tBoc-L-serine benzyl ester 12
(10) ##STR00008##
(11) Some .sup.tBoc-serine 11 (16.60 g, 81.0 mmol) was dissolved in benzene (250 mL) and some DBU (18.90 g, 21.6 mL, 124.0 mmol) was added. Some benzyl bromide (21.25 g, 15 mL, 124.0 mmol) was then added dropwise to the agitated reaction mixture. After agitation at ambient temperature overnight, the reaction was neutralised with a 1M solution of HCl (150 mL). The benzene was eliminated at low pressure, and the residue was taken up in ethyl acetate. The solution was washed with brine, the organic layer was dried on Na.sub.2SO.sub.4, and then the solvent was eliminated under vacuum to give the raw product. After column chromatography (50% petroleum ether, 50% ethyl acetate), tBoc-serine benzyl ester 12 was obtained in the form of a white solid (16.90 g, 57.0 mmol, 71%); melting point 61-63 C. (melting point in the literature 69-70 C. [16]); [Found: C, 60.63; H, 7.16; N, 4.64. C.sub.15H.sub.21NO.sub.5 requires C, 61.00; H, 7.17; N, 4.74%]; .sub.max/cm.sup.1 3417, 3356 (NH and OH), 1756 (CO, ester), 1667 (CO, carbamate), 1523 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.44 (9H, s, C(CH.sub.3).sub.3), 2.24 (1H, br, OH), 3.91 (1H, br d, J=11.1 Hz, CH.sub.a-3), 3.98 (1H, br d, J=11.1 Hz, CH.sub.b-3), 4.41 (1H, br, CH-2), 5.19 (1H, d, J=12.3 Hz, COOCH.sub.a), 5.24 (1H, d, J=12.3 Hz, COOCH.sub.b), 5.44 (1H, br, NH), 7.35-7.37 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 27.9 (CH.sub.3, C(CH.sub.3).sub.3), 55.6 (CH, C-2), 63.3 (CH.sub.2), 67.1 (CH.sub.2), 80.1 (quat., C(CH.sub.3).sub.3), 127.8 (2CH.sub.Ar), 128.1 (CH.sub.Ar), 128.3 (2CH.sub.Ar), 134.9 (quat., C.sub.Ar), 153.0 (quat., CO), 170.3 (quat., CO); MS (ESI) m/z 318.3 (MNa.sup.+).
1.3 Preparation of tBoc--chloro-L-alanine benzyl ester 13
(12) ##STR00009##
(13) Some trichloro-acetonitrile (15.16 g, 10.5 mL, 105.0 mmol) was added to a solution of .sup.tBoc-serine benzyl ester 12 (15.58 g, 52.8 mmol) in dichloromethane (200 mL), under nitrogen, followed by 10 minutes of agitation at ambient temperature. Some triphenylphosphine (27.54 g, 105.0 mmol) was dissolved in dichloromethane (150 mL) under nitrogen, and this solution was added dropwise to the agitated reaction mixture. After agitation at ambient temperature overnight, the reaction was neutralised with brine (250 mL); after separation, the organic layer was extracted with brine (3100 mL). The organic layer was dried on anhydrous sodium sulfate, and the solvent was eliminated under low pressure to give the raw product. Purification by column chromatography (70% petroleum ether, 30% ethyl acetate) gave the product 13 in the form of a white solid (14.94 g, 47.6 mmol, 90%); melting point 54-57 C.; [Found: C, 57.53; H, 6.49; N, 4.38. C.sub.15H.sub.20ClNO.sub.4 requires C, 57.42; H, 6.42; N, 4.46%]; .sub.max/cm.sup.1 3364 (NH), 1725 (CO, ester), 1680 (CO, carbamate), 1518 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.45 (9H, s, C(CH.sub.3).sub.3), 3.84 (1H, dd, J=3.3 and 11.4 Hz, CH.sub.a-3), 4.00 (1H, dd, J=3.3 and 11.4 Hz, CH.sub.b-3), 4.74 (1H, m, CH-2), 5.22 (1H, d, J=12.3 Hz, COOCH.sub.a), 5.27 (1H, d, J=12.3 Hz, COOCH.sub.b), 5.43 (1H, d, J=7.2 Hz, NH), 7.36 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 28.3 (CH.sub.3, C(CH.sub.3).sub.3), 45.5 (CH.sub.2, C-3), 54.6 (CH, C-2), 67.8 (COOCH.sub.2), 80.5 (quat., C(CH.sub.3).sub.3), 128.3 (CH), 128.6 (2CH), 128.6 (2CH), 134.9 (quat., C.sub.Ar), 155.0 (quat., CO), 169.0 (quat., CO); HRMS (nanospray ionisation) calculated for (C.sub.15H.sub.21NO.sub.4Cl).sup.+ 314.1154, found 314.1159.
1.4 Preparation of -chloro-L-alanine HBr benzyl ester 14
(14) ##STR00010##
(15) Some .sup.tBoc--chloro-L-alanine benzyl ester 13 (1.40 g, 4.46 mmol) was dissolved in a minimal quantity of acetic acid (5 mL), and then HBr in AcOH (33% m/m) was added (5.53 mL, 30.7 mmol of HBr) and the reaction mixture was agitated for 10 minutes at ambient temperature. The reaction was neutralised with diethyl ether (200 mL) and the solution was kept in the freezer overnight. At rest, a white solid precipitated out, which was collected by filtration, and washed with cold diethyl ether to give the product 14 (1.10 g, 3.6 mmol, 81%); melting point 131-134 C.; [Found: C, 40.56; H, 4.51; N, 4.68. C.sub.10H.sub.13BrClNO.sub.2 requires C, 40.77; H, 4.45; N, 4.75%]; .sub.max/cm.sup.1 2950, 2875, 2846 (br NH.sub.3.sup.+), 1750 (CO, ester), 1489, 1228, 1208 (CO); .sup.1H NMR (300 MHz, D.sub.2O) .sub.14 4.17 (1H, dd, J=3.3 and 12.6 Hz, CH.sub.a-3), 4.31 (1H, dd, J=3.3 and 12.6 Hz, CH.sub.b-3), 4.81 (1H, m, CH-2), 5.31 (1H, d, J=12.3 Hz, COOCH.sub.a), 5.39 (1H, d, J=12.3 Hz, COOCH.sub.b), 7.55 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 41.8 (CH.sub.2, C-3), 53.9 (CH, C-2), 69.2 (COOCH.sub.2), 128.7 (2CH), 128.9 (2CH), 129.1 (CH), 134.5 (quat., C.sub.Ar), 167.0 (quat., CO); HRMS (nanospray ionisation) calculated for (C.sub.10H.sub.13.sup.35ClNO.sub.2).sup.+ 214.0629, found 214.0630.
1.5 Preparation of the compound tBoc--chloro-L-alanine 15
(16) ##STR00011##
(17) Some tBoc--chloro-L-alanine benzyl ester 13 (1.57 g, 5.0 mmol) was dissolved in methanol (50 mL) and 10% palladium on charcoal (0.16 g) in ethyl acetate (20 mL) was added. The reaction was agitated at a pressure of 1.5 bar H.sub.2 overnight. The catalyst was eliminated by filtration through a Celite plug and washed out with methanol (200 mL). After elimination of the methanol at low pressure, the raw product was purified by column chromatography (95% dichloromethane, 5% methanol) to give the product 15 in the form of a white solid (0.99 g, 4.4 mmol, 88%); melting point 119-123 C. (melting point in the literature 123-125 C. (13)); .sub.max/cm.sup.1 3434 (NH), 2975 (OH), 1752 (CO), 1734 (CO), 1677, 1521 (amide II), 1370, 1212; .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H1.47 (9H, s, C(CH.sub.3).sub.3), 3.90 (1H, dd, J=2.7 and 11.1 Hz, CH.sub.a-3), 4.05 (1H, d, J=11.1 Hz, CH.sub.b-3), 4.78 (1H, m, CH-2), 5.47 (1H, d, J=6.3 Hz, NH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 28.3 (CH.sub.3, C(CH.sub.3).sub.3), 45.2 (CH.sub.2, C-3), 54.3 (CH, C-2), 80.9 (quat., C(CH.sub.3).sub.3), 155.3 (quat., CO, C-4), 173.2 (quat., CO, C-1); HRMS (nanospray ionisation) calculated for (C.sub.8H.sub.13NO.sub.4.sup.35Cl).sup. 222.0539, found 222.0541.
1.6 Preparation of tBoc--chloro-L-alanine pentafluorophenol ester 16
(18) ##STR00012##
(19) Some tBoc--chloro-L-alanine 15 (1.16 g, 5.2 mmol) and pentafluorophenol (0.95 g, 5.7 mmol) was dissolved in ethyl acetate (25 mL) and cooled in an ice bath. Some dicyclohexylcarbodiimide (1.05 g, 5.7 mmol) was added and the solution was agitated for 3 hours. The precipitated urea by-product was eliminated by filtration. The residue was concentrated at low pressure, and any additional precipitate was eliminated by filtration. The remaining ethyl acetate was eliminated by evaporation. The oil thus obtained was triturated with petroleum ether to give the product 16 in the form of a white solid (3.35 g, 8.6 mmol, 69%) which was collected by filtration; melting point 128-131 C.; [Found: C, 43.53; H. 3.49; N, 3.63. C.sub.14H.sub.13.sup.35ClF.sub.5NO.sub.4 requires C, 43.15; H, 3.36; N, 3.59%]; .sub.max/cm.sup.1 3367 (NH), 1780 (CO), 1681 (CO), 1518 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.48 (9H, s, C(CH.sub.3).sub.3), 3.94 (1H, dd, J=3.6 and 11.4 Hz, CH.sub.a-3), 3.96 (1H, dd, J=3.6 and 11.4 Hz, CH.sub.b-3), 5.10 (1H, br t, J=3.6 Hz, CH-2), 5.46 (1H, d, J=7.5 Hz, NH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 28.25 (CH.sub.3, C(CH.sub.3).sub.3), 44.78 (CH.sub.2, C-3), 54.55 (CH, C-2), 81.26 (quat., C(CH.sub.3).sub.3), 118.94 (m, CF.sub.Ar), 154.74 (quat., C-1), 154.79 (quat., CO, C-4), 166.77 (quat., CO, C-1); .sup.19F NMR (282 MHz, CDCl.sub.3) .sub.F 161.67 (2F, t, J=19.8 Hz, CF-3 and 5), 156.73 (1F, t, J=23.1 Hz, CF-4), 151.63 (2F, d, J=18.9 Hz, CF-2 and 6); MS (ESI) m/z 388.8 (M-H).sup..
1.7 Preparation of tBoc--chloro-L-alanyl-L-fosfalin diethyl ester 17
(20) ##STR00013##
(21) Some L-fosfalin diethyl ester (1.10 g, 6.1 mmol) was dissolved in dry DCM (50 mL) and cooled in an ice bath. .sup.tBoc--chloro-alanylpentafluorophenol ester 16 (2.37 g, 6.1 mmol) was added in portions and the reaction mixture was agitated at ambient temperature until completion. The reaction was neutralised with water (150 mL), and then extraction was performed with dichloromethane (3100 mL). The organic layer was dried on anhydrous MgSO.sub.4 and the solvent was eliminated under vacuum. The residue was purified by column chromatography (50% petroleum ether, 50% ethyl acetate up to 90% ethyl acetate, 10% methanol) to give the product 17 in the form of a white solid (1.45 g, 3.8 mmol, 62%); melting point 80-83 C.; [Found: C, 43.46; H, 7.27; N, 7.21. C.sub.14H.sub.28ClN.sub.2O.sub.6P requires C, 43.47; H, 7.30; N, 7.24%]; .sub.max/cm.sup.1 3303, 3215, 3065, 2978 (NH), 1716 (CO), 1667 (CO), 1555, 1518 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.25-1.42 (9H, m, 3CH.sub.3), 1.46 (9H, s, C(CH.sub.3).sub.3), 3.73 (1H, dd, J=4.2 and 11.1 Hz, CH.sub.a-3), 4.00 (1H, dd, J=4.2 and 11.1 Hz, CH.sub.b-3), 4.07-4.18 (4H, m, 2OCH.sub.2), 4.39-4.54 (2H, m, CH-2 and 2), 5.35 (1H, d, J=8.4 Hz, NH), 6.77 (1H, d, J=8.7 Hz, NH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 15.8 (CH.sub.3, C-3), 16.3-16.5 (2CH.sub.3, m, CH.sub.3CH.sub.2O), 28.2 (3CH.sub.3, C(CH.sub.3).sub.3), 40.3 (CH), 42.4 (CH), 44.9 (CH.sub.2, C-3), 62.6 (CH.sub.2, d, J=6.75 Hz, OCH.sub.2CH.sub.3), 62.9 (CH.sub.2, d, J=6.6 Hz, OCH.sub.2CH.sub.3), 80.8 (quat., C(CH.sub.3).sub.3), 155.0 (quat., CO), 168.3 (quat., CO); .sup.31P NMR (121.5 MHz, CDCl.sub.3) .sub.P 24.6 (m); MS (ESI) m/z 387.3 (MH.sup.+), 409.3 (MNa.sup.+), 385.1 (M.sup.).
1.8 Obtaining the compound -chloro-L-alanyl-L-fosfalin 18 (compound C in FIG. 1)
(22) ##STR00014##
(23) Some tBoc--chloro-L-alanyl-L-fosfalin diethyl ester 17 (1.33 g, 3.4 mmol) was agitated in 33% m/m HBr in acetic acid (17 mL) for 24 h. The reaction mixture was poured into diethyl ether (200 mL) and placed in the freezer (15 C.) overnight. The diethyl ether was decanted off and the residual precipitate was taken up in a minimal quantity of methanol (approximately 10 mL). Then propylene oxide was added in large excess (approximately 250 mL). The hygroscopic precipitate was filtered and recrystallised from water and acetone to give the product 18 in the form of a white solid (0.99 g, 3.2 mmol, 93%); melting point 210-212 C.; [Found: C, 25.80; H, 5.21; N, 12.04. C.sub.5H.sub.12ClN.sub.2O.sub.4P requires C, 26.04; H, 5.25; N, 12.15%] .sub.max/cm.sup.1 3258 (br NH), 3100, 2930 (br) (OH), 1654 (CO), 1565, 1514 (amide II), 1038; .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.30 (3H, dd, J=7.2 and 15 Hz, CH.sub.3-3), 3.97-4.11 (3H, m, CH.sub.2-3 and CH-2), 4.39 (1H, m, CH-2); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 15.2 (CH.sub.3, C-3), 42.6 (CH.sub.2, C-3), 44.2 (CH, d, J=147.9 Hz, C-2), 54.2 (CH, C-2), 165.6 (quat., CO, C-1); .sup.31P NMR (121.5 MHz, CDCl.sub.3) .sub.P 18.6 (m); HRMS (nanospray ionisation) calculated for (C.sub.5H.sub.11N.sub.2O.sub.4P.sup.35Cl).sup. 229.0150, found 229.0154.
1.9 Preparation of tBoc--chloro-L-alanyl--chloro-L-alanine benzyl ester 19
(24) ##STR00015##
(25) Some -chloro-L-alanine benzyl ester hydrobromide 14 (1.13 g, 3.9 mmol) was dissolved in DMF (30 mL) and added to a solution of tBoc--chloro-L-alanine pentafluorophenol ester 16 (1.50 g, 3.9 mmol) in dichloromethane (10 mL) at 0 C. Some diisopropylethylamine (0.50 g, 0.66 mL, 3.9 mmol) was added dropwise to this solution. The mixture thus obtained was agitated at ambient temperature for 2 hours, and then heated to 35 C., and the progress of the reaction was monitored by TLC (80% petroleum ether, 20% ethyl acetate). Once the reaction was finished, it was neutralised with a 1M solution of HCl (50 mL) and the organic layer was washed with water (100 mL) and brine (100 mL). The organic layer was dried on anhydrous sodium sulfate and the solvent was evaporated under vacuum. The raw product was purified by gradient column chromatography (from 80% petroleum ether, 20% ethyl acetate; to 50% petroleum ether, 50% ethyl acetate) to give the product 19 in the form of a white solid (1.16 g, 2.8 mmol, 70%); melting point 89-91 C.; [Found: C, 51.83; H, 5.78; N, 6.78. C.sub.18H.sub.24.sup.35Cl.sub.2N.sub.2O.sub.5 requires C, 51.56; H, 5.77; N, 6.68%]; .sub.max/cm.sup.1 3336 (NH), 3321 (NH), 1739 (CO), 1655 (m, CO), 1508 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.48 (9H, s, C(CH.sub.3).sub.3), 3.73 (1H, dd, J=4.8 and 11.1 Hz, CH), 3.89-4.06 (3H, m, 3CH), 4.55 (1H, br, CH), 4.98 (1H, m, CH), 5.24 (2H, m, COOCH.sub.2), 5.35 (1H, br, NH), 7.25 (1H, br, NH), 7.37 (5H, br s, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 28.24 (CH.sub.3, C(CH.sub.3).sub.3), 33.90 (CH.sub.2Ph), 44.5 (CH), 44.60 (CH.sub.2), 53.61 (CH), 68.13 (CH.sub.2), 81.37 (quat., C(CH.sub.3).sub.3), 128.22 (CH.sub.Ar), 128.41 (CH.sub.Ar), 128.69 (CH.sub.Ar), 134.71 (quat.), 168.19 (2quat., CO), 168.90 (quat., CO); HRMS (nanospray ionisation) calculated for (C.sub.18H.sub.25N.sub.2O.sub.5.sup.35Cl).sup.+ 419.1135, found 419.1139.
1.10 Preparation of the compound tBoc--chloro-L-alanyl--chloro-L-alanine
(26) ##STR00016##
(27) Some .sup.tBoc--chloro-L-alanine--chloro-L-alanine benzyl ester 19 (1.00 g, 2.4 mmol) was dissolved in methanol (50 mL) and 10% palladium on charcoal (0.10 g) in ethyl acetate (20 mL) was added. The reaction mixture was agitated in an H.sub.2 atmosphere (1.8 bar) for 24 h, and then filtered through a Celite plug and washed with methanol (200 mL). The solvents were eliminated under vacuum and, after purification by column chromatography (95% DCM, 5% methanol), upon trituration with petroleum ether, the product 20 was obtained in the form of a white solid (0.52 g, 1.6 mmol, 66%); melting point 72-74 C.; .sub.max/cm.sup.1 3320 (NH), 2978 (NH), 1724 (CO), 1665 (CO), 1530 (amide II); .sup.1H NMR (300 MHz, DMSO-d.sub.6) .sub.H 1.39 (9H, s, C(CH.sub.3).sub.3), 3.67 (1H, dd, J=8.4 and 11.1 Hz, CH), 3.79-3.95 (3H, m, CH.sub.2 and CH), 4.36 (1H, br, CH), 4.61-4.67 (1H, m, CH), 7.16 (1H, d, J=8.1 Hz, NH), 8.38 (1H, d, J=7.5 Hz, NH); HRMS (nanospray ionisation) calculated for (C.sub.11H.sub.17.sup.35Cl.sub.2N.sub.2O5).sup. 327.0520, found 327.0517;
1.11 Preparation of tBoc--chloro-L-alanyl--chloro-L-alanine pentaflurophenol ester 22
(28) ##STR00017##
(29) Some .sup.tBoc--chloro-L-alanyl--chloro-L-alanine 20 (0.48 g, 1.46 mmol) was dissolved in ethyl acetate (50 mL) and cooled in an ice bath, which was followed by the addition of pentafluorophenol (0.29 g, 1.56 mmol) and dicyclohexylcarbodiimide (0.33 g, 1.61 mmol). The solution was agitated at 0 C. for 2 hours and the precipitated urea was eliminated by filtration. The residue was concentrated under vacuum and any new precipitate was also eliminated by filtration. The ethyl acetate was eliminated under vacuum and the oil thus obtained was triturated with petroleum ether to give the product 22 in the form of a white solid (0.65 g, 1.3 mmol, 89%), which was collected by filtration and taken on to the next step without any other purification; .sub.max/cm.sup.1 3334 (NH), 3006, 2970, 2939 (NH), 1787 (CO), 1688 (CO), 1664 (CO), 1514 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.46 (9H, s, C(CH.sub.3).sub.3), 3.76 (1H, dd, J=4.7 and 11.3 Hz, CH.sub.a), 3.98 (1H, dd, J=3.5 and 11.7 Hz, CH.sub.c), 4.06 (1H, dd, J=4.4 and 11.3 Hz, CH.sub.b), 4.16 (1H, dd, J=3.2 and 11.7 Hz, CH.sub.d), 4.53 (1H, br, CH), 5.32-5.37 (2H, m, CH and NH), 7.31 (1H, d, J=7.1 Hz, NH);
1.12 Preparation of tBoc--chloro-L-alanyl--chloro-L-alanyl-L-fosfalin diethyl ester 23
(30) ##STR00018##
(31) Some .sup.tBOC--chloro-L-alanyl--chloro-L-alanine pentafluorophenol ester 22 (0.95 g, 1.9 mmol) and diethyl 1-aminoethylphosphonate 3 (0.32 g, 1.9 mmol) was dissolved in dichloromethane (25 mL) at 0 C., and then agitated at ambient temperature until completion of the reaction, followed by a TLC. After extraction with water (100 mL), the organic layer was dried on anhydrous MgSO.sub.4 and the volatile components were eliminated at low pressure. The raw solid was purified by column chromatography (97% DCM, 3% MeOH) to give the product 23 in the form of a white solid (0.62 g, 1.3 mmol, 66%); melting point 154.6-155.9 C.; .sub.max/cm.sup.1 3291, 3265, 2962, 2848 (NH), 1680 (CO), 1639 (CO), 1523 (amide II); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.35-1.41 (9H, m, 3CH.sub.3), 1.48 (9H, s, C(CH.sub.3).sub.3), 3.73-3.80 (2H, m, CH.sub.a-3 and CH.sub.a-3), 4.01-4.2 (6H, m, CH.sub.b-3, CH.sub.b-3 and 2OCH.sub.2), 4.43-4.54 (2H, m, CH-2 and CH-2), 4.79-4.84 (1H, m, CH-2), 5.38 (1H, d, J=6.9 Hz, NH), 7.05 (1H, d, J=9.0 Hz, NH), 7.19 (1H, d, J=7.8 Hz, NH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 15.7 (CH.sub.3, C-3), 16.6 (CH.sub.3, OCH.sub.2CH.sub.3), 16.7 (CH.sub.3, OCH.sub.2CH.sub.3), 28.4 (3CH.sub.3, C(CH.sub.3).sub.3), 40.7 (CH), 44.5 (CH.sub.2), 44.6 (CH.sub.2), 49.4 (CH), 54.1 (CH), 62.8 (CH.sub.2, d, J=15.5 Hz, OCH.sub.2CH.sub.3), 63.1 (CH.sub.2, d, J=15.7 Hz, OCH.sub.2CH.sub.3), 81.6 (quat., C(CH.sub.3).sub.3), 156.9 (quat., CO), 168.9 (quat., CO), 177.3 (quat., CO); MS m/z 514.3, 515.2, 516.2 (MNa.sup.+); HRMS (nanospray ionisation) calculated for (C.sub.17H.sub.33N.sub.2O.sub.7P.sup.35Cl.sub.2).sup.+ 492.1428, found 492.1422.
1.13 Obtaining -chloro-L-alanyl--chloro-L-alanyl-L-fosfalin 24 (compound E in FIG. 1)
(32) ##STR00019##
(33) Some .sup.tBOC--chloro-L-alanyl--chloro-L-alanyl-L-fosfalin diethyl ether 23 (0.36 g, 0.7 mmol) was dissolved in acetic acid (5 mL) and HBr in AcOH (33% m/m (4 mL) was added. The solution was agitated at ambient temperature overnight, and then the reaction was neutralised with diethyl ether (200 mL). After resting in the freezer (15 C.) for 4 h, the brown oil thus obtained was separated by decanting off the diethyl ether. The raw product was then washed with cold diethyl ether (550 mL). The residue was taken up in methanol (3 mL) and, upon addition of propylene oxide (150 mL), a white precipitate formed. The liquid later was decanted off and the residue was triturated with cold diethyl ether (530 mL) to give the product 24 in the form of a white solid (0.18 g, 0.4 mmol, 59%); melting point 157-159 C.; .sub.max/cm.sup.1 3285, 3258 (br NH), 1646 (br) (CO), 1539 (large amide II), 1152, 1044; .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.37 (3H, dd, J=7.2 and 15.3 Hz, CH.sub.3-3), 3.98-4.2 (5H, m, CH.sub.2-3 and CH.sub.2-3 and CH), 4.62 (1H, t, J=4.8 Hz, CH), 4.87-4.90 (1H, m, CH); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 15.4 (CH.sub.3, C-3), 42.4 (CH.sub.2), 43.4 (CH.sub.2), 45.1 (CH, C-2), 53.8 (CH), 55.1 (CH), 166.8 (quat., CO), 168.9 (quat., CO); HRMS (nanospray ionisation) calculated for (C.sub.8H.sub.15N.sub.3O.sub.5P.sup.35Cl.sub.2).sup. 336.0102, found 336.0096.
Example 2: Evaluation of Antibacterial Activity of Antimicrobial Ccompounds C and E Synthesised According to Example 1
2.1 Introduction
(34) This example 2 presents the results of a study aimed at: evaluating the antibacterial activity of the compounds according to the invention, as synthesised in Example 1, namely compounds C and E as represented in
(35) The antibacterial activity of five compounds A-E was evaluated with a large collection of 297 bacteria which included a predominance of multi-resistant strains including carbapenemase-producing enterobacteria (n=128), methicillin-resistant Staphylococci aureus (n=37) and glycopeptide-resistant enterococci (n=43). Fosfomycin, a naturally present antibiotic also containing a phosphonic acid group (F) was included for comparison purposes.
(36) As a reminder, compounds A-F are as follows:
(37) A: L-alanyl-L-1-aminoethylphosphonic acid (alafosfalin);
(38) B: L-alanyl-L-alanyl-L-1-aminoethylphosphonic acid (di-alanyl fosfalin);
(39) C: -chloro-L-alanyl-L-1-aminoethylphosphonic acid (-Cl-alafosfalin);
(40) D: -chloro-L-alanyl--chloro-L-alanine (-Cl-Ala--Cl-Ala);
(41) E: -chloro-L-alanyl--chloro-L-alanyl-L-1-aminoethylphosphonic acid (-Cl-Ala--Cl-alafosfalin);
(42) F: disodium [(2R,3S)-3-methyloxiran-2-yl] phosphonate (fosfomycin).
2.2 Materials and Methods
(43) Antibacterial agents and media. Fosfomycin, alafosfalin, glucose-6-phosphate and all the ingredients of the antagonist-free agar medium were purchased from Sigma Chemical Company, Poole, United Kingdom. The agar medium IsoSensitest was purchased from Oxoid, Basingstoke, United Kingdom.
(44) Bacterial isolates. The enterobacteria (n=197) were obtained from various international sources, and all possessed -lactamases which were defined on a molecular level by benchmark laboratories and/or experts renowned in the field. These included Citrobacter freundii (n=5), other species of Citrobacter (n=4), Enterobacter aerogenes (n=1), Enterobacter cloacae (n=27), Escherichia coli (n=53), Klebsiella oxytoca (n=5), Klebsiella pneumoniae (n=87), Kluyvera spp (n=1), Proteus mirabilis (n=8), Providencia rettgeri (n=2), Salmonella spp (n=3), and Serratia marcescens (n=1). Among these 197 isolates, there were 128 (65%) carbapenemase producers, including; 87 with NDM-1, 9 with IMP, 11 with KPC, 14 with OXA-48 and 7 with VIM. The majority of the carbapenemase producers jointly produced broad-spectrum -lactamases (BSBL) or cephalosporinases (AmpC -lactamases), but the latter are not documented for the sake of clarity. Among the remaining isolates, 47 possessed BSBLs (20 with CTX-M, 19 with an SHV type, and 8 with a TEM type) and 22 possessed AmpC (3 with ACC-1, 6 with a CMY type, 6 with DHA-1, 3 with an FOX type and 4 with an LAT type).
(45) A collection of 50 isolates of Staphylococci aureus included 36 strains of methicillin-resistant S. aureus (MRSA) frequently encountered in Europe, including strains isolated in Belgium, Finland, France, Germany and the United Kingdom. Another strain of MRSA, NCTC 11939, was included as a control, as well as a methicillin-sensitive control (NCTC 6571). Twelve other isolates of methicillin-sensitive Staphylococcus aureus (MSSA) recently collected from blood cultures were also included. Finally, 50 isolates of enterococci included two control strains (Enterococcus faecalis NCTC 755 and Enterococcus faecium NCTC 7171) and 48 isolates originating from clinical samples obtained from at least three different hospitals. The clinical isolates included E. faecalis (n=10), E. faecium (n=33), Enterococcus casseliflavus (n=3), Enterococcus gallinarum (n=2). Among the 50 isolates, 43 were vancomycin-resistant, as demonstrated by the MIC value and the confirmation of the resistance genes by PCR.
(46) Determining the minimum inhibitory concentrations (MIC). All the MICs were determined using an agar dilution method [16]. This required use of a medium without a defined antagonist (peptone-free), prepared as described previously with the inclusion of 2% horse blood lysed with saponin, 25.0 g/mL of NAD and 25.0 g/mL of hemin [17]. The compounds tested were dissolved in sterile deionised water and incorporated into the agar medium in a concentration range of 0.0031 to 8.0 g/mL (0.016 to 32 g/mL for gram-positive bacteria). All the isolates were prepared at a density equivalent to 0.5 McFarland units in sterile deionised water using a densitometer (approximately 1.510.sup.8 CFU/mL), and then diluted to 1 for 15. An aliquot of 1 L of each diluted suspension was then placed onto plates with a multispot inoculator to give the final recommended inoculation of 10 000 CFU/spot [16]. The MICs of fosfomycin were determined by the same method, except that the IsoSensitest medium (Oxoid, Basingstoke, United Kingdom) was used, plus 25.0 g/mL of glucose-6-phosphate (Sigma, Poole, United Kingdom) and an extended range of fosfomycin concentrations. All the dishes (including the antimicrobial-free controls) were incubated for 22 hours at 37 C. All the tests were performed on at least two independent replicates in order to examine the reproducibility.
2.3 Results
(47) The minimum inhibitory concentrations (MIC) 50 and 90 of the five compounds A-E were calculated and are presented in Table 1 below. These values correspond respectively to the smallest antibiotic concentration sufficient to inhibit in vitro 50 and 90% of the growth of a bacteria strain, respectively MIC.sub.50 and MIC.sub.90.
(48) More specifically, Table 1 below indicates the MICs of the six antimicrobials against the main bacteria groups tested. Alafosfalin presented good activity against most of the enterobacteria isolates, although different species exhibited different degrees of sensitivity. A high activity was observed against 53 isolates of E. coli, of which 35 isolates (66%) were carbapenemase producers. The MIC.sub.90 for E. coli was 0.25 g/mL and growth of all the isolates was inhibited by 2 g/mL. It was found that the activity of alafosfalin was approximately four times higher than that of fosfomycin.
(49) Although the antibacterial activity of alafosfalin against E. coli is satisfactory, as indicated previously, the activity of -Cl-alafosfalin against E. coli proved to be at least twice that of alafosfalin and at least eight times higher than that of fosfomycin. The MIC.sub.90 was 0.125 g/mL and all the isolates saw their growth inhibited by 0.5 g/mL.
(50) K. pneumoniae was less sensitive to all the compounds tested when compared to E. coli, although numerous isolates presented relatively low MICs. For example, 87% of K. pneumoniae isolates were inhibited by 8 g/mL alafosfalin and 93% were inhibited by 8 g/mL -Cl-alafosfalin.
(51) All the E. cloacae isolates (n=27) were inhibited by 4 g/mL alafosfalin, which was typically 32 times more active than fosfomycin against this species. As previously, -Cl-alafosfalin proved to be the most active compound with all the isolates inhibited by 1 g/mL. The other enterobacteria species are excluded from Table 1, since there were fewer than 10 isolates tested.
(52) For the taxa Citrobacter (n=9), E. aerogenes (n=1), Kluyvera sp. (n=1), and S. marcescens (n=1), all the isolates were sensitive to 4 g/mL alafosfalin and 2 g/mL -Cl-alafosfalin. One of the five isolates of K. oxytoca required a MIC>8 g/mL alafosfalin, but all were inhibited by 2 g/mL -Cl-alafosfalin, demonstrating once more the highly satisfactory antibacterial activity of the latter.
(53) Eight P. mirabilis isolates and two P. rettgeri isolates required MICs 8 g/mL for all the agents tested (including fosfomycin).
(54) Three Salmonella isolates exhibited MICs 8.0 g/mL for alafosfalin but only from 2.0 to 4.0 g/mL for -Cl-alafosfalin.
(55) -Cl-alafosfalin and -Cl-Ala--Cl-Ala had the greatest activity against Staphylococci aureus, but there was a slight overall difference between the five peptide antimicrobials. 90% of the MRSA isolates, originating from various geographic sources, were inhibited by 8.0 g/mL alafosfalin and all the isolates were inhibited by 2.0 g/mL -Cl-alafosfalin, demonstrating once more, a greater antibacterial activity than that of alafosfalin.
(56) Against enterococci, the most noticeable observation was the high activity of di-alanyl fosfalin, for which the MICs were (on average) 16 times lower than those of alafosfalin and, in certain cases, 256 times lower. Among the 34 E. faecium isolates (including 31 vancomycin-resistant isolates), all were inhibited by 32 g/mL alafosfalin or 4 g/mL di-alanyl fosfalin. In any event, among the enterococci, the antibacterial activity of -Cl-Ala--Cl-alafosfalin (compound according to the invention) proved greater than that of alafosfalin.
2.4 Conclusion
(57) As we have shown in this study, the antimicrobial compounds according to the present invention, and notably -Cl-alafosfalin, present a useful in vitro antibacterial activity (very frequently greater than that of alafosfalin) against the majority of bacteria and notably of multi-resistant bacteria. Thus, by way of example, the MIC.sub.50 and MIC.sub.90 of alafosfalin for the CPEs were respectively 1 g/mL and 4 g/mL, whereas the phosphonopeptide according to the invention, g-Cl-alafosfalin, presented one of the values of 0.5 g/mL and 2 g/mL. Alafosfalin was only moderately active against MRSA presenting an MIC.sub.90 of 8 g/mL, whereas -Cl-alafosfalin was more active with an MIC.sub.90 of 2 g/mL. Compared to alafosfalin, -Cl-Ala--Cl-alafosfalin enables an inhibition gain targeted on certain species. This may be very useful for inhibiting a species without altering the rest of the microbial flora, for example inhibiting E. coli without inhibiting the other enterobacteria, or E. faecalis without inhibiting E. faecium. This becomes particularly advantageous for specifically isolating a species by inhibiting other bacteria, for example for seeking S. aureus.
(58) TABLE-US-00001 TABLE 1 Minimum inhibitory concentrations of various antimicrobial agents against groups of bacteria, including isolates endowed with definite resistance mechanisms. Concentration (g/mL) Modal MIC (most Organism (number tested) frequent and antimicrobial agent MIC) MIC.sub.50 MIC.sub.90 Range Enterobacteriaceae (197) Alafosfalin 2 2 >8 0.031->8.sup. Di-alanyl fosfalin >8 8 >8 0.031->8.sup. -Cl-Alafosfalin 1 0.5 8 0.031->8.sup. -Cl-Ala--Cl-Ala >8 >8 >8 2->8 -Cl-Ala--Cl-Alafosfalin 4 2 >8 0.031->8.sup. Fosfomycin 4 4 >32 0.125->32 E. coli (53) Alafosfalin 0.063 0.125 0.25 0.031-2.sup. Di-alanyl fosfalin 0.25 0.5 2 0.031->8.sup. -Cl-Alafosfalin 0.063 0.063 0.125 0.031-0.5.sup. -Cl-Ala--Cl-Ala 8 8 >8 2->8 -Cl-Ala--Cl-Alafosfalin 0.25 0.25 1 0.031-1.sup. Fosfomycin 0.5 0.5 1 0.125-8 K. pneumoniae (87) Alafosfalin 2 2 >8 0.25->8 Di-alanyl fosfalin >8 >8 >8 0.5->8 -Cl-Alafosfalin 1 1 8 0.125->8 -Cl-Ala--Cl-Ala >8 >8 >8 8->8 -Cl-Ala--Cl-Alafosfalin 4 4 >8 0.5->8 Fosfomycin 4 8 >32 2->32 E. cloacae (27) Alafosfalin 1 1 1 0.125-4 Di-alanyl fosfalin 4 >8 >8 0.25->8 -Cl-Alafosfalin 0.5 0.5 0.5 0.063-1 -Cl-Ala--Cl-Ala >8 >8 >8 8->8 -Cl-Ala--Cl-Alafosfalin 1 1 4 0.25-8 Fosfomycin 16 16 32 4->32 CPE (128) Alafosfalin 2 1 4 0.031->8.sup. Di-alanyl fosfalin >8 8 >8 0.063->8 -Cl-Alafosfalin 1 0.5 2 0.031->8.sup. -Cl-Ala--Cl-Ala >8 >8 >8 2->8 -Cl-Ala--Cl-Alafosfalin 4 2 8 0.031->8.sup. Fosfomycin 16 4 32 0.125->32 BSBL (47) Alafosfalin 2 2 >8 0.031->8.sup. Di-alanyl fosfalin >8 8 >8 0.031->8.sup. -Cl-Alafosfalin 0.063 0.5 >8 0.031->8 -Cl-Ala--Cl-Ala >8 >8 >8 2->8 -Cl-Ala--Cl-Alafosfalin >8 4 >8 0.063->8 Fosfomycin >32 8 >32 0.125->32 AmpC (22) Alafosfalin >8 4 >8 0.063->8 Di-alanyl fosfalin >8 >8 >8 0.063->8 -Cl-Alafosfalin >8 2 >8 0.031->8.sup. -Cl-Ala--Cl-Ala >8 >8 >8 8->8 -Cl-Ala--Cl-Alafosfalin >8 8 >8 0.25->8 Fosfomycin 32 8 >32 0.125->32 All S. aureus (50) Alafosfalin 4 4 8 0.125-16 Di-alanyl fosfalin 4 8 16 0.5-32 -Cl-Alafosfalin 2 1 2 0.125-4 -Cl-Ala--Cl-Ala 2 2 4 0.125-16 -Cl-Ala--Cl-Alafosfalin 16 16 16 .sup.2-16 Fosfomycin 8 4 16 0.5->32 MRSA (37) Alafosfalin 4 4 8 0.125-16 Di-alanyl fosfalin 4 8 16 0.5-32 -Cl-Alafosfalin 2 2 2 0.125-2 -Cl-Ala--Cl-Ala 2 2 4 0.125-16 -Cl-Ala--Cl-Alafosfalin 16 16 16 .sup.2-16 Fosfomycin 8 4 16 0.5->32 MSSA (13) Alafosfalin 4 4 8 0.25-16 Di-alanyl fosfalin 16 8 16 0.5-32 -Cl-Alafosfalin 1 1 2 0.5-4 -Cl-Ala--Cl-Ala 1 1 2 0.5-2 -Cl-Ala--Cl-Alafosfalin 16 16 16 .sup.4-16 Fosfomycin 4 4 16 .sup.2-16 All Enterococci (50) Alafosfalin 8 16 >32 4->32 Di-alanyl fosfalin 0.5 0.5 2 0.016->32.sup. -Cl-Alafosfalin 8 8 16 2->32 -Cl-Ala--Cl-Ala 16 16 32 .sup.2-16 -Cl-Ala--Cl-Alafosfalin 8 8 16 0.125->32 Fosfomycin >32 >32 >32 16->32 E. faecalis (11) Alafosfalin 8 8 32 4->32 Di-alanyl fosfalin 0.031 0.063 0.5 0.016->32.sup. -Cl-Alafosfalin 8 8 16 4->32 -Cl-Ala--Cl-Ala 8 8 16 .sup.4-16 -Cl-Ala--Cl-Alafosfalin 0.25 0.25 4 0.125->32 Fosfomycin 32 32 >32 32->32 E. faecium (34) Alafosfalin 16 16 16 .sup.4-32 Di-alanyl fosfalin 0.5 0.5 2 0.016-4.sup. -Cl-Alafosfalin 4 4 8 .sup.2-32 -Cl-Ala--Cl-Ala 16 16 32 2->32 -Cl-Ala--Cl-Alafosfalin 8 8 8 0.125->32 Fosfomycin >32 >32 >32 16->32 GRE (43) Alafosfalin 16 16 >32 4->32 Di-alanyl fosfalin 0.5 0.5 >32 0.016->32.sup. -Cl-Alafosfalin 4 8 16 2->32 -Cl-Ala--Cl-Ala 16 16 >32 4->32 -Cl-Ala--Cl-Alafosfalin 8 8 16 0.125->32 Fosfomycin >32 >32 >32 32->32 Abbreviations: CPE: carbapenemase-producing Enterobacteriaceae; BSBL: Enterobacteriaceae with a broad-spectrum -lactamase; MRSA: methicillin-resistant Staphylococcus aureus; MSSA: methicillin-sensitive Staphylococcus aureus; GRE: glycopeptide-resistant enterococci.
Example 3: Reaction Medium According to the Invention for Detecting Bacteria of the Genus Salmonella
3.1 Composition of the Medium
(59) The present Example 3 aims to compare the detection specificity of a reaction medium according to the invention, intended to detect bacteria of the genus Salmonella, namely the medium modified chromlD Salmonella against a reference detection medium for the same bacteria, namely the medium chromlD Salmonella.
(60) The respective compositions of the reaction medium according to the present invention and of the reference reaction medium are presented below, within Table 2.
(61) TABLE-US-00002 TABLE 2 Compositions of the reaction medium according to the invention and of the reference medium Modified chromID Salmonella ChromID Salmonella medium = beta medium = IDSalm g/L g/L Peptones 6.25 g Peptones 6.25 g Glucose 0.5 Glucose 0.5 Bile salts 1.5 Bile salts 1.5 NaCl 5.0 NaCl 5.0 Buffer 0.2 Buffer 0.2 Agar 14.0 Agar 14.0 Chromogenic mixture 9.6 Chromogenic mixture 9.6 -Cl-Alafosfalin 0.002
3.2 Results
(62) The results obtained are presented in Table 3 below:
(63) TABLE-US-00003 TABLE 3 Results IDSalm beta Reference # Bacteria Growth Colour Growth Colour Salmonella montevideo ++ ++ Mauve ++ ++ Mauve Salmonella seftenberg ++ ++ Mauve ++ ++ Mauve Salmonella choleraesuis ++ ++ Mauve ++ ++ Mauve Salmonella meleagridis ++ ++ Mauve ++ ++ Mauve NCTC 12023 Salmonella typhimurium ++ ++ Mauve ++ ++ Mauve Salmonella berta ++ ++ Mauve ++ ++ Mauve Salmonella stanley ++ ++ Mauve ++ ++ Mauve NCTC 6676 Salmonella enteritidis ++ ++ Mauve ++ ++ Mauve Salmonella simsburg ++ ++ Mauve ++ ++ Mauve Salmonella lexington ++ ++ Mauve ++ ++ Mauve Salmonella limete ++ ++ Mauve ++ ++ Mauve Salmonella corvalis ++ ++ Mauve ++ ++ Mauve Salmonella haifa ++ ++ Mauve ++ ++ Mauve Salmonella zanzibar ++ ++ Mauve ++ ++ Mauve Salmonella indiana ++ ++ Mauve ++ ++ Mauve Salmonella javiana ++ ++ Mauve ++ ++ Mauve NCTC 11304 Salmonella indiana ++ ++ Mauve ++ ++ Mauve Salmonella oranienburg ++ ++ Mauve ++ ++ Mauve NCTC 4840 Salmonella poona ++ ++ Mauve ++ ++ Mauve Salmonella tennessee ++ ++ Mauve ++ ++ Mauve Salmonella emek ++ ++ Mauve ++ + Mauve Salmonella virohrady ++ ++ Mauve ++ ++ Mauve Salmonella java ++ ++ Mauve ++ ++ Mauve Salmonella augustenborg ++ ++ Mauve ++ ++ Mauve Salmonella alachia ++ ++ Mauve ++ ++ Mauve Salmonella panama ++ ++ Mauve ++ ++ Mauve Salmonella virchow ++ ++ Mauve ++ ++ Mauve Salmonella paratyphi ++ ++ Mauve ++ ++ Mauve Salmonella orthmarschen ++ ++ Mauve ++ ++ Mauve Salmonella vilvorde ++ ++ Mauve ++ ++ Mauve Salmonella gallinarium + +/ Mauve + +/ Mauve NCTC 8385 Salmonella typhi ++ + Mauve + NCTC 9528 K. pneumoniae ++ ++ Blue ++ ++ Blue NCTC 11936 E. cloacae ++ + Blue ++ + Blue NCTC 10322 S. marcescens ++ ++ Blue ++ ++ Blue NCTC 10662 P. aeruginosa NCTC S. maltophilia +/ +/ NCTC 19606 A. baumannii ++ ++ Mauve ++ ++ Mauve NCTC 7475 P. rettgeri WILD 462213 M. morganii ++ ++ 69052 Escherichia coli ++ ++ 68957 Escherichia coli ++ ++ 68958 Escherichia coli ++ +/ Blue ++ 68805 Escherichia coli ++ +/ Blue + 69367 Escherichia coli + +/ Blue + 69371 Escherichia coli ++ ++ 69035 Escherichia coli ++ V. Blue ++ 69051 Escherichia coli + +/ Blue + 69017 Escherichia coli ++ ++ 69148 Escherichia coli 68880 Escherichia coli ++ ++ 68886 Escherichia coli ++ ++ 69102 Escherichia coli ++ ++ 69157 Escherichia coli 1 colony +/ 69285 Escherichia coli 2 colonies +/ 69130 Escherichia coli ++ ++ 69134 Escherichia coli ++ ++ 69135 Escherichia coli + + 69174 Escherichia coli 69176 Escherichia coli +/ +/ Blue +/
3.3 Conclusion
(64) The addition of -Cl-Alafosfalin to the chromlD Salmonella medium makes it possible to selectively inhibit the osidase activities of the strains of Escherichia coli. Insofar as it proves easier to detect one or more mauve colonies in a medium of colourless colonies than in a medium of blue colonies, if a salmonella strain is present in low concentration in mixture with one or more Escherichia coli strains, it becomes significantly easier to detect it in the reaction medium of the present invention.
Example 4: Synthesis Method of Antimicrobial Compound G According to the Invention (FIG. 2): L-Norvalinyl--chloro-L-alanyl-D/L-fosfalin
(65) L-Norvalinyl--chloro-L-alanyl-D/L-fosfalin (L-Nva--Cl-L-Ala-D/L-fosfalin) is represented by the following formula:
(66) ##STR00020##
(67) The synthesis of this antimicrobial compound is detailed below. The corresponding synthesis method is moreover illustrated in
(68) As represented in
4.1 Synthesis of the Reaction Intermediate -chloro-L-alanine O-benzyl ester 32 from the Compound tBOC-L-serine 41 (As Illustrated in FIG. 5)
4.1.1 Synthesis of (S)-benzyl 2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate (tBOC-L-serine O-benzyl ester) 43 [9]
(69) ##STR00021##
(70) Some .sup.tBOC-L-serine (commercially available) 41 (30 mmol, 6.16 g) was dissolved in dry benzene (100 mL), to which some 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) (36 mmol, 5.5 mL) and some benzyl bromide 42 (36 mmol, 4.4 mL) was then added. The solution was agitated overnight at ambient temperature under nitrogen, and the solvent was later eliminated under low pressure to give an off-white liquid residue. Some ethyl acetate (200 mL) was added, and the contents of the flask were ultrasound-treated, then washed with a 1M solution of HCl (250 mL), a 10% weight/volume aqueous solution of K.sub.2CO.sub.3 (250 mL) and brine (250 mL). The organic layer was dried on MgSO.sub.4, filtered, concentrated under vacuum and purified by column chromatography [petrol/ethyl acetate (1:1)] to give the product 43 in the form a white solid (8.05 g, 27.3 mmol, 91%); m.p. 61-66 C. (lit. m.p. [10] 59-60 C.); [].sup.21.sub.D18.5 (c 1.0, CH.sub.3OH); .sub.max/cm.sup.1 3419 (NH), 3361 (OH), 2978 (CH), 1758 (CO), 1668 (CO), 1524 (NH bend), 1155 (CO), 1068 (CO); .sup.1H NMR (300 MHz, DMSO) .sub.H 1.38 (9H, s, C(CH.sub.3).sub.3), 3.68 (2H, t, J=6.0 Hz, CH.sub.2OH), 4.10-4.16 (1H, m, CH-2), 4.91 (1H, t, J=6.0 Hz, OH), 5.10 (1H, d, J=12.0 Hz, OCH.sub.2aAr), 5.17 (1H, d, J=12.0 Hz, OCH.sub.2bAr), 6.97 (1H, d, J=9.0 Hz, NH), 7.32-7.38 (SH, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, DMSO) .sub.C 28.6 (C(CH.sub.3).sub.3), 57.0 (CH-2), 61.8 (CH.sub.2-3), 66.2 (OCH.sub.2Ar), 78.8 (C(CH.sub.3).sub.3), 128.0 (CH.sub.Ar), 128.4 (CH.sub.Ar), 128.8 (CH.sub.Ar), 136.5 (CH.sub.Ar quat), 155.8 (C-4, quat), 171.4 (C-1, quat).
4.1.2 Synthesis of (R)-benzyl 2-((tert-butoxycarbonyl)amino)-3-chloropropanoate (tBOC--chloro-L-alanine O-benzyl ester) 44 [11]
(71) ##STR00022##
(72) ((S)-benzyl 2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate 43 (25 mmol, 7.39 g), obtained in step 4.1.1 above, was dissolved in dry DCM (100 mL), and then some trichloro-acetonitrile (50 mmol, 5.0 mL) was added. The solution was agitated at ambient temperature for 2 hours. Some triphenylphosphine (50 mmol, 13.15 g) in dry DCM (50 mL) was added slowly to this solution. The solution thus obtained was agitated overnight at ambient temperature under nitrogen. Some brine (100 mL) was added to stop the reaction. After separation, the organic layer was washed with brine (360 mL), dried on MgSO.sub.4, filtered and concentrated under vacuum to give a liquid orange residue. The residue was purified by column chromatography [petrol/ethyl acetate (7:3)] to give the product 44 in the form of an off-white solid (7.41 g, 23.6 mmol, 95%); m.p. 53-58 C.; [].sup.22.sub.D23.0 (c 1.0, CH.sub.3OH); .sub.max/cm.sup.1 3365 (NH), 2917 (CH), 1727 (CO), 1679 (CO), 1522 (NH bend), 1181 (CO), 1158 (CO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.45 (9H, s, C(CH.sub.3).sub.3), 3.85 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2a-3), 3.99 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2b-3), 4.74 (1H, m, CH-2), 5.20 (1H, d, J=12.0 Hz, OCH.sub.2aAr), 5.25 (1H, d, J=12.0 Hz, OCH.sub.2bAr), 5.44 (1H, d, J=6.0 Hz, NH), 7.33-7.38 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 27.3 (C(CH.sub.3).sub.3), 44.5 (CH.sub.2-3), 53.5 (CH-2), 66.8 (OCH.sub.2Ar), 79.5 (C(CH.sub.3).sub.3), 127.3 (CH.sub.Ar), 127.6 (CH.sub.Ar), 127.6 (CH.sub.Ar), 133.9 (CH.sub.Ar quat), 154.0 (C-4, quat), 168.0 (C-1, quat); CHN [Found: C, 57.71; H, 6.46; N, 4.18. C.sub.15H.sub.20ClNO.sub.4 requires C, 57.42; H, 6.42; N, 4.46%].
4.1.3 Synthesis of (R)-1-(benzyloxy)-3-chloro-1oxopropan-2-aminium chloride (-chloro-L-alanine hydrochloride) 32
(73) ##STR00023##
(74) Compound 44 (10 mmol, 3.14 g), obtained in step 4.1.2. above, was dissolved in a 2M solution of HCl in ether (200 mL). The solution was agitated at ambient temperature overnight. The solid thus obtained was filtered and washed with diethyl ether to give the product 32 in the form of a white solid (2.36 g, 9.5 mmol, 95%); m.p. 145 C. (secondary); .sub.max/cm.sup.1 2848 (CH), 1749 (CO), 1593 (Ar CC), 1490 (Ar CC), 1231 (CO); .sup.1H NMR (300 MHz, DMSO) .sub.H 4.16 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2a-3), 4.22 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2b-3), 4.77 (1H, t, J=3.0 Hz, CH-2), 5.26 (1H, d, J=12.0 Hz, OCH.sub.2aAr), 5.31 (1H, d, J=15.0 Hz, OCH.sub.2bAr), 7.33-7.46 (5H, m, 5CH.sub.Ar), 9.09 (3H, br, NH.sub.3.sup.+); .sup.13C NMR (75 MHz, DMSO) .sub.C 43.3 (CH.sub.2-3), 53.5 (CH-2), 68.0 (OCH.sub.2Ar), 128.6 (CH.sub.Ar), 128.8 (CH.sub.Ar), 128.9 (CH.sub.Ar), 135.4 (CH.sub.Ar quat), 167.0 (C-1, quat); CHN [Found: C, 47.16; H, 5.43; N, 5.43. C.sub.10H.sub.13Cl.sub.2NO.sub.2.0.2H.sub.2O requires C, 47.34; H, 5.32; N, 5.52%].
4.2 Synthesis of (R)-benzyl 2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoate (tBOC-L-Norvalinyl--chloro-L-alanine O-benzyl ester) 33
(75) ##STR00024##
(76) Some .sup.tBOC-L-Norvaline (commercially available) 31 (6.0 mmol, 1.31 g) was dissolved in dry THF (50 mL) and some N-methyl morpholine (6.0 mmol, 0.66 mL) was added. The solution was then cooled to 0 C. and some isobutyl chloroformate (6.0 mmol, 0.78 mL) was added dropwise. The mixture was agitated at 0 C. under nitrogen for 1 hour. Some (R)-1-(benzyloxy)-3-chloro-1oxopropan-2-aminium chloride (chloride salt of -Cl-L-alanine benzyl ester) 32 (5.4 mmol, 1.36 g), obtained in step 4.1.3 above, in dry DCM (30 mL) previously neutralised by N-methyl morpholine (5.4 mmol, 0.59 mL) at 0 C. was added to the agitated solution. The solution thus obtained was agitated overnight under nitrogen at ambient temperature. The solution was filtered and concentrated under vacuum. The residue was dissolved in DCM (60 mL) and washed with a 10% weight/volume solution of citric acid (225 mL), then with water (25 mL). The organic layer was dried on MgSO.sub.4, filtered and concentrated under vacuum to give a yellow liquid which was purified by column chromatography [petrol/ethyl acetate (7:3)] to give the product 33 in the form of a white solid (1.74 g, 4.2 mmol, 78%); m.p. 95-98 C.; [].sup.25.sub.D25.0 (c 1.0, CH.sub.3OH); .sub.max/cm.sup.1 3327 (NH), 2960 (CH), 1738 (CO), 1668 (CO), 1518 (NH bend), 1169 (CO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 0.92 (3H, t, J=9.0 Hz, CH.sub.3-8), 1.35-1.45 (11H, [m, CH.sub.2-7], [s, C(CH.sub.3).sub.3]), 1.52-1.65 (1H, m, CH.sub.2a-6), 1.75-1.82 (1H, m, CH.sub.2b-6), 3.89 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2a-3), 3.99 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2b-3), 4.11-4.15 (1H, m, CH-5), 4.96-5.00 (2H, [m, CH-2], [m, HNCO.sub.2]), 5.20 (1H, d, J=12.0 Hz, OCH.sub.2a Ar), 5.25 (1H, d, J=12.0 Hz, OCH.sub.2aAr), 6.97 (1H, d, J=6.0 Hz, HNCO), 7.33-7.37 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 12.7 (CH.sub.3-8), 17.8 (CH.sub.2-7), 27.3 (C(CH.sub.3).sub.3), 33.4 (CH.sub.2-6), 43.8 (CH.sub.2-3), 52.2 (CH-2), 53.4 (CH-5), 67.0 (OCH.sub.2Ar), 79.3 (C(CH.sub.3).sub.3), 127.4 (CH.sub.Ar), 127.6 (CH.sub.Ar), 127.7 (CH.sub.Ar), 133.8 (CH.sub.Ar), quat), 154.5 (C-9, quat), 167.5 (C-1, quat), 171.2 (C-4, quat); CHN [Found: C, 58.49; H, 7.22; N, 6.81. C.sub.20H.sub.29ClN.sub.2O.sub.5 requires C, 58.18; H, 7.08; N, 6.78%]; HRMS (nanospray ionisation) calculated for (C.sub.20H.sub.30ClN.sub.2O5)+413.1838, found MH.sup.+ 413.1837.
4.3 Synthesis of (R)-2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoic acid (tBOC-L-norvalinyl--chloro-L-alanine) 34
(77) ##STR00025##
(78) The product 33, (1.8 mmol, 0.75 g), obtained in step 4.2 above, was dissolved in methanol (60 mL) and added to the stainless steel pressure vessel. 10% palladium on charcoal (0.0941 g) was added and the solution thus obtained was agitated at 3.5 bar H.sub.2 pressure at ambient temperature for 72 hours. The catalyst was eliminated by filtration on Celite, washed with methanol and concentrated under vacuum to give the product 34 in the form of a light yellow solid (0.573 g, 1.78 mmol, 99%); m.p. 59-63 C.; [].sup.25.sub.D 12.0 (c 1.0, CH.sub.3OH); .sub.max/cm.sup.1 3313 (NH), 2964 (CH), 1655 (CO), 1509 (NH bend), 1161 (CO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 0.87 (3H, t, J=4.2 Hz, CH.sub.3-8), 1.38 (11H, [m, CH.sub.2-7], [s, C(CH.sub.3).sub.3]), 1.51-1.62 (1H, m, CH.sub.2a-6), 1.67-1.79 (1H, m, CH.sub.2b-6), 3.91 (2H, m, CH.sub.2-3), 4.19 (1H, m, CH-5), 4.85 (1H, m, CH-2), 5.20 (1H, m, NH carbamate), 6.45 (1H, br, OH), 7.25 (1H, m, NH amide); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 12.7 (CH.sub.3-8), 17.9 (CH.sub.3-7), 27.3 (C(CH.sub.3).sub.3), 33.6 (CH.sub.2-6), 43.4 (CH-3), 52.4 (CH-2), 53.2 (CH-5), 79.7 (C(CH.sub.3).sub.3), 155.1 (C-9, quat), 170.7 (C-1/4, quat), 172.0 (C-1/4, quat); CHN [Found: C, 48.77; H, 7.61; N, 8.22. C.sub.13H.sub.23ClN.sub.2O.sub.5 requires C, 48.37; H, 7.18; N, 8.68%].
4.4 Synthesis of the reaction intermediate D/L-fosfalin diethyl ester 35
(79) As represented in
4.4.1 Synthesis of ()-(1-aminoethyl)phosphonic acid (D/L-fosfalin) 54 [12]
(80) ##STR00026##
(81) Some N-phenylthio-urea 53 (40 mmol, 6.10 g) was dissolved in glacial acetic acid (20 mL). Acetaldehyde 51 (60 mmol, 3.40 mL) was added dropwise, and then triphenyl phosphite 52 (40 mmol, 11 mL) was added. The solution was agitated at ambient temperature for 5 mins, and then heated to reflux at 85 C. for 1 hour. A mixture of glacial acetic acid (2 mL) and hydrochloric acid (37%, 20 mL) was added and the reaction was heated to reflux overnight at 145 C. The solution was cooled at ambient temperature, transferred and washed with ethanol in a 500 mL round-bottomed flask. A small quantity of fosfalin hydrochloride salt was obtained by filtration and the filtrate was concentrated under vacuum to give a dark orange liquid residue. The residue was dissolved in a minimum quantity of ethanol (20 mL) and propylene oxide (120 mL) was added to produce a white precipitate. The white solid was filtered under nitrogen and dried in a desiccator (on phosphorus pentoxide) for 3 days, which was followed by recrystallisation from hot water/ethanol to give the zwitterion product 54 in the form of a white solid (4.39 g, 35 mmol, 88%); m.p. 265-268 C. (s) (lit. m.p. [13] 271-275 C.); .sub.max/cm.sup.1 2910 (br OH), 1616 (POH), 1532 (NH bend), 1143 (PO), 1035 (PO), 930 (PO); .sup.1H NMR (500 MHz, D.sub.2O) .sub.H 1.47 (3H, dd, J.sub.PH=14.9 Hz and J.sub.HH=7.3 Hz, CH.sub.3), 3.40 (1H, m, CH); .sup.13C NMR (125 MHz, D.sub.2O) .sub.C 13.5 (CH.sub.3, d, J.sub.PC=2.7 Hz), 44.7 (CH, d, J.sub.PC=144.2 Hz).
4.4.2 Synthesis of diethyl (1-(2,2,2-trifluoro-acetamido)ethyl)phosphonate 56 [14]
(82) ##STR00027##
(83) Some 1-aminoethylphosphonic acid 44 (40 mmol, 6.47 g), obtained in step 4.4.1. above, was added to a mixture of trifluoroacetic acid (5 mL) and trifluoroacetic anhydride (25 mL). The solution was agitated and heated to reflux at 60 C. After 1 hour, the solution was cooled and triethyl orthoformate (150 mL) was added slowly. The solution was heated to reflux at 110 C. for 2 hours, and then cooled to ambient temperature. The solvent was eliminated under vacuum to give a brown solid. The solid was redissolved in DCM and purified by column chromatography [DCM/MeOH (9:1)] to give the product 56 in the form of an off-white solid (11.00 g, 39.6 mmol, 99%); m.p. 98-103 C. (s) (lit m.p..sup.3 101-102 C.); .sub.max/cm.sup.1 3202 (NH), 1715 (CO), 1565 (NH bend), 1210 (PO), 1011 (CF), 968 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.23 (3H, t J=6.0 Hz, OCH.sub.2CH.sub.3-a), 1.28 (3H, t J=9.0 Hz, OCH.sub.2CH.sub.3-b), 1.38 (3H, dd, J.sub.PH=15.0 Hz and J.sub.HH=6.0 Hz, CH.sub.3-2), 3.98-4.13 (4H, m, 2OCH.sub.2CH.sub.3), 4.32-4.47 (1H, m, CH-1), 8.11 (1H, d, J=9.0 Hz, NH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 13.7 (CH.sub.3-2), 15.2 (OCH.sub.2CH.sub.3, d, J.sub.PC=2.3 Hz), 15.3 (OCH.sub.2CH.sub.3, d, J.sub.PC=1.5 Hz), 40.8 (CH-1, d, J.sub.PC=159.0 Hz), 61.8 (OCH.sub.2CH.sub.3, d, J.sub.PC=6.8 Hz), 62.2 (OCH.sub.2CH.sub.3, d, J.sub.PC=7.5 Hz), 114.9 (CF.sub.3, q, J.sub.FC=285.8 Hz), 156.0 (CO, q, J.sub.FC=6.0 Hz); .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 23.0; .sup.19F-.sup.1H.sub.decoup NMR (282 MHz, CDCl.sub.3) .sub.P75.5.
4.4.3 Synthesis of ()-diethyl (1-aminoethyl)phosphonate (D/L-fosfalin diethyl ester) 35 [15]
(84) ##STR00028##
(85) Diethyl (1-(2,2,2-trifluoro-acetamido)ethyl)phosphonate 56 (20 mmol, 5.55 g), obtained in step 4.4.2. above, was dissolved in ethanol (200 mL) and sodium borhydrate (200 mmol, 7.57 g) was slowly added. The mixture thus obtained was agitated at ambient temperature for 1 hour, and then heated to reflux (90 C.) for 3 hours. The solution was cooled to ambient temperature and the solvent was eliminated under low pressure to give a white solid residue. The residue was treated with a saturated solution of NaHCO.sub.3 (96 g/L) (150 mL) and extracted in DCM (650 mL). The organic layer was dried on MgSO.sub.4 and filtered. The filtrate was concentrated under vacuum to give a light yellow liquid, and purified by column chromatography [DCM/MeOH (9.0:1.0)] to give the product 35 in the form of a yellow liquid (2.52 g, 13.9 mmol, 70%); .sub.max/cm.sup.1 3431 (NH), 2980 (CH), 1215 (PO), 1020 (PO), 957 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H1.19-1.30 (9H, [dd, J.sub.PH=17.4 Hz, 7.2 Hz, CH.sub.3-2], [t, J=7.2 Hz, 2OCH.sub.2CH.sub.3), 1.65 (2H, br, NH.sub.2), 2.99-3.09 (1H, m, CH-1), 4.02-4.14 (4H, m, 2OCH.sub.2CH.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 15.5 (OCH.sub.2CH.sub.3-a), 15.6 (OCH.sub.2CH.sub.3-b), 16.3 (CH.sub.3-2), 43.3 (CH-1, d, J.sub.PC=148.5 Hz), 61.1 (OCH.sub.2CH.sub.3-a, d, J.sub.PC=1.5 Hz), 61.2 (OCH.sub.2CH.sub.3-b, d, J.sub.PC=1.5 Hz); .sup.31P-.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P29.6.
4.5 Synthesis of tert-butyl ((2S-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamate (tBOC-L-norvalinyl--chloro-L-alanyl-D/L-fosfalin diethyl ester) 36
(86) ##STR00029##
(87) (R)-2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)-3-chloropropanoic acid (.sup.tBOC-L-norvalinyl--chloro-L-alanine) 34 (1.8 mmol, 0.58 g), obtained in step 4.3 above, was dissolved in dry THF (35 mL), then N-methyl morpholine (1.9 mmol, 0.21 mL) was added. The solution was cooled to 0 C. and some isobutyl chloroformate (1.9 mmol, 0.25 mL) was added dropwise. The mixture was agitated at 0 C. for 1 hour. Some diethyl 1-aminoethylphosphonate (D/L-fosfalin diethyl ester) 35 (1.8 mmol, 0.33 g)obtained in step 4.4.3. abovein dry THF (10 mL) was added to the agitated solution, and the solution thus obtained was agitated overnight under nitrogen at ambient temperature. The solution was filtered and concentrated under vacuum. The residue was dissolved in DCM (60 mL) and washed with a 10% weight/volume solution of citric acid (230 mL), 10% weight/volume potassium carbonate (30 mL) and water (30 mL). The organic layer was dried on MgSO.sub.4, filtered and concentrated under vacuum to give a light yellow liquid residue. The residue was purified by column chromatography [ethyl acetate/methanol (96:4)] to give the product 36 in the form of a sticky white solid (0.45 g, 0.93 mmol, 52%); m.p. 196 C. (decomposition); [].sup.24.sub.D22.5 (c 1.0, CH.sub.3OH); .sub.max/cm.sup.1 3272 (NH), 2977 (CH), 1709 (CO), 1644 (CO), 1530 (NH bend), 1229 (CO), 1165 (PO), 1019 (PO), 972 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 0.86 (3H, t, J=6.0 Hz, CH.sub.3-12), 1.17-1.38 (20H, [m, 2OCH.sub.2CH.sub.3], [m, CH.sub.3-2], [s, C(CH.sub.3).sub.3], [m, CH.sub.2-11]), 1.53-1.59 (1H, m, CH.sub.2a-10), 1.70-1.86 (1H, m, CH.sub.2b-10), 3.69 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2a-6), 3.91 (1H, dd, J=12.0 Hz, 3.0 Hz, CH.sub.2a-6), 3.97-4.13 (5H, [m, 2OCH.sub.2CH.sub.3], [m, CH-9]), 4.35-4.46 (1H, m, CH-1), 4.73-4.79 (1H, m, CH-5), 4.97-5.03 (1H, m, NH-13), 7.00-7.10 (1H, 2d, J=9.0 Hz, 9.0 Hz, NH-7, diastereo-isomers L,L,L and L,L,D), 7.23-7.34 (1H, 2d, J=9.0 Hz, 9.0 Hz, NH-3, diastereo-isomers L,L,L and L,L,D); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 12.7 (CH.sub.3-12), 14.5 (CH.sub.3-2), 15.3, 15.5 (2OCH.sub.2CH.sub.3), 17.9 (CH.sub.2-11), 27.3 (C(CH.sub.3).sub.3), 33.2 (CH.sub.2-10), 40.4 (d, J.sub.PC=157.5 Hz, CH-1), 43.4 (CH.sub.2-6), 52.7 (CH-5), 61.4, 61.9 (2OCH.sub.2CH.sub.3), 79.4 (C(CH.sub.3).sub.3), 155.0 (C-14, quat), 166.8 (C-4, quat), 171.4 (C-8, quat); .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 24.8; HRMS (nanospray ionisation) calculated for (C.sub.19H.sub.38ClN.sub.3O.sub.7P).sup.+ 486.2130, found 486.2124; CHN [Found: C, 46.51; H, 7.76; N, 8.21. C.sub.19H.sub.37ClN.sub.3O.sub.7P requires C, 46.96; H, 7.67; N, 8.65%].
4.6 Synthesis of hydrogeno (1-((R)-2-((S)-2-ammoniopentanamido)-3-chloropropanamido)ethyl)phosphonate (L-Norvalinyl--chloro-L-alanyl-D/L-fosfalin) 37
(88) ##STR00030##
(89) Some tert-Butyl ((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamate (.sup.tBOC-L-norvalinyl--chloro-L-alanyl-D/L-fosfalin diethyl ester) 36 (2.0 mmol, 0.99 g), obtained in step 4.5. above, was dissolved in HBr and acetic acid (33%) (3.0 mL). The solution was agitated overnight at ambient temperature. Dry diethyl ether (150 mL) was added and the mixture was stored at 20 C. overnight. The solvent was decanted off and the oily brown raw product was triturated with dry diethyl ether (560 mL). The orange-brown hygroscopic residue was dissolved in dry methanol (5 mL), followed by the addition of excess propylene oxide. The solution was filtered and washed with diethyl ether to give a pale green solid which was later filtered to give the end product 37 in the form of a pale green solid (0.64 g, 1.94 mmol, 97%); m.p. 175 C. (secondary) [].sup.21.sub.D2.50 (c 1.0, H.sub.2O+DIEA, 9.9:0.1); .sub.max/cm.sup.1 3294 (NH.sup.+), 2963 (CH), 1668 (CO), 1645 (CO), 1538 (NH bend), 1132 (PO), 1039 (PO), 998 (PO);.sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.01 (3H, t, J=9.0 Hz, CH.sub.3-12), 1.30-1.37 (3H, m, CH.sub.3-2), 1.44-1.54 (2H, m CH.sub.3-11), 1.90-1.98 (2H, m, CH.sub.2-10), 3.91-4.15 (4H, [m, CH.sub.2-6], [m, CH-9], [m, CH-1]), 4.79 (1H, m, CH-5); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 12.9 (CH.sub.3-12), 15.7 (CH.sub.3-2), 17.6 (CH.sub.2-11), 33.0 (CH.sub.2-10), 43.3 (CH.sub.2-6), 53.1 (CH-1, CH-9), 55.0 (CH-5), 170.4 (C-4, C-8, quat); .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 18.5; HRMS (nanospray ionisation) calculated for (C.sub.10H.sub.20ClN.sub.3O.sub.5P).sup. 328.0835, found 328.0833.
Example 5: Synthesis Method of Antimicrobial Compound H According to the Invention (FIG. 2): L-methionyl--chloro-L-alanyl-D/L-fosfalin
(90) L-methionyl--chloro-L-alanyl-D/L-fosfalin (L-Met--Cl-L-Ala-D/L-fosfalin) is represented by the following formula:
(91) ##STR00031##
(92) The synthesis of this antimicrobial compound is detailed below.
5.1. Synthesis of the reaction intermediate Tert-butyl((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)carbamate (tBOC--Cl-L-Ala-D/L-Fos diethyl ester)
(93) .sup.tBoc--Cl-L-Ala-D/L-Fos diethyl ester is represented by the following formula:
(94) ##STR00032##
(95) Some N-methylmorpholine (7.8 mmol, 0.90 mL) was added to a suspension of .sup.tBOC--Cl-L-Ala-OH (7.8 mmol, 1.74 g) in dry THF (60 mL) at 5 C. Some isobutyl chloroformate (7.8 mmol, 1.00 mL) was added slowly and the resulting mixture was agitated at 5 C. for 1 hour. Some diethyl 1-aminoethylphosphonate (8.6 mmol, 1.57 g) in dry THF (20 mL) was added to the agitated mixture at 5 C. The resulting mixture was agitated under nitrogen at 5 C. for 30 minutes, and then at ambient temperature overnight. The mixture was filtered and the solvent was eliminated under vacuum to give a pale yellow liquid, which was washed with 10% weight/volume citric acid (225 mL), 10% weight/volume potassium carbonate (25 mL) and water (25 mL). The combined organic layers were dried on magnesium sulfate, filtered and concentrated under vacuum to give a pale yellow liquid. The liquid was purified by column chromatography, using 100% DCM, and then increasing progressively to 90:10 DCM/MeOH, to give the product in the form of a yellow syrup consisting of 2 diastereoisomers, .sup.tBOC--Cl-L-Ala-L-Fos diethyl ester and .sup.tBOC--Cl-L-Ala-D-Fos diethyl ester (2.70 g, 7.0 mmol, 90%); .sub.max/cm.sup.1 3261 (NH), 1713 (CO), 1670 (CO), 1517 (NH bend), 1225 (PO), 1164 (PO), 1020 (PO), 970 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.15-1.39 (18H, [s, C(CH.sub.3).sub.3], [m, CH.sub.3-2], [m, 2OCH.sub.2CH.sub.3]), 3.64-3.71 (1H, m, CH..sub.a/b-6), 3.89-3.96 (1H, m, CH..sub.a/b-6), 4.01-4.12 (4H, m, 2OCH.sub.2CH.sub.3), 4.42-4.49 (2H, [m, CH-1], [m, CH-5]), 5.40 (1H, d, J=3.0 Hz, NH-3 or NH-7-A), 5.43 (1H, d, J=6.0 Hz, NH-3 or NH-7-A), 7.00 (1H, d, J=9.0 Hz, NH-3 or NH-7-A), 7.07 (1H, d, J=9.0 Hz, NH-3 or NH-7-B); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 15.6 (d, J.sub.PC=5.3 Hz, CH.sub.3-2), 16.3 (d, J.sub.PC=2.3 Hz, OCH.sub.2CH.sub.3), 16.5 (d, J.sub.PC=2.3 Hz, OCH.sub.2CH.sub.3), 28.3 (C(CH.sub.3).sub.3) 41.2 (d, J.sub.PC=157.5 Hz, CH-1-A), 41.3 (d, J.sub.PC=156.8 Hz, CH-1-B), 44.9 (CH.sub.2-6-A), 45.0 (CH.sub.2-6-B), 55.3 (CH-5), 62.5 (d, J.sub.PC=3.0 Hz, OCH.sub.2CH.sub.3-A), 62.6 (d, J.sub.PC=3.8 Hz, OCH.sub.2CH.sub.3B), 62.9 (d, J.sub.PC=3.0 Hz, OCH.sub.2CH.sub.3-A), 63.0 (d, J.sub.PC=3.0 Hz, OCH.sub.2CH.sub.3B), 80.7 (C(CH.sub.3).sub.3), 154.9 (CO-8), 168.3 (CO-4); .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 24.8.
5.2. Synthesis of the reaction intermediate (2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-aminium chloride (-Cl-L-Ala-D/L-Fos diethyl ester hydrochloride)
(96) -Cl-L-Ala-D/L-Fos diethyl ester hydrochloride is represented by the following formula:
(97) ##STR00033##
(98) A solution of .sup.tBOC--Cl-L-Ala-D/L-Fos diethyl ester (obtained in step 5.1.; 6.7 mmol, 2.59 g) in a 2M HCl solution in diethyl ether (100 mL) was agitated under nitrogen at ambient temperature overnight. The mixture was then filtered and the off-white hygroscopic solid was washed with diethyl ether. The solid was then dried overnight in a desiccator containing phosphorus (V) oxide and triturated with petrol to give the product in the form of a pale green solid consisting of 2 diastereoisomers, -Cl-L-Ala-L-Fos diethyl ester hydrochloride and -Cl-L-Ala-D-Fos diethyl ester hydrochloride (1.51 g, 4.7 mmol, 70%); m.p. 127-131 C. (decomp.); .sub.max/cm.sup.1 3204 (NH), 1687 (CO), 1562 (NH bend), 1204 (PO), 1010 (PO), 961 (PO); .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.28 (3H, t, J=6.0 Hz, OCH.sub.2CH.sub.3), 1.29 (3H, t, J=6.0 Hz, OCH.sub.2CH.sub.3), 1.37 (3H, dd, .sup.3J.sub.PH=18.0 Hz, .sup.3J.sub.HH=6.0 Hz, CH.sub.3-2), 3.92-4.04 (2H, m, CH.sub.2-6), 4.07-4.21 (4H, m, 2OCH.sub.2CH.sub.3), 4.38-4.48 (2H, [m, CH-1], [m, CH-5]); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 13.7 (CH.sub.3-2), 14.0 (CH.sub.3-2), 15.7 (OCH.sub.2CH.sub.3), 15.7 (OCH.sub.2CH.sub.3), 41.7 (d, J.sub.PC=158.3 Hz, CH-1), 42.0 (d, J.sub.PC=157.5 Hz, CH-1), 42.4 (CH.sub.2-6), 53.7 (CH-5), 53.8 (CH-5), 64.3 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3), 64.5 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3B), 165.7 (CO-4-A), 165.8 (CO-4-B); .sup.31P-.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.p 26.1.
5.3. Synthesis of the reaction intermediate Tert-butyl((2S)-1-(((2R)-3-chloro-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino)-4-(methylthio)-1-oxobutan-2-yl)carbamate (tBOC-L-Met--Cl-L-Ala-D/L-Fos diethyl ester)
(99) .sup.tBoc-L-Met--Cl-L-Ala-D/L-Fos diethyl ester is represented by the following formula:
(100) ##STR00034##
(101) Some N-methylmorpholine (3.4 mmol, 0.40 mL) was added to a solution of .sup.tBOC-L-Met-OH (3.4 mmol, 0.85 g) in dry THF (60 mL). The solution was cooled to 5 C. and some isobutyl chloroformate (3.4 mmol, 0.45 mL) was added dropwise. The mixture was agitated at 5 C. for 1 hour. Some -Cl-L-Ala-D/L-Fos diethyl ester hydrochloride obtained in step 5.2. (3.4 mmol, 1.10 g) in dry DCM (20 mL), which had been neutralised with N-methylmorpholine (3.4 mmol, 0.40 mL) at 5 C. was added dropwise to the agitated mixture. The resulting mixture was agitated under nitrogen at 5 C. for 30 minutes, and then at ambient temperature overnight. The mixture was then filtered and concentrated under vacuum, then washed with 10% weight/volume citric acid (225 mL), 10% weight/volume potassium carbonate (25 mL), water (25 mL) and brine (30 mL). The organic layer was dried on MgSO.sub.4, filtered and the solvent was eliminated under vacuum to give a yellow liquid, which was purified by column chromatography [DCM/MeOH (95:5)] to give a colourless liquid. Recrystallisation from diethyl ether/petrol gave the product in the form of a white solid consisting of 2 diastereoisomers, .sup.tBOC-L-Met--Cl-L-Ala-L-Fos diethyl S ester and .sup.tBOC-L-Met--Cl-L-Ala-D-Fos diethyl ester (0.88 g, 1.7 mmol, 50%); m.p. 96-99 C.; .sub.max/cm.sup.1 3278 (NH), 1709 (CO), 1639 (CO), 1523 (NH bend), 1228 (PO), 1018 (PO), 970 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.17-1.36 (9H, [m, CH.sub.3-2], [m, 2OCH.sub.2CH.sub.3]), 1.38 (9H, s, C(CH.sub.3).sub.3), 1.87-2.07 (5H, [s, CH.sub.3-12], [m, CH.sub.2-10]), 2.48-2.54 (2H, m, CH.sub.2-11), 3.71 (1H, dd, J=12.0 Hz, 6.0 Hz, CH.sub.a/b-6), 3.88 (1H, dd, J=12.0 Hz, 6.0 Hz, CH.sub.a/b-6), 3.99-4.13 (4H, m, 2OCH.sub.2CH.sub.3), 4.20 (1H, m, CH-9), 4.37-4.47 (1H, m, CH-1), 4.78-4.84 (1H, m, CH-5), 5.39 (1H, d, J=6.0 Hz, NH-13-A), 5.41 (1H, d, J=6.0 Hz, NH-13-B), 7.15 (1H, d, J=6.0 Hz, NH-7-A), 7.24 (1H, d, J=6.0 Hz, NH-7-B), 7.52 (1H, m, NH-3); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 14.4 (CH.sub.3-2 or CH.sub.3-12), 14.5 (CH.sub.3-2 or CH.sub.3-12), 15.4 (OCH.sub.2CH.sub.3), 15.5 (OCH.sub.2CH.sub.3), 27.3 (C(CH.sub.3).sub.3), 29.2 (CH.sub.2-11-A), 29.3 (CH.sub.2-11-B), 30.2 (CH.sub.2-10-A), 30.4 (CH.sub.2-10-B), 40.3 (d, J.sub.PC=159.0 Hz, CH-1), 43.5 (CH.sub.2-6-A), 43.7 (CH.sub.2-6-B), 52.7 (CH-5), 53.1 (CH-9), 61.6 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3-A), 61.7 (d, J.sub.PC=6.0 Hz, OCH.sub.2CH.sub.3B), 62.0 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3-A), 62.1 (d, J.sub.PC=7.5 Hz, OCH.sub.2CH.sub.3B), 79.6 (C(CH.sub.3).sub.3), 154.8 (CO-14), 166.7 (CO-4-A), 166.8 (CO-4-B), 170.7 (CO-8-A), 170.8 (CO-8-B); .sup.31P-.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 24.8; CHN [Found: C, 44.08; H, 7.47; N, 8.18. C.sub.19H.sub.37ClN.sub.3O.sub.7PS requires C, 44.06; H, 7.20; N, 8.11%].
5.4. Synthesis of antimicrobial compound H (FIG. 2), namely the compound Hydrogeno(1-((R)-2-((S)-2-ammonio-4-(methylthio)butanamido)-3-chloropropanamido)ethyl)phosphonate (L-Met--Cl-L-Ala-D/L-Fos)
(102) The antimicrobial compound L-Met--Cl-L-Ala-D/L-Fos is represented by the following formula:
(103) ##STR00035##
(104) A solution of .sup.tBOC-L-Met--Cl-L-Ala-D/L-Fos diethyl ester obtained in step 5.3. (1.4 mmol, 0.71 g) in hydrogen bromide/glacial acetic acid (33%) (8.0 mL) was agitated overnight at ambient temperature. Some dry diethyl ether (70 mL) was then added and the mixture was placed in a freezer overnight. The solvent was decanted off and the raw product was triturated with dry diethyl ether (550 mL). The brownish-orange raw product was dissolved in methanol (5 mL) and excess propylene oxide was added. The mixture was filtered and washed with dry diethyl ether to give a green solid, which was dried in a desiccator containing phosphorus (V) oxide and recrystallised from hot water/ethanol to give the product in the form of a pale green solid consisting of 2 diastereoisomers, L-Met--Cl-L-Ala-L-Fos and L-Met--Cl-L-Ala-D-Fos (0.17 g, 0.48 mmol, 35%); m.p. 175-179 C. (decomp.); .sub.max/cm.sup.1 3264 (NH.sup.+), 2829 (large OH), 1641 (CO), 1546 (NH bend), 1149 (PO), 1041 (PO), 921 (PO); .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.10-1.50 (3H, m, CH.sub.3-2), 2.08-2.32 (5H, [m, CH.sub.3-12], [m, CH.sub.2-10]) 2.61 (2H, CH.sub.2-11), 3.37-4.16 (4H, [m, CH.sub.2-6], [m, CH-1], [m, CH-9]), 4.48 (1H, m, CH-5); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 14.0 (CH.sub.3-12), 15.5 (CH.sub.3-2), 28.2 (CH.sub.2-11), 30.0 (CH.sub.2-10), 43.3 (CH.sub.2-6), 44.8 (CH-1), 52.3 (CH-9), 55.0 (CH-5), 169.4 (CO-4 and CO-8); .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 18.7.
Example 6: Evaluation of the antibacterial activity of L-Norvalinyl--chloro-alanyl-D/L-fosfalin (compound G; cf. FIG. 2) and of L-Methionyl--chloro-alanyl-D/L-fosfalin (compound H; cf. FIG. 2) synthesised respectivelyaccording to Examples 4 and 5
6.1. Introduction
(105) The minimum inhibitory concentrations of compounds G and H for 12 strains of Gram-negative bacteria and 6 strains of Gram-positive bacteria were determined after 22 hours of incubation using an agar dilution method as described in example 2 (cf. notably section 2.2 Materials and methods).
6.2 Results
(106) The results obtained are presented in Table 4 below
(107) TABLE-US-00004 TABLE 4 Minimum inhibitory concentrations of the antimicrobial compounds G and H against various bacteria species Minimum inhibitory Minimum inhibitory Strain concentration (MIC) of concentration (MIC) of Species reference compound G (g/mL) compound H (g/mL) Acinetobacter baumannii ATCC 19606 >8 >8 Burkholderia cepacia ATCC 25416 >8 >8 Enterobacter cloacae NCTC 11936 4 4 Escherichia coli NCTC 10418 1 0.5 Escherichia coli NCTC 12241 0.5 0.5 Klebsiella pneumoniae NCTC 9528 0.5 0.5 Providencia rettgeri NCTC 7475 >8 >8 Pseudomonas aeruginosa NCTC 10662 >8 >8 Salmonella typhimurium NCTC 74 >8 >8 Salmonella enteritidis NCTC 6676 >8 >8 Serratia marcescens NCTC 10211 0.5 0.25 Yersinia enterocolitica NCTC 11176 0.25 0.25 Enterococcus faecalis NCTC 775 0.063 0.032 Enterococcus faecium NCTC 7171 1 1 Listeria monocytogenes NCTC 11994 >8 >8 Staphylococci epidermidis NCTC 11047 2 1 Staphylococcus aureus NCTC 6571 4 2 Staphylococcus aureus NCTC 11939 >8 >8 (SARM)
(108) According to the data presented in table 4 above, it clearly appears that compounds G and H are strongly inhibiting against the tested strains of certain Gram-negative bacteria species and of certain Gram-positive bacteria species, notably concerning Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Yersinia enterocolitica, Enterococcus faecalis, and less inhibiting against other Gram-negative and Gram-positive bacteria species, notably with regard to Acinetobacter baumannii, Burkholderia cepacia, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis, Listeria monocytogenes.
6.3 Conclusion
(109) The very significant differences in terms of inhibitory concentration observed between various bacteria species make compounds G and H particularly well suited compounds to be incorporated in reaction media making it possible to selectively seek and/or isolate certain bacteria species in biological samples, and notably selectively seek: Gram-negative bacteria (Gram-negative target bacteria) such as Acinetobacter baumannii, Burkholderia cepacia, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis; or Gram-positive bacteria (Gram-positive target bacteria) such as Listeria monocytogenes.
Example 7: Synthesis method of the Antimicrobial Compound I According to the Invention (FIG. 2): L-norvalinyl-L-alanyl-D/L-fosfalin
(110) L-norvalinyl-L-alanyl-D/L-fosfalin (L-Nva-L-Ala-D/L-fosfalin) is represented by the following formula:
(111) ##STR00036##
(112) The synthesis of this antimicrobial compound is detailed below.
7.1. Synthesis of the reaction intermediate (S)-Benzyl 2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)propanoate (tBOC-L-Nva-L-Ala-OBzl)
(113) The compound .sup.tBOC-L-Nva-L-Ala-OBzl is represented by the following formula:
(114) ##STR00037##
(115) Some N-methylmorpholine (15.0 mmol, 1.65 mL) was added to a solution of .sup.tBOC-L-Nva-OH (10.0 mmol, 2.17 g) in dry THF (60 mL). The solution was cooled to 5 C. and some isobutyl chloroformate (15.0 mmol, 1.95 mL) was added dropwise. The mixture was agitated at 5 C. for 1 hour. Some L-alanine benzyl ester p-toluenesulfonate salt (10.0 mmol, 3.52 g) in dry DCM (30 mL) which had been neutralised with diisopropylethylamine (15.0 mmol, 2.60 mL) at 5 C., was added dropwise to the agitated mixture. The resulting solution was agitated under nitrogen at 5 C. for 30 minutes, and then overnight at ambient temperature. The solution was filtered and concentrated under vacuum, then washed with 10% weight/volume citric acid (225 mL), 10% weight/volume potassium carbonate (25 mL) and water (25 mL). The organic layer was dried on MgSO.sub.4, filtered and concentrated under vacuum to give a yellow hygroscopic liquid, which was purified by column chromatography [40-60 petrol/ethyl acetate (7:3)] to give the product in the form of an off-white solid (2.40 g, 6.3 mmol, 63%); m.p. 60-63 C.; .sub.max/cm.sup.1 3299 (NH), 1743 (CO), 1655 (CO), 1527 (NH bend), 1245 (CO), 1162 (CO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 0.83 (3H, t, J=9.0 Hz, CH.sub.3-8), 1.25-1.36 (14H, [d, J=6.0 Hz, CH.sub.3-3], [m, CH.sub.2-7], [s, C(CH.sub.3).sub.3]), 1.42-1.54 (1H, m, CH.sub.a/b-6), 1.64-1.73 (1H, m, CH.sub.a/b-6), 4.02 (1H, m, CH-5), 4.54 (1H, pentet, J=6.0 Hz, CH-2), 4.96 (1H, d, J=9.0 Hz, NHCO.sub.2), 5.07 (1H, d, J=12.0 Hz, OCH.sub.a/bAr), 5.12 (1H, d, J=12.0 Hz, OCH.sub.a/bAr), 6.56 (1H, d, J=6.0 Hz, NHCO), 7.27 (5H, m, 5CH.sub.Ar); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 12.7 (CH.sub.3-8), 17.3 (CH.sub.3-3), 17.8 (CH.sub.2-7), 27.3 (C(CH.sub.3).sub.3), 33.7 (CH.sub.2-6), 47.1 (CH-2), 53.4 (CH-5), 66.1 (OCH.sub.2Ar), 79.0 (C(CH.sub.3).sub.3), 127.1-127.6 (5CH.sub.Ar), 134.3 (CH.sub.Ar quat.), 154.6 (CO-9), 170.8 (CO-4), 171.5 (CO-1); CHN [Found: C, 63.75; H, 8.37; N, 7.86. C.sub.20H.sub.30N.sub.2O.sub.5 requires C, 63.47; H, 7.99; N, 7.40%]; HRMS (nanospray ionisation) calculated for (C.sub.20H.sub.31N.sub.2O.sub.5).sup.+ 379.2227, found 379.2222.
7.2. Synthesis of the reaction intermediate (S)-2-((S)-2-((tert-butoxycarbonyl)amino)pentanamido)propanoic acid (tBOC-L-Nva-L-Ala-OH)
(116) The compound .sup.tBOC-L-Nva-L-Ala-OH is represented by the following formula:
(117) ##STR00038##
(118) Some .sup.tBOC-L-Nva-L-Ala-OBzl obtained in step 7.1. (6.0 mmol, 2.27 g) was dissolved in methanol (60 mL) and hydrogenated in the presence of 5% palladium on charcoal (0.23 g) ata pressure of 3.5 bar H.sub.2 at ambient temperature overnight. The catalyst was eliminated by filtration through Celite and washed with methanol. The solution was concentrated under vacuum to give the product in the form of a white solid (1.66 g, 5.7 mmol, 96.0%); m.p. 53-58 C. (decomp.); .sub.max/cm.sup.1 3500-2500 (br, OH), 3300 (NH), 2961 (NH), 1688 (CO), 1655 (CO), 1522 (NH bend), 1245 (CO), 1164 (CO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 0.85 (3H, t, J=9.0 Hz, CH.sub.3-8), 1.27-1.39 (14H, [m, CH.sub.3-3], [m, CH.sub.2-7], [s, C(CH.sub.3).sub.3]), 1.48-1.53 (1H, m, CH.sub.a/b-6), 1.67-1.71 (1H, m, CH.sub.a/b-6), 4.10 (1H, m, CH-5), 4.50 (1H, m, CH-2), 5.27 (1H, m, NHCO.sub.2), 6.93 (1H, m, NHCO), 8.87 (1H, br, OH); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 13.7 (CH.sub.3-8), 18.0 (CH.sub.3-3), 18.8 (CH.sub.2-7), 28.3 (C(CH.sub.3).sub.3), 34.5 (CH.sub.2-6), 48.1 (CH-2), 54.3 (CH-5), 80.4 (C(CH.sub.3).sub.3), 156.0 (CO-9), 172.5 (CO-4), 175.5 (CO-1); CHN [Found: C, 54.18; H, 8.78; N, 9.62. C.sub.13H.sub.24N.sub.2O.sub.5 requires C, 54.15; H, 8.39; N, 9.72%]; HRMS (nanospray ionisation) calculated for (C.sub.13H.sub.25N.sub.2O.sub.5).sup.+ 289.1758, found 289.1758.
7.3. Synthesis of the reaction intermediate tert-Butyl((2S)-1-(((2S)-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamate (tBOC-L-Nva-L-Ala-D/L-Fos diethyl ester)
(119) .sup.tBoc-L-Nva-L-Ala-D/L-Fos diethyl ester is represented by the following formula:
(120) ##STR00039##
(121) Some N-methylmorpholine (5.3 mmol, 0.58 mL) at 5 C. was added to a solution of .sup.tBOC-L-Nva-L-Ala-OH (5.0 mmol, 1.45 g; obtained from step 7.2.) in dry THF (50 mL). Some isobutyl chloroformate (5.3 mmol, 0.70 mL) was added slowly and the resulting mixture was agitated at 5 C. for 1 hour. Some diethyl 1-aminoethylphosphonate (4.8 mmol, 0.87 g; the synthesis of which is described in example 4.4 and represented in
7.4. Synthesis of compound I, namely the compound Hydrogeno(1-((S)-2-((S)-2-ammoniopentanamido)propanamido)ethyl)phosphonate (L-Nva-L-Ala-D/L-Fos)
(122) As indicated previously, the compound L-Nva-L-Ala-D/L-Fos is represented by the following formula:
(123) ##STR00040##
(124) The .sup.tBOC-L-Nva-L-Ala-D/L-Fos diethyl ester obtained in step 7.3. (1.6 mmol, 0.72 g) was dissolved in glacial acetic acid (30 mL) and some hydrogen bromide/acetic acid (33%) (25 mL) was added. The solution was agitated overnight at ambient temperature. Some dry diethyl ether (150 mL) was added and the mixture was placed in a freezer overnight. The solvent was decanted off and the raw product was triturated with dry diethyl ether (5100 mL). The yellowish-orange raw product was dissolved in methanol (5 mL) and excess propylene oxide was added. The solution was filtered and washed with diethyl ether to give a pale green solid, which was recrystallized from hot water/acetone to give the product in the form of a pale green solid consisting of 2 diastereoisomers, L-Nva-L-Ala-L-Fos and L-Nva-L-Ala-D-Fos (0.22 g, 0.75 mmol, 47%); m.p. 200-210 C. (decomp.); .sub.max/cm.sup.1 3280 (NH.sup.+), 1643 (CO), 1552 (NH bend), 1149 (PO), 1037 (PO), 922 (PO); .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 0.96 (3H, t, CH.sub.3-12), 1.29 (3H, m, CH.sub.3-6) 1.42-1.40 (5H, [m, CH.sub.3-2], [m, CH.sub.2-11]), 1.88-1.86 (2H, m, CH.sub.2-10), 4.02-4.00 (2H, [m, CH-5], [m, CH-9]), 4.34-4.39 (1H, m, CH-1); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 13.4 (CH.sub.3-12), 16.0 (CH.sub.3-2), 17.1 (CH.sub.3-6), 17.2 (CH.sub.3-6), 18.1 (CH.sub.2-11), 33.5 (CH.sub.2-10), 50.7 (d, J.sub.PC=22.5 Hz, CH-1), 53.5 (CH-5 or CH-9), 170.4 (CO-8), 174.7 (CO-4); HRMS (nanospray ionisation) calculated for (C.sub.10H.sub.23N.sub.3O.sub.5P).sup.+ 296.1370, found 296.1373; .sup.31P.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 18.5; CHN [Found: C, 37.61; H, 7.51; N, 12.91. C.sub.10H.sub.22N.sub.3O.sub.5P.1.4H.sub.2O requires C, 37.48; H, 7.80; N, 13.11%].
Example 8: Synthesis method of antimicrobial compound J (FIG. 2): L-methionyl-L-alanyl-D/L fosfalin
(125) The compound L-methionyl-L-alanyl-D/L fosfalin is represented by the following formula:
(126) ##STR00041##
(127) The synthesis of this antimicrobial compound is detailed below.
8.1. Synthesis of the reaction intermediate Tert-butyl ((S)-1-(((R)-1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)carbamate (tBOC-L-Ala-D/L-Fos diethyl ester)
(128) .sup.tBoc-L-Ala-D/L-Fos diethyl ester is represented by the following formula:
(129) ##STR00042##
(130) Some N-methylmorpholine (15.0 mmol, 1.65 mL) at 5 C. was added to a solution of .sup.tBOC-L-Ala-OH (10.0 mmol, 1.90 g) in dry THF (60 mL). Some isobutyl chloroformate (15.0 mmol, 1.90 mL) and the resulting mixture were agitated at 5 C. for 1 hour. Some diethyl 1-aminoethylphosphonate (10.0 mmol, 1.84 g) in dry THF (20 mL) at 5 C. was added to the agitated mixture. The resulting mixture was agitated under nitrogen at 5 C. for 30 minutes, then at ambient temperature overnight. The solution was filtered and concentrated under vacuum to give a pale yellow syrup, which was redissolved in DCM (60 mL) and washed with 10% weight/volume citric acid (225 mL), 10% weight/volume potassium carbonate (25 mL) and water (25 mL). The combined organic layers were dried on magnesium sulfate, filtered and concentrated under vacuum to give a pale yellow syrup, which was purified by column chromatography, using initially 100% DCM and increasing to 95:5 DCM/methanol, to give the product in the form of an off-white solid consisting of 2 diastereoisomers, .sup.tBOC-L-Ala-L-Fos diethyl ester and .sup.tBOC-L-Ala-D-Fos diethyl ester (2.49 g, 7.1 mmol, 71%); m.p. 102-105 C.; .sub.max/cm.sup.1 3280 (NH), 1710 (CO), 1652 (CO), 1556 (NH bend), 1229 (PO), 1173 (PO), 1013 (PO), 973 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.23-1.44 (21H, [s, C(CH.sub.3).sub.3], [m, CH.sub.3-2], [m, CH.sub.3-6], [m, 2OCH.sub.2CH.sub.3]), 4.06-4.23 (5H, [m, 2OCH.sub.2CH.sub.3], [m, CH-5]), 4.40-4.52 (1H, m, CH-1), 5.11-5.15 (1H, 2d, J=1.5 Hz, 1.5 Hz, NH-7), 6.70-6.78 (1H, 2d, J=2.3 Hz, 2.3 Hz, NH-3);.sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 15.6 (CH.sub.3-2), 16.3 (d, J=3.0 Hz, OCH.sub.2CH.sub.3), 16.4 (d, J=2.3 Hz, OCH.sub.2CH.sub.3), 16.4 (d, J=3.8 Hz, OCH.sub.2CH.sub.3), 16.5 (d, J=1.5 Hz, OCH.sub.2CH.sub.3), 18.4 (CH.sub.3-6), 28.3 (C(CH.sub.3).sub.3) 40.8 (d, J.sub.PC=156.8 Hz, CH-1), 41.0 (d, J.sub.PC=156.8 Hz, CH-1), 50.0 (CH-5), 62.4-62.8 (4d, J.sub.PC=6.8 Hz, 6.8 Hz, 6.8 Hz, 6.8 Hz, 2OCH.sub.2CH.sub.3), 80.0 (C(CH.sub.3).sub.3), 155.2 (CO-8), 172.1 (CO-4); CHN [Found: C, 48.22; H, 8.58; N, 7.87. C.sub.14H.sub.29N.sub.2O.sub.6P requires C, 47.72; H, 8.30; N, 7.95%].
8.2. Synthesis of the reaction intermediate (S)-1-(((R)-1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-aminium chloride (L-Ala-D/L-Fos diethyl ester hydrochloride)
(131) L-Ala-D/L-Fos diethyl ester hydrochloride is represented by the following formula:
(132) ##STR00043##
(133) A solution of .sup.tBOC-L-Ala-D/L-Fos diethyl ester (obtained in step 8.1.; 6.0 mmol, 2.13 g) in 2M HCl in diethyl ether (100 mL) was agitated under nitrogen at ambient temperature overnight. The resulting solid was collected by filtration and washed with dry diethyl ether. The off-white hygroscopic solid was dried overnight in a desiccator containing phosphorus (V) oxide and washed with petrol to give the product in the form of a pale green solid consisting of 2 diastereoisomers, L-Ala-L-Fos diethyl ester hydrochloride and L-Ala-D-Fos diethyl ester hydrochloride (1.46 g, 5.1 mmol, 84%); m.p. 102-105 C.; .sub.max/cm.sup.1 2986 (NH.sup.+), 1673 (CO), 1555 (NH bend), 1017 (PO), 950 (PO); .sup.1H NMR (300 MHz, d.sub.4-CH.sub.3OH) .sub.H 1.29-1.44 (9H, [m, 2OCH.sub.2CH.sub.3], [m, CH.sub.3-2], 1.51 (3H, d, J=6.0 Hz, CH.sub.3-6), 3.90-3.98 (1H, m, CH-5), 4.08-4.22 (4H, m, 2OCH.sub.2CH.sub.3), 4.28-4.47 (1H, m, CH-1); .sup.13C NMR (75 MHz, d.sub.4-CH.sub.3OH) .sub.C 13.7 (CH.sub.3-2-A), 14.0 (CH.sub.3-2-B), 15.4 (2OCH.sub.2CH.sub.3), 16.3 (CH.sub.3-6), 41.1 (d, J.sub.PC=158.3 Hz, CH-1), 41.4 (d, J.sub.PC=158.3 Hz, CH-1), 48.8 (CH-5), 48.9 (CH-5), 62.7-63.0 (2OCH.sub.2CH.sub.3), 169.0 (CO-4).
8.3. Synthesis of the reaction intermediate Tert-butyl((2S)-1-(((2S)-1-((1-(diethoxyphosphoryl)ethyl)amino)-1-oxopropan-2-yl)amino-4-(methylthio)-1-oxobutan-2-yl)carbamate (tBOC-L-Met-L-Ala-D/L-Fos diethyl ester)
(134) The compound .sup.tBOC-L-Met-L-Ala-D/L-Fos diethyl ester is represented by the following formula:
(135) ##STR00044##
(136) Some N-methylmorpholine (5.1 mmol, 0.60 mL) was added to a solution of .sup.tBOC-L-Met-OH (3.4 mmol, 0.88 g) in dry THF (50 mL). The solution was cooled to 5 C. and some isobutyl chloroformate (5.1 mmol, 0.70 mL) was added dropwise. The mixture was agitated at 5 C. for 1 hour. Some L-Ala-D/L-Fos diethyl ester hydrochloride obtained in step 8.2. (3.4 mmol, 0.97 g) in dry THF (15 mL), which had been neutralised with diisopropylethylamine (5.1 mmol, 1.00 mL) at 5 C., was added dropwise to the agitated solution. The resulting solution was agitated under nitrogen at 5 C. for 30 minutes, and then overnight at ambient temperature. The solution was filtered and concentrated under vacuum, then washed with 10% weight/volume citric acid (225 mL), 10% weight/volume potassium carbonate (25 mL) and water (25 mL). The organic layer was dried on MgSO.sub.4, filtered and concentrated under vacuum to give a yellow solid, which was purified by column chromatography [DCM/MeOH (95:5)] to give the product in the form of an off-white solid consisting of 2 diastereoisomers, .sup.tBOC-L-Met-L-Ala-L-Fos diethyl ester and .sup.tBOC-L-Met-L-Ala-D-Fos diethyl ester (0.53 g, 1.1 mmol, 32%); m.p. 172-176 C.; .sub.max/cm.sup.1 3272 (NH), 1708 (CO), 1637 (CO), 1530 (NH bend), 1226 (PO), 1165 (PO), 1020 (PO), 966 (PO); .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.H 1.16-1.36 (12H, [m, CH.sub.3-2], [m, CH.sub.3-6], [m, 2OCH.sub.2CH.sub.3]), 1.36 (9H, s, C(CH.sub.3).sub.3), 1.82-2.01 (2H, m, CH.sub.2-10), 2.04 (3H, s, CH.sub.3-12), 2.49 (2H, t, J=9.0 Hz, CH.sub.2-11), 4.00-4.12 (4H, m, 2OCH.sub.2CH.sub.3), 4.21 (1H, m, CH-9), 4.33-4.43 (1H, m, CH-1), 4.45-4.53 (1H, m, CH-5), 5.40 (1H, d, J=9.0 Hz, NH-13-A), 5.44 (1H, d, J=6.0 Hz, NH-13-B), 6.85 (1H, d, J=6.0 Hz, NH-7-A), 6.92 (1H, d, J=6.0 Hz, NH-7-B), 7.07 (1H, d, J=9.0 Hz, NH-3-A), 7.16 (1H, d, J=9.0 Hz, NH-3-B); .sup.13C NMR (75 MHz, CDCl.sub.3) .sub.C 14.3 (CH.sub.3-2-A), 14.3 (CH.sub.3-2-B), 15.4-15.5 (2OCH.sub.2CH.sub.3), 17.7 (CH.sub.3-6), 27.3 (C(CH.sub.3).sub.3), 29.2 (CH.sub.2-11-A), 29.3 (CH.sub.2-11-B), 30.8 (CH.sub.2-10-A), 30.8 (CH.sub.2-10-B), 39.9 (d, J.sub.PC=156.8 Hz, CH-1-A), 40.0 (d, J.sub.PC=156.8 Hz, CH-1-B), 47.9 (CH-5-A), 48.0 (CH-5-B), 52.6 (CH-9), 61.5 (d, J.sub.PC=4.5 Hz, OCH.sub.2CH.sub.3-A), 61.6 (d, J.sub.PC=4.5 Hz, OCH.sub.2CH.sub.3B), 61.7 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3-A), 61.9 (d, J.sub.PC=6.8 Hz, OCH.sub.2CH.sub.3B), 79.1 (C(CH.sub.3).sub.3), 154.6 (CO-14), 170.3 (CO-4 or CO-8-A), 170.4 (CO-4 or CO-8-B), 170.5 (CO-4 or CO-8-A), 170.6 (CO-4 or CO-8-B).
8.4. Synthesis of compound J according to the invention, namely the compound Hydrogeno(1-((S)-2-((S)-2-ammonio-4-(methylthio)butanamido)propanamido)ethyl)phosphonate (L-methionyl-L-alanyl-D/L fosfalin)
(137) As indicated previously, the compound L-methionyl-L-alanyl-D/L-fosfalin is represented by the following formula:
(138) ##STR00045##
(139) A solution of .sup.tBOC-L-Met-L-Ala-D/L-Fos diethyl ester (obtained in step 8.3; 0.9 mmol, 0.43 g) in hydrogen bromide/glacial acetic acid (33%) (10 mL) was agitated overnight at ambient temperature. Some dry diethyl ether (150 mL) was added and the mixture was placed in a freezer overnight. The solvent was decanted off and the raw product was triturated with dry diethyl ether (5100 mL). The orange brownish raw solid was dissolved in methanol (5 mL), followed by the addition of excess propylene oxide. The solution was filtered and washed with diethyl ether to give a green solid, which was recrystallized from hot ethanol and further dried in a desiccator containing phosphorus (V) oxide to give the product in the form of a pale green solid consisting of 2 diastereoisomers, L-Met-L-Ala-L-Fos and L-Met-L-Ala-D-Fos (0.13 g, 0.41 mmol, 46%); m.p. 213-217 C. (decomp.); .sub.max/cm.sup.1 3263 (NH.sup.+), 2834 (large OH), 1641 (CO), 1552 (NH bend), 1150 (PO), 1041 (PO), 919 (PO); .sup.1H NMR (300 MHz, D.sub.2O) .sub.H 1.12-1.29 (3H, m, CH.sub.3-2), 1.38 (3H, d, J=6.0 Hz, CH.sub.3-6), 2.11-2.34 (5H, [s, CH.sub.3-12], [m, CH.sub.2-10]), 2.62 (2H, m, CH.sub.2-11), 3.95-4.11 (2H, [m, CH-1], [m, CH-9]), 4.32-4.46 (1H, m, CH-5); .sup.13C NMR (75 MHz, D.sub.2O) .sub.C 14.2 (CH.sub.3-12), 15.5 (CH.sub.3-2), 16.6 (CH.sub.3-6), 28.5 (CH.sub.2-11), 29.5 (CH.sub.2-10), 49.9 (CH-1, CH-5 and CH-9), 169.4 (CO-4 and CO-8); .sup.31P-.sup.1H.sub.decoup NMR (121 MHz, CDCl.sub.3) .sub.P 20.7.
Example 9: Evaluation of the antibacterial activity of L-Norvalinyl-L-alanyl-D/L-fosfalin (compound I in FIG. 2) and of L-Methionyl-L-alanyl-D/L-fosfalin (compound J in FIG. 2) synthesised respectively according to examples 7 and 8
9.1. Introduction
(140) As for example 6, the minimum inhibitory concentrations (MIC) of compounds I and J against 12 strains of Gram-negative bacteria and 6 strains of Gram-positive bacteria were determined after 22 hours of incubation using an agar dilution method as described in example 2 (cf. notably section 2.2 Materials and methods).
(141) The 18 bacteria strains tested for the purposes of the present example are identical to those tested in example 6.
9.2 Results
(142) The results obtained are presented in Table 5 below
(143) TABLE-US-00005 TABLE 5 Minimum inhibitory concentrations of antimicrobial compounds I and J against various bacteria species Minimum inhibitory Minimum inhibitory Strain concentration (MIC) of concentration (MIC) of Species reference compound I (g/mL) compound J (g/mL) Acinetobacter baumannii ATCC 19606 >8 >8 Burkholderia cepacia ATCC 25416 >8 >8 Enterobacter cloacae NCTC 11936 >8 >8 Escherichia coli NCTC 10418 1 2 Escherichia coli NCTC 12241 1 2 Klebsiella pneumoniae NCTC 9528 0.25 0.5 Providencia rettgeri NCTC 7475 >8 >8 Pseudomonas aeruginosa NCTC 10662 >8 >8 Salmonella Typhimurium NCTC 74 >8 >8 Salmonella Enteritidis NCTC 6676 >8 >8 Serratia marcescens NCTC 10211 0.25 0.5 Yersinia enterocolitica NCTC 11176 0.125 0.5 Enterococcus faecalis NCTC 775 0.063 0.125 Enterococcus faecium NCTC 7171 1 4 Listeria monocytogenes NCTC 11994 >8 >8 Staphylococci epidermidis NCTC 11047 1 2 Staphylococcus aureus NCTC 6571 1 1 Staphylococcus aureus NCTC 11939 >8 >8 (SARM)
(144) According to the data presented in table 5 above, it clearly appears that compounds I and J are strongly inhibiting against the tested strains of certain Gram-negative bacteria species and certain Gram-positive bacteria species, notably concerning Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Yersinia enterocolitica, Enterococcus faecalis, and less inhibiting against other Gram-negative and Gram-positive bacteria species, notably with regard to Acinetobacter baumannii, Burkholderia cepacia, Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis, Listeria monocytogenes.
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