Gold (I)-Phosphine Compounds as Anti-Bacterial Agents

Abstract

A compound of formula (I): for use in the prevention or treatment of a bacterial infection wherein: A is either S or Se; RA is selected from: wherein: each of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is independently selected from CH or N, wherein at least three of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is CH; V is selected from O, CH—OR.sup.01, N—CO.sub.2—R.sup.C2 or N—R.sup.N2; one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; X is selected from NH, S or O; R.sup.C1 is selected from O—R.sup.O2 or NHR.sup.N1; R.sup.O1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.O2 is C.sub.1-3 unbranched alkyl; R.sup.N1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.N2 is C.sub.1-3 unbranched alkyl; R.sup.C2 is either C.sub.1-3 unbranched alkyl or C.sub.3-4 branched alkyl; R.sup.C3 is selected from C.sub.1-3 unbranched alkyl and C.sub.2H.sub.4CO.sub.2H; R.sup.C4 is either H or Me; R.sup.C5 is either H or Me; R.sup.C6 represents one or two optional methyl substituents; and n is an integer from 2 to 8.

##STR00001##

Claims

1. A compound of formula (I): ##STR00048## for use in the prevention or treatment of a bacterial infection wherein: A is S; R.sup.A is selected from: ##STR00049## wherein: each of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is independently selected from CH or N, wherein at least three of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is CH; V is selected from O, CH—OR.sup.O1, N—CO.sub.2—R.sup.C2 or N—R.sup.N2; one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; X is selected from NH, S or O; R.sup.C1 is selected from O—R.sup.O2 or NHR.sup.N1; R.sup.O1 is selected from H and C.sub.1-3 unbranched alkyl; is C.sub.1-3 unbranched alkyl; R.sup.N1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.N2 is C.sub.1-3 unbranched alkyl; R.sup.C2 is either C.sub.1-3 unbranched alkyl or C.sub.3-4 branched alkyl; R.sup.C3 is selected from C.sub.1-3 unbranched alkyl and C.sub.2H.sub.4CO.sub.2H; R.sup.C4 is either H or Me; R.sup.C5 is either H or Me; R.sup.C6 represents one or two optional methyl substituents; and n is an integer from 2 to 8.

2. A compound according to claim 1, wherein R.sup.A is A1: ##STR00050## and either: (a) one of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is N, or (b) two of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 are N, or (c) R.sup.A is phenyl.

3-5. (canceled)

6. A compound according to claim 1, wherein R.sup.A is A2: ##STR00051## and either: (a) V is O; or (b) V is CH—OR.sup.O1; or (c) V is N—CO.sub.2—R.sup.C2; or (d) V is N—R.sup.N2.

7-13. (canceled)

14. A compound according to claim 2, wherein there are no optional methyl substituents.

15. A compound according to claim 2, wherein there is a single methyl substituent represented by R.sup.C6.

16. A compound according to claim 2, wherein there are two methyl substituents represented by R.sup.C6.

17. A compound according to claim 1, wherein R.sup.A is A3: ##STR00052## and either: (a) X is O and one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is N; or (b) X is NH and Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 are CH.

18-19. (canceled)

20. A compound according to claim 1, wherein R.sup.A is R.sup.A is A4: ##STR00053## and either: (a) R.sup.C1 is O—R.sup.O2 where R.sup.O2 is methyl; or (b) R.sup.C1 is NHR.sup.N1, and R.sup.N1 is H.

21-22. (canceled)

23. A compound according to claim 20, wherein either: (a) R.sup.C4 and R.sup.C5 are both H; or (b) R.sup.C4 is H and R.sup.C5 is Me; or (c) R.sup.C4 and R.sup.C5 are both Me.

24-25. (canceled)

26. A compound according to claim 1, wherein R.sup.A is A5: ##STR00054## and either: (a) R.sup.C3 is methyl; or (b) R.sup.C3 is C.sub.2H.sub.4CO.sub.2H.

27-28. (canceled)

29. A compound according to claim 26, wherein n is an integer from 4 to 8.

30. A compound according to claim 29, wherein the bacterial infection prevented and/or treated is infection by one or more Gram-positive bacteria.

31. A compound according to claim 29, wherein the bacterial infection prevented and/or treated is infection by one or more Gram-negative bacteria.

32-39. (canceled)

40. A method of removing or eliminating an existing biofilm, inhibiting biofilm formation, reducing the biomass of a biofilm, promoting the dispersal of microorganisms from a biofilm, sensitizing a microorganism in a biofilm to an antimicrobial agent, killing a microorganism within a biofilm, treating or preventing an infection, disease or disorder caused by a biofilm, inhibiting the growth of a microbial persister cell, killing a microbial persister cell, or treating or preventing an infection, disease or disorder caused by or associated with a microbial persister cell; the method comprising exposing the biofilm to an effective amount of a compound as described in claim 1.

41-42. (canceled)

43. The method according to claim 40, wherein the biofilm comprises bacteria, or the microbial persister cells are bacteria.

44-47. (canceled)

48. The method according to claim 40, comprising further administering at least one additional antimicrobial agent.

49. A medical device coated or impregnated with a compound as described in claim 1.

50. A compound of formula (I): ##STR00055## wherein A and R.sup.A are as defined in claim 1, with the proviso that the compound is not: ##STR00056##

51. A compound according to claim 50, wherein if A is S, R.sup.A is (A3), then one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is N.

52. A compound according to claim 50, wherein if A is S, R.sup.A is (A1), then Y.sup.1 and Y.sup.2 are CH and Y.sup.3, Y.sup.4 and Y.sup.9 are independently selected from N and CH.

53. A pharmaceutical composition comprising a compound according to claim 50.

54. A pharmaceutical composition according to claim 53, which also comprises a pharmaceutical acceptable diluent or excipient.

55-59. (canceled)

Description

EXAMPLES

[0222] Analytical Methods

[0223] MeCN-FA Method: Phenomenex Luna C18(2) 3 μm, 4.6×50 mm; H.sub.2O+0.1% formic acid; B=MeCN+0.1% formic acid; 45° C.; 0 min 5%, 1 min 37.5%, 3 min 95%, 3.5 min 95%, 3.51 min 5%, 4.5 min 5%; 2.2-2.3 mL/min.

[0224] MeOH-Bicarbonate Method: Phenomenex Luna C18(2) 3 μm, 4.6×50 mm; H.sub.2O+10 mmol ammonium bicarbonate; B=MeOH; 45° C.; 0 min 5%, 1 min 37.5%, 3 min 95%, 3.5 min 95%, 3.51 min 5%, 4.5 min 5%; 2.2-2.3 mL/min.

[0225] Synthesis of Key Intermediates

(R)-2-Acetylamino-3-((R)-2-acetylamino-2-methoxycarbonyl-ethyldiselenyl)-propionic acid methyl ester (I1) (Used During Synthesis of 9)

[0226] ##STR00020##

[0227] Anhydrous MeOH (15 mL) was cooled to 0° C. and acetyl chloride (1.6 mL, 22.5 mmol) added dropwise over the course of 5 minutes. The colourless solution was stirred at 0° C. for 10 minutes whereupon L-selenocysteine (500 mg, 1.5 mmol) was added in one portion. The resultant yellow reaction mixture was warmed to rt and stirred at this temperature for 24 h before concentrating in vacuo to give the crude di-selenide ester hydrochloride as a yellow solid. The crude material was re-suspended in DCM (15 mL) and cooled to 0° C. at which point Et.sub.3N (1 mL, 7.5 mmol) was added followed by acetyl chloride (0.3 mL, 4.5 mmol). The reaction was stirred at rt for 4 h (reaction complete by LC-MS) before DCM (30 mL) and H.sub.2O (30 mL) were added. The layers were separated and the aqueous phase extracted with DCM (2×20 mL). The combined organic extracts were passed through a phase separator cartridge and the solvent removed in vacuo to give the crude product as a yellow oil which was purified by column chromatography (Biotage Isolera 4) eluting with neat EtOAc to provide the title compound as a colourless oil (270 mg, 0.6 mmol, 41%).

(R)-2-Acetylamino-3-((R)-2-acetylamino-2-methoxycarbonyl-ethyldiselenyl)-propionic acid ethyl ester (I4) (Used During Synthesis of 18)

[0228] ##STR00021##

[0229] Procedure as described for (R)-2-Acetylamino-3-((R)-2-acetylamino-2-methoxycarbonyl-ethyldiselenyl)-propionic acid methyl ester I1, except that anhydrous EtOH was used instead of MeOH. The method provided the title compound as a clear oil (412 mg, 0.87 mmol, 58%).

(R)-2-Acetylamino-3-mercapto-propionamide (I2) (Used During Synthesis of 4)

[0230] ##STR00022##

[0231] (R)-2-Acetylamino-3-mercapto-propionic acid methyl ester (177 mg, 1.0 mmol) was dissolved in NH.sub.4OH (28% aq., 5 mL) and the reaction mixture stirred at rt for 7 days. The solvent was evaporated in vacuo to give a white solid (150 mg, 0.93 mmol, 93%).

(R)-2-Acetylamino-3-mercapto-propionic acid ethyl ester (I3) (Used During Synthesis of 8)

[0232] ##STR00023##

[0233] N-Acetyl-L-cysteine (4.7 g, 28.8 mmol) was dissolved in ethanol (140 mL) and the reaction mixture degassed and flushed with N.sub.2 before cooling to 0° C. SOCl.sub.2 (2.4 mL) was then added drop wise before allowing the reaction to warm to rt and stir at this temperature for 4 h. The solvent was removed in vacuo to give a yellow oil which was then diluted with water and EtOAc. The layers were separated and the aqueous phase extracted with EtOAc (3×). The combined organic extracts were dried over MgSO.sub.4 and evaporated to dryness before purifying by flash column chromatography (Biotage Isolera Four, 100 g KPSiI column, EtOAc) to afford the desired product as a pale yellow oil which crystallized to give a white solid (2.59 g, 13.5 mmol, 47%).

S-pyrimidin-5-yl N,N-dimethylcarbamothioate I5 (Used During Synthesis of 20)

[0234] ##STR00024##

(a) Dimethyl-thiocarbamic acid pyrimidin-5-yl ester

[0235] NaH (60% dispersion, 450 mg, 11.25 mmol) was added to 5-hydroxypyrimidine (1.03 g, 10.72 mmol) in dry DMF (10 mL) at rt. The reaction mixture was stirred for 10 min, then N,N-dimethylthiocarbamoyl chloride (1.39 g, 11.25 mmol) was added. The reaction mixture was heated to 90° C. for 1 h, then stirred at rt for 18 h. The reaction mixture was poured into brine (40 mL) and was extracted with DCM (3×30 mL). The combined organic extracts were passed through a phase separator cartridge and the solvent removed in vacuo to give the crude product which was purified by flash column chromatography (Biotage Isolera 4) eluting with neat iso-hexane to 50% EtOAc/iso-hexane to give the title compound as a yellow solid (212 mg, 1.16 mmol, 11%).

(b) S-pyrimidin-5-yl N,N-dimethylcarbamothioate I5

[0236] Dimethyl-thiocarbamic acid pyrimidin-5-yl ester (120 mg, 0.66 mmol) was dissolved in DMSO. The reaction mixture was heated to 200° C. for 3 h in a microwave reactor. Purification was achieved by preparative HPLC (MeOH—H.sub.2O, pH2) to afford the product as an orange solid (37 mg, 0.21 mmol, 31%).

Example 1

[0237] ##STR00025##

[0238] Method A: To a stirred suspension of the chloro (trimethylphosphine) gold (I) compound (125 mg, 0.41 mmol) in EtOH (1 mL) at 0° C., was slowly added the appropriate thiol (0.41 mmol) as a solution in 10% K.sub.2CO.sub.3 (aq., 1 mL) and EtOH (1 mL). The reaction was stirred at 0° C. for 1 hour before warming to rt and allowing to stir at this temperature for 3 h. Once the reaction had gone to completion (by TLC) the reaction was diluted with H.sub.2O (5 mL) and the solution extracted with DCM (3×15 mL). The combined organic extracts were passed through a phase separator cartridge and the solvent evaporated to provide the title compound.

[0239] Method B: As Method A, except after stirring at 0° C. the reaction was heated at 50° C. for 16 h whereupon a thick white ppt had formed. The solid was collected by filtration, washed with EtOH (1 mL) and H.sub.2O (2 mL) before drying under high vacuum for 24 h to give the title compound.

[0240] Method C: As Method A, except the reaction is stirred at 0° C. for 1 hour only

[0241] Method D: As Method C, except the reaction mixture is acidified with aq. 1M KHSO.sub.4 to pH=3-4 before extraction.

[0242] The following compounds were made using these methods:

TABLE-US-00002 TABLE 2 Compound Analytical Data Structure Number Method Yield/Physical appearance [00026] 1 B .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.58 (d, 9H, J = 11.4 Hz), 6.97 (m, 2H), 7.24 (br s, 2H), 12.16 (br s, 1H). .sup.13C-NMR (100 MHz, DMSO-d6): δ ppm 14.68 (d, J = 37.3 Hz), 120.22 (s)..sup.31P- NMR (162 MHz, DMSO-d6): δ ppm −0.02 (s). White solid, 133 mg, 78% [00027] 2 C .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.55 (d, 9H, J = 10.4 Hz), 2.05 (s, 3H), 3.31 (dd, 1H, J = 13.1, 4.5 Hz), 3.43 (dd, 1H, J = 13.1, 4.5 Hz), 3.74 (s, 3H), 4.78 (dt, 1H, J = 7.8, 4.5 Hz), 6.70 (d, 1H, J = 7.8 Hz). .sup.13C-NMR (100 MHz, CDCl3): δ ppm 15.99 (d, J = 36.6 Hz), 23.36 (s), 30.29 (s), 52.35 (s), 54.75 (s), 169.82 (s), 171.69 (s). .sup.31P- NMR (162 MHz, CDCl3): δ ppm −1.76 (s). White solid; 144 mg, 79% [00028] 3 C .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.68 (d, 9H, J = 11.6 Hz), 7.17 (dd, 1H, J = 8.1, 5.1 Hz), 7.84 (dd, 1H, J = 8.1, 1.5 Hz), 8.27 (dd, 1H, J = 5.1, 1.5 Hz). .sup.13C-NMR (100 MHz, DMSO-d6): δ ppm 14.55 (d, J = 38.8 Hz), 116.20 (s), 118.12 (s), 143.57 (s), 144.64 (s), 156.80 (s), 174.45 (s). .sup.31P-NMR (162 MHz, DMSO-d6): δ ppm −0.09 (s). Pale brown solid; 115 mg, 83% [00029] 4 C .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.59 (d, 9H, J = 10.4 Hz), 2.05 (s, 3H), 3.08 (dd, 1H, J = 12.9, 7.8 Hz), 3.44 (dd, 1H, J = 12.9, 4.5 Hz), 4.50 (m, 1H), 5.45 (br s, 1H), 6.85 (d, 1H, J = 6.3 Hz), 7.10 (bs, 1H). .sup.13C-NMR (100 MHz, CDCl3): δ ppm 15.98 (d, J = 35.0 Hz), 23.46 (s), 30.71 (s), 57.19 (s), 170.25 (s), 173.56 (s). .sup.31P-NMR (162 MHz, CDCl3): δ ppm −3.12 (s) White solid; 65 mg, 52% [00030] 5 A .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.55 (d, 9H, J = 10.4 Hz), 3.09 (m, 2H), 3.37 (s, 3H), 3.54 (m, 2H), 3.61-3.66 (m, 24H). .sup.13C-NMR (100 MHz, CDCl3): δ ppm 16.17 (d, J = 35.9 Hz), 26.88 (s), 29.71 (s), 59.06 (s), 69.95 (s), 70.51 (s), 70.56 (s), 70.59 (s), 71.92 (s), 76.44 (s). .sup.31P-NMR (162 MHz, CDCl3): δ ppm −1.77 (s) Clear gum; 35 mg, 56% [00031] 6 A .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.56 (d, 9H, J = 10.4 Hz), 2.61 (t, 2H, J = 6.1 Hz), 3.1 (t, 2H, J = 7.6 Hz), 3.60-3.66 (m, 30H), 3.76 (t, 2H, J = 6.1 Hz). .sup.13C-NMR (100 MHz, CDCl3): δ ppm 16.03 (d, J = 36.6 Hz), 29.70 (s), 35.14 (s), 66.68 (s), 69.98 (s), 70.17 (s), 70.26 (s), 70.45 (s), 70.50 (s), 70.53 (s), 70.58 (s), 174.17 (s). .sup.31P-NMR (162 MHz, CDCl3): δ ppm −1.52 (s) Clear gum; 42 mg, 57% [00032] 7 C .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.64 (d, 9H, J = 11.4 Hz), 7.00 (m, 1H), 7.57 (bs, 1H), 8.06 (bs, 1H), 12.50 (br s, 1H). .sup.13C-NMR (100 MHz, DMSO-d6): δ ppm 16.67 (d, J = 38.1 Hz). .sup.31P-NMR (162 MHz, DMSO-d6): δ ppm −0.17 (bs) Brown solid; 152 mg, 89% [00033] 8 C .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.31 (t, 3H, J = 7.3 Hz), 1.58 (d, 9H, J = 10.1 Hz), 2.07 (s, 3H), 3.34 (dd, 1H, J = 13.4, 4.5 Hz), 3.44 (dd, 1H, J = 13.4, 4.5 Hz), 4.21 (m, 2H), 4.77 (dt, 1H, J = 7.5, 4.5 Hz), 6.73 (d, 1H, J = 7.5 Hz). .sup.13C-NMR (100 MHz, CDCl3): δ ppm 14.34 (s), 16.01 (d, J = 36.6 Hz), 23.38 (s), 30.29 (s), 54.73 (s), 61.26 (s), 169.81 (s), 171.18 (s)..sup.31P-NMR (162 MHz, CDCl3): δ ppm −2.38 (s) Clear gum; 443 mg, 89% [00034] 10 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.58 (d, 9H, J = 10.6 Hz), 6.97 (tt, 1H, J = 7.3, 1.3 Hz), 7.06-7.12 (m, 2H), 7.51-7.55 (m, 2H). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −1.94 (s) White solid; 121 mg, 97% [00035] 11 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.54 (d, 9H, J = 10.6 Hz), 6.77 (ddd, 1H, J = 7.4, 5.0, 0.8 Hz), 7.23 (td, 1H, J 7.4, 2.0 Hz), 7.39 (br d, 1H, J = 8.1 Hz), 8.18 (dd, J 5.0, 2.0 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −2.37 (s) Pale yellow solid; 122 mg, 98% [00036] 12 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.61 (d, 9H, J = 10.6 Hz), 6.80 (t, 1H, J = 4.8 Hz), 8.33 (d, 1H, J = 4.8 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −2.35 (s) Off white solid; 118 mg, 95% [00037] 13 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.56 (d, 9H, J = 10.1 Hz), 1.70-1.85 (m, 2H), 1.90-2.05 (m, 2H, J = 4.8 Hz), 2.11 (br d, 2H, J = 12.9 Hz), 2.24 (s, 3H), 2.85 (br d, 2H, J = 12.1 Hz), 3.18 (br t, 1H, J = 9.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −2.05 (s) White solid; 49 mg, 38% [00038] 14 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.66 (d, 9H, J = 10.9 Hz), 7.05 (dd, 1H, J = 7.8, 4.8 Hz), 7.87 (dd, 1H, J = 7.8, 1.8 Hz), 8.47 (dd, 1H, J = 4.8, 1.8 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −2.36 (s) Off-white solid; 113 mg, 79% [00039] 15 C .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.09-1.22 (m, 2H), 1.28-1.40 (m, 2H), 1.55 (d, 9H, J = 10.6 Hz), 1.76 (br d, 2H, J = 12.6 Hz), 1.95 (br d, 2H, J = 12.8 Hz), 2.96 (tt, 1H, J = 11.4, 3.8 Hz), 3.31 (m, 1H), 4.43 (d, 1H, J = 4.3 Hz). .sup.31P-NMR (162 MHz, DMSO-d6): δ ppm 1.85 (s) White solid; 50 mg, 38% [00040] 16 C .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.55 (d, 9H, J = 10.4 Hz), 1.74 (ddd, 2H, J = 15.9, 11.6, 4.3 Hz), 2.04 (br d, 2H, J = 13.1 Hz), 3.32-3.42 (m, 3H), 3.95 (br d, 2H, J = 13.1 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm 0.47 (s) White solid; 150 mg, 86% [00041] 17 D .sup.1H-NMR (400 MHz, CDCl3): δ ppm 1.30 (s, 3H), 1.55 (s, 3H), 1.60 (d, 9H, J = 10.4 Hz), 2.07 (s, 3H), 4.95 (br s, 1H), 6.63 (br s, 1.0 Hz) .sup.13C-NMR (100 MHz, CDCl3): δ ppm 14.34 (s), 16.01 (d, J = 36.6 Hz), 23.38 (s), 30.29 (s), 54.73 (s), 61.26 (s), 169.81 (s), 171.18 (s). .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −0.42 (s) White solid; 81 mg, 54%

Example 2

[0243] ##STR00042##

[0244] The appropriate diselenide (0.085 mmol) was dissolved in EtOH (1 mL) and the reaction cooled to 0° C. NaBH.sub.4 (7 mg, 0.17 mmol) was added in one portion and the pale yellow solution stirred at 0° C. for 20 minutes. Chloro(trimethyl phosphine) gold (I) (53 mg, 0.0.17 mmol) was then added in one portion and the reaction warmed to rt and stirred at this temperature for 3 h. The reaction mixture was diluted with DCM (30 mL) and subsequently washed with saturated NH.sub.4Cl (aq., 20 mL), saturated NaHCO.sub.3 (aq., 20 mL) and finally water (20 mL). The organic phase was passed through a phase separator cartridge and the solvent removed in vacuo to give a brown oil which was purified by column chromatography (Biotage Isolera 4) eluting with neat EtOAc to 1:1 EtOAc-WIPE 129 to provide the title compound.

[0245] The following compounds were made using this method:

TABLE-US-00003 Compound Analytical Data Structure Number Yield/Physical appearance [00043] 9 .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.55 (d, 9H, J = 11.1 Hz), 1.86 (s, 3H), 2.85 (dd, 1H, J = 11.9, 7.8 Hz), 2.98 (dd, 1H, J = 11.9, 5.8 Hz), 3.62 (s, 3H), 4.37 (dt, 1H, J = 7.6, 5.8 Hz), 8.1 (d, 1H, J = 7.6 Hz). .sup.13C-NMR (100 MHz, DMSO- d6): δ ppm 14.80 (d, J = 35.1 Hz), 18.09 (s), 21.73 (s), 22.41 (s), 51.78 (s), 169.08 (s), 171.64 (s). .sup.31P-NMR (162 MHz, DMSO-d6): δ ppm 3.05 (s) Colourless gum (58 mg, 0.12 mmol, 69%) [00044] 18 .sup.1H-NMR (400 MHz, DMSO-d6): δ ppm 1.19 (t, 3H, J = 7.1 Hz), 1.54 (d, 9H, J = 10.9 Hz), 1.86 (s, 3H), 2.84 (dd, 1H, J = 11.9, 7.6 Hz), 2.98 (dd, 1H, J = 11.9, 5.6 Hz), 4.07 (dq, 2H, J = 7.1, 1.5 Hz), 4.33 (dt, 1H, J = 7.6, 5.6 Hz), 8.04 (d, 1H, J = 7.6 Hz). .sup.31P-NMR (162 MHz, DMSO- d6): δ ppm −2.84 (s) Orange solid; 71 mg, 67%

Example 3

[0246] ##STR00045##

[0247] The appropriate sulfanyl formamide (0.329 mmol) was dissolved in a mixture of MeOH (1 mL) and 10% aq NaOH (0.3 mL). The reaction mixture was heated to 100° C. for 1 hour in a microwave reactor, cooled to 0° C. and chloro(trimethylphosphine)gold(I) (101 mg, 0.33 mmol) added in one portion. The reaction mixture was stirred at 0° C. for 1 h, then poured into H.sub.2O (10 mL) and extracted with DCM (3×20 mL). The combined organic extracts were passed through a phase separator cartridge and the solvent removed in vacuo to give the title compound.

TABLE-US-00004 Num- Analytical Data Structure ber Yield/Physical appearance [00046] 19 .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.60 (d, 9H, J = 10.6 Hz), 7.00 (ddd, 1H, J = 5.6, 4.8, 0.8 Hz), 7.75 (ddd, 1H, J = 8.1, 4.6, 0.8 Hz), 7.75 (ddd, 1H, J = 8.1, 2.5, 1.5 Hz), 8.16 (dd, J = 4.8, 1.2 Hz), 8.81 (dd, J = 2.5, 0.8 Hz) .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −1.04 (s) Off-white solid; 116 mg, 92% [00047] 20 .sup.1H-NMR (400 MHz, CDCl.sub.3): δ ppm 1.62 (d, 9H, J = 10.6 Hz), 8.75 (s, 1H), 8.81 (s, 1H) .sup.31P-NMR (162 MHz, CDCl.sub.3): δ ppm −1.95 (s) Off-white solid; 67 mg, 91%

Example 4

[0248] Growth Media

[0249] Tryptic Soy Broth

TABLE-US-00005 Formula/Litre Pancreatic Digest of Casein 17.0 g  Enzymatic Digest of Soybean 3.0 g Sodium Chloride 5.0 g Di-potassium hydrogen Phosphate 2.5 g Glucose 2.5 g

[0250] Directions for use: Dissolve 30 g of the medium in one litre of purified water, mix thoroughly, and then autoclave at 121° C. for 15 minutes.

[0251] Luria Broth

TABLE-US-00006 Formula/Litre Tryptone 10.0 g  Yeast Extract 5.0 g NaCl 5.0 g

[0252] Directions for use: Dissolve components in 1 litre of distilled or deionized water and sterilize by autoclaving at 121° C. for 15 minutes.

[0253] Mueller Hinton II Broth (Cation-Adjusted)

TABLE-US-00007 Formula/Litre Beef Extract 3.0 g Acid Hydrolysate of Casein 17.5 g  Starch 1.5 g *Adjusted and/or supplemented as required with appropriate salts to provide 20-25 mg/L of calcium and 10-12.5 mg/L of magnesium and as additionally required to meet performance criteria.

[0254] Directions for use: Dissolve components in 1 litre of distilled or deionized water andand sterilize by autoclaving at 121° C. for 15 minutes.

[0255] Brain Heart Infusion Broth

TABLE-US-00008 Formula/Litre Brain Heart Infusion solids 12.5 g   Beef heart infusion solids 5 g Proteose peptone 10 g  Glucose 2 g Sodium Chloride 5 g Di-sodium Phosphate 2.5 g  

[0256] Directions for use: Dissolve components in 1 litre of purified water. Heat the mixture with frequent agitation to completely dissolve the medium, and sterilize by autoclaving at 121° C. for 15 minutes.

[0257] Growth Assay for S. aureus. (NCTC8325)

[0258] Stock solution of the test compounds (20 mg/ml) in dimethyl sulfoxide (DMSO) were serially diluted in DMSO and each diluted compound added in duplicate to a 96-well plate to a final DMSO concentration of 2% (v/v). An overnight culture of S. aureus (Oxford strain) grown in tryptic soy broth (TSB) was diluted to approximately 5×10.sup.7 cfu/ml and 150 μl of this sample was added to each well of the 96-well plates. Control wells included an ‘untreated’ control with bacteria in TSB in the presence of 2% DMSO and a negative sample (containing 150 μl TSB growth media in the presence of 2% DMSO). Plates were incubated in a shaking incubator at 37° C. for 22 h and bacterial growth assessed by absorbance at a wavelength of 595 nm. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound that inhibited growth compared to the no-treatment control.

[0259] Variation of Growth Assays for:

[0260] Klebsiella pneumoniae (NCTC 13443), Vibrio cholerae, E. coli (ATCC 25922), Acinetobacter baumannii (ATCC BAA-747), Klebsiella oxytoca, Proteus vulgaris (ATCC 6380 or Enterobacter cloacae: use of 1/100 overnight dilution to set up assay, medium used: Luria broth (LB); incubation without shaking.

[0261] P. aeruginosa (ATCC 27853): use of 1/100 overnight dilution to set up assay, medium used: Cation adjusted Mueller Hinton broth (CaMHB); incubation without shaking.

[0262] Enterococcus feacalis (ATCC29212): use of 1/100 overnight dilution to set up assay, medium used: brain heart infusion broth containing 0.5% yeast extract; incubation without shaking.

TABLE-US-00009 S. aureus E. faecalis K. pneumoniae A. baumannii E. coli MIC MIC MIC MIC MIC Compound (μg/mL) (μg/mL) (μg/mL) (μg/mL) (μg/mL) 1 0.8-1.6 1.6-3.1 3.1 1.6 2 0.2-0.7 2    6.3-12.5 3.1 3.1 3 0.8-1.6 1.6 3.1-6.3 1.6 1.6 4 1.6 12.5 3.1  6.3-12.5 5 3.1 25 6.3 6 3.1 50  6.3-12.5 7 1.3 2.5 8 0.8-1.6 6.3 3.1  6.3-12.5 9 1.6 3.1-6.3 10 0.8 1.6 0.8 1.6 11 0.8 1.6 0.8-1.6 1.6 12 0.8 1.6-3.1 0.8-1.6 1.6-3.1 13 1.6-3.1 6.3 6.3 6.3 14 ≦0.8 1.6 1.6 15 ≦0.8 3.1 1.6-3.1 3.1-6.3 16 1.6 6.3 1.6 3.1-6.3 17 1.6 25 6.3 6.3 18 >100 >100 >100    19 ≦0.8 3.1 3.1 20 3.1 3.1 3.1 P. aeruginosa V. cholerae K. oxytoca P. vulgaris E. cloacae MIC MIC MIC MIC MIC Compound (μg/mL) (μg/mL) (μg/mL) (μg/mL) (μg/mL) 1 12.5 0.8 2 12.5 0.8 3.1-6.3 3.1-6.3 3.1-6.3 3 12.5 0.8-1.6 1.6 0.8-1.6 3.1-6.3 4 12.5 1.6 3.1 3.1 6.3 8  6.3-12.5 0.8-1.6 3.1 3.1 3.1-6.3 10 3.1 11 3.1 12 3.1 13 6.3 14 3.1 16 6.3 19 3.1 20 3.1

[0263] CHO Toxicity Assay

[0264] Cell counting kit-8 (Sigma, CCK-8) assays were performed to assess the effect of compounds on cell viability. The assay is based on the reduction of a water-soluble tetrazolium salt (WST-8) by cellular dehydrogenases to a formazan dye which can be detected spectroscopically. 96-well plates were seeded with chinese hamster ovary cells (CHO) cells at 7×10.sup.3 cells per well in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (containing 15 mM HEPES, NaHCO.sub.3, pyridoxine and L-glutamine) supplemented with 10% fetal bovine serum (FBS). The following day serial dilutions of compounds (dissolved and diluted in DMSO) were added to the cells in duplicates. Control included an ‘untreated’ control where cells were grown in the presence of 1% DMSO and a medium only control (plus 1% DMSO). After 24 hours CCK-8 reagent (10 μl) was added to each well and cell viability was assessed by measuring the absorbance at a wavelength of 450 nm after 2.5-3 hours. Only living cells can reduce the tetrazolium salts into coloured formazan products. Results were expressed as 50% growth inhibition (TD.sub.50) values compared to ‘untreated’ control.

[0265] The therapeutic index was calculated as the ratio of the dose that produces growth inhibition in 50% of CHO cells divided by the dose where 50% of S. aureus growth is inhibited.

TABLE-US-00010 CHO cell Therapeutic Index Compound TD.sub.50 (μg/mL) (CHO) 1 3.2 9.4 2 6.4 26 3 5.0 9.4

[0266] HepG2 Cell Inhibition Assay

[0267] Cell counting kit-8 (Sigma, CCK-8) assays were performed to assess the effect of compounds on cell viability. The assay is based on the reduction of a water-soluble tetrazolium salt (WST-8) by cellular dehydrogenases to a formazan dye which can be detected spectroscopically. 96-well plates were seeded with the human hepatocyte cell line (HepG2) at approximately 8×10.sup.3 cells per well in Minimum Essential Medium Eagle (EMEM) with Earle's salts and sodium bicarbonate supplemented with 10% heat-inactivated foetal bovine serum 2 mM glutamine and 1% non-essential amino acids (NEAA). The following day serial dilutions of compounds (dissolved and diluted in DMSO) were added to the cells in duplicates. Control included an ‘untreated’ control where cells were grown in the presence of 1% DMSO and a medium only control (plus 1% DMSO). After 24 hours CCK-8 reagent (10 μl) was added to each well and cell viability was assessed by measuring the absorbance at a wavelength of 450 nm after 2-3 h hours. Only living cells can reduce the tetrazolium salts into coloured formazan products. Results were expressed as 50% growth inhibition (TD.sub.50) values compared to ‘untreated’ control. The therapeutic index was calculated as the ratio of the dose that produces growth inhibition in 50% of HepG2 cells divided by the dose where 50% of S. aureus or E. coli growth is inhibited.

TABLE-US-00011 HepG2 cell Therapeutic Index Therapeutic Index Compound TD.sub.50 (μg/mL) (HepG2)/S. aureus (HepG2)/E. coli 1 4 10 4 2 11 31 2.5 3 9 10 8 4 22 24 5 5 6.6 6 1 6 22 7 12.5 18 5 8 9.5 32 2 10 2 >2.5 2 11 4.5 >6 5.5 12 4.5 >6 3 13 15 >19 4 14 7 >9 10 15 4.5 >6 5 16 4.5 >6 2 18 >50 19 3 >4 4

[0268] Efficacy Studies in the Galleria mellonella Model

[0269] G. mellonella larvae at 5.sup.th or 6.sup.th instar stage were purchased from a commercial supplier and used within 3 days. Prior to infection larvae were kept at room temperature. Larvae were infected with bacteria (various Gram positive and negative bacteria, including S. aureus, K. pneumoniae, E. coli and P. aeruginosa) using a sterile Hamilton syringe. Bacteria cultures were grown overnight, washed ×3 in PBS and resuspended in PBS. Larvae were wiped with 70% ethanol and 10 μl of bacteria solution (to cause 80% death within 3-4 days) was injected into the bottom right proleg of the larvae. Larvae injected with 10 μl of PBS were used as negative controls. Larvae were then placed in petri dishes (1 dish per condition) containing filter paper at the bottom of the dish at 37° C. After various time points post infection (1-6 h), larvae were taken from the incubator wiped again with 70% ethanol and injected with 10 μl of various concentrations of compound, dissolved in either 5% dimethyl sulfoxide, 5% ethanol or 5% 1-methyl-2-pyrrolidinone into a proleg on the left hand-side. Control larvae received 10 μl of 5% solvent. Ten larvae were injected for each condition. To assess the toxicity of the compound, larvae were injected with various concentrations of compound alone. Larvae were returned to a 37° C. incubator and checked daily. Larvae were considered dead when no movement occurred when touched with a blunt pair of forceps. Black or discoloured larvae which still showed movement were considered to be alive. Numbers of dead larvae were recorded each day.

[0270] Survival Proportions Following Dosing with 10 μl of Compound 8 Solutions of Varying Strengths

TABLE-US-00012 mg/kg Day 1 Day 2 Day 3 Day 4 0 100 100 100 100 3.75 100 100 100 100 7.5 100 100 100 100 15 100 100 100 100 30 100 90 90 90 60 100 80 60 50

[0271] Survival Proportions Following Infection with 10 μl of a 5×10.sup.7 cfu/ml S. aureus Suspension and Treated 4 Hours Later with 10 μl of Compound 8 Corresponding to Concentrations of 2 mg/kg and 4 mg/kg

TABLE-US-00013 mg/kg Day 1 Day 2 Day 3 Day 4 0 90 10 0 0 2 90 60 40 20 4 90 50 40 40

[0272] Compound 8 significantly increased mean survival rates at both 2 mg/kg (p=0.02) and 4 mg/kg (p=0.04).

[0273] Biofilm Prevention Assay

[0274] The effect of a test compound on the formation of a S. aureus biofilm may be assessed using a biofilm prevention assay as described by Merritt et al. Current Protocols in Microbiology, 2011, 1B.1.1-1B1.18 with slight modifications. Briefly, S. aureus NCTC 8325, MRSA (RPAH18) and MRSA (MW2) are grown overnight in Tryptic soy broth (TSB) and diluted to between 1/50 and 1/100 before 150 μL is added to the wells of a flat bottomed 96-well plate. Three microliters of auranofin at the appropriate dilution in DMSO are added to the wells in duplicate. Controls included a serial dilution of lincomycin in ethanol (to assess plate to plate variation), a positive control with bacteria alone in TSB with 2% DMSO and a negative (no bacteria) control with 150 μL TSB containing 2% DMSO. Plates are sealed with AeraSeal™ and incubated at 37° C. for 24 hours. The plates are then washed three times with PBS, dried at 60° C. for 1 hour and stained with crystal violet for 1 hour. The plates are again washed three times with water, dried and scanned prior to the addition of 33% acetic acid to re-solubilize the crystal violet stain bound to the adherent cells. Absorbance is then measured at 595 nm and expressed as a percentage of the bacteria only control.

[0275] The effect of a test compound on preformed S. aureus biofilms can also be assessed. Briefly S. aureus NCTC 8325 is plated in 96-well plates as described in above and incubated 37° C. for 24 hours. Biofilms are then washed 3 times with TSB and 150 μL of fresh TSB and 3 μL of auranofin at the appropriate dilution in DMSO was added to the wells in duplicate. Plates are again sealed with AeraSeal™ and reincubated 37° C. for 24 hours. Biofilm is then detected as described above. Compounds 2, 3, 4, 7, 8, 10, 11, 12, 14 and 15 all disrupted the biofilm.

[0276] Persister Cell Assay

[0277] To determine whether S. aureus persister cells are susceptible to treatment with a test compound, a persister cell (or SCV) isolate hemB mutant of NCTC 8325-4 may be used (Von Eiff et al., (1997) J Bacteriol 179:4706-4712). This persister cell variant displays varying resistance to erythromycin and the aminoglycosides gentamicin and kanamycin. Growth assays are performed essentially as described above with the bacteria being grown in TSB. Disc assays were also performed by plating bacteria on TSB agar. Discs impregnated with an amount of test compound were placed on top of the agar. The plates were incubated overnight at 37° C. and any zone of bacterial inhibition was observed.

[0278] Abbreviations

[0279] aq. Aqueous

[0280] br Broad

[0281] d Doublet

[0282] DCM Dichloromethane

[0283] DMSO Dimethyl sulfoxide

[0284] Et Ethyl

[0285] EtOAc Ethyl acetate

[0286] EtOH Ethanol

[0287] FA Formic acid

[0288] g Gram

[0289] h Hours

[0290] J Coupling constant

[0291] LC-MS Liquid chromatography-mass spectrometry

[0292] Me Methyl

[0293] MeCN Acetonitrile

[0294] MeOH Methanol

[0295] mg Milligram

[0296] min Minutes

[0297] mL Millilitre

[0298] mmol Millimole

[0299] ppm Parts per million

[0300] ppt Precipitate

[0301] q Quartet

[0302] rt Room temperature

[0303] s Singlet

[0304] TLC Thin layer chromatography

[0305] t Triplet

[0306] WIPE Water/isopropanol/Ethyl acetate (1:2:9)

REFERENCES

[0307]

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