Antimicrobial compounds

Abstract

A compound for use as an antimicrobial having a formula (A). ##STR00001##

Claims

1. A compound having a structure selected from the group consisting of: ##STR00080## wherein each of W.sub.1, W.sub.2, W.sub.3, and W.sub.4 is the same and is selected from the group consisting of C.sub.2 alkyl and C.sub.2 alkene; each of Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4 is the same and each is: ##STR00081## each of R.sub.1, R.sub.2, R.sub.4, and R.sub.5 is H; R.sub.3 is (CH.sub.2).sub.nCOOH or an ester thereof and n is 0 or 1; with the proviso that when the structure is ##STR00082## R.sub.3 is not COOCH.sub.3, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein each of W.sub.1, W.sub.2, W.sub.3, and W.sub.4 is a C.sub.2 alkene.

3. The compound of claim 1, having a structure ##STR00083## or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, having a structure ##STR00084## or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, having a structure ##STR00085## or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, having a structure ##STR00086## or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1, having a structure ##STR00087## or a pharmaceutically acceptable salt thereof.

8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is (CH.sub.2).sub.nCOOH or an ester thereof and n is 0 or 1.

9. The compound of claim 7, having a structure ##STR00088## or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is (CH.sub.2).sub.nCOOH or an ester thereof and n is 0 or 1.

11. The compound of claim 1, having a structure ##STR00089## or a pharmaceutically acceptable salt thereof.

12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is (CH.sub.2).sub.nCOOH or an ester thereof and n is 0 or 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1 and 2 provide general pathways for synthesis of the target compounds through the Heck Cross coupling approach.

(2) FIG. 3 is a disk diffusion analysis for TSB007 (135B) showing zone inhibition at 48 hours for Staphylococcus aureus NCTC 10442 MRSA. The disk on the left contained 20 l of 10 mg/ml 135B in DMSO and the disk on the right contained 20 l of DMSO.

(3) FIG. 4 is a disk diffusion analysis for TSB007 (135B), showing the inhibition at 48 hours for Streptococcus pneumoniae ATCC 49619. The disk on the left contained 20 l of 10 mg/ml 135B in DMSO and the disk on the right contained 20 l of DMSO.

(4) FIG. 5 is a disk diffusion analysis for TSB007 (135B), showing the inhibition at 48 hours for Clostridium difficile NCTC 43593. The disk on the left contained 20 l of 10 mg/ml 1356 in DMSO and the disk on the right contained 20 l of DMSO.

(5) FIG. 6 is a graph of the time-kill experiments of S. aureus NCTC 6571 with TSB007 (NAL135B) at concentrations of 1.8 g/ml and 3.6 g/ml. Error bars represent standard deviations of triplicate experiments.

(6) FIG. 7a-i shows tristyrylbenzene structural analogues of TSB007.

(7) FIG. 8 shows distyrylbenzene structural analogues of TSB007.

(8) FIG. 9 shows tetrastyrylbenzene structural analogues of TSB007.

(9) FIG. 10 shows triacryloylbenzene structural analogues of TSB007.

(10) FIG. 11 shows tricinnamylbenzene structural analogues of TSB007.

(11) FIG. 12 shows tricinnamyltriazine structural analogues of TSB007.

EXAMPLES

(12) The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

Example 1

(13) Synthesis of Compounds

(14) The compounds of the present invention are synthesised using the Heck Cross Coupling method.

(15) Generally, the pathways for synthesis are as shown in FIGS. 1 and 2the synthesis of the target compounds through the Heck Cross coupling approach. The reagents and reaction conditions for the chemical transformations shown in these figures are denoted by a letter for each reaction. The key for this labelling is: For FIG. 1: (a). Pd.sub.2(dba).sub.3/[(t-Bu).sub.3PH]BF.sub.4, Tetrahydrofuran (THF), reflux, 17 h (82-97%) (b) LiOH, H.sub.2O/EtOH, reflux, 3 hrs (c) Pd/CH.sub.2, CH.sub.2Cl.sub.2/EtOH, 17 h (d) LiOH, H.sub.2O/EtOH, reflux, 3 hrs For FIG. 2: (a) NaOMe, MeOH, 22, rt. 17 h, 84% (b) Pd/C (10% w/w), H.sub.2, 1:1 CH.sub.2Cl.sub.2/MeOH, 17 h, rt, 93% (c) LiOH, MeOH/H.sub.2O, reflux, 97% (d) Pd.sub.2(dba).sub.3.CHCl.sub.3, [(t-Bu).sub.3PH]BF.sub.4, Cy.sub.2NMe, THF, reflux, 17 h, 84% from 26, 72 h, 29% from 27 (e) LiOH, 9:1 EtOH/H.sub.2O, reflux, 17 h, 66% (f) Pd/C, H.sub.2, 1:1 CH.sub.2Cl.sub.2/EtOH, 17 h, 96% (g) LiOH, EtOH/H.sub.2O, reflux, 17 h, 82%.
General Conditions

(16) Terephthaldehyde, tributylphosphonium tetrafluoroborate, 1,3,5-tribromobenzene and 1,2,4-tribromobenzene were purchased from the Sigma-Aldrich Chemical Company. Dry THF was distilled from sodium benzophenone ketyl radical and stored over a sodium mirror. N-Methyldicyclohexylamine was distilled under reduced pressure and stored under argon. Dess-Martin Periodinane, Herrmann-Beller paladacycle [Herrmann et al. Chem. Int. Ed. Engl. 1995, 34, 1844-1849] (methyl 4-carboxybenzyl)triphenylphosphonium bromide, Pd.sub.2(dba).sub.3.CHCl.sub.3, and Pd(PPh3).sub.4 were prepared as described previously [Ukai et al. Organomet. Chem. 1974, 65, 253-266; Coulson, D. R. Inorg. Synth. 1972, 13, 121-123]. 1,3-di(hydroxymethyl)benzene was prepared by the LiAlH.sub.4 reduction of dimethyl 1,3-benzenedicarboxylate in a similar procedure to the 1,2-isomer [Sharpless, K. B.; Oi, R. Org. Synth. 1996, 73, 1-13] Ethyl 4-vinylbenzoate was prepared by the Fischer esterification of 4-vinyl benzoic acid [Broos et al. J. Chem. Ed. 1978, 55, (12), 813. 33; Tullen et al. J. Chem. Ed. 1971, 48, (7)].

(17) NMR spectra were acquired on either a Bruker AV500 (.sup.1H at 500.13 MHz, .sup.13C at 125.8 MHz) or a Bruker AV600 (.sup.1H at 600.13 MHz, .sup.13C at 150.9 MHz) and all signals are reported in parts per million (ppm). .sup.1H and .sup.13C assignments were made with the aid of DEPT, COSY, HSQC and HMBC sequences where appropriate. .sup.1H spectra were referenced to residual (partially) undeuterated solvents, CDCl.sub.3 (CHCl.sub.3 at 7.26 ppm) and d.sub.6-DMSO (d.sub.5-DMSO at 2.50 ppm (pentet)). .sup.13C spectra were referenced to the deuterated solvents, CDCl.sub.3 at 77.16 ppm and d.sub.6-DMSO at 39.52 ppm.

(18) Infrared spectra samples were prepare using the KBr disc method and samples acquired on a Perkin Elmer. Spectrum One spectrometer at 2 cm.sup.1 resolution. Electronic spectra were collected using a HP8452 spectrophotometer in 1 cm quartz cells at 110.sup.5 or 110.sup.6 MolL.sup.1 in the solvents indicated: Fluorescence spectra were recorded at 110.sup.7 MolL.sup.1.

(19) Mass spectra were acquired on a VG Autospec employing the electron impact (EI) ionization mode.

(20) Standard Conditions for Heck Cross-Coupling Procedure

(21) To a flame-dried schlenk flask was added the halobenzene (1 equiv), Pd.sub.2(dba).sub.3.CHCl.sub.3 (2-15 mol %) and [(t-Bu).sub.3PH]BF.sub.4 (10-60 mol %) which were subsequently dried under vacuum for 15 minutes before being dissolved in dry THF. N-Methyldicyclohexylamine (4 equiv) and ethyl 4-vinylbenzoate 7 (3.3 equiv) were added via syringe and the reaction monitored by TLC (neat CH.sub.2Cl.sub.2). Upon completion of the reaction the residual THF was removed in vacuo, the crude material redissolved in CH.sub.2Cl.sub.2 and filtered to remove any insoluble material before being absorbed onto fine silica and eluting with 0:100 to 2:98 MeOH/CH.sub.2Cl.sub.2.

(22) Standard Method for Reduction of Alkenes

(23) The alkene was loaded into a glass autoclave tube and dissolved/suspended in 1:1 CH.sub.2Cl.sub.2/MeOH or 1:1 CH.sub.2Cl.sub.2/EtOH depending upon the ester present. Argon was bubbled through the mixture for 10 minutes before 10% Pd/C (10 wt % of alkene) was added, and the flask pressurized with H.sub.2 (50 atm). The reaction was allowed to proceed for 17 h before being depressurized, purged with argon, filtered through a pad of celite and concentrated under reduced pressure. Further purification is described for each compound when necessary.

(24) Standard Method for Saponification Reactions

(25) The ester (1 equiv) and LiOH (2 equiv per ester) were dissolved in 9:1 H.sub.2O/MeOH or H.sub.2O/EtOH depending upon the ester and refluxed overnight. After cooling to room temperature the solvent was removed under reduced pressure, the remaining solution diluted with H.sub.2O, cooled in an ice-bath and the pH adjusted to 3 by the addition of HCl (1M). The precipitate was collected filtered and product dried under vacuum.

1,3,5-Tris[(1E)-2-(ethyl 4-benzoate)vinyl]benzene (10)

(26) Prepared as per, the standard procedure using 1,3,5-tribromobenzene 8 (1010 mg, 3.21 mmol), Pd.sub.2(dba).sub.3.CHCl.sub.3 (882 mg, 0.85 mmol), t-Bu.sub.3PHBF.sub.4 (560 mg, 1.93 mmol), Cy.sub.2NMe (3.0 mL), ethyl 4-vinylbenzoate 7 (1870 mg, 10.61 mmol) and THF (40 mL). The product was eluted with 2:98 MeOH/CH.sub.2Cl.sub.2 and recrystallized from CH.sub.2Cl.sub.2/EtOH to give 10 as an off white powder, 1.86 g (97%).

(27) .sup.1H NMR (600 MHz, CDCl.sub.3): 1.42 (t, J=7.1 Hz, 9H, CH.sub.3), 4.40 (q, J=7.1 Hz, 6H, CH.sub.2), 7.24 (AB quartet, 6H, vinyl), 7.60 (d, J=8.3 Hz, 6H, ArH), 7.61 (s, 3H, ArH), 8.06 (d, J=8.3 Hz, 6H, ArH).

(28) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.51 (CH.sub.3), 61.13 (CH.sub.2), 125.0 (CH), 126.5 (CH), 128.7 (CH), 129.7 (C), 130.2 (CH), 130.5 (CH), 137.9 (C), 141.5 (C), 166.5 (CO).

(29) IR (KBr): (cm.sup.1) 2979, 2929, 1713, 1604, 1279, 1178, 1105, 762, 698

(30) HR-EI.sup.+-MS: C.sub.39H.sub.36O.sub.6 requires 600.2512 amu, found 600.2513.

(31) EI.sup.+-MS: MI=C.sub.39H.sub.36O.sub.6; m/z: 600.3 (100%)=MI.sup.+, 555.2 (7%)=[MI-EtO].sup.+.

(32) UV-Vis (CH.sub.2Cl.sub.2): (nm) [log (M.sup.1cm.sup.1)] 258 [4.49], 330 [4.28]

(33) Fluorescence (CH.sub.2Cl.sub.2): excitation (nm) [emission (nm)] 258 [397, 418, 518], 330 [397, 418]; (cyclohexane) 328 [393, 413]

1,3,5-Tris[(1E)-2-(4-benzoic acid)vinyl]benzene (5)

(34) Using the standard saponification procedure, 10 (252.1 mg, 0.42 mmol), LiOH.H.sub.2O (112.0 mg, 2.7 mmol) and 1:9 H.sub.2O/EtOH (20 mL) gave an gelatinous precipitate that was collected and recrystallised from THF/H.sub.2O and dried to give the triacid 5 as a pale brown powder, 209 mg (95%).

(35) .sup.1H, NMR (500.1 MHz, d.sub.6-DMSO): 7.49 (m, 6H, vinyl CH), 7.76 (d, J=8.5 Hz, 6H, ArH), 7.88 (s, 3H, core ArH), 7.98 (d, J=8.5 Hz, 6H, ArH).

(36) .sup.13C NMR (125.8 MHz, d.sub.6-DMSO): (ppm) 125.0, 126.5, 128.4, 129.7, 129.9, 130.50, 137.6, 141.3, 167.1.

(37) IR (KBr): (cm.sup.1) 3067, 3026, 1684 (.sub.CO), 1604, 1566, 1420, 1384, 1312, 1286, 1179.

(38) HR-EI.sup.+-MS: C.sub.33H.sub.24O.sub.6 requires 516.1573 amu. found 516.1564.

(39) EI.sup.+-MS: MI=C.sub.33H.sub.24O.sub.6; m/z: 516.1 (100%)=MI.sup.+, 472.1 (11.3%)=[MI-CO.sub.2].sup.+

1,3,5-Tris[(1E)-2-(ethyl 4-benzoate)ethyl]benzene (11)

(40) Conducted as per the standard reduction procedure with trimester 10 (251 mg, 0.42), Pd/C (20 mg) and 1:1 CH.sub.2Cl.sub.2/EtOH (15 mL). The crude product was recrystallized from CH.sub.2Cl.sub.2/EtOH to give the triester 11 237 mg (93%) of a white solid.

(41) .sup.1H-NMR (600 MHz, CDCl.sub.3): 1.38 (t, J=7.1 Hz, 9H, CH.sub.3), 2.87 (m, 12H, bridge CH.sub.2), 4.36 (q, J=7.1 Hz, 6H, CH.sub.2), 6.76 (s, 3H), 7.20 (d, J=8.1 Hz, 6H, ArH), 7.97 (d, J=8.1 Hz, 6H, ArH).

(42) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.4, 37.5, 38.0, 60.8, 126.5, 128.3, 128.6, 129.7, 141.4, 147.2, 166.7.

(43) HR-EI-MS: C.sub.39H.sub.42O.sub.6 requires 606.2981 amu. found 606.2994.

1,3,5-Tris[2-(4-benzoic acid)ethyl]benzene (12)

(44) Using the standard procedure triester 11 (252.0 mg, 0.42 mmol), LiOH.H.sub.2O (107.2 mg, 2.6 mmol) and 1:9 H.sub.2O/EtOH (20 mL) gave triacid 3 202 mg (93%) as a white powder.

(45) .sup.1H-NMR (500 MHz, d.sub.6-DMSO): 2.82 (cm, 12H, CH.sub.2), 6.83 (s, 3H, ArH), 7.28 (d, J=8.2 Hz, 6H, ArH), 7.85 (d, J=8.2 Hz, 6H, ArH).

(46) .sup.13C NMR (125 MHz, CDCl.sub.3): 36.7, 37.1, 126.2, 128.4, 126.2, 129.3, 140.9, 146.9, 167.3.

(47) IR (KBr): (cm.sup.1) 3067, 2929, 1686, 1610, 1422, 1315, 1288, 1179.

(48) HR-EI.sup.+-MS: C.sub.33H.sub.30O.sub.6 requires 522.2042 amu. found 522.5897.

(49) EI.sup.+-MS: MI.sup.+=C.sub.33H.sub.30O.sub.6; m/z: 504.2 (90%)=[MI-H.sub.2O].sup.+, 387.1 (100%)=[MI-CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+.

1,2,4-Tris[(1E)-2-(ethyl 4-benzoate)vinyl]benzene (14)

(50) Using the standard Heck cross-coupling procedure, 1,2,4-tribromobenzene 13 (1.018 g, 3.2 mmol), ethyl 4-vinylbenzoate 9 (1.843 g, 10.5 mmol), Pd.sub.2(dba).sub.3.CHCl.sub.3 (87.6 mg, 0.08 mmol), [(t-Bu).sub.3PH]BF.sub.4 (122.6 mg, 0.42 mmol), Cy.sub.2NMe (3 mL) in THF (40 mL) were heated for 17 h. The crude mixture was subjected to flash chromatography eluting with neat CH.sub.2Cl.sub.2. The crude product was recrystallised from CH.sub.2Cl.sub.2/EtOH to give 1.681 g (88%) of a pale yellow solid.

(51) .sup.1H NMR (600 MHz, CDCl.sub.3): 1.409, 1.413, 1.416 (3t, J=7.1 Hz, 9H, CH.sub.3), 4.392, 4.394, 4.400 (3q, J=7.1 Hz, 6H, CH.sub.2) 7.06-7.11 (m, 2H, vinyl CH), 7.17-7.28 (m, 2H, vinyl CH), 7.51-7.67 (cm, 9H), 7.73 (m, 1H, core ArH), 8.04-8.09 (cm, 6H, ArH).

(52) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.5 (CH.sub.3), 61.1, 61.13, 61.1 (CH.sub.2), 125.8 (CH), 126.5 (CH), 126.5 (CH), 126.6 (CH), 126.4 (CH), 127.3 (CH), 128.3 (CH), 128.4 (CH), 128.7 (CH), 129.6 (C), 129.8 (C), 129.8 (C), 130.2 (CH), 130.2 (CH), 130.3 (CH), 130.5 (C), 130.7 (C), 131.3 (C), 135.6 (C), 136.4 (C), 136.9 (C), 141.62 (CO), 141.66 (CO), 141.7 (CO).

(53) IR (KBr): (cm.sup.1) 2981, 1713 (.sub.CO), 1604, 1278, 1178, 1107

(54) HR-EI.sup.+-MS: C.sub.39H.sub.36O.sub.6 requires 600.2512 amu. found 600.2504.

(55) EI.sup.+-MS: MI=C.sub.39H.sub.36O.sub.6; Mk: 600.2 (100%)=MI.sup.+, 555.2 (13.3%)=[MI-EtO].sup.+, 437.1 (70.1%)=[MI-2OEt-EtO.sub.2CH].sup.+

(56) UV-Vis (Solv): (nm) [, (M.sup.1cm.sup.1)] 258 [4.53], 338 [4.83], 362 [4.76, shoulder].

(57) Fluorescence (CH.sub.2Cl.sub.2): excitation (nm) [emission (nm)] 258 [436 (shoulder), 450], 338 [436 (shoulder), 450]; (cyclohexane) 334 [416, 440]

1,2,4-Tris[(1E)-2-(4-benzoic acid)vinyl]benzene (6)

(58) Triester 14 (250 mg, 0.42 mmol) and LiOH.H.sub.2O (70.2 mg, 1.67 mmol) in 9:1 EtOH/H.sub.2O were treated as described in the general saponifiaction procedure giving the triacid 6 186.6 mg (86%) as a, yellow/brown solid.

(59) IR (KBr): (cm.sup.1) 2929, 1684 (.sub.CO), 1603, 1419, 1315, 1287, 1178, 1125, 763.

1,2,4-Tris[2-(ethyl 4-benzoate)ethyl]benzene (15)

(60) Triester 14 (306 mg, 0.51 mmol) and Pd/C (10% w/w, ca 40 mg) in 1:1 EtOH/CH.sub.2Cl.sub.2 (20 mL) was treated as described. The crude product was recrystallised from THF and hexane to give ester 15 305 mg (98%).

(61) .sup.1H-NMR (600 MHz, CDCl.sub.3): 1.376, 1.381, 1.393 (3t, 3CH.sub.3, 9H) 2.79-2.95 (m, 12H), 4.33-4.40 (m, 6H), 6.86 (s, 1H), 6.95 (d, J=7.8 Hz, 1H), 7.04 (d, J 7.8 Hz, 1H), 7.17 (AB d, J=8.0 Hz, 2H), 7.19 (AB d, J=8.0 Hz, 2H) 7.21 (AB d, J=8.1 Hz, 2H), 7.95 (cm, J=7.7 Hz, 6H)

(62) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.48, 14.50, 34.0, 34.4, 37.2, 37.7, 38.1, 61.0, 61.0, 126.6, 128.43, 128.54, 128.56, 128.7, 129.5, 129.7, 129.8, 129.9, 136.7, 139.0, 139.3, 147.2, 147.2, 147.3, 166.7, 166.8, 166.8.

(63) IR (KBr): (cm.sup.1) 2981, 2942, 1713, 1610, 1283, 1177, 1123, 1108.

(64) HR-EI.sup.+-MS: C.sub.39H.sub.42O.sub.6 requires 606.2981 amu. found EI.sup.+-MS: MI=C.sub.39H.sub.42O.sub.6; m/z: (%)=(%)=[MI-].sup.+

1,2,4-Tris[2-(4-benzoic acid)ethyl]benzene (16)

(65) Triester 15 (200 mg, 0.33 mmol) and LiOH.H.sub.2O (92.2 mg, 2.15 mmol) in 9:1 EtOH/H.sub.2O (25 mL) was treated as described in the general saponification procedure, giving triacid 16 161 mg (94%).

(66) .sup.1H NMR (500.1 MHz, d.sub.6-DMSO): 2.77-2.91 (m, 12H, methylene), 6.960 (AB, J=8.5 Hz, 1H), 6.967 (s, 1H), 7.07 (AB, J=8.5 Hz, 1H), 7.271 (AB, J=8.4 Hz, 2H), 7.296 (AB, J=8.4 Hz, 2H), 7.299 (AB, J=8.4 Hz, 2H), 7.82-7.87 (m, 6H), 12.83 (br s, CO.sub.2H).

(67) .sup.13C NMR (125.8 MHz, d.sub.6-DMSO): 33.1, 33.5, 36.3, 36.7, 37.0, 126.2, 128.5, 128.6, 128.6, 128.6, 129.0, 129.3, 129.4, 136.4, 138.6, 138.7, 146.9, 146.9, 167.3, 167.3.

(68) IR (KBr): (cm.sup.1), 1688, 1610, 1422, 1315, 1289, 1178.

(69) HR-EI.sup.+-MS: C.sub.33H.sub.30O.sub.6 requires 522.2042 amu. found 522.2045.

(70) EI.sup.+-MS: MI=C.sub.33H.sub.30O.sub.6; m/z: 522.2 (9%)=MI.sup.+, 504.2 (86%)=[MI-H.sub.2O].sup.+, 387.1 (100%)=[MI-CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+.

1,2,4,5-Tetrakis[(1E)-2-(ethyl 4-benzoate)vinyl]benzene (18)

(71) 1,2,4,5-tetrabromobenzene 17 (255.8 mg, 0.66 mmol), ethyl-4-vinylbenzoate 9 (498.6 mg, 2.83 mmol), Pd.sub.2(dba).sub.3.CHCl.sub.3 (69.2 mg, 0.07 mmol), [(t-Bu).sub.3PH]BF.sub.4 (75.4 mg, 0.26 mmol), Cy2NMe (0.8 mL) in THF (8 mL) were treated under the aforementioned cross-coupling procedure. The product was purified by flash chromatography with CH.sub.2Cl.sub.2/MeOH (100:0-99:1) an the eluent. The crude material was recrystallized from CH.sub.2Cl.sub.2/EtOH to give triester 18 541.2 mg (82%) as a bright yellow solid.

(72) .sup.1H NMR (600 MHz, CDCl.sub.3): 1.41 (t, J=7.1 Hz, 12H, CH.sub.3), 4.40 (q, J=7.1 Hz, 8H, CH.sub.2), 7.13 (d, J=16 Hz, 4H, vinyl CH), 7.56 (d, J=16.1 Hz, 4H, vinyl CH), 7.60 (d, J=8.3 Hz, 8H, ArH), 7.83 (s, 2H, core ArH), 8.06 (d, J=8.3 Hz, 4H, ArH).

(73) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.3 (CH.sub.3), 61.0 (CH.sub.2), 125.2 (ArCH, core), 126.5 (ArCH), 128.1 (CH, vinyl), 129.7 (C.sub.q), 130.1 (ArCH), 131.0 (CH, vinyl), 135.6 (C), 141.4 (C), 166.3 (CO).

(74) IR (KBr): (cm.sup.1) 2981, 1710, 1637, 1617, 1604, 1282, 1180, 1107.

(75) HR-EI.sup.+-MS: C.sub.50H.sub.46O.sub.8 required 774.3193 amu. found 774.3506 amu.

(76) EI.sup.+-MS: MI=C.sub.50H.sub.46O.sub.8; m/z: 774.4 (100%)=MI.sup.+, 729.1 (16%)=[MI-OEt].sup.+, 611.1 (45%)=[MI-CH.sub.2(C.sub.6H.sub.4CO.sub.2Et)].sup.+.

(77) UV-Vis (Solv): (nm) [log (M.sup.1 cm.sup.1)] 258 [4.62], 312 [4.73], 346 [4.82].

(78) Fluorescence (CH.sub.2Cl.sub.2): excitation (nm) [emission (nm)] 258 [455, 518], 312 [362, 381, 452], 346 [381, 454]; (cyclohexane) 336 [442].

1,2,4,5-Tetrakis[(1E)-2-(4-benzoic acid)vinyl)]benzene (7)

(79) Triester 18 (151.7 mg, 0.20 mmol), LiOH.H.sub.2O (86.6 mg, 2.06 mmol) in 9:1 EtOH/H.sub.2O (30 mL) were treated as described in the general saponification procedure. The crude product was recrystallized from THF and MeOH to give triacid 7 111.3 mg (85%) as a tan powder.

(80) .sup.1H NMR (500 MHz, CDCl.sub.3): (d, J=Hz, 6H, ArH), (s, 3H, ArH), (d, J=Hz, 6H, Ark

(81) .sup.13C NMR (125.8 MHz, CDCl.sub.3): (ppm), 124.2, 127.1, 127.6 (CH), 129.5, 129.9, 130.7, 135.3, 141.5, 167.2.

(82) IR (KBr): (cm.sup.1)

(83) HR-EI.sup.+-MS: C.sub.42H.sub.30O.sub.8 requires 662.1941 amu,

1,2,4,5-Tetrakis[(2-(ethyl 4-benzoate)ethyl)benzene (19)

(84) Triester 18 (200.4 mg, 0.24 mmol) and Pd/C (10% w/w, c.a. 30 mg) in 1:1 EtOH/CH.sub.2Cl.sub.2 (40 mL) was treated as described. The crude product was recrystallized from CH.sub.2Cl.sub.2 and EtOH to give compound 19 147.1 mg (79%) as a white solid.

(85) .sup.1H NMR (600 MHz, CDCl.sub.3): 1.38 (t, J=7.2 Hz, 12H), 2.82 (m, 16H), 4.36 (q, J=7.2 Hz, 8H), 6.81 (s, 2H), 7.18 (d, J=8.0 Hz, 8H, ArH), (s, 3H, ArH), (d, J=Hz, 6H, ArH).

(86) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.8, 34.0, 37.8, 61.0, 128.5, 128.6, 129.8, 130.6, 136.9, 147.2, 166.7.

(87) IR (KBr): (cm.sup.1) 2980, 2935, 1716, 1611, 1285, 1176, 1107, 1022

(88) HR-EI.sup.+-MS: C.sub.50H.sub.54O.sub.8 requires 782.3819 amu,

1,2,4,5-Tetrakis[2-(4-benzoic acid)ethyl]benzene (20)

(89) Triester 19 (101.4 mg, 0.13 mmol), LiOH.H.sub.2O (51 mg, 1.22 mmol) in 9:1 EtOH/H.sub.2O (25 mL) were treated as described in the general saponification procedure, to give tetraacid 20 87.3 mg (97%) as a white solid.

(90) .sup.1H NMR (500.1 MHz, d.sub.6-DMSO): 2.77 (s, 16H, CH.sub.2CH.sub.2), 6.86 (s, 2H, H2), 7.26 (AB, J=8.3 Hz, 8H, H2) 7.84 (AB, J=8.3 Hz, 8H, H3).

(91) .sup.13C NMR (125.8 MHz, d.sub.6-DMSO): 33.1, 36.8, 128.5, 128.6, 129.4, 136.3, 147.0, 167.3.

(92) IR (KBr): (cm.sup.1) 2945, 2863, 1688, 1610, 1422, 1315, 1288, 1178.

(93) HR-EI.sup.+-MS: C.sub.42H.sub.38O.sub.8 requires 670.2567 amu. found 670.2562.

(94) EI.sup.+-MS: MI=C.sub.42H.sub.38O.sub.8; m/z: 517.0 (100%)=[MI-H.sub.2O(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+, 499.0 (12%)=[MI-2(H.sub.2O)(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+, 381.0 (19.5%)=[MI-H.sub.2O-2(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+, 135.0=[(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+.

1,4-Bis[(1E)-2-(ethyl 4-benzoate)vinyl]benzene (28)

(95) Method A: 1,4-Dibromobenzene 26 (101.5 mg, 0.43 mmol), ethyl 4-vinyl benzoate 7 (166 mg, 0.94 mmol), Pd.sub.2(dba).sub.3.CHCl.sub.3 (45.1 mg, 0.04 mmol), [(t-Bu).sub.3PH]BF.sub.4 (52.2 mg, 0.18 mmol), Cy.sub.2NMe (300 l) in THF (5 mL) were heated at reflux overnight. The THF was removed under reduced pressure, the crude product purified using flash chromatography with CH.sub.2Cl.sub.2 as the eluent. Additional recrystallisation from CH.sub.2Cl.sub.2 and EtOH, gave 28 152.2 mg (84%). Method B: 1,4-dichlorobenzene 27 (59.9 mg, 0.41 mmol), ethyl 4-vinyl benzoate 7 (160 mg, 0.91 mmol), Pd.sub.2(dba).sub.3.CHCl.sub.3 (42.1 mg, 0.04 mmol), [(t-Bu).sub.3PH]BF.sub.4 (47.7 mg, 0.16 mmol), Cy.sub.2NMe (300 l) in THF (5 mL) were heated at reflux for 3 days. The reaction mixture was concentrated under reduced pressure, the crude product was purified by chromatography with CH.sub.2Cl.sub.2 as the eluent. Additional recrystallisation from CH.sub.2Cl.sub.2 and EtOH, gave 28 49.8 mg (29%).

(96) .sup.1H NMR (600 MHz, CDCl.sub.3): 1.41 (t, J=7.1 Hz, 6H), 4.39 (q, J=7.1 Hz, 4H), 7.14 (AB, J=16.3 Hz, 2H, vinyl CH), 7.24 (AB, J=16.3 Hz, 2H, vinyl CH), 7.55 (s, 4H), 7.57 (d, J=8.1 Hz, 4H), 8.04 (d, J=8.1 Hz, 4H).

(97) .sup.13C NMR (150 MHz, CDCl.sub.3): 14.5 (CH.sub.3), 61.1 (CH.sub.2), 126.5 (CH), 127.4 (CH), 128.0 (CH), 129.5 (C), 130.2 (CH), 130.7 (CH), 136.9 (C), 141.8 (C), 166.5 (CO).

(98) IR (KBr): (cm.sup.1) 2984, 2925, 1716 (CO), 1708 (CO), 1279, 1179, 1107.

(99) HR-EI.sup.+-MS: C.sub.28H.sub.26O.sub.4 requires 426.1831 amu. found 426.1824 UV-Vis (Solv): (nm) [log (M.sup.1 cm.sup.1)] 254 [3.85], 372 [4.74].

(100) Fluorescence (CH.sub.2Cl.sub.2): excitation (nm) [emission (nm)] 254 [414, 437], 372 [414, 437]; (cyclohexane) 328 [393, 413].

1,4-Bis[(1E)-2-(4-benzoic acid)vinyl]benzene (3)

(101) Using the standard procedure 28 (150.7 mg, 0.35 mmol), LiOH.H.sub.2O (62.3 mg, 1.48 mmol) and 1:9 H.sub.2O/EtOH (20 mL) gave an impure dark brown/black precipitate. The crude product was recrystallized from H.sub.2O and EtOH to give acid 3 as a dark brown powder 86.2 mg (66%).

(102) HR-EI.sup.+-MS: C.sub.24H.sub.18O.sub.4 requires 370.1205 amu. found 370.1205.

1,4-Bis[2-(methyl 4-benzoate)vinyl]benzene (23)

(103) (Methyl 4-carboxybenzyl)triphenylphosphonium bromide (22) (4.43 g, 9.02 mmol) was dissolved in MeOH (100 mL) and treated with NaOMe (45 mL 0.222M in MeOH). The ensuing yellow solution was treated with terephthalaldehyde (512 mg, 3.82 mmol) in one portion and the resultant mixture was heated at reflux for 17 h. The resulting yellow precipitate formed was collected and washed with MeOH to give 23 1.27 g (84%). 40:60 mixture of the E/E and E/Z products.

(104) .sup.1H NMR (500.1 MHz, CDCl.sub.3): 3.834 (s, CH.sub.3, EE), 3.837 (s; CH.sub.3, EZ), 3.85 (s, CH.sub.3, EZ), 6.691 (AB, J=12.3 Hz, vinyl CH, EE), 6.726 (AB, J=12.3 Hz, vinyl CH, EZ), 6.735 (AB, J=12.3 Hz, vinyl CH, EE), 6.774 (AB, J=12.3 Hz, vinyl CH, EZ), 7.10 (s, core ArH, EE), 7.23 (AB, J=8.3 Hz, ArH, EZ), 7.326 (AB, J=16.4 Hz, vinyl CH, EZ), 7.345 (AB, J=8.4 Hz, ArH, EE), 7.389 (AB, J=16.4 Hz, vinyl CH, EZ), 7.393 (AB, J=8.4 Hz, ArH, EZ), 7.54 (AB, J=8.3 Hz, ArH, EZ), 7.72 (AB, J=8.5 Hz, ArH, EZ), 7.84 (AB, J=8.4 Hz, ArH, EE), 7.87 (AB, J=8.4 Hz, ArH, EZ), 7.85 (AB, J=8.5 Hz, ArH, EZ).

(105) .sup.13C NMR (125.8 MHz, CDCl.sub.3): 52.1 (CH.sub.3), 52.1 (CH.sub.3), 126.6 (CH), 126.9 (CH), 127.6 (CH), 128.2 (C), 128.2 (C), 128.3 (C), 128.7 (CH), 128.8 (CH), 129.1 (CH), 129.2 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 130.7 (CH), 131.6 (CH), 135.6 (C), 136.0 (C), 136.1 (C), 141.8 (C), 141.8 (C), 142.0 (C).

(106) IR (KBr): (cm.sup.1) 3011, 2959, 1716, 1606, 1436, 1276, 1182, 1109.

(107) HR-EI-MS: C.sub.26H.sub.22O.sub.4 requires 398.1518 amu. found 398.1515 amu.

1,4-Bis[2-(methyl 4-benzoate)ethyl]benzene (24)

(108) Diester 23 (1.27 g, 3.19 mmol) and Pd/C (10% w/w c.a 100 mg) in 1:1 MeOH/CH.sub.2Cl.sub.2 (30 mL) was treated as described in the general procedure section. The crude product was recrystallized from CH.sub.2Cl.sub.2 and MeOH to give 1.20 g (93%) as a white solid.

(109) .sup.1H NMR (600 MHz, CDCl.sub.3): 2.93 (AABB, 8H, CH.sub.2CH.sub.2), 3.91 (s, 6H, OCH.sub.3), 7.05 (s, 4H, H2/H3), 7.21 (d, J=7.6 Hz, 4H, H2), 7.95 (d, J=7.6 Hz, 4H, H3).

(110) .sup.13C NMR (150 MHz, CDCl.sub.3): (ppm) 37.2, 38.0, 52.1, 128.1, 128.6, 128.7, 129.8, 139.0, 147.3, 167.3.

(111) IR (KBr): (cm.sup.1) 2944, 2923, 1726, 1609, 1431, 1280, 1176, 1105.

(112) HR-EI.sup.+-MS: C.sub.26H.sub.26O.sub.4 requires 4021831 amu. found 402.1834 amu.

(113) EI.sup.+-MS: MI=C.sub.26H.sub.26O.sub.4; m/z: 402.2 (8%)=MI.sup.+, 370.1 (33.2%)=[MI-MeOH].sup.+, 253.1 (100%)=[MI-CH.sub.2C.sub.6H.sub.4CO.sub.2Me].sup.+, 149.1=[CH.sub.2C.sub.6H.sub.4CO.sub.2Me].sup.+.

1,4-Bis[2-(4-benzoic acid)ethyl]benzene (25)

(114) Method 1: Diester 24 (308.6 mg, 0.77 mmol), LiOH.H.sub.2O (125 mg, 3.0 mmol) in MeOH/H.sub.2O (9:1, 20 mL) were treated as described in the general saponification procedure, to provide diacid 25 280 mg (97%). Method 2: Diacid 3 (107.2 mg, 0.25 mmol) and Pd/C (10% w/w c.a. 10 mg) in 1:1 EtOH/CH.sub.2Cl.sub.2 (10 mL) was treated as described. The crude mixture containing 29 (103 mg, 96%) was suspended in 9:1 EtOH/H.sub.2O (10 mL) and LiOH.H.sub.2O (26.3 mg, 0.63 mmol) added and treated under the general saponification procedure described earlier to afford diacid 3 (73.2 mg, 82%)

(115) .sup.1H NMR (500.1 MHz, d.sub.6-DMSO): 2.88 (AABB, 8H, CH.sub.2CH.sub.2), 7.11 (s, 4H, H2/H3), 7.32 (d, J=8.2 Hz, 6H, ArH), 7.85 (d, J=8.2 Hz, 6H, ArH).

(116) .sup.13C NMR (125.8 MHz, d.sub.6-DMSO): 36.2, 37.0, 128.3, 128.6, 129.3, 138.7, 146.8, 167.3.

(117) IR (KBr): (cm.sup.1) 2944, 2923, 1685 (CO), 1610, 1425, 1318, 1292, 1180, 537.

(118) HR-EI.sup.+-MS: C.sub.24H.sub.22O4 requires 374.1518 amu.

1,3-Bis[2-(methyl 4-benzoate)vinyl]benzene (4)

(119) Isophthaldehyde 30 (460 mg, 3.43 mmol), xx (4.4 g, 8.95 mmol), NaOMe (20 mL, 1.0 M) in MeOH (100 mL) were treated under analogous conditions to those described for the preparation of alkene 23. The white precipitate was filtered, washed with MeOH, and dried to give 4 (0.85 g, 62%). The product was a 44:56 mixture of the E/E and E/Z products.

(120) .sup.1H NMR (600.1 MHz, CDCl.sub.3): 3.82 (s, CH.sub.3, ct), 3.83 (s, CH.sub.3, E/E), 3.85 (s, CH.sub.3, E/Z), 6.64 (AB, J=12.4 Hz, trans vinyl CH, E/E), 6.67 (AB, J=12.4 Hz, trans vinyl CH, E/E), 6.76 (AB, J=12.3 Hz, trans vinyl CH, E/Z), 6.81 (AB, J=12.3 Hz, trans' vinyl CH, E/Z), 7.05 (dd, J.sub.1=7.6 Hz, J.sub.2=1.4 Hz, H4, ee), 7.08 (s, H2, ee), 7.10 (d, J=7.7 Hz, H4/H6, E/Z) 7.13 (AB, J=16.5 Hz, cis vinyl CH, E/Z), 7.17 (t, J=7.6 Hz, H5, E/E), 7.25 (AB, J=8.2 Hz, H2, E/Z), 7.27 (t, J=7.7 Hz, H5, E/Z), 7.33 (AB, J=16.5 Hz, cis vinyl CH, E/Z), 7.36 (AB, J=8.2 Hz, ArH, E/Z), 7.46 (s, H2, E/Z), 7.50 (d, J=7.7 Hz, H4/H6, E/Z), 7.66 (AB, J=8.3 Hz, ArH, E/Z), 7.76 (AB, J=8.4 Hz, H3, E/E), 7.84 (AB, J=8.3 Hz, ArH, E/Z), 7.92 (AB, J=8.4 Hz, ArH, E/Z).

(121) .sup.13C NMR (150.9 MHz, CDCl.sub.3): 52.1 (CH.sub.3), 52.1 (CH.sub.3), 126.2 (CH), 126.6 (CH), 127.0 (CH), 127.6 (CH), 127.9 (CH), 128.1 (C), 128.2 (C), 128.3 (C), 128.3 (CH), 128.3 (CH), 128.7 (CH), 128.9 (CH), 128.9 (CH), 129.2 (CH), 129.3 (CH), 129.3 (CH), 129.5 (CH), 129.6 (CH), 130.9 (CH), 131.7 (CH), 131.8 (CH), 136.6 (C), 136.8 (C), 136.8 (C), 141.6 (C), 141.9 (C), 165.9 (C), 165.9 (C), 166.0 (C).

(122) IR (KBr): (cm.sup.1) 1720, 1606, 1435, 1280, 1179, 1109.

(123) HR-EI.sup.+-MS: C.sub.26H.sub.22O.sub.4 requires 398.1518 amu. found 398.1518

1,3-Bis[2-(methyl 4-benzoate)ethyl]benzene (31)

(124) The E/Z isomeric mixture 4 (499.3 mg, 1.25 mmol) and Pd/C (10% w/w, c.a.50 mg) in MeOH/CH.sub.2Cl.sub.2 (40 mL, 1:1) was treated as described previously. The crude product was recrystallized from CH.sub.2Cl.sub.2/MeOH to give ester 31 (489.7 mg, 98%) as a white solid.

(125) .sup.1H-NMR (600 MHz, CDCl.sub.3): 2.8-2.96 (AABB, 8H, CH.sub.2CH.sub.2), 3.90 (s, 6H, Me), 6.91 (s, 1H, H2), 6.99 (dd, 2H, J=7.6 and 1.4 Hz, 2H, H4/H6), 7.16-7.25 (cm, 5H, ArH), 7.96 (d, J=8.2 Hz, 4H, H3).

(126) .sup.13C NMR (150 MHz, CDCl.sub.3): 37.5, 38.0, 52.1, 126.3, 128.0, 128.5, 128.7, 128.8, 129.8, 141.3, 147.3, 167.2.

(127) IR (KBr): (cm.sup.1) 1715, 1607 (m), 1438, 1279, 1109.

(128) HR-EI.sup.+-MS: C.sub.26H.sub.26O.sub.4 requires 402.1831 amu. found 402.1840.

1,3-Bis[2-(4-benzoic acid)vinyl]benzene (32)

(129) Ester 25 (202.6 mg, 0.50 mmol), LiOH.H.sub.2O (92.2 mg, 2.20 mmol) and 9:1 MeOH/H.sub.2O (30 mL) were treated as described in the general saponfication procedure, giving 175.8 mg (94%) as a white solid.

(130) .sup.1H-NMR (500.1 MHz, d.sub.6-DMSO): 2.87 (AABB, 8H, CH.sub.2CH.sub.2), 6.99-7.05 (m, 3H, ArH) 7.15 (t, J=7.4 Hz, 1H, H5), 7.31 (AB, J=8.4 Hz, 4H, H2), 7.84 (AB, J=8.4 Hz, 4H, H3), 12.8 (br s, 2H, CO.sub.2H).

(131) 13C NMR (125.8 MHz, d.sub.6-DMSO): 36.5, 37.0, 126.0, 128.1, 128.4, 128.6, 128.6, 129.3, 141.0, 146.9, 167.3.

(132) HR-EI.sup.+-MS: C.sub.24H.sub.22O.sub.4 requires 374.1518 amu. found 374.1523.

(133) EI.sup.+-MS: MI=C.sub.24H.sub.22O.sub.4; m/z: (%)=MI.sup.+, 239.0 (100%)=[MI-(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)].sup.+: 193.0 (34%)=[MI-(CH.sub.2(C.sub.6H.sub.4CO.sub.2H)CO.sub.2HH.sup.+].sup.+.

Example 2

(134) Toxicity of a Compound of Formula C-TSB007 (135B)

(135) (A) Haemolytic Activity

(136) In a preliminary study using disk diffusion methodology, a compound of Formula C (TSB007 also known as 135B) (10 mg/ml) had antibacterial activity mostly against Gram-positive bacteria.

(137) As an indicator of toxicity to human cells, haemolysis experiments on sheep erythrocytes were undertaken with TSB007.

(138) 10 mg/ml stock solution of TSB007 was prepared in 100% DMSO. Master stock solutions of TSB007 (10 mg/ml) were made by dissolving 20 mg dehydrated compound in 2 ml 100% DMSO. They were stored in foil-covered glass bottles at 20 C. Master stocks stored in this way retained full antimicrobial activity for a minimum period of 6 weeks (results not shown).

(139) Serial, 10-fold dilutions were performed in PBS to make solutions of 1000, 100, 10 and 1 mg/L of TSB007. In microcentrifuge tubes, 500 l of each dilution was combined with 480 l PBS and 20 l washed sheep erythrocytes (100%) so that the final concentration of erythrocytes was 2% and the final concentrations of compound were 500, 50, 5 and 0.5 mg/L. Dilutions of DMSO without. TSB007 were prepared and tested as above to check for haemolysis due to DMSO. A 100% haemolysis control was prepared with 980 l water and 20 l erythrocyte suspension. A negative control was prepared with 980 l PBS and 20 l erythrocyte suspension. Dilutions and controls were prepared and tested in duplicate.

(140) Tubes were incubated at 37 C. for 2 h on a rocker then centrifuged at 12 000 g for 5 mins. The OD.sub.540 of the supernatant was determined using 100 l volumes transferred to a microtitre tray. % haemolysis was determined by blanking the OD against that of the negative control and presenting the resulting OD as a proportion of the OD of the positive control (blanked with water).

(141) The haemolytic activity of TSB007 was very low (Table 1). No haemolysis was observed for the equivalent tests of DMSO.

(142) TABLE-US-00001 TABLE 1 Haemolysis of sheep erythrocytes exposed to 0.5-500 mg/L of compound TSB007 Concentration of Average (SD) % haemolysis compound (mg/L) (n = 2) 500 5.053 (0.595) 50 2.316 (0.099) 5 2.175 (0.099) 0.5 2.316 (0.695)
(B) Cytotoxic Activity

(143) L929 cells grown to approximately 80% confluency in HGM-M were washed with Hanks, trypsonised to detach the cells from the flask, then diluted to 10.sup.5 cells/ml in HGM-M and 200 l used to inoculate the wells of a 96-well microtitre tray. After incubation for 24 h at 37 C., adherent cells were washed with Hanks and then 100 l HGM-M was added to the wells. Fresh TSB007 master stock solution (10 mg/ml) was serially 10-fold diluted in HGM-M to make solutions of 1000, 100, 10 and 1 g/ml of TSB007. To the wells of the microtitre tray, 100 l of these TSB007 solutions were also added, giving final concentrations of 500, 50, 5 and 0.5 g/ml of TSB007. The same dilutions of DMSO were prepared and tested to check that cytotoxic activity was not due to the DMSO. A negative control was prepared containing only HGM-M, and a positive control was prepared by adding 100 l of carboplatin (10 mg/ml; Mayne Pharma Pty Ltd, Australia) to wells containing 100 l HGM-M. Controls and dilutions of TSB007 and DMSO were prepared in duplicate.

(144) After 24 h shaking at 37 C., cytotoxicity was quantified using the neutral red assay. The cells were washed with Hanks then 200 l HGM-M and 20 l Neutral Red (3.3 g/L; Sigma-Aldrich) were added to each well. After 2 h shaking at 37 C., cells were washed twice with PBS then 200 l of 1% acetic acid in 50% EtOH was added to each well to solubilise the stain. After 15 min shaking at 37 C. the OD.sub.690 nm of the wells was measured and subtracted from the OD.sub.540 nm. Results were then blanked against wells to which no cells had been added, and converted to a ratio of the OD of the negative control. Ratios 50.5 indicated a cytostatic effect. Testing was performed on two separate occasions.

(145) At concentrations of 0.5-50 g/ml, TSB007 was not significantly cytostatic to L929 cells (Table 2). The compound was cytostatic at 500 g/ml. The concentrations of DMSO present in the different TSB007 dilutions were not significantly cytostatic (average ratios 0.515). The controls performed as expected.

(146) TABLE-US-00002 TABLE 2 Cytotoxicity of 0.5-500 g/ml TSB007 to L929 cells Concentration of compound (g/ml) Average (SD) (n = 2).sup.a 500 0.384 (0.009) 50 0.867 (0.030) 5 0.882 (0.021) 0.5 0.846 (0.285) .sup.aThe ratio of the well's OD compared to the OD of the negative control. Ratios 0.5 indicate a cytostatic effect and are highlighted.
(C) Ames Test for Mutagenic Activity

(147) To perform the Ames Test on TSB007, a method based on that published by Zeiger and Mortelmans in Current Protocols in Toxicology (1999; Section 3.1.1-3.1.29) was used, in the absence of a metabolic activation system.

(148) Briefly, single colonies from overnight BA cultures of three commonly used Salmonella tester strains, S. typhimurium TA98, TA100 and TA1535, were used to inoculate 10 ml of nutrient broth. After incubation for 15-18 h at 37 C. with shaking, the concentration of the culture was appropriate for use in the test (1-210.sup.9 cfu/ml).

(149) Glucose minimal agar plates with a volume of 20 ml were dried thoroughly. Molten top agars (2 ml) were prepared, supplemented with biotin and trace histidine, and maintained at 43-48 C. To these, 50 l of the bacterial broth culture (110.sup.8 cells) and 100 l of test solution (see, below) were added. The molten top agar was then poured directly over the surface of the glucose minimal agar and gently swirled to ensure even distribution of the agar. Once solidified, plates were incubated at 37 C. for 48 h then colony counts were performed. Plates were prepared in duplicate.

(150) Test solutions were prepared as follows and included three concentrations of TSB007, a negative solvent control and a positive control (selected from the recommended positive control chemicals and test concentrations) for each strain. Master stock solution (10 mg/ml) was incorporated directly into molten top agar to test the compound at 1000 g/plate. Dilutions of the stock were prepared in sterile distilled water to also test 100 g/plate and 300 g/plate. DMSO (100%) was incorporated into molten top agar as the negative solvent control. This was equivalent to the highest amount of DMSO incorporated into the molten top agar when testing TSB007. The positive controls used were 4-nitro-o-phenlenediamine at 2.5 g/plate (for TA98), and sodium azide at 5 g/plate (for TA100 and TA1535).

(151) Colony counts that were two to three times greater than on the negative solvent control plate indicated a mutagenic effect and were regarded as positive. In these cases, the increase in colonies is usually dose related. A positive result in this test is highly predictive of rodent carcinogenicity.

(152) TSB007 gave a negative result (Table 3). In general, the number of colonies on plates containing TSB007 was not greater than on the negative control plates. The positive control performed as expected. The decrease in colony counts at 1000 g/plate TSB007 may be due to antimicrobial activity against the tester strains.

(153) TABLE-US-00003 TABLE 3 Colony counts of three S. typhimurium tester strains exposed to different concentrations of TSB007 under Ames Test conditions Number of colonies Nega- Posi- tive tive TSB007 100 TSB007 300 TSB007 1000 Strain Control Control g/plate g/plate g/plate TA98 14 300 13 11 2 12 285 16 11 3 TA100 113 350 111 110 88 96 360 92 105 89 TA1535 28 600 17 16 0 29 600 29 24 0

Example 3

(154) Antimicrobial Activity of Formula C

(155) (A) Initial Screening by Disk Diffusion

(156) Studies using disk diffusion methodology showed that the compound of Formula C, (TSB007) (10 mg/ml) had antibacterial activity against a range of organisms, mostly Gram-positive bacteria.

(157) Briefly, blood agar plate (BA) cultures of the 38 organisms listed in Table 5 were prepared over 24 and 48 h for normal and slow-growing organisms, respectively. Pre-dried (30 mins) Mueller Hinton agar plates (MHA; from PathWest Media) were swabbed with 0.5 McFarland suspensions of the BA cultures in saline (0.85% NaCl) as per CLSI guidelines. Anaerobes were instead inoculated onto pre-reduced BA plates, and Streptococcus spp. onto MHA containing 5% sheep blood. Two Whatman 6 mm Antibiotic Assay disks were placed onto each inoculated plate. One disk was impregnated with 20 l 100% DMSO and the second with 20 l of 10 mg/ml TSB007 in 100% DMSO. Plates were incubated at 35 C. (with 5% CO.sub.2 where mentioned in the Table). Zones were measured after 24 and 48 h.

(158) TSB007 resulted in zones of inhibition of all Gram-positive bacteria tested (Table 5). Zones halved with S. aureus strains, and were no longer seen with S. xylosus, following extended incubation (24 h vs. 48 h), mostly due to the growth of subpopulations of discrete colonies. No zones of inhibition were seen with C. albicans nor with Gram-negative bacteria except M. catarrhalis. Y. enterocolitica had a negligible zone of inhibition after 24 h that was not seen after 48 h.

(159) Photographs were taken after 48 h of all plates where TSB007 produced zones of inhibition. FIGS. 3-5 show a selection of these.

(160) TABLE-US-00004 TABLE 5 Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20 l) and a DMSO control tested against a panel of Gram-negative bacteria Zone diameters (trim) Special growth 24 h 48 h 24 h 48 h Repeat Organism conditions? TSB007 TSB007 DMSO DMSO Results Gram-negative Acinetobacter baumannii ATCC 15308 No N/A N/A N/A N/A N/A Acinetobacter baumannii ATCC 19606 No N/A N/A N/A N/A Aeromonas hydrophila NCTC 8049/ No N/A N/A 8 ATCC 7966 Burkholderia cepacia ATCC 25416 No N/A N/A N/A Citrobacter freundii NCTC 9750/ No N/A N/A N/A N/A 9 ATCC 8090 Enterobacter cloacae NCTC 10005/ No N/A N/A N/A N/A ATCC 13047 Enterobacter aerogenes ATCC 13048 No N/A N/A N/A N/A Escherichia coli NCTC 10538 No N/A N/A N/A N/A 7.5? Klebsiella edwardsii NCTC 10896 No N/A N/A N/A N/A 9 Klebsiella pneumoniae ATCC 13883 No N/A N/A N/A N/A Moraxella catarrhalis ATCC 25238 Incubated in 25 25 N/A N/A 36 (instead of 5% CO.sub.2 (NCTC NCTC 3625) 3625) Moraxella catarrhalis NCTC 3622 Incubated in No growth 5% CO.sub.2 Morganella morganii NCTC 235/ No N/A N/A N/A N/A ATCC 25830 Proteus mirabilis NCTC 10975 No N/A N/A N/A N/A 9? Proteus vulgaris NCTC 4635 No N/A N/A N/A N/A Pseudomonas aeruginosa NCTC 10662 No N/A N/A N/A N/A N/A Salmonella enterica subsp. ATCC 13311 No N/A N/A N/A N/A 11 enterica serovar Typhimurium Serratia marcescens NCTC 1377 No N/A N/A N/A N/A Shigella flexneri NCTC 8192 No N/A N/A N/A N/A 8.5 Stenotrophomonas ATCC 13637 No N/A N/A N/A N/A 9 maltophilia (instead of (M2495) M2495) Vibrio cholerae M3695 No N/A N/A 7 7 7 (non-toxigenic) (other strain?) Yersinia enterocolitica ATCC 9610 No 7 N/A N/A N/A 8 (instead of (clinical clinical 26609) 26609) Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20 l) and a DMSO control tested against a panel of Gram-positive bacteria and Candida albicans Gram-positive Bacillus cereus ATCC 13061 No 10 9 N/A N/A 9 Bacillus subtilis ATCC 6633 No 8 8 N/A N/A N/A Enterococcus faecalis NCTC 775 No 10 10 N/A N/A 8 Enterococcus faecium ATCC 19434 No 11 10 N/A N/A Listeria monocytogenes NCTC 7973 No 9 9 N/A N/A 15? Micrococcus luteus ATCC 10240 No 26 26 N/A N/A 32 Staphylococcus aureus NCTC 6571/ No 20 9 N/A N/A 21 ATCC 9144 Staphylococcus aureus NCTC 10442 No 18 9 N/A N/A 23 (methicillin resistant) Staphylococcus epidermidis ATCC 12228 No 22 22 N/A N/A 20 Staphylococcus xylosus ATCC 29971 No 17 N/A N/A N/A 18 Streptococcus pneumoniae ATCC 49619 Mueller Hinton + 16 16 N/A N/A 26 Blood & (strain Incubated in 5% 12213) CO.sub.2 Streptococcus pyogenes NCTC 8191 Mueller Hinton + 14 14 N/A N/A 17 Blood & Incubated in 5% CO.sub.2 Anaerobes Bacteroides fragilis ATCC 23745 BA - prereduced N/A N/A N/A N/A (Gram-neg) Clostridium difficile NCTC 43593 BA - prereduced 10 10 N/A N/A (Gram-pos) Clostridium perfringens ATCC 2734 BA - prereduced 12 12 N/A N/A (Gram-pos) Yeast Candida albicans ATCC 24433 MHA used N/A N/A N/A N/A N/A (instead of ATCC 10231) Note. N/A indicates no activity. No result indicates the organism was not tested.
(B) Anaerobic Screening

(161) While the above study showed compound TSB007 to be active against Gram-positive bacteria, the zones of inhibition were smaller for the anaerobic Gram-positives. The Gram-negative anaerobe was not inhibited by TSB007. The compound may therefore be less active in an anaerobic environment. Staphylococcus aureus is a facultative anaerobe. The membrane potential is reduced for this organism when grown anaerobically and therefore can affect susceptibility to certain antibiotics.

(162) To confirm the compound was also active against S. aureus in an anaerobic environment, zones of inhibition of S. aureus grown aerobically and anaerobically were compared.

(163) The method described above was used for this investigation except that two MHA plates were prepared for each organism with one examined after aerobic incubation and the other after anaerobic incubation. The organisms tested included S. aureus NCTC 6571, S. aureus NCTC 10442, and S. epidermidis ATCC 12228. The results are shown in Table 6. Zones of inhibition were larger when these organisms were incubated anaerobically compared to aerobically. This was largely due to a lack, when tested anaerobically, of the creeping zones (a zone of faint growth within the main zone of inhibition) seen on plates incubated aerobically. No zones of inhibition were seen for DMSO only.

(164) TABLE-US-00005 TABLE 6 Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20 l) against Staphylococcus spp. incubated aerobically and anaerobically Zone diameters (mm) 24 h 48 h Organism O.sub.2 AnO.sub.2 O.sub.2 AnO.sub.2 S. aureus NCTC 6571 14 18 No zone 18 S. aureus NCTC 10442 14 17 11 17 S. epidermidis ATCC 12228 18 22 18 22

Example 4

(165) MIC and MBC by Broth Microdilution

(166) (A) MIC and MBC by Broth Microdilution

(167) The method used was based on CLSI protocols for broth microdilution testing of these species. As recommended by CLSI, inocula were prepared by direct colony suspension, and the media used were cation-adjusted Mueller Hinton broth (CA-MHB) for Staphylococcus spp. and CA-MHB containing 5% lysed horse blood (CA-MHB+LHB) for Streptococcus spp. Briefly, 24 h blood agar plate (BA) cultures of Staphylococcus aureus strains NCTC 6571, ATCC 29213, ATCC 33592 (MRSA) and NCTC 10442 (MRSA), Staphylococcus epidermidis ATCC 29971, Staphylococcus xylosus ATCC 29971, Streptococcus pneumoniae strains ATCC 49619 and ATCC 6305, and Streptococcus pyogenes strains NCTC 8191 and NCTC 8302 were used to prepare 0.5 McFarland suspensions in saline. Two 640 g/ml working stocks of TSB007 were prepared from a fresh master stock, one in CA-MHB for testing against the Staphylococcus spp. and the other in CA-MHB+LHB for testing against Streptococcus spp. The working stocks were further diluted 1/10 in these broths for adding to microtitre trays to test MICs of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.06 g/ml.

(168) The wells of two 96-well microtitre trays (one for Staphylococcus spp. and one for Streptococcus spp.) were filled with 100 l of the appropriate broth, except for the first column. The last column, a sterility control containing no TSB007 or inoculum, was filled with an additional 100 l broth. Then 100 l of the 64 g/ml TSB007 solution was added to the first two columns, the contents of column 2 was mixed, then 100 l of this was used in two-fold serial dilutions along the rest of the tray except the last two columns. The final 100 l was discarded. The second last column was used as a growth control so did not have TSB007 added. Finally, to the first 11 wells of one row per strain, 100 l of a 10.sup.2 dilution (in the appropriate broth) of the 0.5 McFarland suspension was added. The microtitre trays were then incubated at 35 C. for 24 h before the MICs were read visually. Wells were further subcultured (10 l drop onto BA then incubated at 35 C. for 24 h) and colonies subsequently counted to determine minimum bactericidal concentrations (MBCs). The concentration of the initial inoculum was confirmed on BA by counting colonies from 210 l spots of 10.sup.5 dilutions of the 0.5 McFarland suspensions. Testing was performed on two separate occasions.

(169) The concentrations of inocula were largely as expected, equating to 1.010.sup.5-6.010.sup.5 CFU/ml. Slightly lower concentrations were achieved for S. xylosus ATCC 29971 (5.0-7.510.sup.4 CFU/ml) and S. pneumoniae ATCC 49619 (2.5-5.010.sup.4 CFU/ml). TSB007 was more active against Staphylococcus spp. (particularly S. aureus) than Streptococcus spp. This was consistent with disc diffusion results above. S. aureus and S. epidermidis had the lowest MICs (4 g/ml). In general, MBCs were significantly higher than MICs.

(170) TABLE-US-00006 TABLE 4 MIC and MBC of compound TSB007 tested against Staphylococcus spp. and Streptococcus spp..sup.a Final inoculum MIC MBC Organism (10.sup.5 CFU/ml) (g/ml) (g/ml) S. aureus NCTC 6571 3.25-3.75 2-4 >32 S. aureus ATCC 29213 4.50-5.00 4 >32 S. aureus ATCC 33592 (MRSA) 4.75-6.00 2-4 >32 S. aureus NCTC 10442 (MRSA) 2.25-3.25 2-4 >32 S. epidermidis ATCC 12228 2.00-3.25 2-4 >32 S. xylosus ATCC 29971 0.50-0.75 8-16 8-16 S. pneumoniae ATCC 49619 0.25-0.50 32 >32 S. pneumoniae ATCC 6305 1.00-2.00 >32 >32 S. pyogenes NCTC 8191 1.00-2.50 >32 >32 S. pyogenes NCTC 8302 1.00-1.50 >32 >32 .sup.aResults are from two independent replicates
(B) Expansion of Broth Microdilution MIC and MBC Testing Range for S. aureus NCTC 6571 and E. coli NCTC 10418

(171) The method described above, with some modifications, was used to determine the MIC and MBC for S. aureus NCTC 6571 and E. coli NCTC 10418. Concentrations of the compound were previously too low to determine the MBC of this S. aureus strain. Modifications were the use of Mueller-Hinton broth (MHB) instead of CA-MHB, the preparation of bacterial inoculum by the growth method instead of direct colony suspension, and the use of more concentrated working stocks and test concentrations of TSB007. Hence, overnight 10 ml MHB cultures prepared from BA cultures were used to make the 0.5 McFarland suspensions in MHB, and doubling dilutions of the working stock at 2048 g/ml TSB007 were made in MHB across the tray to test MICs of 1024 g/ml to 0.5 g/ml. Testing was performed on two separate occasions.

(172) Subsequently, an extended range of concentrations of TSB007 was tested for inhibition of S. aureus NCTC 6571 and E. coli NCTC 10418. The MIC of S. aureus NCTC 6571 was 2 g/ml, confirming the previous MIC result for this strain, and the MBC was 128-256 g/ml. The MIC and MBC for E. coli NCTC 10418 were 512 g/ml and >1024 g/ml, respectively.

Example 5

(173) Time-Kill Experiments

(174) An overnight 10 ml MHB culture of S. aureus NCTC 6571 was diluted to 0.5 McFarland in MHB then 1 ml was used to inoculate each of four 100 ml flasks prepared as follows: 1) 9.0 ml MHB (control) 2) 9.99 ml MHB plus 9.7 l of TSB007 working stock at 2048 g/ml (1.8 g/ml) 3) 9.98 ml MHB plus 19.5 l of TSB007 working stock at 2048 g/ml (3.6 g/ml) 4) 6.74 ml MHB plus 256 l of a TSB007 master stock (320 g/ml). This equated to 3.2% DMSO which did not have any effect on the viability of S. aureus NCTC 6571 (data not shown).

(175) The flasks were incubated at 37 C. for 10 min before inoculation. Samples (0.1 ml) were taken immediately after inoculation (time 0) and then at 15, 30, 60, 120 and 240 min. The samples were immediately diluted 10.sup.1 in 0.01 M phosphate buffered saline at pH 7.0 (PBS). Further 10-fold dilutions in PBS were made down to 10.sup.4 until the 60 min time point, and then down to 10.sup.6 for the remaining time points. Viable counts were estimated using 320 l drops from each dilution on MHA which was then incubated at 37 C. overnight before counting. These were normalised against the initial counts from each flask. Testing was performed on three separated occasions.

(176) The viability of S. aureus NCTC 6571 cultures containing TSB007 at 1.8 g/ml (NB. The MIC was 2 g/ml), 3.6 g/ml (1.8MIC) and at 320 g/ml (1.25MBC) was tested. The initial concentration of microorganism in each flask was 0.5-1.010.sup.7 CFU/ml. At both 1.8 g/ml and 3.6 g/ml, TSB007 was inhibitory for the 4 h of the experiment (FIG. 6). At 24 h, the cells with TSB007 at 1.8 g/ml had grown to counts of 10.sup.8 CFU/ml, and those with 3.6 g/ml to 10.sup.3-10.sup.6 CFU/ml. The cells with 320 g/ml at the first time point of 15 min were below the limit of detection with a >99.9% kill.

Example 6

(177) Analogues of TSB007

(178) Various analogues of TSB007 were synthesised and tested in DMSO in disk diffusion assays against S. aureus NCTC 6571 using the protocol of Example 3. A number of compounds were also tested against E. coli NCTC 10418.

(179) TABLE-US-00007 TABLE 5 Compounds tested (see FIGS. 8 to 12 for structure information) Zone of inhibition (mm) Compound S. aureus NCTC 6571 E. coli NCTC 10418 TSB049 0 0 TSB063 0 0 TSB037 0 0 TSB041 0 0 TSB019 0 0 TSB023 0 0 TSB025 0 0 TSB065 14 0 TCT003 0 0 TSB001 0 TSB009 0 TSB053 0 TAB001 0 TCB001 11 TCB003 0 0 Not tested

(180) Compounds TSB065 and TCB001 were successful at inhibiting growth of S. aureus NCTC 6571, with TSB065 giving a zone of inhibition of 14 mm and TCB001 giving a zone of inhibition of 11 mm.

(181) Several analogues were also tested against a range of microorganisms as shown in Table 6.

(182) TABLE-US-00008 TABLE 6 Effect of some analogues of TSB007 on growth of various microoganisms. Zone of Inhibition (mm) of compounds Microorganism TSB068 TSB067 QSB001 Gram ve bacteria A. calcoaceticus ATCC 15308 0 0 0 A. hydrophila NCTC 8049 7 7 7 C. freundii NCTC 9750 0 0 8.5 E. coli NCTC 10418 0 6.5 7 E. coli NCTC 10538 0 7.5 7.5 K. pneumoniae NCTC 10896 0 0 8 M. catarrhalis NCTC 3625 32 32 18 P. mirabilis NCTC 10975 0 0 0 P. aeruginosa NCTC 10662 0 0 0 S. typhimurium ATCC 13311 0 0 8.5 S. flexneri NCTC 8192 0 0 0 S. maltophilia ATCC 13637 0 0 8 V. cholerae non-01 clinical 0 0 8 Y. enterocolitica clinical 26609 0 0 7.5 Gram +ve bacteria B. cereus ATCC 13061 0 0 0 B. subtilis ATCC 6633 0 0 0 E. faecalis NCTC 775 0 0 0 L. monocytogenes NCTC 7973 15 12 0 M. luteus ATCC 10240 8 20 0 S. aureus NCTC 6571 8 14 8.5 S. aureus NCTC 10442 0 12 0 S. epidermidis ATCC 1228 0 11 0 S. xylosus ATCC 29971 0 0 0 S. pyogenes NCTC 8191 20 14 8 S. pneumoniae ATCC 49619 23 16 9 Fungi C. albicans ATCC 90028 0 0 0

(183) Componds DSB001, DSD002, DSB004, TSB008 and QSB002 showed no zones of inhibition with S aureus NCTC6571 when applied at 10 mg/ml.