ANTIBIOTICS

Abstract

The invention provides a compound of Formula (I) or a pharmaceutically acceptable salt; wherein L is a substituted or non-substituted aromatic ligand; Z is a halogen or coordinating ligand; M is a group 8 or group 9 transition metal selected from; Fe, Ru, Os, Co, Rh and Os; A and B are independently selected from a substituted or non-substituted indole or pyridine, may be the same or different and coordinate to the transition metal M through the nitrogen atom of the indole or pyridine. The compounds have been shown to have use against antibiotic-resistant bacteria. A further aspect of the invention is directed towards treating or preventing bacterial infections.

Claims

1. A method of treating or prophylaxis of a bacterial infection caused by a bacterium comprising administering to a subject an effective amount of a compound or pharmaceutically acceptable salt of a compound of Formula I: ##STR00003## wherein L is a substituted or non-substituted aromatic ligand; Z is a halogen or coordinating ligand; M is a group 8 or group 9 transition metal selected from; Fe, Ru, Os, Co, Rh or Os; A and B are independently selected from a substituted or non-substituted indole or pyridine, may be the same or different and coordinate to the transition metal M through the nitrogen atom of the indole or pyridine.

2. The method of treating or prophylaxis of claim 1, wherein one of A or B is a substituted or non-substituted, especially non-substituted, indole; and the second of B or A is a substituted or non-substituted, especially a non-substituted, pyridine.

3. The method of treating or prophylaxis of claim 1, wherein L comprises 1 or 2 aromatic rings.

4. The method of treating or prophylaxis of claim 1, wherein L is a substituted or non-substituted cyclopentadienyl ring or a substituted or non-substituted benzene.

5. The method of treating or prophylaxis of claim 1, wherein L is substituted by one or more side groups comprising a sugar, hydroxyl, halogen, carboxylate, triazole, amide or a C.sub.1 to C.sub.4 straight or branched chain alkyl group.

6. (canceled)

7. The method of treating or prophylaxis of claim 1, wherein L is selected from para-cymene, pentamethylcyclopentadienyl, hexamethyl benzene, biphenyl or cyclopentadienyl.

8. (canceled)

9. The method of treating or prophylaxis of claim 1, wherein the compound of Formula I is selected from FKB3, FKB10 or FKB11: ##STR00004##

10. The method of treating or prophylaxis of claim 1 wherein the bacterial infection is caused by a Mycobacterium, a Gram-positive bacterium or a Gram-negative bacterium.

11. (canceled)

12. The method of treating or prophylaxis of claim 1 wherein the bacterium is antibiotic resistant.

13. The method of treating or prophylaxis of claim 1, wherein the bacterium produces a β-lactamase or carbapenumase.

14. The method of treating or prophylaxis of claim 1, wherein the bacterium is from the genus Mycobacterium, Escherichia, Salmonella, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Klebsiella, Acinetobacter or Enterobacter.

15. The method of treating or prophylaxis of claim 1, wherein the bacterium is Mycobacterium absessus, or Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumonia.

16. A compound of Formula I ##STR00005## or a pharmaceutically acceptable salt; wherein L is a substituted or non-substituted aromatic ligand; Z is a halogen or coordinating ligand; M is a group 8 or group 9 transition metal selected from; Fe, Ru, Os, Co, Rh or Os; A and B are independently selected from a substituted or non-substituted indole or pyridine, may be the same or different and coordinate to the transition metal M through the nitrogen atom of the indole or pyridine.

17. The compound or pharmaceutically acceptable salt of claim 16, wherein one of A or B is a substituted or non-substituted, especially non-substituted, indole; and the second of B or A is a substituted or non-substituted, especially a non-substituted, pyridine.

18. The compound or pharmaceutically acceptable salt of claim 16, wherein L comprises 1 or 2 aromatic rings.

19. The compound or pharmaceutically acceptable salt of claim 16, wherein L is a substituted or non-substituted cyclopentadienyl ring or a substituted or non-substituted benzene.

20. The compound or pharmaceutically acceptable salt of claim 16, wherein L is substituted by one or more side groups comprising a sugar, hydroxyl, halogen, carboxylate, triazole, amide or a C.sub.1 to C.sub.4 straight or branched chain alkyl group.

21. (canceled)

22. The compound or pharmaceutically acceptable salt of claim 16, wherein L is selected from para-cymene, pentamethylcyclopentadienyl, hexamethyl benzene, biphenyl or cyclopentadienyl.

23. (canceled)

24. The compound or pharmaceutically acceptable salt of claim 16, selected from FKB3, FKB10 or FKB11 ##STR00006##

25. The compound or pharmaceutically acceptable salt of claim 16 in combination with a pharmaceutically acceptable carrier.

26. The compound or pharmaceutically acceptable salt of claim 16 in the form of an oral tablet, an oral suspension and solution, an injectable formulation, eye drops, a topical lotion, a topical cream or an aerosol.

27. A method of killing or inhibiting the growth of a bacterium comprising contacting the bacterium with an effective amount of the compound or pharmaceutically acceptable salt of claim 16.

Description

[0027] The invention will now be described by way of example only with reference to the following figures:

[0028] FIG. 1 provides stability of FKB10 in MeOD (A) and MeOD/D.sub.2O(B), FKB11 in MeOD (C) and MeOD/D.sub.2O (D) and FKB3 in MeOD (E) and MeOD/D.sub.2O (F);

[0029] FIG. 2 start point and end point data for compounds tested against M. abscessus measured at 570 nm. A FKB3, B FKB10, C FKB11;

[0030] FIG. 3 start point and end point data for compounds tested against E. coli ATCC measured at 570 nm. A FKB3, FKB10, C FKB11;

[0031] FIG. 4 start point and end point data for compounds tested against E. coli J53 2138E measured at 570 nm. A FKB3, FKB10, C FKB11;

[0032] FIG. 5 start point and end point data for compounds tested against E-coli J53 2140E measured at 570 nm. A FKB3, B FKB10, C FKB11;

[0033] FIG. 6 start point and end point data for compounds tested against E. coli 1496 ESBL measured at 570 nm. A FKB3 B FKB10, C FKB11;

[0034] FIG. 7 start point and end point data for compounds tested against P. aeruginosa ATCC measured at 570 nm. A FKB3, B FKB10, C FKB11;

[0035] FIG. 8 start point and end point data for compounds tested against K. pneumoniae H467 measured at 570 nm. A FKB3, B FKB10, C FKB11.

[0036] 1.1 Synthesis and Stability of FKB3, FKB10 and FKB11

[0037] FKB3: p-cymene ruthenium dimer (118.2 mg, 0.19 mmol) and 2-(2-pyrindyl)-1H-Indole (75 mg, 0.38 mmol) were placed in a 100 mL 2 neck round bottom flask and dissolved in 15 ml dichloromethane. Triethylamine (54 μl, 0.38) was added to the reaction mixture. The dark orange solution was then left stirring overnight at room temperature under nitrogen. The solvent was removed under vacuum. The orange crude was dissolved in ethyl acetate and washed with 0.1 M HCl (3×10 mL) and brine (1×10 mL). The combined organic layer was dried over MgSO.sub.4, filtered and dried over vacuum to obtain an orange powder. The product was purified by chromatography (acetone/dichloromethane 10:90 v/v). Yield: 72.1 mg (40.2%). HRMS-ESI+: calculated (M-Cl).sup.+429.0905 m/z, found 429.0906 m/z. 1H NMR (400 MHz, CDCl.sub.3): δ.sub.H 8.94 ppm (1H, d, J=5.8 Hz), 7.73 (1H, d, J=8.2 Hz), 7.68 (1H, d, J=7.0 Hz), 7.63 (1H, d, J=8.2 Hz), 7.52 (1H, d, J=8.2 Hz), 7.15 (2H, t, J=7.5 Hz), 7.01 (3H, m), 5.95 (1H, d, J =6.1 Hz), 5.59 (1H, d, J=6.1 Hz), 5.53 (15 H, s), 2.35 (1 H, sept, J=6.9 Hz), 2.31v(3H, s), 0.88 (3H, d, J=6.9 Hz), 0.86 (3H, d, J=6.9 Hz).

[0038] FKB10: Rh dimer [(Cp*)RhCl.sub.2].sub.2 (110 mg, 0.18 mmol) and 2-(2-pyrindyl)-1H-Indole (78 mg, 0.38 mmol) were placed in a 2 neck round bottom flask and dissolved in 25 mL of dichloromethane to obtain an orange solution. Triethylamine (66 μL, 0.47 mmol) was added and the reaction mixture left to react for 24 h at room temperature. The solvent of the reaction mixture was removed under vacuum and the orange solid dissolved in dichloromethane (20 mL) and extracted with 0.1 M HCl solution (1×10 mL). The combined organic layer was dried over magnesium sulfate, filtered and dried over vacuum to obtain an orange powder. The product was purified by column chromatography in silica, acetone/dichloromethane (1:9 v/v) and recrystallised in dichloromethane to obtain crystalline orange needles. Yield: 63.4 mg (38.1%). HRMS-ESI+: calculated (M-Cl).sup.+431.1000 m/z, found 431.0833 m/z. 1H NMR (400 MHz, CDCl.sub.3): δ.sub.H 8.56 ppm (1H, d, J=5.7 Hz), 7.74 (1H, d, J=8.2 Hz), 7.68 (1H, t, J=7.8 Hz), 7.61 (1H, d, J=7.8 Hz), 7.46 (1H, d, J=8.2 Hz), 7.08 (2H, m), 7.03 (1H, s), 6.93 (1H, t, J =7.6 Hz), 1.66 (15 H, s).

[0039] FKB11: FKB11 was synthesised following the preparation described for compound FKB10 with the following modifications. Ir dimmer [(Cp*)IrCl.sub.2].sub.2 (102 mg, 0.128 mmol), 2-(2-pyrindyl)-1H-Indole (50 mg, 0.257 mmol). The product was obtained as a yellow powder. Yield: 64.5 mg (27.2%). HRMS-ESI+: calculated (M-Cl).sup.+ 521.1569 m/z, found 521.1564 m/z. 1H NMR (400 MHz, CDCl.sub.3): δ.sub.H 8.55 ppm (1H, d, J=5.6 Hz), 7.80 (1H, d, J=8.3 Hz), 7.67 (1H, t, J=7.8 Hz), 7.59 (1H, d, J=7.8 Hz), 7.44 (1H, d, J=8.5 Hz), 7.05 (2H, m), 6.94 (1H, m), 1.66 (15 H, s).

[0040] Stability of FKB3, FKB10 and FKB11: Each complex was dissolved in MeOD (2.2 mM) and diluted to a final concentration of 1.1 mM with either MeOD or D.sub.2O. .sup.1H NMR spectra was recorded at t=<10 min, 12 h and 24 h.

[0041] Each complex was also dissolved in MeOD (2.2 mM) and diluted to a final concentration of 1.1 mM with D.sub.2O. The sample was then reacted with 1 mol. equiv. of silver nitrate to obtain the fully hydrolyzed specie.

[0042] From the NMR spectra of FIG. 1 it can be observed that FKB10 is stable over 24 h both in methanol and in aqueous solution. Furthermore, addition of silver nitrate does not produce any shift of the .sup.1H NMR resonances, suggesting that ligand substitution occurs replacing the chloride ligand by either methanol or water.

[0043] The iridium compound FKB11 shows no changes in the .sup.1H NMR spectra, which indicates stability; as a side note, the compound is quite insoluble, and it precipitates over time. The ruthenium complex FKB3 is fully stable. Interestingly, when the compound is dissolved in 100% MeOD, two species can be observed, corresponding to the chloride and the methoxide adducts.

[0044] FIG. 1: Stability of FKB10 in MeOD (A) and MeOD/D.sub.2O (B), FKB11 in MeOD (C) and MeOD/D.sub.2O (D) and FKB3 in MeOD (E) and MeOD/D.sub.2O (F).

[0045] 1.2. Chemosensitivity Assays Against Human Cell Lines

[0046] In vitro chemosensitivity tests were performed against HCT116 (colorectal cancer cell line), A2780 and A2780cisR (cisplatin sensitive and cisplatin resistant ovarian cancer cell lines, respectively). Cancer cell lines were routinely maintained as monolayer cultures in RPMI medium supplemented with 10% foetal calf serum, penicillin (100 I.U./ml) and streptomycin (100 μg/ml), sodium pyruvate (1 mM) and L-glutamine (2 mM). For chemosensitivity studies, cells were incubated in 96-well plates at a concentration of 7.5×10.sup.3 cells per well and the plates were incubated for 24 hours at 37° C. and a 5% CO2 humidified atmosphere prior to drug exposure.

[0047] Complexes were dissolved in dimethylsulfoxide (DMSO) to provide stock solutions which were further diluted with media to provide a range of final concentrations. Drug-media solutions were added to cells (the final concentration of DMSO was less than 0.5% (v/v) in all cases) and incubated for 24 hours at 37° C. and 5% CO.sub.2 humidified atmosphere. The drug-media was removed from the wells and the cells were washed with PBS (100 μL, twice), and 200 μL of complete fresh media were added to each well. The plates were further incubated for 72 hours at 37° C. in a humidified atmosphere of 5% CO2 to allow for a period of recovery. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (40 μL, 2.5 mg/mL) was added to each well and incubated for 2 hours at 37° C. and 5% CO.sub.2 humidified atmosphere. All solutions were then removed and 100 μL of DMSO was added to each well in order to dissolve the purple formazan crystals. A Thermo Scientific Multiskan EX microplate photometer was used to measure the absorbance in each well at 570 nm. Cell survival was determined as the absorbance of treated cells divided by the absorbance of controls and expressed as a percentage. The IC.sub.50 values were determined from plots of % survival against drug concentration. Each experiment was repeated in duplicate of triplicates and a mean value was obtained and stated as IC.sub.50 (μM)±SD. Cisplatin was used as a positive control. As a side note, the higher the IC.sub.50 values are, the less cytotoxic the compounds are.

TABLE-US-00001 TABLE 1 IC.sub.50 values/μM ± SD for cisplatin and compounds FKB3, FKB10, and FKB11 against HCT116, A2780, and A2780 cell lines. IC.sub.50/μM ± SD Complex Metal HCT116 A2780 A2780cisR FKB3 Ru 107.9 ± 11.5 45.4 ± 6.9 71.9 ± 11.7 FKB10 Rh 104.2 ± 9.8 23.5 ± 1.3 41.0 ± 4.7 FKB11 Ir 64.81 ± 4.6 22.2 ± 2.1 32.6 ± 1.7 cisplatin Pt  59.8 ± 7.5  5.7 ± 0.4 11.9 ± 2.2

[0048] 2. Antimicrobial Activity

[0049] 2.1 Methods

[0050] Growth of Organisms

[0051] All Mycobacterium abscessus (M. abs) cultures were grown in Middlebrook 7H9 media, supplemented with glycerol (0.5% v/v), ADC (acid-dextrose-glucose) (10% v/v) and tween80 (0.05% w/v), at 30° C. with shaking at 180 rpm for 72 h. The optical density (OD.sub.600 nm) was adjusted to 0.1 prior to microtiter plate set-up.

[0052] The gram negative organisms selected were Escherichia coli (E. coli ATCC, J53 2138E, J53 2140E and 1496 ESBL), Pseudomonas aeruginosa ATCC and Klebsiella pneumoniae H467. All organisms were grown in nutrient broth at 37° C. with 180 rpm shaking, for 18 h. The optical density (OD.sub.600 nm) was adjusted to 0.1 prior to microtiter plate set-up.

[0053] M.abscessus is an example of a mycobacterium capable of causing lung infections. J53 2138 E, J532140 E and I496ESBL are β-lactamase producing strains. H467 produces a carbapenumase.

[0054] Compounds

[0055] Each compound was made in DMSO (dimethyl sulfoxide) to a stock concentration of 100 mM. A master plate was used to generate the desired concentrations for testing. This was achieved by serial 5-fold dilution (repeated 5 times, the final row was DMSO only (0 mM)).

[0056] Experimental Set-up

[0057] For each compound, 95 μl of culture (OD 0.1) was added to the microtiter plate and 5 μl of serially diluted compound added. The concentrations tested were: 1 mM, 200 μM, 40 μM, 8 μM, 1.6 μM and 320 nM. All plates were read in the plate reader at 570 nm (T=0). For M. abs the plates were incubated at 30° C. for 96 h with reads being taken every 24 h. For gram negative organisms the plates were incubated at 37° C. and read at 0 h and 24 h. After the final read for all organisms the experiment was plated out to observe bactericidal activity. M. abs was plated onto Middlebrook 7H11 agar supplemented with glycerol (0.5%) and incubated at 30° C. for 72-96 h. The gram negative organisms were plated onto nutrient agar and incubated at 37° C. for 24 h. Colony forming units were counted for all organisms.

[0058] Data Analysis

[0059] All OD readings were transferred to excel and adjusted to account for OD of the broth and compounds. The data was then transferred to GraphPad Prism 8 for data analysis.

[0060] 2.2 Results

[0061] Out of the compounds tested, activity was observed for a total of 5 compounds. Three of the compounds, FKB3, FKB10 and FKB11, exhibited varying levels of growth inhibition against all organisms tested.

[0062] M. abscessus ATCC

[0063] Reduction in optical density reading was observed for all compounds tested against M. abs at 1 mM (FIG. 2). Uniquely, FKB10 showed a further reduction in optical density at both 200 μM and 40 μM, suggesting the highest potency against M. abs (FIG. 2B).

[0064] E. coli ATCC

[0065] All compounds tested against E. coli ATCC showed activity down to 40 μM (FIG. 3). Both FKB10 and FKB11 were the most potent, showing the least change in optical density over 24 h at 1 mM concentration (FIG. 3B/C). FKB3 and FKB11 both have minimum bactericidal concentrations (MBC) of 1 mM.

[0066] E. coli J53 2138

[0067] All of compounds tested against E. coli J53 2138E showed a reduction in optical density. FKB3 and FKB11 were the only compounds with an observable MBC of 1 mM respectively.

[0068] E. coli J53 2140E

[0069] All of the compounds tested show a reduction of optical density over 24 h, with the largest reduction in change of optical density seen for FKB11 at 200 μM (FIG. 5C). FKB3 and FKB11 were the only compounds with an observable MBC of 1 mM respectively.

[0070] E. coli 1496 ESBL

[0071] All of the compounds tested show a reduction of optical density over 24 h, with the largest reduction in change of optical density seen for FKB3 at 1 mM, as well as FKB10 at 200 μM (FIG. 6A/B). FKB3 and FKB11 were the only compounds with an observable MBC of 1 mM respectively.

[0072] P. aeruginosa ATCC

[0073] All of the compounds tested caused a reduction in change of optical density at 1 mM (FIG. 7). The largest of these reductions occurred when P. aeruginosa was treated with FKB11 (FIG. 7C). However, despite this, there was no observable MBC for any compound against P. aeruginosa, suggesting these compounds are bacteriostatic, rather than bactericidal against this organism.

[0074] K. pneumoniae H467

[0075] Each compound tested against K. pneumoniae H467 showed a reduction in optical density over 24 h. The largest reduction occurred with the addition of FKB11 at 1 mM (FIG. 8C). Both FKB3 and FKB11 gave an MBC of 1 mM.

CONCLUSIONS

[0076] These compounds show activity against a wide range of bacterial species, including highly antimicrobial Gram negative strains. Although the optimal concentration for activity is high (1 mM), a number of these compounds are bactericidal against the majority of the organisms screened. Therefore, these compounds represent an intriguing avenue for use and future optimisation and drug development.