5-ACETAMIDOMETHYL-OXAZOLIDINONE DERIVATIVES FOR USE IN THE TREATMENT OF CANCER

Abstract

The disclosure provides a compound, or a pharmaliorating or preventing cancer.

##STR00001##

Claims

1. A method of treating, preventing or ameliorating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I): ##STR00007## wherein X is O, S, SO or SO.sub.2; R.sup.1 is hydrogen, except when X is O then R.sup.1 can be hydrogen, CN, CO.sub.2R.sup.6 or a C.sub.1-2 alkyl, optionally substituted with OR.sup.6, OCOR.sup.6, N(R.sup.6).sub.2 or NHCOR.sup.6; R.sup.2 is hydrogen, except when X is O and R.sup.1 is CH.sub.3 then R.sup.2 can be H or CH.sub.3; R.sup.3 and R.sup.4 are independently hydrogen, F or Cl; R.sup.5 is hydrogen, C.sub.1-8 alkyl optionally substituted with one or more of R.sup.7; C.sub.3-6 cycloalkyl, amino, C.sub.1-8 alkylamino, C.sub.1-8 dialkylamino or C.sub.1-8 alkoxy; each R.sup.6 is independently hydrogen, C.sub.1-8 alkyl optionally substituted with one or more of R.sup.7, C.sub.3-6 cycloalkyl, amino, C.sub.1-8 alkylamino, C.sub.1-8 dialkylamino or C.sub.1-8 alkoxy; each R.sup.7 is independently F, Cl, OH, C.sub.1-8 alkoxy, C.sub.1-8 acyloxy or O—CH.sub.2—Ph; and n is 0, 1 or 2; or a pharmaceutically acceptable salt or solvate thereof.

2. The method of claim 1, wherein X is O.

3. The method of claim 1, wherein R.sup.1 is hydrogen, CN, CO.sub.2R.sup.6 or a C.sub.1-2 alkyl, optionally substituted with OR.sup.6, OCOR.sup.6, N(R.sup.6).sub.2 or NHCOR.sup.6, optionally wherein R.sup.1 is hydrogen, CN, CO.sub.2H or a C.sub.1-2 alkyl, optionally substituted with OH, OCOH, NH.sub.2 or NHCOH.

4. (canceled)

5. The method of claim 1, wherein R.sup.1 is hydrogen or a C.sub.1-2 alkyl.

6. The method of claim 1, wherein R.sup.2 is hydrogen.

7. The method of claim 1, wherein at least one of R.sup.3 and R.sup.4 is F or Cl.

8. The method of claim 1, wherein one of R.sup.3 and R.sup.4 is F or Cl and the other is hydrogen, optionally one of R.sup.3 and R.sup.4 is F and the other is hydrogen.

9. The method of claim 1, wherein R.sup.5 is hydrogen or a C.sub.1-8 alkyl optionally substituted with one or more of R.sup.7.

10. The method of claim 1, wherein R.sup.5 is hydrogen or a C.sub.1-5 alkyl optionally substituted with one or more of R.sup.7, optionally wherein R.sup.5 is CH.sub.3.

11. (canceled)

12. The method of claim 1, wherein n is 1.

13. The method of claim 1, wherein the compound of formula (I) is a compound of formula (Ia): ##STR00008## or a pharmaceutically acceptable salt or solvate thereof.

14. The method of claim 1, wherein the cancer is a solid tumour or solid cancer.

15. The method of claim 1, wherein the cancer is bowel cancer, brain cancer, breast cancer, endometrial cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer, optionally wherein: (i) the bowel cancer is colon cancer or rectal cancer; (ii) the brain cancer is a glioma or a glioblastoma; (iii) the breast cancer is a HER2 positive breast cancer or HER2 negative breast cancer; (iv) the liver cancer is hepatocellular carcinoma; (v) the lung cancer is non-small cell lung cancer or small cell lung cancer; or (vi) the skin cancer is a melanoma.

16. (canceled)

17. The method of claim 1, wherein the compound of formula (I) is used in combination with one or more chemotherapy drugs, optionally wherein the compound of formula (I) is administered after the one or more chemotherapy drugs.

18. The method of claim 17, wherein the chemotherapy drug comprises bleomycin, capecitabine, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, doxorubicin, epirubicin, eribulin, etoposide, 5-fluorouracil, folinic acid, gemcitabine, methotrexate, mustine, oxaliplatin, paclitaxel, prednisolone, procarbazine, vinblastine, vincristine and/or vinorelbine.

19. The method of claim 1, wherein the compound of formula (I) is used in combination with a drug that damages DNA or which interferes with the DNA damage response process (DDR).

20. The method of claim 19, wherein the compound of formula (I) is used in combination with a Poly (ADP-ribose) polymerase (PARP) inhibitor, an ATM inhibitor, an ATR inhibitor, a checkpoint inhibitor, a vascular endothelial growth factor (VEGF) inhibitor or a wee1 inhibitor.

21. The method of claim 20, wherein (i) the PARP inhibitor is a PARP1 inhibitor; or (ii) the checkpoint inhibitor is a programmed cell death protein 1 (PD-1) inhibitor, a programmed death-ligand 1 (PD-L1) inhibitor or a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor.

22. The method of claim 21, wherein the PARP1 inhibitor is aurothiomalate, aurothioglucose (ATG), rucaparib, olaparib, nirparib, talazoparib, veliparib, pamiparib, 2X-121 or auranofin.

23. The method of claim 22, wherein the PARP1 inhibitor comprises a gold complex, optionally wherein the PARP1 inhibitor comprises aurothiomalate, ATG or auranofin.

24.-29. (canceled)

Description

[0070] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0071] FIG. 1 is a graph showing the absorbance values BRCA1 deficient ovarian cancer cells, UWB1.289, exposed to cisplatin 1 μM for 24 hours followed by Minocycline, Aurothiomalate (ATM), Aurothioglucose (ATG), Rucaparib, Olaparib, Nirparib, Auranofin or Linezolid for 6 days;

[0072] FIG. 2 is a graph showing the percentage proliferation of the UWB1.289 cell line shown in FIG. 1 for selected experiments;

[0073] FIG. 3 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Olaparib and Linezolid for 6 days;

[0074] FIG. 4 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Olaparib and AZD6738 (AZD) for 6 days;

[0075] FIG. 5 is a graph showing the percentage proliferation of the UWB1.289 cell line shown in FIGS. 3 and 4 for selected experiments;

[0076] FIG. 6 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Auranofin and Linezolid for 6 days;

[0077] FIG. 7 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Auranofin and AZD6738 (AZD) for 6 days;

[0078] FIG. 8 is a graph showing the percentage proliferation of the UWB1.289 cell line shown in FIGS. 6 and 7 for selected experiments;

[0079] FIG. 9 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Aurothiomalate (ATM) and Linezolid for 6 days;

[0080] FIG. 10 is a graph showing the absorbance values of the UWB1.289 cell line after it was exposed to cisplatin 1 μM for 24 hours and followed by a combination of Aurothiomalate (ATM) and AZD6738 (AZD) for 6 days;

[0081] FIG. 11 is a graph showing the percentage proliferation of the UWB1.289 cell line shown in FIGS. 9 and 10 for selected experiments; and

[0082] FIG. 12 is a graph showing the size of tumours in mice in a MDA-MB-436 (BRCA1 mutation) breast cancer cell xenograft experiment, where the mice are either untreated or are treated with 100 mg/kg of linezolid BID.

EXAMPLE 1

Comparison of Ability of PARPis and ATR Inhibitors to Reduce Proliferation of Cancer Cells

[0083] Methods [0084] Cell morphology, viability and proliferation rate was assessed by visual and counting method. [0085] Day 0: Cells were split and around 1000 cells/well were seeded in normal complete media. [0086] Day 1: Cisplatin was added at 1 μM to the test and controls wells. Untreated cells were left as control. [0087] Day 2: medium containing cisplatin was discarded and new fresh media containing bromodeoxyuridine (BrdU) and the drugs at the concentrations shown in table 1, below, was added. [0088] Day 6: medium was discarded, and cells fixed. BrdU assay was performed according to the manufacturer. [0089] Controls comprised cells without BrdU (Blank), untreated cells (UN) and Cisplatin treated cells for 24 hours only (CIS). [0090] All experiments were conducted in triplicate.

TABLE-US-00001 TABLE 1 Concentrations of drugs in medium added to cells Conc Conc Conc Conc Conc Conc Conc Compound 1 2 3 4 5 6 7 Minocycline 0.5 nM 5 nM 50 nM 500 5 50 μM 500 nM μM μM Aurothiomalate 0.05 0.5 nM 5 nM 50 500 5 μM 50 (ATM) nM nM nM μM ATG 0.0012 0.012 0.12 1.2 12 120 1.2 nM nM nM nM nM nM μM Rucaparib 0.01 0.1 nM 1 nM 10 100 1 μM 10 nM nM nM μM Olaparib 1 nM 10 nM 100 1 10 100 1 nM μM μM μM mM Niraparib 0.00002 0.0002 0.002 0.02 0.2 2 nM 20 nM nM nM nM nM nM Auranofin 0.01 0.1 nM 1 nM 10 100 1 μM 10 nM nM nM μM Linezolid 0.01 0.1 nM 1 nM 10 100 1 μM 10 nM nM nM μM

[0091] BrdU Proliferation Assay [0092] After 6 days of incubation with drugs, the media was discarded, and cells were fixed for 30 minutes, at room temperature, with FixDenat Solution. [0093] FixDenat Solution was then removed and substituted with Anti-BrdU-POD working solution for 2 hours at room temperature. [0094] Plates were then washed 3 times with Washing buffer. [0095] Substrate solution was added. [0096] The reaction was stopped by adding H2SO4 and immediately read at 450 nm.

[0097] Data Analysis

[0098] Data was analysed using Excel and Prism software. The average of the absorbances and the standard error were calculated using the technical triplicate for each condition.

[0099] Results

[0100] Olaparib, rucaparib and niraparib are all known and approved PARPis. Unsurprisingly, the results show that these compounds were able to effectively reduce proliferation of the cancer cells. However, these approved PARPis do not reduce proliferation close to zero.

[0101] ATM, ATG and auranofin are all gold complexes that can act as PARPis. FIGS. 1 and 2 show that these compounds were also able to effectively reduce proliferation of the cancer cells. The proliferation reduction caused by the gold complexes is approximately equivalent to the proliferation reduction achieved by the approved PARPis.

[0102] Meanwhile, minocycline is a selective PARP2 inhibitor. As shown in FIGS. 1 and 2, this compound was not able to effectively reduce proliferation of the cancer cells, except at high concentrations. This is consistent with the observation that PARP1 is required for DDR.

[0103] Finally, the results show that linezolid achieves a similar decrease in proliferation of cancer cells to the approved PARPis and the gold complexes.

EXAMPLE 2

Combining PARPis and ATR Inhibitors to Reduce Proliferation of Cancer Cells

[0104] Methods

[0105] The methods were the same as described in example 1 except that the new fresh media added on day 2 contained bromodeoxyuridine (BrdU) and the drugs at the concentrations shown in table 2, below. All possible combinations of concentrations for compounds A and B were tested.

TABLE-US-00002 TABLE 2 Concentrations of drugs in medium added to cells Compound Concentration Compound Concentration A of compound A B of compound B Olaparib 2 nM, 20 nM, Linezolid 1 nM, 10 nM and 200 nM, 2 μM and 100 nM 20 μM Olaparib 200 nM, 2 μM AZD 7.4 nM, 74 nM and 20 μM and 740 nM Aurothiomalate 0.3 nM, 3 nM and Linezolid 1 nM, 10 nM and (ATM) 30 nM 100 nM Aurothiomalate 0.3 nM, 3 nM and AZD 7.4 nM, 74 nM (ATM) 30 nM and 740 nM Auranofin 1 nM, 10 nM and Linezolid 1 nM, 10 nM and 100 nM 100 nM Auranofin 1 nM, 10 nM and AZD 7.4 nM, 74 nM 100 nM and 740 nM

[0106] Results

[0107] The results are shown in FIGS. 3 to 11. Due to the fact that approved PARPis, gold complexes and ATR inhibitors do not reduce proliferation close to zero, the inventors have considered combination treatments in order to evaluate the possibility of additional, synergistic proliferation reduction of combination regimes in comparison to individual drug therapy.

[0108] AZD6738 (AZD) is a known ATR inhibitor. As expected, the combination of olaparib and AZD resulted in a further decreased proliferation when compared to the degree of proliferation observed for olaparib alone, see FIG. 5. The addition of linezolid, instead of AZD, also showed a similar decrease in proliferation.

[0109] The combination of auranofin or Aurothiomalate (ATM) with linezolid or AZD showed a particularly marked improvement over the gold complexes when used alone, see FIG. 11. In fact, proliferation was reduced to 2% when Aurothiomalate (ATM) was present at a concentration of 30 nM and linezolid was present at a concentration of 100 nM.

EXAMPLE 3

MDA-MB-436 (BRCA1 Mutation) Breast Cancer Cell Xenograft Experiment

[0110] Animal Maintenance

[0111] Animals were quarantined for 7 days before the study. The general health of the animals was evaluated by a veterinarian, and complete health checks were performed. Animals with abnormalities were excluded prior the study.

[0112] Housing

[0113] General procedures for animal care and housing were in accordance with the standard, Commission on Life Sciences, National Research Council, Standard operating procedures (SOPs) of Pharmaron, Inc. The mice were kept in laminar flow rooms at constant temperature and humidity with 3-5 mice in each cage. Animals were housed in polycarbonate cage which is in the size of 300×180×150 mm.sup.3 and in an environmentally monitored, well-ventilated room maintained at a temperature of (22±3° C.) and a relative humidity of 40% to 70%. Fluorescent lighting provided illumination approximately 12 hours per day. The bedding material was soft wood, which was changed once per week.

[0114] Diet

[0115] Animals had free access to irradiation sterilized dry granule food during the entire study period except for time periods specified by the protocol.

[0116] Water

[0117] Sterile drinking water in a bottle was available to all animals ad libitum during the quarantine and study periods. The bottle and the stopper with attached sipper tube was autoclaved prior to use. Samples of water from the animal facility were analyzed and results of water analysis were reviewed by the veterinarian, or designee, to assure that no known contaminants were present that could have interfered with or affected the outcome of studies.

[0118] Method for Tumour Inoculation

[0119] The MDA-MB-436 tumour cell line was maintained in vitro as monolayer culture in DMEM medium modified supplemented with 10% heat inactivated foetal bovine serum at 37° C. in an atmosphere of 5% CO.sub.2 in air. The tumour cells were routinely sub-cultured once a week by trypsin-EDTA treatment, not to exceed 4-5 passages. The cells growing in an exponential growth phase were harvested and counted for tumour inoculation.

[0120] All mice were inoculated subcutaneously on the right flank with MDA-MB-436 tumour cells (1×10.sup.7) in 0.1 ml of DMEM with Matrigel mixture (1:1 ratio) for tumour development. The treatment started when the mean tumour size reached is approximately 100-150 mm.sup.3. Mice were then be assigned to groups such that the mean tumour volume is the same for each treatment group. The treatments were administered to the tumour-bearing mice orally at 12 hour intervals.

[0121] Formulation

[0122] 560 mg linezolid was dissolved in 1.4 ml ethanol and 12.6 ml PEG400. The solution was vortexed and sonicated with high energy ultrasonic probe to get a uniform solution. The resultant solution was used for 1 day.

[0123] Tumour Measurements

[0124] The measurement of tumour size was conducted twice weekly with a calliper and recorded. The tumour volume (mm3) was estimated using the formula: TV=a×b.sup.2/2, where “a” and “b” are long and short diameters of a tumour, respectively.

[0125] Results

[0126] As shown in FIG. 12, the mice treated with 100 mg/kg of linezolid BID had a reduced tumour size of 38% compared to the control group.

[0127] It is noted that the dose 100 mg/kg BID of linezolid administered to the mice is equivalent to a human dose. This is generally a dosage of 600 mg BID, either orally or by i.v., in humans.

[0128] Conclusions

[0129] Linezolid has been shown to reduce proliferation of cancer cells when used alone, for both in vitro and in vivo experiments. Furthermore, a synergistic effect is observed when linezolid is used with a PARPi. A particularly noticeable synergistic effect was observed for the combination of ATM and linezolid. The inventors conclude that proliferation reduction due to linezolid is equivalent to proliferation reduction achieved by AZD6738, arguably the ATR inhibitor that is currently the most advanced in its clinical trial programme.