Use of sanglifehrin macrocyclic analogues as anticancer compounds

Abstract

The present invention relates to sanglifehrin macrocyclic analogues according to Formula (I), ##STR00001##
particularly for use in treatment of cancer, especially liver cancer.

Claims

1. A sanglifehrin analogue having the structure ##STR00008## or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising a sanglifehrin analogue as defined in claim 1 together with one or more pharmaceutically acceptable excipients.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Structure of cyclosporine A and analogues

(2) FIG. 2: Structure of sanglifehrin A and analogues

(3) FIG. 3: In vitro evaluation of anticancer activity of sanglifehrin macrocyclic analogue NV556, and cyclosporine based cyclophilin inhibitors cylcosporin A (CsA) and alisporivir (ALV) on the hepatocellular carcinoma cell line HepG2. Viable cell counts were determined from triplicate samples after four days of incubation in the presence of test compounds.

(4) FIG. 4: shows results of in vitro evaluation of combined anticancer activity of a sanglifehrin macrocyclic analogue with the anticancer agent sorafenib against the SK-Hep-1 tumour cell line in a monolayer proliferation assay

(5) FIG. 5: shows results of in vitro evaluation of combined anticancer activity of a sanglifehrin macrocyclic analogue with the anticancer agent doxorubicin against the SK-Hep-1 tumour cell line in a monolayer proliferation assay

(6) FIG. 6: shows results of in vivo evaluation of anticancer activity of a sanglifehrin macrocyclic analogue against the HuH-7 tumor cell line implanted subcutaneously into mice

(7) FIG. 7: shows a synergistic growth inhibitory effect by simultaneous treatment of the HepG2 tumor cell line with Compound 1 and the kinase inhibitor sorafenib at different doses in a monolayer proliferation assay

EXAMPLES

(8) Materials & Methods

(9) Materials

(10) Unless otherwise indicated, all reagents used in the examples below were obtained from commercial sources.

(11) Compound Generation

(12) Compounds of the invention are generated using methods as described in WO2011/098809, WO2011/144924, WO2011/098808, WO2012/085553, WO2011/098805, WO2012/131371 and WO2012/131377.

(13) In Vitro Bioassays of Anticancer Activity

(14) The cells were seeded in a 96-well plate, test compound or DMSO was added and the plate was incubated for additional four days or 7 days (Calcein AM assay). Viable cell counts were determined after detachment by treatment with trypsin either by trypan blue exclusion, flow cytometry, indirectly by measuring ATP content by addition of an ATP measuring reagent (CellTiter Glo), or Calcein AM, prepared according to the instruction of the manufacturer directly.

(15) Percentage inhibition in the ATP test was calculated by the formula described below:
Growth inhibition %: 100100(ExperimentalBlank)/(DMSOBlank)

(16) XLfit software ((Fit model: Dose response one site/.sup.F(x)[fit=(A+((BA)/(1+((C/x){circumflex over ()}D))))]) for curve fitting and IC50 calculation) was used for curve fitting and IC50 calculation.

(17) In Vivo Assessment of Anticancer Activity

(18) The HUH-7 (Human Liver tumor) cell or another cell line was cultured in DMEM (Low glucose)+2 mM L-glutamine with fetal bovine serum added to a final concentration of 10%. The cell line was sub-cultured by removing and discarding the culture medium. The cell layer was rinsed briefly with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor. Thereafter, 2.0 to 3.0 mL of Trypsin-EDTA solution was added to the flask and the cell layer was observed under an inverted microscope until detached. The cells were aspirated by gentle pipetting after addition of 6.0 to 8.0 mL of complete growth medium. A sub-cultivation ratio of 1:1 to 1:2 was used and the culture flasks were incubated at 37 C. with medium renewal two to three times per week.

(19) BALB/c nude female mice, 6-8 weeks, weighing approximately 18-20 g were maintained in a special pathogen-free environment and in individual ventilation cages (4-6 mice per cage). An acclimation period of approximately one week was used between animal receipt and tumor inoculation in order to accustom the animals to the laboratory environment. All cages, bedding, and water were sterilized before use. The mice were inoculated subcutaneously in the right flank with HUH-7 tumor cells (110.sup.6 cells/mouse) in 0.2 mL mixture of base media with 50% Matrigel. The treatments were started when the average tumor size reached approximately 150 mm.sup.3-180 mm.sup.3. Body weight was determined and tumor size measured with a caliper two times per week.

(20) In vitro evaluation of compounds for anticancer activity in a panel of human tumour cell lines in a monolayer proliferation assay were carried out at Shanghai Chempartner. The characteristics of the selected cell lines is summarised in Table 1.

(21) TABLE-US-00001 TABLE 1 Test cell lines # Cell line Origin 1 A-375 skin, malignant melanoma 2 U251 brain, human glioma 3 HCT 116 colon, carcinoma 4 A2780 ovary, carcinoma 5 Hep G2 Liver, carcinoma 6 Huh-7 Liver, carcinoma, 7 SK-Hep-1 Liver, adenocarcinoma 8 A549 lung, carcinoma 9 PANC-1 pancreas, ductal carcinoma 10 RPMI 8226 myeloma, plasmacytoma, B lymphocyte 11 PC-3 prostate, adenocarcinoma 12 HT-29 colon, adenocarcinoma 13 MDA-MB-231 Breast, carcinoma 14 Hep3B2.1-7- Liver, carcinoma Luc 15 LIXC-003 Liver, carcinoma 16 LIXC-004 Liver, carcinoma 17 LIXC-006 Liver, carcinoma 18 LIXC-011 Liver, carcinoma 19 LIXC-012 Liver, carcinoma 20 LIXC-066 Liver, carcinoma 21 LIXC-086 Liver, carcinoma

Example 1. Generation of Compound 1

(22) ##STR00007##

(23) To a mixture of compound 2 (Hansson et al., 2015, Chemistry & Biology 22, 285-292 and WO 2012/131377, 50 mg, 0.066 mmol), tert-butyl diethylphosphonoacetate (50 mg, 0.198 mmol) and LiCl (8.3 mg, 0.198 mmol) in CH.sub.3CN (1 mL) was added dropwise a solution of DBU (20 mg, 0.132 mmol) in CH.sub.3CN (0.2 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography twice (first time using petroleum ether/EtOAc as eluent and then DCM/MeOH) to afford compound 1 as light yellow solid.

Example 2. In Vitro Bioassay for Anticancer Activity

(24) In vitro evaluation of anticancer activity of sanglifehrin macrocyclic analogue NV556, and cyclosporine based cyclophilin inhibitors cyclosporine A (CsA) and alisporivir (ALV) on the hepatocellular carcinoma cell line HepG2. Viable cell counts were determined from triplicate samples after four days of incubation in the presence of test compounds. The results are shown in FIG. 3.

Example 3. In Vitro Bioassays for Anticancer Activity

(25) In vitro evaluation of a sanglifehrin macrocyclic analogue (NV556) for anticancer activity in a panel of 11 human tumour cell lines in a monolayer proliferation assay was carried out as described in the general methods above. Data are means from two independent experiments testing 10 concentrations (3 nM-25 M) in duplicate.

(26) TABLE-US-00002 Maximum Concentration giving 50% Cell line inhibition (%) of maximum effect (M) A375 87.6 2.1 U251 62.0 16.9 HCT 116 88.7 0.1 A2780 83.7 0.1 HepG2 80.6 0.1 Huh-7 22.4 0.03 SK-Hep-1 94.6 0.1 A549 45.2 0.1 PANC-1 51.0 1.3 RPMI 8226 87.1 0.2 PC-3 45.3 0.3 HT-29 56.8 41.6 MDA-MB-231 49.9 0.5

Example 4. In Vitro Bioassays for Anticancer Activity

(27) In vitro evaluation of the combined anticancer activity of a sanglifehrin macrocyclic analogue with the anticancer agents sorafenib or doxorubicin against the SK-Hep-1 tumour cell line in a monolayer proliferation assay was carried out as described in the general methods above. The results are shown in FIGS. 4 and 5.

(28) As can be seen from the data, the combination of NV556 and sorafenib or NV556 and doxorubicin displayed a stronger reduction in cell numbers than either compound alone over certain concentration ranges.

Example 5. In Vitro Bioassays for Anticancer Activity

(29) In vitro evaluation of a sanglifehrin macrocyclic analogue (1) for anticancer activity against human hepatocyte cancer cell lines in comparison with NV556 (10) a 7-day proliferation assay. The cell numbers were determined by Calcein AM staining as described in the methods section. Data are from one experiment with each cell line testing duplicate samples of 9 concentrations of each compound. Both compounds were included in each experiment.

(30) TABLE-US-00003 IC.sub.50 (nM) Cell line NV556 (10) Compound 1 Hep3B2.1-7-Luc 54 11 LIXC-002 39 <3 LIXC-006 62 9 LIXC-066 244 93 LIXC-086 9491 905

(31) As can be seen from the data, compound 1 was more potent (3-10-fold) than NV556 (10) in all cases.

Example 6. In Vitro Bioassays for Anticancer Activity

(32) In vitro evaluation of a sanglifehrin macrocyclic analogue (1) for anticancer activity against human hepatocyte cancer cell lines in comparison with the cyclophilin inhibitor alisporivir and the kinase inhibitor sorafenib in a 7-day proliferation assay. The cell numbers were determined by Calcein AM staining as described in the methods section. Data are from one single experiment or means of two independent experiments testing duplicate samples of 9 concentrations of each compound. All three compounds were included in each experiment.

(33) TABLE-US-00004 IC.sub.50 (nM) Cell line Compound 1 Alisporivir Sorafenib n HepG2 7 3131 2964 2 Hep3B2.1-7-Luc 9 2885 3899 1 LIXC-003 8 4729 4 2 LIXC-004 4 3809 2191 2 LIXC-006 6 993 3962 2 LIXC-011 12 8710 538 1 LIXC-012 65 3221 1152 2 LIXC-066 169 1639 1654 1 LIXC-086 686 2251 1897 1

(34) As can be seen from the data, compound 1 was more potent (3-1000-fold) than alisporivir in all cases and more potent (3-600-fold) than sorafenib in 8/9 cases.

Example 7. In Vivo Assessment of Anticancer Activity

(35) NV556 was evaluated for anticancer effect on human HuH-7 hepatocellular carcinoma cell growth in nude mice. The tumor cells were injected subcutaneously in the right flank and treatment with NV556 (10) was initiated when the tumor size had reached 150 mm.sup.3-180 mm.sup.3. NV556 was administered once daily by oral gavage (PO) or intraperitoneal injections (IP) at a dose of 100 mg/kg (N=8). The results of biweekly tumor measurements (mean and standard error) are shown in FIG. 6.

Example 8. In Vitro Combination of Compound 1 with Sorafenib

(36) To test for potential synergistic activity with sorafenib, compound 1 (A) was added at a range of concentrations along with a range of concentrations of sorafenib (B) against a liver cancer cell line, HepG2 in a 7-day proliferation assay (Calcein AM assay, described in the methods section). Data are from one single experiment representative of two independent experiments testing duplicate samples. The data was plotted in a normalized isobologram (Chou and Martin. CompuSyn for Drug Combinations: PC Software and User's Guide: A Computer Program for Quantitation of Synergism and Antagonism in Drug Combinations, and the Determination of IC50 and ED50 and LD50 Values, ComboSyn Inc, Paramus, (NJ), 2005) and is shown in FIG. 7. Values at the lower left below the hypotenuse indicate synergism.