WO2015060738A1

WO2015060738A1 - N-{2- [4- (4-methoxyophenyl)-1,3-thiazol-2-yl] ethyl}-2-oxo-2, 5, 6, 7-tetrahydro-1h-cyclopenta [b] pyridine-3-carboxamide for use in the treatment of cancer

Info

Publication number: WO2015060738A1
Application number: PCT/PL2014/050067
Authority: WO
Inventors: Wiesław ŚWIĘTNICKI
Original assignee: WROCŁAWSKIE CENTRUM BADAŃ EIT+ Sp z o.o.
Priority date: 2013-10-23
Filing date: 2014-10-23
Publication date: 2015-04-30
Also published as: PL405743A1; PL226024B1

Abstract

The subject of the invention is N-{2-[4-(4-methoxyphenyl)-1,3- thiazol-2-yl]ethyl}-2-oxo-2,5,6,7-tetrahydro-1H- cyclopenta[b]pyridine-3-carboxamideto be used as an anti- cancer agent or efflux pump inhibitor.

Description

N-{2- [4- (4-METHOXYOPHENYL)-1 ,3-THIAZOL-2-YL] ETHYL}-2-OXO-2, 5, 6, 7-TETRAHYDRO-1 H-CYCLOPENTA [B]

PYRIDINE-3-CARBOXAMIDE FOR USE IN THE TREATMENT OF CANCER

5 The subject of the invention is N- { 2- [ 4- ( 4-methoxyphenyl ) -1 , 3- thiazol-2-yl ] ethyl }-2-oxo-2, 5, 6 , 7-tetrahydro-lH- cyclopenta [b] pyridine-3-carboxamide as an anti-cancer agent or efflux pump inhibitor. The invention finds application in medicine .

10 An active efflux is a mechanism responsible for the extrusion of toxic substances and antibiotics from within cells to the external environment. The said mechanism is important in medicine, as it may contribute to bacterial immunity to antibiotics or chemotherapy during cancer

15 treatment. ABC type (ATP-binding cassette) efflux pumps are known in the form of protein transporters located in the cellular membranes of all cell types. As active transporters, they require a source of chemical energy, which may be provided by, amongst others, adenosine triphosphate hydrolysis

20 as the energy source. From the US patent US6586424 and the international patent application WO2012/16934 Al tyrosine kinases inhibitors are known, which may be useful during treatment of, amongst others, cancers. The search for efflux pump inhibitors applicable in cancer treatment is still on.

25 The subject of the invention is N- { 2- [ 4- ( 4-methoxyphenyl ) -

1 , 3-thiazol-2-yl] ethyl }-2-oxo-2, 5, 6, 7-tetrahydro-lH- cyclopenta [b] pyridine-3-carboxamide to be used as an anticancer agent .

The second subject of the invention is N-{2-[4-(4- 30 methoxyphenyl ) -1, 3-thiazol-2-yl] ethyl }-2-oxo-2, 5, 6, 7- tetrahydro-l/i-cyclopenta [b] pyridine-3-carboxamide to be used as an efflux pump inhibitor, preferably type ABC class Bl, B8, CI and G2.

Example 1 synthesis of N- { 2- [ 4- ( 4-methoxyphenyl ) -1 , 3-thiazol-

2-yl ] ethyl }-2-oxo-2, 5, 6 , 7-tetrahydro-l/i-cyclopenta [b] pyridine-

3-carboxamide N-[2- [4- (4-methoxyphenyl ) -1 , 3-thiazole-2-yl ] ethyl ] -2-oxo-l , 5 , 6 , 7-tetrahydro-lH-cyclopenta [b] pyridine-3- carboxamide

The following were added to glass test tubes (10ml) : 2-oxo- 2,5,6, 7-tetrahydro-lH-cyclopenta [b] pyridine-3-carboxylic acid (92 mg, 0.51 mmol), dry DMF (2ml), 2 - (4 - (4 methoxyphenyl ) -1 , 3-thiazol-2-yl ) ethanamine hydrochloride (125 mg, 0.46 mmol), Et3N (56 mg, 0.55 mmol), Nl- ( ( ethylimino ) methylene ) -N3 , N3-dimethylpropane-l , 3-diamine (87 mg, 0.56 mmol) and benzotriazol-l-ol (93 mg, 0.69 mmol) . The obtained mixture was mixed at room temperature for two days and then diluted with water (3 ml) and CHC13 (3 ml) . The obtained sediment was filtered and dried in vacuum, obtaining 52 mg (26% efficiency) .

Example 2

The compound obtained in example 1 was dissolved in 100% DMSO, which in the test constituted solvent control whereas cisplatin was the reference compound. Starting solutions (stock solutions) of the tested substance from example 1 with a concentration of 10 mM were prepared for each experiment ex tempore, dissolving 0.8 μΐ of the compound in 400 μΐ test medium OR ( OptiMEM+RPMI , 5% FBS, antibiotics ( SP ( streptomycin, penicillin) ) . Using a series of dilutions method, solutions of the tested substances were prepared together with a solvent control, obtaining final compound concentrations of 10, 1, 0.1, 0.01 μΜ and 0.1, 0.01, 0.001, 0.0001 % DMSO. Prior to every solution sampling, the solutions were centrifuged at revs 300 x g, for 5 min. Cisplatin [1 mg/ml] was used in the tests, with final concentrations 10, 1, 0.1, 0.01 g/ml respectively. To determine cytotoxic activity of tested inhibitors, the following cell lines were used: BAL/B3T3 - mice fibroblasts, LoVo - human colon carcinoma, LoVoDX - doxorubicin resistant human colon cancer. The lines were obtained from the IITD PAN, Wroclaw cell line collection. The cells were grown in an incubator, in humid atmosphere of 5% CO₂ and 95% air at 37°C. OR medium was used to prepare a number of dilutions. The cells were grown in media dedicated to particular lines given in Table 1.

Table 1 Growing media used in tests Example 3 Determining cytotoxicity

Cytotoxic activity was determined as follows. Approximately 10,000 cells in 100 μΐ dedicated culture medium were placed into a 96-well storage plate, medium without cells placed into 3 wells. After 24h incubation at 37°C in 5% CO₂ and 95 % air, solutions of tested compounds were applied (3 times for each dilution) to the last row of the 96-well plate which constituted control cells and control medium to which only OR test medium was added. Half of a 96-well storage plate was prepared in a similar manner for solvent control (DMSO) and cisplatin. After 72 h, 20 μΐ of MTT solution was added to each well, then after 4 h of incubation 80 μΐ of lysis buffer was added and a reading was made using spectrophotometer set at 545 nm.

Each experiment was repeated three times.

BAL/B3T3, Lovo, L0V0DX, IC50 (μΜ) IC50 (μΜ) IC50 (μΜ)

Compound from IC50 IC50 8.68±0.78 example 1 undetectable undetectable

DMSO IC50 IC50 IC50

undetectable undetectable undetectable

Cis-Pt 2.65±1.19 3.30±2.25 2.26±1.52 DMSO solvent control did not affect tested cells and proliferation inhibition did not exceed 15% in any used cell line. However, the addition of compound from example 1 led to significant toxicity for LoVoDX line, but not for LoVo line. 5 Toxicity was caused by doxorubicin, which the LoVoDx line may remove aided by active efflux pumps. Blocking efflux pumps by the compound from example 1 is the reason for doxorubicin toxicity for LoVoDX line. Toxicity for cisplatin is caused by the specificity of efflux pumps overexpression .

10

Table 3 shows cell proliferation inhibition following DMSO [%] and Cis-Pt [ug/ml] treatment.

% proliferation inhibition concentration 0.0001 0.001 0.01 0.1 1 10

DMSO average 0.00 0.00 3.90 0.00

SD 0.00 0.00 6.75 0.00

BALB/3T3

Cis-Pt average 3.96 14.68 32.86 77.26

SD 6.86 10.51 10.66 11.99

DMSO average 3.47 7.25 4.13 3.47

SD 6.00 6.72 5.40 6.00

LoVo

Cis-Pt average 1.79 11.26 31.02 74.24

SD 3.10 7.85 12.98 14.47

DMSO average 0.49 1.26 1.99 2.94

SD 0.69 2.03 3.45 5.10

LoVoDX

Cis-Pt average 0.00 3.48 33.50 89.40

SD 0.00 3.76 29.86 9.23 Table 3

Cisplatin toxicity for cell lines is caused by a lack of mechanism removing cisplatin from cells. BALB/3T3 line normally does not contain overexpressed ABC type efflux pumps which are able to remove cisplatin. LoVoDx does contain such overexpressed transporters but these may be unspecific for cisplatin .

Table 4 shows cell proliferation inhibition relations to the used compound concentration

Table 4

Cell proliferation inhibition by the compound obtained in example 1 indicates compound toxicity. For BALB/3T3 and LoVo lines proliferation inhibition does not exceed 25%. LoVoDX line exhibits proliferation inhibition of more than 50% in the presence of doxorubicin in the same compound concentration obtained in example 1, which indicates a lack of active doxorubicin removal mechanism. The lack of mechanism is caused by inhibition of ABC type efflux pump which remove doxorubicin as normally the LoVoDX line overexpresses such pumps. Efflux pumps blocked by the compound obtained in example 1 as ABC type Bl, B8, CI and G2 class pumps.

Claims

Patent claims

1. N-{ 2- [4- ( 4-methoxyphenyl) -1, 3-thiazol-2-yl ] ethyl } -2-oxo- 2,5,6, 7-tetrahydro-l/i-cyclopenta [b] pyridine-3 -carboxamide to be used as an anti-cancer agent.

2. N- { 2- [ 4- ( 4-methoxyphenyl ) -1, 3-thiazol-2-yl] ethyl } -2-oxo- 2,5,6, 7-tetrahydro-l/i-cyclopenta [b] pyridine-3 -carboxamide to be used as an efflux pump inhibitor, preferably type ABC class Bl, B8, CI and G2.