RU2522548C2 - Uridine phosphorylase inhibitor - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a new derivative of uracil described by the following structural formula, and its pharmaceutically acceptable salt. The compound has the structural formula according to the R-enantiomer form:

The given invention also refers to a method for preparing the above compound and to a pharmaceutical composition on the basis of the above compound. The method for preparing consists in a reaction of 5-fluoruracil and R-tert-butyl-3-iodopropane-1.2-diyldicarbamate.

EFFECT: compound possesses the properties of uridine phosphorylase inhibitors and can be used as an active ingredient for preparing a drug for treating cancer, such as an antimetabolite of the uridine phosphorylase inhibitors of tumour cells.

4 cl, 1 tbl, 1 ex

Description

The object of the present invention is a new derivative of uracil, which is an inhibitor of uridine phosphrylase. This invention also relates to a method for producing the named compound and to a pharmaceutical composition based on this compound, which is an anti-metabolite of uracil.

Uridine phosphorylase (UPh, UVase) is a key enzyme of pyrimidine metabolism, catalyzing the reversible phosphorylation of uridine with the formation of uracil and ribose-1-phosphate [1, 2]. The enzyme has been identified in prokaryotes, yeast, and higher organisms [3]. The physiological activity of uridine phosphorylase is present in all living organisms from bacteria to humans, although the structure of the gene and the spatial structure of the protein molecule of UVase vary over a wide range [4, 5, 6, 7]. Protein is expressed in various cells and tissues of humans and animals. Uridine phosphorylase plays a decisive role in the so-called rapid clearance, that is, the elimination of excess plasma uridine from the body. [8] Induction of uridine phosphorylase activity was shown for colon carcinoma cells [9, 10, 11], melanoma [3], breast adenocarcinoma [12], hepatoma and Ehrlich ascites carcinoma [13]. The authors [9] found an increase in uridine phosphorylase activity in 9 out of 12 studied samples of colon carcinoma. In three samples, the enzyme activity did not change or slightly decreased. Katsumata et al. [10] found a positive correlation between uridine phosphorylase activity and the stage of the process in colon cancer.

Inhibition of uridine phosphorylase leads to the death of some protozoa - pathogens of humans and animals (for example, Giardia lamblia, Schistosoma mansoni) [14, 15, 16], because in protozoa, uridine phosphorylase is involved in the metabolism of uridine and thymidine [16], and most of the pyrimidine bases are synthesized by resynthesis.

M. Iigo et al. [17] showed that uridine phosphorylase inhibitors increase the activity of the antitumor pharmaceutical drug 5-fluorouracil (5FU) during chemotherapy of induced breast cancer in BDF ₁ mice and human squamous cell transplant grafts in nude mice. In experiments on mice of the CD ₈ F _{1 line} , it was found that the introduction of BAU (240 mg / kg) allowed us to reduce the dose of 5FU by 1.5 times with the same therapeutic efficacy [18]. Thus, the possibility of changing the activity of 5FU by specific uridine phosphorylase inhibitors in order to reduce the toxicity of the main drug was shown. This effect is associated with a decrease in 5FU catabolism under the action of an uridine phosphorylase inhibitor.

The antitumor drug 5-fluorouracil (5FU) is an antimetabolite of uracil due to competition for thymidyl synthetase. As a result of the action of 5FU, the synthesis of nucleic acids is disrupted and the proliferation and / or death of tumor cells are stopped. 5FU - an important component of modern chemotherapy - is successfully used in cancer of the colon, breast, digestive organs, genitourinary system, as well as skin tumors. To manifest a cytostatic effect, 5FU must be converted into one of the active metabolites: 5-fluoruridine-5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) or 5-fluorodeoxyuridine-5'- triphosphate (FdUTP) [19].

Currently used in clinical practice or undergoing preclinical or clinical trials, uridine phosphorylase inhibitors are also pyrimidine derivatives [20, 21, 22, 23]. The main compounds used are those synthesized on the basis of acycluridine or anhydrouridin (2.2'- or 2.3'-). These inhibitors were synthesized without the use of computer-aided rational design of molecules of biologically active compounds and do not form bonds with the phosphate anion in the phosphate binding center. The ethylene diamine group of the compound described in the patent forms hydrogen and ionic bonds with the phosphate anion, thereby improving the affinity of the compound for uridine phosphorylase. It should also be noted the low cost and ease of synthesis of the compound, since the scheme is one-stage, and all precursors are commercially available.

The present invention relates to a new class of potent uridine phosphorylase inhibitors.

Thus, an object of the invention is a compound of the formula

in the R-enantiomeric form, as well as a pharmaceutically acceptable salt of the compound, for example dihydrochloride.

Laboratory method for the preparation of a pharmaceutically acceptable salt of (R) -1- (2,3-Diaminopropyl) -5-fluoropyrimidine-2,4-dione

(R) -1- (2,3-Diaminopropyl) -5-fluoropyrimidin-2,4-dione dihydrochloride 3 can be prepared according to the following scheme:

Example 1

To a solution of 1.3 g (10 mmol) of 5-fluorouracil 1 in 20.0 ml of DMSO was added 2.0 g (5 mmol) of (R) -tert-butyl 3-iodopropane-1,2-diyl dicarbamate (obtained from (D) -2.3- diaminopropionic acid according to the method of [Wu W., Li R., Malladi SS, Warshakoon HJ, Kimbrell MR, Amolins MW, Ukani R., Datta A., David SAJ Med. Chem., 2010, vol. 53 (8), p .3198]) and 2.8 g (0.02 mol) of anhydrous potassium carbonate and the mixture is stirred under argon for 2 hours at 40 ° C. The reaction mass is poured into water and, with stirring, carefully neutralized with a 10% hydrochloric acid solution, the products are extracted with ethyl acetate, the extract is washed three times with water, dried and evaporated in vacuo. The residue was purified by chromatography (Silica Gel Merck 60, chloroform-methanol, 10: 0 → 10: 1) and crystallized from 1,4-dioxane.

The yield of the di (Boc) derivative is 0.8 g (39%).

T. pl.> 206-8 ° C. ¹ H NMR spectrum (DMSO-d ₆ , 400 MHz), δ, ppm: 10.99 (1H, br s, NH); 7.77 (1H, d, J = 5.7 Hz, H-6); 6.86 (1H, t, J = 6.1 Hz, NH); 6.43 (1H, d, J = 8.8 Hz, NH); 3.92-3.92 (2H, m, NCH ₂ ); 3.67-3.60 (1H, m, CH); 3.07-2.97 (2H, m, CH ₂ NH); 1.37 (9H, s, t-Bu); 1.27 (9H, s, t-Bu). Found: m / z (ESI), 403.1979 [M + H] ⁺ . C ₇ H ₁₂ FN ₄ O ₂ . Calculated: 403.1987.

To a solution of 0.7 g (1.7 mmol) of the di (Boc) derivative, 30.0 ml of methanol-dioxane (1: 1) are dissolved in a warm mixture, 30.0 ml of a solution of (2N) HCl in Et ₂ O are added and the mixture is stirred for 4 hours. The precipitate is filtered off. washed with Et ₂ O and dried in vacuo.

Yield 0.4 g (84%), colorless crystals of dihydrochloride 2. T. pl.> 260 ° C (decomp.). HPLC (LW = 267 nm, eluent H ₂ O-MeCN-HCO ₂ NH ₄ (98-2-0.2)) t _R = 4.80 min, purity 98.8%. ¹ H NMR spectrum (CD ₃ OD, 400 MHz), δ, ppm: 7.72 (1H, d, J = 5.13 Hz, H-6); 4.36-4.34 (2H, m, NCH ₂ ); 4.99-4.93 (1H, m, CH); 3.37-3.24 (2H, m, CH ₂ NH ₃ ). ¹³ C NMR spectrum (CD ₃ OD, 100 MHz), δ, ppm: 160.7 (C = O, J = 26 Hz); 152.6 (C = O); 141.1 (CF, J = 230 Hz); 126.5 (CH, J = 33 Hz); 153.7 (C = O); 50.3 (NCH ₂ ); 41.5 (CH); 40.0 (CH ₂ NH ₃ ). Found: m / z (ESI), 203.0921 [M + H] ⁺ . C ₇ H ₁₂ FN ₄ O ₂ . Calculated: 203.0939.

Waters systems were used for liquid chromatography, including a linear degasser, a Waters 600 quaternary nasal pump, a Gilson 233 plate sample applicator, and a Waters 996 PDA UV detector.

For mass spectrometry, a unified quadrupole mass spectrometer (Micromass, Platform model) was used, equipped with an electrospray source with a resolution of 0.8 Da with a 50% valley. Calibration was carried out monthly in the mass region of 80-1000 Da, using a calibration mixture of sodium iodide and rubidium iodide in a solution of a mixture of isopropanol / water (1/1 vol./about.).

Physical state and color under standard conditions: colorless crystals.

T _pl. > 200 ° C.

Applications

The compounds of the present invention can have wide therapeutic use. They can be effectively used in the treatment of a number of oncological diseases such as colon and rectal cancer, breast cancer, esophagus, stomach, pancreas, primary liver cancer, ovarian cancer, cancer of the cervix, bladder, malignant tumors of the head and neck, cancer prostate, adrenal cancer, vulvar cancer, penile cancer, carcinoid, skin cancer (for ointment).

The object of the present invention is also the products of the above formula, having the properties of an inhibitor of uridine phosphorylases as an active component in the manufacture of a medicinal product, as well as salts of adding pharmaceutically acceptable inorganic or organic acids of these products of the formula, as well as pharmaceutical compositions containing a drug as an active ingredient, the above, in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions containing a compound of this invention must be in liquid form in the form of a sterile solution for intravascular and intracavitary administration. Water for injection may be a suitable liquid carrier; weak solutions of hydrochloric acid and sodium chloride may act as stabilizers.

The meanings of all the special and scientific terms used in this document are well understood by a person skilled in the art. Moreover, all patents (or patent applications), as well as other bibliographic references, are incorporated by reference.

Modeling the binding of a compound to UVase from Salmonella typhimurium (StUPh) and Homo sapiens (hUPP1).

To study the mechanism of inhibition of uridine phosphorylases, the molecular docking method was used. The search for optimal solutions for ligand incorporation into the binding site of the target proteins StUPh and hUPP1 was carried out in accordance with the protocol worked out on the structure of the StUPh complex with 5-FUra (ID PDB: 3NSR), solved by X-ray diffraction. Molecular docking was performed by AutoDock 4.2 in a variant of the genetic algorithm [24].

According to the results of X-ray diffraction analysis of the StUPh support structure, the region of the uracil-binding site can be described by a rectangular parallelepiped with a side length of 15.1 16 × 16.4 Ǻ × 13.6 Ǻ. Based on these data, the ligand enzyme binding region was chosen in the form of a cube with an edge length of 22.5 Ǻ at its 60-fold discreteness.

A docking decision group was clustered if r.m.s.d. the atomic coordinates of the revealed conformers did not exceed 1.5 Ǻ. After molecular docking, the geometry of the complex was optimized in the Gromacs program (Gromos 96 force field, SPC 216 water model).

The putative mechanism of inhibition of uridine phosphorylases

5FU Patented compound Stuph

hUPP1

The propyl diamine group of the patented compound is coordinated by the phosphate anion via ionic and hydrogen bonds. In addition, an additional hydrogen bond is formed between the nitrogen atom of the propyldiamine group and the carboxyl oxygen atom of the side chain of glutamic acid (198 - StUPh, 250 - hUPP1). Thereby, a high affinity of the patented compound for the active center of uridine phosphorylase is achieved.

Information sources

1. Leer J.C., Hammer-Jespersen K., Schwartz M. // Eur J Biochem. 1977. V. 75. I.1. P.217-24.

2. Vita A., Amici A., Cacciamani T. et al. // Int J Biochem. 1986. V. 18. I.5. P.431-5.

3. Leyva A., Kraal I., Lankelma J. et al. // Anticancer Res. 1983. V.3. I.4. P.227-31.

4. Liu M., Cao D., Russell R. et al. // Cancer Res. 1998. V. 58. I.23. P.5418-24.

5. Takehara M., Ling F., Izawa S. et al. // Biosci Biotechnol Biochem. 1995. V. 59. I.10. P.1987-90.

6. Walton L., Richards C.A., Elwell L.P. // Nucleic Acids Res. 1989. V.17. I.16. P.6741.

7. Watanabe S., Hino A., Wada K. et al. // J Biol Chem. 1995. V.270. I.20. P.12191-6.

8. Moyer J.D., Henderson J.F. // Biochem Pharmacol. 1985. V.34. I.1. P.101-5.

9. Finan P.J., Koklitis P.A., Chisholm E.M. et al. // Br J Cancer. 1984. V.50. I.5. P.711-5.

10. Katsumata K., Tomioka H., Sumi T. et al. // Cancer Chemother Pharmacol. 2003. V. 51. I.2. P.155-60.

11. Luccioni C., Beaumatin J., Bardot V. et al. // Int J Cancer. 1994. V. 58. I.4. P.517-22.

12. Kanzaki A., Takebayashi Y., Bando H. et al. // Int J Cancer. 2002. V. 97. I.5. P.631-5.

13. Ishitsuka H., Miwa M., Takemoto K. et al. // Gann. 1980. V. 71. I.1. P.112-23.

14. el Kouni M.H., Naguib F.N., Niedzwicki J.G. et al. // J Biol Chem. 1988. V.263. I.13. P.6081-6.

15. Jimenez B.M., Kranz P., Lee C.S. et al. // Biochem Pharmacol. 1989. V.38. I.21. P.3785-9.

16. Lee C.S., Jimenez B.M., O'Sullivan W.J. // Mol Biochem Parasitol. 1988. V.30. I.3. P.271-7.

17. Iigo M., Nishikata K., Nakajima Y. et al. // Biochem Pharmacol. 1990. V.39. I.7. P.1247-53.

18. Martin D.S., Stolfi R.L., Sawyer R.C. // Cancer Chemother Pharmacol. 1989. V.24. I.1. P.9-14.

19. Iigo M., Nishikata K., Hoshi A. // Jpn J Cancer Res. 1992. V.83. I.4. P.392-6.

20. Niedzwicki J.G., Chu S.H., el Kouni M.H. et al. // Biochem Pharmacol. 1982. V.31. I.10. P.1857-61.

21. Drabikowska A.K., Lissowska L., Draminski M. et al. // Z Naturforsch C. 1987. V. 42. I.3. P.288-96.

22. Drabikowska A.K., Lissowska L., Veres Z.et al. // Biochem Pharmacol. 1987. V. 36. I.23. P.4125-8.

23. Pizzorno G., Yee L., Burtness B.A. et al. // Clin Cancer Res. 1998. V.4. I.5. P.1165-75.

24. Morris G. M., Huey R., Lindstrom W. et al. // J Comput Chem. 2009. V.30. I.16. P.2785-91.

Claims (4)

1. The compound of the formula

in the R-enantiomeric form, as well as a pharmaceutically acceptable salt of the compound. 2. The compound according to claim 1. having the properties of an inhibitor of uridine phosphorylase as an active component in the manufacture of a medicament suitable for the treatment of cancer, an antimetabolite inhibitor of uridine phosphorylase of tumor cells. 3. A pharmaceutical composition having the properties of an uridine phosphorylase inhibitor suitable for treating cancer, comprising the compound of claim 1 together with a pharmaceutically acceptable diluent or carrier. 4. A method for producing a compound of formula (I) as defined in claim 1, comprising reacting 5-fluorouracil with R-tert-butyl-3-iodopropane-1,2-diyl dicarbamate.