GB2321454A - Gemcitabine esters and amides - Google Patents

Abstract

Gemcitabine esters or amides in which the 3'- and/or 5'-OH group and/or the N<SP>4</SP>-amino group is derivatised with a C 18 - and/or C 20 -saturated or monounsaturated acyl group, preferably an acyl group selected from oleoyl, elaidoyl, cis-eicosenoyl and trans-eicosenoyl, are useful as anti-cancer and anti-viral agents.

Description

GEMCITABINE DERIVATIVES This invention relates to certain long chain saturated and monounsaturated fatty acid derivatives of 2',2'-difluorodeoxycytidine (Gemcitabine), and to pharmaceutical compositions containing them.

Gemcitabine has the formula:

Gemcitabine is a nucleoside analogue which has shown effect for the treatment of neoplastic conditions in both in vitro and in in vivo studies. (New anticancer agents, Weiss et al, Cancer Chemotherapy and Biological Response Modifiers Annual 16, editors Pinedo, Longo and Chabner, 1996. Elsevier Science B.V., Supplement to Seminars in Oncology, Vol. 22, No. 4, Suppl. 11, 1995, editors Yarbro et al. Gemcitabine Hydrochloride: Status of Preclinical Studies). A beneficial effect has also been observed in the clinical development of Gemcitabine. In these studies both the clinical and side effects of Gemcitabine are highly schedule dependent. (Seminars in Oncology, Vol. 22, No. 4, Suppl. 11, 1995, pp 42-46).

Gemcitabine is activated inside the cell by deoxycytidine kinase to its active form, the triphosphate of Gemcitabine (dFdCTP). Parallel to this Gemcitabine is deactivated by deoxycytidine deaminase to the corresponding uracil derivative (inactive).

We have now surprisingly found that certain fatty acid derivatives of Gemcitabine have a totally altered pharmacokinetics and tissue distribution.

Especially will this be the case with malignant diseases in the RES, lungs, pancreas, intestines, esophagus, uterus, ovaries, melanoma and mammae.

The compounds of the present invention can be represented by the formula I:

wherein R1, R2 and R3 are independently selected from hydrogen and C18- and C20- saturated and monounsaturated acyl groups, with the proviso that R1, R2 and R3 cannot all be hydrogen.

Gemcitabine has three derivatisable functions, namely the 5'- and 3'-hydroxyl groups and the N4 amino group. Each group can selectively be transformed into an ester or amide derivative, but di-adducts (di-esters or ester-amides) and tri-adducts may be formed as well.

In the case of the di- and tri-adducts the acyl substituent groups need not necessarily be the same.

Currently, the mono-acyl derivatives of this invention, i.e. with two of Rl, R2 and R3 being hydrogen, are preferred. It is especially preferred that the monosubstitution with the acyl group should be in the 3'-O and 5'-O positions of the sugar moiety, with 5'-O substitution being most preferred.

The double bond of the mono-unsaturated acyl groups may be in either the cis or the trans configuration, although the therapeutic effect may differ depending on which configuration is used.

The position of the double bond in the monounsaturated acyl groups also seem to affect the activity.

Currently, we prefer to use esters or amides having their unsaturation in the -9 position. In the system of nomenclature, the position U of the double bond of a monounsaturated fatty acid is counted from the terminal methyl group, so that, for example, eicosenoic acid (C20:1 w-9) has 20 carbon atoms in the chain and a single double bond is formed between carbon 9 and 10 counting from the methyl end of the chain. We prefer to use esters, ester-amides and amides derived from oleic acid (C18:1 c-9, cis), elaidic acid (C18:l -9, trans), eicosenoic acid(s) (C20:l -9, cis) and (C20:1 0-9, trans), and the amides and 5'-esters are currently the most preferred derivatives of this invention.

Esters, ester-amides and amides of Gemcitabine derived from stearic acid (Cl8: ) and eicosanoic acid (C20:0) are advantageously used in some cases.

The derivatives of Gemcitabine according to this invention may generally be prepared according to the following reaction equation:

wherein Nu-YH stands for Gemcitabine, Y is oxygen at the 3' and/or 5' position of the sugar moiety or nitrogen at the 4 position of the pyrimidine moiety of Gemcitabine, Fa is, an acyl group of a monounsaturated C18 or C23 fatty acid, and X is a leaving group, for example Cl, Br, 3-thiazolidine-2-thione or OR' wherein R' is Fa, COCH3, COEt or COCF3. Thus, the reaction proceeds by acylation of the nucleoside. This is accomplished by the use of suitable reactive derivatives of fatty acids, especially acid halides or acid anhydrides.

Generally, a proton scavenger needs to be present in order to mop up the acid HX which is liberated by the reaction. Thus, a base may be added to the reaction mixture. For example, when an acid halide such as an acid chloride is used, a tertiary amine base, such as triethylamine, N,N-dimethylaniline, pyridine or N,N-dimethylaminopyridine can be added to the reaction mixture to bind the liberated hydrohalic acid. In other cases, a solvent used in the reaction may serve as the proton scavenger.

Normally, the acylation reaction proceeds without the need for a catalyst.

The reactive fatty acid derivative FaX may, in some cases, be formed in situ by means of coupling reagents such as N,N'-dicyclohexylcarbodiimide (DCC), N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC) or O-(lH-benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate(TBTU).

The reactions are preferably carried out in an unreactive solvent such as N,N-dimethylformamide or a halogenated hydrocarbon, such as dichloromethane.

If desired any of the above mentioned tertiary amine bases may be used as solvent, taking care that a suitable excess is present. In this case a separate proton scavenger is not needed. The reaction should preferably be kept between 50C and 25"C. After a period of 1 to 60 hours, the reaction will be essentially completed.

The progress of the reaction can be followed using thin layer chromatography (TLC) and appropriate solvent systems. When the reaction is completed as determined by TLC, the product can be extracted with an organic solvent and purified by chromatography and/or recrystallization from an appropriate solvent system. As more than one hydroxyl group and also an amino group are present in Gemcitabine, a mixture of acylated compounds may be produced. If required, the individual mono- and multiacylated derivatives required may be separated by, for instance, chromatography, crystallization, supercritical extraction, etc.

When it is desired to prepare a multi-acyl compound of formula I, in which Rl and/or R2 and/or R3 are the same acyl group, it is preferred to employ the above method(s) using the appropriate acyl-reagent(s) in excess.

In order to prepare multi-acyl compounds of formula I, in which R1 and/or R2 and/or R3 are different, it is preferred to employ the above methods in a stepwise manner with the appropriate choice of reagent. It is also possible to employ properly chosen protecting groups to ensure a specific reaction. A selection of these methods is shown in Scheme 1 below. Any combination of the methods may be employed to prepare a specific product.

NHCOR' N II NH2 R's 0F NH 'F RCOCI OH RCOO < X I RCOO ON NHPG, NH, NHPGI NH2 NA NA l l R"COOH PG,O - R"COOH HO 1" OH R"COO ~ A PG3O BDC OH HOOF F Deprotect F F R..coO Scheme 1.

The following Examples illustrate the preparation of two preferred Gemcitabine derivatives of this invention.

EXAMPLE 1 Elaidic acid (5')-Gemcitabine ester To a solution of 2',2'-difluorodeoxyribofuranosylcytosine (Gemcitabine) (0.42g, 1.6 mmol) in 30 ml DMF was added 0.81 ml DMF containing 1.6 mmol HCl(g) followed by a solution of elaidic acid chloride (0.51g, 1.7 mmol) in 3 ml DMF and the reaction mixture was stirred at ambient temperature for 12 hours.

The solvent was evaporated at high vacuum and the crude product was purified on a column of silica gel with 15% methanol in chloroform as the eluent system. The impure fractions were repurified to give a total of 0.25g (30%) of the title compound.

1H NMR (DMSO-d6 300 MHz) b: 7.5(1H, d, ArH), 7.45(2H, br.

s, NH2), 6.45(1H, d, -OH), 6.17(1H, t, CH-1'), 5.8(1H, d, ArH), 5.35(2H, m, CH=CH), 4.4-4.05(3H, m, CH2-5' and CH-4'), 3.95(1H, m, CH-3'), 2.35 (2H, t, CH2-COO), 1.95(2H, m, CH2-CH=) , l.55(2H, m, CH2-C-COO) , 1.25(20H, m, CH2) , 0.85(3H, t, CH3).

EXAMPLE 2 Elaidic acid (N4)-Gemcitabine amide To a solution of 2',2'-difluorodeoxyribofuranosylcytosine (Gemcitabine) (0.38g, 1.3 mmol) in 5 ml pyridine was added elaidic acid chloride (0.57g, 1.9 mmol) and the reaction mixture was stirred at ambient temperature for 2.5 hours. The solvent was evaporated at high vacuum and the crude product was purified on a column of silica gel with 15% methanol in chloroform as the eluent system. Product containing fractions were evaporated, and the residue was treated with ether/hexan in an ultra-sound bath. The crystalline material was dried to give 0.lg (15%) of the title compound.

1H NMR (DMSO-d6 300 MHz) 6: 10.95(1H, s, NHCO), 8.25(1H, d, ArH), 7.25(1H, d, ArH), 6.30(1H, d, -OH), 6.15(1H, t, CH-1'), 5.35(2H, m, CH=CH), 5.30(lH, t, -OH), 4.2(lH, m, CH-4'), 3.9-3.6(3H, m, CH-3' and CH2-5'), 2.35(2H, t, CH2-CON), 1.95(2H, m, CH2-C=), l.55(2H, m, CH2-C-COO), 1.25(20H, m, CH2) , 0.85(3H, t, CH3).

Preferred Gemcitabine derivatives of this invention have a higher therapeutic value for treating malignant diseases than Gemcitabine itself.

Gemcitabine has an optimal effect at a plasma concentration of about 20 HM but higher concentrations, above 35 HM, inhibit the anti-cancer effect due to saturation of the phosphoralation mechanism.

(Gandhi, Cellular Pharmacology of Gemcitabine in Gemcitabine: Rationales for Clinical Trial Design and Evaluation, Mini Symposium, 12.3.96, Vrije Universiteit Amsterdam) . In contrast, preferred Gemcitabine derivatives of the invention yield an optimal plasma level of Gemcitabine for a prolonged time without reaching inhibitory concentrations ( > 35 jiM).

This may be because the derivatives are not subject to phosphorylation and probably not an inhibitor of the mechanism either.

A main problem in cancer treatment is development of resistance to therapy. Multi drug resistance (MDR) is one of the principal reasons for failure of otherwise effective drugs. We have found that the preferred derivatives of this invention somehow block the MDR-pump, and hence circumvent this problem.

The half-life of Gemcitabine in plasma is approximately 10 minutes, due to rapid deamination by the endogenous enzyme deoxycytidine deaminase to the corresponding uracil derivative (P.G. Johnston et al, Cancer Chromatography and Biological Response Modifiers, Annual 16, 1996, Chap. 1, ed. Pinedo H.M. et al.).

In contrast, the derivatives of this invention are poor substrates for the deactivating enzyme, and therefore their half-life is increased. Consequently, the derivatives of this invention are more suited than Gemcitabine itself for systemic or local treatment of malignant tumours.

The new compounds of this invention are not only potentially useful in the treatment of cancer, but also have activity as anti-viral agents..

BIOLOGY Experimental The cytoxicity activity of Gemcitabine-N4elaidic amide and Gemcitabine-5'-elaidic ester were investigated in 2 pairs of rodent and human tumour cell lines, each consisting of a parent line and a subline either resistant or cross-resistant to Gemcitabine.

The cell lines were the human ovarian tumour line A2780 and subline AG6000 which is resistant to Gemcitabine and has a deficiency of deoxycytidine kinase, and the murine colon tumour line C26A and the subline C26G with no altered deoxycytidine kinase but a 10-fold decrease in thymidine kinase I. The cytotoxicity of each compound was evaluated following continuous drug exposure for 72 hours. The cell numbers were determined by SRB assay, and percentage growth inhibition was calculated for each tumour line as IC50 value, given in HM, that is the concentration of the compound giving rise to a 50% growth inhibition compared to control.

Results The IC50 value in UM of cytotoxicity activity of Gemcitabine itself in comparison to cytoxicity activity of Gemcitabine-N4-elaidic amide and Gemcitabine-5'elaidic ester are shown in the table below.

The acitivity of the derivatives of the Gemcitabine is much greater than the cytotoxic activity of Gemcitabine in the cell lines tested.

Table The cytotoxicity of Gemcitabine, Gemcitabine N4-elaidic amide and Gemcitabine-5'-elaidic ester in IC50 (jiM) values in the cell lines C26-A, C26-G, A2780 and AG6000

C26-A C26-G A2780 AG6000 Gemcitabine 0.0055 0.0075 0.0005 100 Gemcitabine-N4 elaidic amide < 0.0001 c0.0001 < 0.0001 35 Gemcitabine-5' elaidic ester 0.0003 0.0005 < 0.0001 100 The Gemcitabine esters or amides of the present invention may be administered systemically, either enterally or parenterally.

For enteral administration, the active compounds of the present invention may be presented as, e.g. soft or hard gelatine capsules, tablets, granules, grains or powders, drags, syrups, suspensions or solutions.

When administered parenterally, preparations of the Gemcitabine esters or amides as injection or infusion solutions, suspensions or emulsions are suitable.

The preparation can contain inert or pharmacodynamically active additives, as well known to those skilled in the formulation arts. For instance, tablets or granulates can contain a series of binding agents, filler materials, emulsifying agents, carrier substances or dilutes. Liquid preparations may be present, for example in the form of a sterile solution.

Capsules can contain a filler material or thickening agent in addition to the active ingredient.

Furthermore, flavour-improving additives as well as the substances usually used as preserving, stabilising, moisture-retaining and emulsifying agents, salts for varying the osmotic pressure, buffers and other additives may also be present.

The dosage in which the preparations according to this invention are administered will vary according to the mode of use and route of use, as well as to the requirements of the patient. In general a daily dosage for a systemic therapy for an adult average patient will be about 0.1-150 mg/kg body weight/day, preferably 1-40 mg/kg/day. For topical administration, an ointment, for instance, can contain from 0.1-10k by weight of the pharmaceutical formulation, especially 0.5-5k by weight.

If desired, the pharmaceutical preparation containing the Gemcitabine esters or amides can contain an antioxidant, e.g. tocopherol, N-methyl-tocophermine, butylated hydrocyanisole, ascorbic acid or butylated hydroxytoluene.

Combination therapies, i.e. in which the administration of a Gemcitabine ester or amide of this invention is carried out in conjunction with other therapies, e.g. surgery, radiation treatment and chemotherapy, are also contemplated. For example, the preferred treatment of brain tumours seems likely to be a combination of surgery and treatment with a Gemcitabine ester or amide of this invention by systemic or local administration.

Claims (12)

CLAIMS:

1. A Gemcitabine derivative having the formula (I):

wherein R1, R2 and R3 are independently selected from hydrogen and C18- and C20- saturated and monounsaturated acyl groups, with the proviso that R1, R2 and R3 cannot all be hydrogen.

2. A compound according to Claim 1, wherein only one of R1, R2 and R3 is a said acyl group.

3. A compound according to Claim 2, wherein said mono-acyl substitution is at the 3'-O or 5'-O position of the sugar moiety.

4. A compound according to Claim 3, wherein said mono-acyl substitution is at the 5'-O position of the sugar moiety.

5. A compound according to any preceding claim, wherein R1, R2 and R3 are selected from oleoyl, elaidoyl, cis-eicosenoyl and trans-eicosenoyl.

6. Elaidic acid (5')-Gemcitabine ester.

7. Elaidic acid (N4)-Gemcitabine amide.

8. A pharmaceutical composition, comprising a Gemcitabine ester or amide according to any preceding claim and a pharmaceutically acceptable carrier or excipient.

9. A Gemcitabine ester or amide according to any one of Claims 1-7 for use as an anti-cancer agent.

10. A Gemcitabine ester or amide according to any one of Claims 1-7 for use as an anti-viral agent.

11. The use of a Gemcitabine ester or amide according to any one of Claims 1-7 in the manufacture of a pharmaceutical composition having anti-cancer activity.

12. A process for preparing a Gemcitabine derivative as defined in Claim 1, characterized by reacting Gemcitabine with a compound of the formula: FaX wherein Fa is an acyl group of a monounsaturated C18 or C20 fatty acid, and X is a leaving group.