NO318934B1 - gemcitabine - Google Patents

Description

GEMCITABINDER DERIVATIVES

This origin relates to certain long-chain, saturated and monounsaturated fatty acid derivatives of 2\2'-difluorodeoxycytidine (gemcitabine) and to pharmaceutical compositions in which they form part. Gemcitabine has the formula:

Gemcitabine is a nucleoside analogue that has shown efficacy in the treatment of neoplasia according to investigations both in vitro and in vivo. (New Anticancer agents, Weiss et al., Cancer Chemotherapy and Biological Response Modifiers Annual 16, ed. Pinedo, Longo and Chabner, 1996. Elsevier Science B.V., Supplement to Seminars in Oncology, vol. 22, no. 4, suppl. 11, 1995, eds 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 effects and side effects of gemcitabine are highly dependent on the treatment regimen. (Seminars in Oncology, vol. 22, no. 5, suppl. 11, 1995, pp. 42-46).

Gemcitabine is activated intracellularly by the enzyme deoxycytidine kinase to the active form, gemcitabine triphosphate (dFdCTP). In parallel, 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 completely different pharmacokinetics and tissue distribution. In particular, this will be the case with malignant diseases of the reticuloendothelial system, lungs, pancreas, intestines, esophagus, uterus, ovaries, as well as melanoma and breast cancer.

The compounds according to the present invention can be represented by

formula I:

where R 1 , R 2 and R 3 are each either hydrogen or a saturated or monounsaturated Cl 8 or C 20 acyl group, with the proviso that R 1 , R 2 and R 3 cannot all be hydrogen.

Gemcitabine has three derivatizable functions, namely the 5'- and 3'-hydroxyl groups and the N<4->amino group. Each of the groups can be selectively transferred to an ester or amide derivative, but di-adducts (diesters or ester amides) and tri-adducts can also be formed. If di- or triadducts are formed, the acyl substituent groups do not necessarily have to be identical.

Today, the monoacyl derivatives according to this invention are preferred, i.e. that two of R1, R2 and R3 are hydrogen. It is particularly preferred that the monosubstitution with the acyl group is located in the 3'-0 and 5'-0 positions on the sugar group, and the 5'-0 substitution is the most preferred.

The double bond of the monounsaturated acyl groups may be in either the cis or 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 appears to have an effect on activity. Today, we prefer to use esters or amides that are unsaturated at the æ-9 position. In the co-nomenclature system, the æ position of the double bond in a monounsaturated fatty acid is counted from the outermost methyl group, so that, for example, eicosenoic acid (C20:1 CD-9) has 20 carbon atoms in the chain and a single double bond is formed between carbons 9 and 10 counted from the methyl end of the chain. We prefer to use esters, esteramides and amides derived from oleic acid (C18:l æ-9, cis), elaidic acid (C18:l cd-9, trans), the eicosenoic acids (C20:l (u-9, cis) and (C20 :l Cu-9, trans), and the amides and 5'-esters are today the most preferred derivatives according to this invention.

Esters, esteramides and amides of gemcitabine derived from stearic acid (Cl8:0) and eicosanoic acid (C20:0) are used with advantage in some cases.

The derivatives of gemcitabine according to this invention can generally be prepared according to the following reaction equation:

where Nu-YH stands for gemcitabine, Y is the oxygen at the 3'- and/or 5'-positions of the sugar group or the nitrogen at the 4-position of the pyrimidine group of gemcitabine, Fa is an acyl group of a monounsaturated Cl8 or C20 fatty acid and X is a group that is replaced, for example Cl, Br, 3-thiazolidine-2-thione or OR', where R' is Fa, COCH 3 , COEt or COCF 3 . In other words, the reaction involves acylating the nucleoside. This is achieved by using suitable reactive derivatives of fatty acids, especially acid halides or acid anhydrides.

In general, a proton renovator must be present to scavenge the acid HX released during the reaction. So a base must be added to the reaction mixture. If, for example, one uses an acid halide, e.g. an acid chloride, a base in the form of a tertiary amine, for example triethylamine, N,N-dimethylaniline, pyridine or N,N-dimethylaminopyridine, can be added to the reaction mixture to bind the liberated hydrogen halide. In other cases, a solvent used in the reaction can be used as a proton renovator.

Normally, the acylation reaction proceeds without the need for a catalyst. The reactive fatty acid derivative FaX can in some cases be formed in situ using binding reagents such as N,N'-dicyclohexylcarbodiimide (DCC), N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) or O-dH-benzotriazole-l -yO-N.N.N'^'-tetramethyluronium tetrafluoroborate (TBTU).

The reactions are preferably carried out in a non-reactive solvent such as N,N-dimethylformamide or a halogenated hydrocarbon such as dichloromethane. If desired, one of the above-mentioned tertiary amines can be used as solvent, provided that a suitable excess is measured. In this case, no special proton renovator is necessary. The reaction temperature should preferably be kept between 5 °C and 25 °C. After a period of 1 to 60 hours, the reaction will be almost complete. The progression of the reaction can be followed using thin layer chromatography (TLC) and suitable solvent systems. When the reaction is complete according to the TSK analysis, the product can be extracted with an organic solvent and purified by chromatography and/or recrystallization from a suitable solvent system. Since there is more than one hydroxyl group and also one amino group in gemcitabine, a mixture of acylated compounds can be formed. If necessary, the individual mono- and multi-acylated derivatives can be separated, for example, by chromatography, crystallization, supercritical extraction, etc.

If it is desired to prepare a multi-acyl compound of formula I where Ri and/or R2 and/or R3 are the same acyl group, it is preferred to use the above-mentioned method(s) with an excess of corresponding acyl reagent(s).

In order to prepare multi-acyl compounds of formula I where R 1 and/or R 2 and/or R 3 are different, it is preferred to use the above methods in a stepwise manner with appropriate choice of reagents. It is also possible to use properly chosen protecting groups to ensure a specific reaction. A selection of these methods is shown in form 1 below. Any combination of the methods can be used to produce a specific product.

The following examples illustrate the preparation of two preferred gemcitabine derivatives according to this invention.

EXAMPLE 1

Elaidic acid-(5' )-gemcitabine ester

To a solution of 2\2'-difluorodeoxyirbofuranosylcytosine (gemcitabine) (0.42 g, 1.6 mmol) in 30 mL of DMF was added 0.81 mL of DMF containing 1.6 mmol of HCl(g) and then a solution of elaidic acid chloride (0.51 g, 1.7 mmol) in 3 mL DMF and the reaction mixture was stirred at room temperature for 12 h. The solution was evaporated in high vacuum and the crude product was purified on a column of silica gel with 15% methanol in chloroform as the eluent system. The crude fractions were purified again to give a total of 0.25 g (30%) of the title compound.

1H NMR (DMSO-d6 300 MHz) δ: 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(4H, m, CH2-CH=), 1.55(2H, m, CH2-C-COO), 1.25(20H, m, CH2), 0.85(3H, t, CH3).

<13>C NMR (DMSO-4, 75 MHz) δ: 172.67(COO), 165.63(C-4), 154.51(C-2), 141.12(C-6), 130 .08 and 130.03(C-9'7C-10"), 126.09, 122.67 and 119.24(t, C-2'), 94.86(C-5), 83.90(C-r) , 77.36(C-4'), 70.41,70.11 and 69.80(t, C-3'), 62.53(C-5'), 33.24,31.95, 31 ,29,29,00, 28,94,28,84, 28,72,28,50, 28,43,28,33, 24,34,22,11(CH 2 ), 13.94(CH 3 ).

In addition, a small amount (0.05 g) of the elaidic acid (3'-)-gemcitabine ester was isolated from impure fractions.

<1>H NMR (DMSO-de 300 MHz) δ: 7.65(1H, d, ArH), 7.40(2H, d, NH2), 6.25(1H, t, t, CH-1' ), 5.82(1H, d, ArH), 5.4-5.2(4H, m, CH=CH and CH-3'), 4.15(1H, m, CH-4'), 3 .85-3.55(2H, m, CH-5'), 2.45(2H, t, CH2-COO), 1.95(2H, m, CH2-CH=), 1.55(2H, m, CH2-C-COO), 1.25(20H, m, CH2), 0.85(3H, t, CH3).

,<3>C NMR (DMSO-de, 75 MHz) δ: 171.70(COO), 165.69(C-4), 154.46(C-2), 141.30(C-6), 130.10 and 130.03(C-9'7C-10"), 125.17, 121.72 and 118.27(t, C-2'), 94.78(C-5), 83.78(C -1'), 78.41(C-4'), 69.93,69.60 and 69.30(t, C-3'), 59.15(C-5'), 32.95, 31 ,93, 31,26, 28,98,28,90,28,81, 28,69,28,46,28,28, 28,23,24,26,22,09(CH2), 13,95( CH3).

EXAMPLE 2

Elaidic acid-(N<4>)-gemcitabinamide

To a solution of 2',2'-difluorodeoxyirbofuranosylcytosine (gemcitabine) (0.38 g, 1.3(?) mmol) in 5 mL of pyridine was added elaidic acid chloride (0.57 g, 1.9 mmol) in 3 mL of DMF and the reaction mixture was stirred at room temperature for 2.5 hours. The solution was evaporated in high vacuum and the crude product was purified on a column of silica gel with 15% methanol in chloroform as the eluent system. The product-containing fractions were evaporated and the evaporation residue treated with ether/hexane in an ultrasonic bath. The crystalline material was dried to give 0.1 g (15%) of the title compound.

1H NMR (DMSO-de 300 MHz) δ: 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(1H, t, -OH), 4.2( 1H, m, CH-4'), 3.9-3.6(3H, m, CH-3' and CH2-5'), 2.35(2H, t, CH2-CON), 1.95( 4H, m, CH2-CH=), 1.55(2H, m, CH2-C-COO), 1.25(20H, m, CH2), 0.85(3H, t, CH3).

<13>C NMR (DMSO-de, 75 MHz) δ: 174.06(CONH), 162.89(C-4), 154.22(C-2), 144.69(C-6), 130 .04(C-9'7C-10"), 122.94(JCF=259Hz( C-2'), 95.91(C-5), 84.11(JCF= 31Hz, C-1'), 81.02(C-4'), 68.35(JCF= 22Hz, C-3'), 58.76(C-5'), 36.38, 31.94, 31.28, 28.99, 28,83,28,71, 28,56,28,48, 28,30,24,34,22,10(CH2), 13.94(CH3).

Preferred gemcitabine derivatives according to this invention have a higher therapeutic value for the treatment of cancer than gemcitabine itself. This has been shown in two different in vivo models both with single and repeated doses. In the case of single-dose treatment, the effect of the derivatives is better than or comparable to gemcitabine. This is particularly clear for the amide derivative where a much better effect is achieved with only 25% of the gemcitabine dose.

With repeated dosing, the difference between the derivatives and gemcitabine is even more striking. This shows itself both in increased survival time and in the number of long-term survivors. Another striking feature is the toxicity observed with gemcitabine even at both the highest and the average repeated dose. Although the effect achieved at the non-toxic low dose (1 mg/kg) is good, it is surpassed by both the N<4->amide and 5'-ester derivatives. Gemcitabine has an optimal effect at a plasma concentration of about 20 µM, and higher concentrations, above 35 µM, inhibit its anticancer effect due to saturation of the phosphorylation 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, the preferred gemcitabine derivatives according to the invention provide an optimal plasma concentration of gemcitabine for a long time without reaching any concentration that inhibits effectiveness (> 35 µM). The reason for this may be that the derivatives are not phosphorylated and probably do not inhibit the mechanism either.

A major problem with cancer treatment is the development of resistance to the treatment. Resistance to several drugs (multidrug resistance, MMR) is one of the most important reasons why otherwise effective drugs fail. We have found that the preferred derivatives of this invention somehow block the MMR pump and therefore circumvent this problem.

The cellular uptake of nucleosides and nucleoside analogues such as gemcitabine occurs mainly through the selective nucleoside transport receptor (NT).

Modulation/inhibition of this receptor can manifest as resistance to the drug in a clinical situation. This phenomenon can be observed in vitro by the addition of NT inhibitors. We have surprisingly found that our derivatives are not affected by the presence of NT inhibitors, the cytostatic activity of the preferred derivatives being the same in the presence of such inhibitors.

The half-life of gemcitabine in plasma is approximately 10 minutes, due to the fact that the endogenous enzyme deoxycytidine deaminase rapidly aminates it to the corresponding uracil derivative (P.G. Johnston et al, Cancer Chromatography and Biological Response Modifiers, Annual 16,1996, ch. 1, ed. Pinedo H.M. et al.).

The derivatives according to this invention, on the other hand, are poor substrates for the deactivating enzyme, and therefore have longer half-lives. Thus, the derivatives according to the invention are better suited than gemcitabine even for systemic or local treatment of malignant tumors.

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

BIOLOGY

Experimental

The cytotoxic activity of Gemcitabine-N-elaidic acid amide and gemcitabine-5'-elaidic acid ester was investigated in two pairs of rodent and human tumor cell lines, each consisting of a parental line and a daughter line either resistant to gemcitabine or to gemcitabine and a variety of other drugs .

The cell lines used were the human ovarian cancer line A2780 and the daughter line AG6000 which is resistant to gemcitabine and has a defective deoxycytidine kinase, and the colon cancer line C26A from mice, and the daughter line C26G without altered deoxycytidine kinase, but with thymidine kinase I reduced to 1/10 of the normal amount. The cytotoxicity of each compound was assessed after continuous drug exposure for 72 hours. The cell number was determined by SRB evaluation, percent growth inhibition was calculated for each tumor line as the IC 50 value, given in µM, that is the concentration of the compound which gives 50% growth inhibition compared to the control.

Results

The IC50 value in µM of the cytotoxic activity of gemcitabine itself compared to the cytotoxic activity of gemcitabine-N<4->elaidic acid amide and gemcitabine-5'-elaidic acid ester is shown in the table below. The activity of the derivatives of gemcitabine is much greater than the cytotoxic activity of gemcitabine against the tested cell lines.

The cytostatic activity of gemcitabine and gemcitabine-5'-elaidic acid ester in CEM cells was determined with and without the nucleoside transport modifiers nitrobenzylthioinosine (NBMPR) or persanthin (pyridamole). As can be seen from the table below, the IC50 for gemcitabine is more than twice as high as for gemcitabine-5<*->elaidic acid ester. On addition of the NT inhibitors, the IC50 values for gemcitabine increase tenfold, while little effect is seen on the values for gemcitabine 5'-elaidic acid ester (an increase of a factor of 1.3-1.5). In the "resistance" situation, the preferred derivative is 15-20 times stronger than the parent drug.

The antitumor effect of gemcitabine-N-elaidic acid amide or gemcitabine-5'-elaidic acid ester was investigated in vivo in mice with two different tumor types, both with single and repeated doses.

The effect of gemcitabine-N<4->elaidic acid amide or gemcitabine-5'-elaidic acid residues on Co-26 implanted in the spleen of mice

Balb/c female mice implanted with the mouse colon cancer Co-26 in the spleen on day 0.1 this model the tumors develop mainly in the liver. Intraperitoneal treatment was started on day 1. Single doses of the compounds were tested and compared with gemcitabine at a single dose. Saline was used as a control.

The average survival time for the animals that died was in the same range for the compounds tested. Gemcitabine-N<4->elaidic acid amide was superior to gemcitabine-5'-elaidic acid ester and gemcitabine in this respect with 5/8 survivors at a dose of only 25 mg/kg compared to 4/7 for gemcitabine at 100 mg/kg.

In a parallel experiment, the animals were given repeated doses on days 1-11

In this experiment, the results with gemcitabine-N<4->elaidic acid amide and low-dose gemcitabine-5'-elaidic acid ester were better than or equal to the results with low-dose gemcitabine. Although the high dose of gemcitabine 5'-elaidic acid ester is slightly toxic, it is less toxic than the high dose of gemcitabine itself.

The effect of gemcitabine-N<4->elaidic acid amide or gemcitabine-5'-elaidic acid ester on P-388 ip in mice, single doses or repeated doses

B6D2F1 female mice were implanted intraperitoneally with P 388 murine lymphocytic leukemia cells. The treatments were started on day 1 after the intraperitoneal implantation of the cells. The average survival time, long-term survivors and the number of deaths from the toxic effect were recorded after single-dose treatment, treatment with a repeated dose for 5 days and for 10 days. The results are shown in the tables below. Single-dose treatment with gemcitabine 5'-elaidic acid ester was effective in terms of prolonged survival time and long-term survival compared with the same dose of gemcitabine.

Single dose treatment

Treatment with repeated dose, day 1-4.

The activity of gemcitabine-N<4->elaidic acid amide was evident at repeated doses on days 1-4 with regard to observed long-term survivors and prolonged survival time at both 1 and 4 mg/kg. In the control group treated with 15 mg/kg gemcitabine, all the animals died from the toxic effect.

Treatment with repeated dose, treatment days 1-11

Treatment for 10 days increased the antitumor effect compared to the shorter treatment. Gemcitabine toxicity was more severe on a mg/kg basis, as 6/6 died from toxicity at 4 mg/kg. Long-term survival was observed after the repeated treatment both with gemcitabine-N<4->elaidic acid amide and gemcitabine-5'-elaidic acid ester, and a significantly prolonged mean survival time was observed both for gemcitabine-N^elaidins<y>reainide and gemcitabine-5' -elaidic acid ester.

The gemcitabine esters or amides according to the present invention can be administered systemically, either enterally or parenterally.

If they are given enterally, the active compounds according to the present invention can be given e.g. as soft or hard gelatin capsules, tablets, granules, grains or powder, dragee, syrup, suspension or solution.

Preparations of the gemcitabine esters or amides as injection or infusion solutions,

suspensions or emulsions are suitable for parenteral use.

The preparation may contain known inert or pharmacodynamically active additives. For example, tablets or granules may contain a series of binders, fillers, emulsifiers, carrier substances or diluents. Liquid preparations may be present, for example in the form of a sterile solution.

Capsules may contain a filler or thickener in addition to the active ingredient. They may also contain flavor-enhancing additives and such substances commonly used as preservatives, humectants, stabilizers, emulsifiers, salts to vary the osmotic pressure, buffers and other additives.

The dose with which the preparations according to this invention can be given will vary according to the method of use and the route of administration, as well as the patient's needs. In general, a daily dose for systemic therapy for an average adult patient will be approximately 0.1-150 mg/kg body weight/day, preferably 1-40 mg/kg/day. For local use, for example in an ointment, one can use from 0.1-10% by weight of the pharmaceutical formulation, especially 0.5-5% by weight.

If desired, the pharmaceutical preparation containing the gemcitabine esters or amides may contain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

Combination therapies, i.e. where gemcitabine esters or amides according to this invention are given simultaneously with other forms of therapy, e.g. surgery, radiotherapy and chemotherapy are also considered. For example, it seems likely that the preferred treatment for brain tumors is a combination of surgery and local or systemic treatment with a gemcitabine ester or amide according to this invention.

Claims (12)

1. A gemcitabine derivative of formula (I): where Ri, R2 and R3 are each either hydrogen or saturated or monounsaturated acyl groups with 18 or 20 carbon atoms in the chain, with the proviso that R1, R2 and R3 cannot all be hydrogen at the same time. 2. A compound according to claim 1 where only one of the units Ri, R2 and R3 is such an acyl group. 3. A compound according to claim 2 where said monoacyl substitution is made in the 3'-0 or 5'-0 position on the sugar group. 4. A compound according to claim 3 where the mentioned monoacyl substitution is made in the 5'-0 position on the sugar group. 5. A compound according to one or more of the preceding claims where Ri, R2 and R3 are either oleoyl, elaidoyl, cis-icosenoyl or trans-icosenoyl. 6. Elaidic acid-(5' )-gemcitabine esters. 7. Elaidic acid-(N<4>)-gemcitabinamide. 8. A pharmaceutical composition comprising a gemcitabine ester or a gemcitabinamide according to one or more of the preceding claims and a pharmaceutically acceptable carrier or excipient. 9. A gemcitabine ester or a gemcitabinamide according to one of claims 1-7 for use as an anticancer agent. 10. A gemcitabine ester or a gemcitabinamide according to one of claims 1-7 for use as an agent against viruses. 11. Use of a gemcitabine ester or a gemcitabinamide according to one of claims 1-7 in the preparation of a pharmaceutical composition which has activity against cancer. 12. A process for producing a gemcitabine derivative according to claim 1, characterized in that gemcitabine is reacted with a compound of the formula: where Fa is an acyl radical of a monounsaturated fatty acid containing 18 or 20 carbon atoms and X is a group that is replaced during the reaction.