MXPA99006790A - Gemcitabine derivatives - Google Patents

Abstract

The invention provides Gemcitabine esters or amides in which the 3'- and/or 5'-OH group and/or the N4-amino group is derivatised with a C18- and/or C20- saturated or mono-unsaturated acyl group, preferably an acyl group selected from oleoyl, elaidoyl, cis-eicosenoyl and trans-eicosenoyl. The Gemcitabine esters and amides are useful as anti-cancer and anti-viral agents.

Description

GEMCITABIN DERIVATIVES DESCRIPTION OF THE INVENTION This invention relates to certain long chain, saturated and monounsaturated fatty acid derivatives of 2 ', 2' - difluorodeoxycytidine gemcitabine or (Gemcitabine) and with pharmaceutical compositions containing them. Gemcitabine has the formula: Gemcitabine is a nucleoside analogue which has shown an effect for the treatment of neoplastic conditions in both in vitro and in vivo studies (New anticancer agents, Weiss et al, Cancer Chemotherapy and Biological Response REF .: 30828 odifiers 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 on the clinical development of gemcitabine has also been observed. In these studies, the clinical and collateral effects of gemcitabine are highly dependent on the protocol: (Seminars in Oncology, Vol.22, No. 4, Suppl 11, 1995, pp. 42-46). Gemcitabine is activated inside cells by deoxycytidine kinase to its active form, gemcitabine triphosphate (dFdCTP). Parallel with this gemcitabine is deactivated by deoxycytidine deaminase to the corresponding uracil derivative (inactive). We have now surprisingly found that certain gemcitabine fatty acid derivatives have completely altered pharmacokinetics and tissue distribution. Especially this will be the case with malignant cancer diseases in RES, lungs, pancreas, intestine, esophagus, uterus, ovaries, melanoma and in the breasts. The compounds of the present invention can be represented by the formula I: wherein R-, R2 and R3 are independently selected from hydrogen and C18 and C20 saturated and monounsaturated acyl groups, with the proviso that Rx, R2 and R3 can not all be hydrogen. Gemcitabine has three derivatizable functions (which can form derivatives) specifically the 5 'and 3' hydroxyl groups and the amino group N4. Each group can be selectively transformed into an ester or amide derivative, but the diaducts (diesters or ester-amides) and the by-products will also be formed. In the case of the diaducts and triaductos, the acyl substituent groups do not necessarily need to be the same. Currently, the acyl derivatives of this invention, that is, with two of Rl f R2 and R3 which are hydrogen, are the ones that prefer. It is especially preferred that the monosubstitution with the acyl group be at the 3'-O and 5'-O positions of the sugar portion, with the 5'-O substitution being further preferred. The double bond of the monounsaturated acyl groups may be in the cis or trans configuration, although the therapeutic effect may differ, based on which configuration is used. The position of the double bond in the monounsaturated acyl groups also seems to affect the activity. Currently, we prefer to use esters or amides that have their unsaturation in position? -9. In the system of nomenclature 0, the position? in double bond of a monounsaturated fatty acid is counted from the terminal methyl group so that, for example, the eicosenoic acid (C20: l? -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, esters-amides and amides derived from oleic acid (C18: l? -9, cis), elaidic acid (C18: l? -9, trans), acid or eicosenoic acids (C20: l? -9, cis ) and C20: 1? -9 trans) and the amides and 5'-esters are currently the most preferred derivatives of this invention. The ester, ester-amide and gemcitabine amides derived from stearic acid (C18: 0) and eicasanoic acid (C20: 0) are advantageously used in some cases.

The gemcitabine derivatives according to this invention can be prepared generally according to the following reaction equation: Base Nu-YH + FaX > Nu-Y-Fa -HX wherein Nu-YH denotes gemcitabine, Y is oxygen at the 3 'and / or 5' position of the nitrogen sugar portion and position 4 of the pyrimidine portion of gemcitabine, Fa is an acyl group of a C18 monounsaturated fatty acid or C20 and X is a leaving group, for example Cl, Br, 3-thiazolidin-2-thione or OR1, wherein R1 is Fa, COCH3, COEt or COCF3. Therefore, the reaction is carried out by acylation of the nucleoside. This is carried out by the use of suitable reactive derivatives of fatty acids,. especially acid halides or acid anhydrides. Generally, a proton eliminator needs to be present in order to eliminate the HX acid which is released by the reaction. Therefore, a base can be enlarged 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 be attached to the reaction mixture. the hydrochloric acid released.

In other cases, a solvent used in the reaction can serve as the proton eliminator. Normally, the acylation reaction proceeds without the need for a catalyst. In some cases, the FaX reactive fatty acid derivative can be formed in situ by means of coupling reagents such as N, N'-dicyclohexy-1-carbodiimide (DCC), N-ethi-N '- (3-dimethylaminopropyl) carbodiimide (EDC) or 0- (IH-benzotriazol-1-yl) -N, N, N ', N'-etramethyluronium tetrafluoroborate (TBTU). Preferably the reactions are carried out in a non-reactive solvent such as N, N-dimethylformamide or a halogenated hydrocarbon such as dichloromethane. If desired, any of the tertiary amine bases mentioned above, "" - can be used as a solvent, taking care that it presents an adequate excess. In this case, a separate proton eliminator is not necessary. The reaction can preferably be maintained between 5 ° C and 5 ° C. After a period of 1 to 60 hours, the reaction will essentially be completed. The progress of the reaction can be monitored using thin layer chromatography (CCD) and appropriate solvent systems. When the reaction is complete, as determined by CCD, the product can be extracted with an organic solvent and purified by chromatography and / or recrystallization from an appropriate solvent system. Since more than one hydroxyl group and also an amino group are present in gemcitabine, a mixture of acylated compounds can be produced. If required, the necessary individual monoacylated and multiaclylated derivatives can be prepared, for example by chromatography, crystallization, supercritical extraction, etc. When it is desired to prepare a multiacyl compound of formula I in which Rx and / or R2 and / or R3 are the same acyl group, it is preferred to use the above methods using an excess of the appropriate acyl reagents. In order to prepare multiacyl compounds of formula I, in which Ri and / or R2 and / or R3 are different, it is preferred to use the above methods in a gradual manner with the choice of suitable reagent. It is also possible to suitably use protective groups chosen to ensure a specific reaction. In scheme 1 below, a selection of these methods is shown. Any combination of the methods can be used to prepare a specific product.

Scheme 1 The following examples illustrate the preparation of two preferred gemcitabine derivatives of this invention.

EXAMPLE 1 Ester (5 ') -gemcitabine of elaidic acid To a solution of 2', 2'-difluoro-deoxyribiburanosyl-cytosine (gemcitabine) (0.42 g, 1.6 mmol) in 30 ml of DMF is added 0.81 ml of DMF containing 1.6 mmoles of HCl (g) followed by a solution of elaidic acid chloride (0.51 g, 1.7 mmol) in 3 ml of DMF and the reaction mixture is stirred at room temperature for 12 hours. The solvent is evaporated under high vacuum and the untreated product is purified on a column of silica gel with 15% methanol in chloroform as the eluent system. The impure fractions are purified to give a total of 0.25 g (30%) of the title compound. XH NMR (DMSO-d6 300 MHz) d 7. 5 (1H, d, ArH), 7.45 (2H, broad S, NH2), 6.45 (1H, d, -OH), 6.17 (1H, t, CH-11), 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-d6, 75 MHz) d 172.67 (COO), 165.63 (C-4), 154.51 (C-2), 141.12 (C-6), 130.08 and 130.03 (C-9 '* / C- 10 ''), 126.09, 122.67 and ll9.24 (t, C-2 '), 94.86 (C-5), 83.90 (C-1'), 77.36 (C- •), 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 (CH2), 13-94 (CH3).

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

X H NMR (DMS0-d 6, 300 MHz) d: 7.65 (1 H, d, Ar H), 7.40 (2 H, d, NH 2), 6.25 I 1 H, t, CH-1 '), 5.82 (1 H, d, Ar H), 5.4-5.2 (4H, m, 0H-5 ', CH = CH and CH-3'), 4.15 (1H, m, CH-4 •), 3.85-3.55 (2H, m, CH2-5 '), 2.45 (2H, t, CH2-C00), 1.95 (2H, m, CH2C =), 1.55 (2H, m, CH2-C-C00), 1.25 (20H, m, CH2), 0.85 (3H, t, CH3) . 3C NMR (DMSO-d6, 75 MHz) d: 171.70 (COO), 165.69 (C-4), 154.46 (C-2), 141.30 (C-6), 130.10 and 130.03 (C-9 '' / C- 10"), 125.17, 121.72 yll8.27 (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 (N4) -gemcitabine elaide of elaidic acid To a solution of 2 ', 2' -difluorodeoxyribiburanosyl-cytosine (gemcitabine) (0.38 g, 1.3 mmol) in 5 ml of pyridine is added elaidic acid chloride (0.57 g, 1.9 mmol) and the reaction mixture is stirred at room temperature. environment for 2.5 hours. The solvent is evaporated under high vacuum and the crude product is purified on a column of silica gel with 15% methanol in chloroform as the eluent system. The fractions containing the product are evaporated, and the residue is treated with ether / hexane in an ultrasound bath. The crystalline material is dried to provide 0.1 g (15%) of the title compound.

X H NMR (DMSO-d 6 300 MHz) d: 10.95 (1 H, S, NHCO), 8.25 (1 H, d, Ar H), 7.25 (1 H, d, Ar H), 6.30 (1 H, 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 (2H, m, CH2-C =), 1.55 (2H, m, CH2-C-COO), 1.25 ( 20H, m, CH2), 0.85 (3H, t, CH3). 13 C NMR (DMSO-d 6, 75 MHz) d: 174. 06 (CONH), 162.89 (C-4) 154.22 (C-2), 144.69 (C-6), 130.04 (C-9 '' / C-10 ''), 122.94 (JCP = 259Hz, C-2"), 95.9KC-5), 84.11 (JCF = 3lHz, C-11), 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).

The preferred gemcitabine derivatives of this invention have a superior therapeutic value for treating malignancies compared to gemcitabine itself. This has been demonstrated in two different in vivo models with both single and repeated dosages. For the single dose treatment, the effect of the derivatives is better or comparable to that of gemcitabine. This is especially pronounced especially for the amide derivative where a superior effect is obtained with only 25% of the dose of gemcitabine. 'X At repeated dosing, the differences between the derivatives and gemcitabine are even more surprising. This is reflected both in an increased survival time and in long-term survivors. Another surprising feature is the toxicity observed with gemcitabine itself in repeated dosing in the high range as a medium.

Although the effect obtained from the non-toxic low-range dosage (1 mg / kg) is good, it is exceeded by both the N4-amide and the 5'-ester derivatives. Gemcitabine has an optimal effect at a plasma concentration of approximately μM, but higher concentrations, higher than 35 μM, inhibit the anticarcinogenic effect due to the 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 of the invention provide an optimal plasma level of gemcitabine for a prolonged time without reaching inhibitory concentrations (>; 35 μM). This may be because the derivatives do not undergo phosphorylation and probably not a mechanism inhibitor either. A major problem in the treatment of cancer is the development of resistance to therapy. Multiple drug resistance (MDR) is one of the main reasons for the failure of otherwise effective medications. We have found that the preferred derivatives of this invention somehow block the pumping of MDR, and therefore eliminate this problem. The cellular uptake of nucleosides and nucleoside analogues such as gemcitabine is mainly via the selective nucleoside transport (NT) receptor. The modulation / inhibition of this receptor can be observed as drug resistance in a clinical situation. This phenomenon can be observed in vitro by the addition of NT inhibitors. We have surprisingly seen that our derivatives are not affected by the presence of NT inhibitors, since the cytostatic activity of the preferred derivatives is preserved in the presence of such inhibitors. The half-life (average duration) of gemcitabine in plasma is approximately 10 minutes, due to a rapid deamination by the endogenous enzyme deoxycytidine deaminase to the corresponding uracil derivative (PG Johnston et al, Cancer Chromatography and Biological Response Modifiers, Annual 16, 1996, Chap. 1, ed. Pinedo HM et al.).

In contrast, the derivatives of this invention are poor substrates for the deactivating enzyme, and therefore their half-life is increased. Accordingly, the derivatives of this invention are more suitable than gemcitabine itself for systemic or local treatment of malignant tumors. The novel compounds of this invention are not only potentially useful in the treatment of cancer but also have activity as antiviral agents.

BIOLOGY Experimental part The cytotoxicity activity of gemcitabine N4-elaidic amide and the 5'elaidic ester of gemcitabine was investigated in 2 pairs of tumor cell lines from both rodent and human, each consisting of a parent or original line and a subline already be resistant or cross-resistant to gemcitabine. The cell lines were the human ovarian tumor line A2780 and the sub line AG6000 which is resistant to gemcitabine and has a deoxycytidine kinase deficiency, and the C26A mouse colon tumor line and the C26G subline without altered deoxycytidine kinase, but a 10-fold decrease in thymidine kinase I. The cytotoxicity of each compound was evaluated after continuous exposure to the drug for 72 hours. The numbers of cells were determined by SRB assay, and the percentage of growth inhibition for each tumor line was calculated as the IC 50 value, given in μM, which is the concentration of the compound that gives rise to 50% inhibition, in comparison with control.

Results The IC50 value, in μM, of the gemcitabine cytotoxicity activity itself in comparison to the gemcitabine N4-elaidic cytotoxicity activity of gemcitabine and gemcitabine 5'-elaidic ester are shown in the table below. The activity of the gemcitabine derivatives is much greater than the cytotoxicity activity of gemcitabine in the cell lines tested.

Table Cytotoxicity of gemcitabine, gemcitabine N4-elaidic amide and gemcitabine 5'elaidic ester in IC50 values (μM) in cell lines C26-A, C26-G, A2780 and AG6000 The cytostatic activity of gemcitabine and 5'-elactic acid ester of gemcitabine was determined in CEM cells with and without modifications of nucleoside transport, nitrobenzylthio-inosine (NBMPR) or persantin (pyridamole). As you can see from the following table, gemcitabine CIS0 is > twice as high as the IC50 of the 5'-elaidic acid ester of gemcitabine. With the addition of NT inhibitors, there is a tenfold increase in the IC50 values of gemcitabine, while the IC50 of the 5'-elaidic acid ester of gemcitabine is little affected (increase of 1.3-1.5). In the "resistant" situation the preferred derivative is 15-20 times more potent than the original drug.

Compound IC50, μM, No Cijo, μM of NBMPR, IC50, μM of inhibitor 100 μM persantin, 4 μg / ml Gemdtabine 0.11 ± 0.01 1.11 ± 0.08 1.26 ± 0.04 acid ester 5 '0.047 ± 0.006 0.072 ± 0.034 0.065 ± 0.023 elaidic gemdtabine The antitumor effect of the N4-elaidic amide of gemcitabine or gemcitabine 5'-elaidic ester is investigated in vivo in mice in two different tumor types, under single as well as repeated dosing.

Effect of Gemcitabine Elaid N4 Amide or Gemcitabine 5 'Elaidic Ester on Co-26 Inoculated into the Vessel in Mice Female Balb / c mice were inoculated with Co-26 mouse colon cancer in the vessel, on day 0. In this model, the tumor develops mainly in the liver. The intraperitoneal treatment is started on day 1. The single doses of the compounds are tested in comparison with gemcitabine at a single dose. Saline solution is used as control.

The average survival time for the animals that died is in the same range for the compounds tested. The elastomeric N-amide of gemcitabine is superior to the elaidic 5'-ester of gemcitabine and to gemcitabine with 5/8 survivors at a dose of only 25 mg / kg, compared to gemcitabine at 100 mg / kg. In a parallel experiment, the dosage was repeated on days 1-11.

In this experiment, the results obtained with gemcitabine elaidic N4 amide and a low dose of gemcitabine 5'-elaidic ester were better or equal to the results obtained with a low dose of gemcitabine. Although the high dose of gemcitabine 5'-elaidic ester is slightly toxic, it is less than a high dose of gemcitabine itself.

Effect of Gemcitabine Elaid N4 Amide or Gemcitabine 5 'Elaidic Ester in P-388 ip in mice, in single doses or in repeated doses In female B6D2F1 mice, P388 cells from mouse lymphatic leukemia were implanted intraperitoneally. The treatments were started on day 1 after the implant of cells intraperitoneally. The mean survival time, long-term survivors and toxic deaths after single-dose treatment, repeat dose treatment for 5 days and repeat dose treatment for 10 days were recorded. The results are presented in the tables below. Single-dose treatment with gemcitabine 5'-elaidic ester is effective with a prolonged survival time and the long-term survivors observed compared to the same dose of gemcitabine.

Treatment with a single dose Repeated dose treatment, days 1 The N4-elaidic amide activity of gemcitabine is clear at repeated dose on days 1-4 with observed long-term survivors and a prolonged median survival time at both one and 4 mg / kg. In the control group treated with gemcitabine at 15 mg / kg, all animals died of toxicity.

Repeated dose treatment, treatment days 1-11 The treatment for 10 days increases the antitumor activity compared to a shorter treatment. The toxicity of gemcitabine is greater on an mg / kg basis, with 6/6 toxic deaths at 4 mg / kg. Long-term survivors were observed after repeated treatment with both gemcitabine elaidic N4 amide and gemcitabine 5'-elaidic ester, and substantially increased average survival times were observed for both gemcitabine N-elaidic amide and 5'-ester elaidic gemcitabine. The gemcitabine esters or amides of the present invention can be administered systemically, either enterally or parenterally. For enteral administration, the active compounds of the present invention can be presented, for example, as soft or hard gelatin capsules, tablets, granules, grains or powders, dragees, syrups, suspensions or solutions. When administered parenterally, preparations of gemcitabine esters or amides such as their injection or infusion solutions, suspensions or emulsions are suitable. The preparation may contain inert or pharmacodynamically active additives, as is well known to those familiar in the formulation arts. For example, the tablets or granules may contain a series of binding agents, fillers, emulsifying agents, carrier substances or diluents. Liquid preparations may be present, for example, in the form of a sterile solution. The capsules may contain a filler or a thickening agent in addition to the active ingredient. In addition, the additives that improve the taste as well as the substances commonly used as preserving agents, stabilizers, moisture retention and emulsifiers, 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 day of use, as well as to the requirements of the patient. In general, a daily dosage for a systemic therapy for an average adult patient will be about 0.1-150 mg / kg body weight / day, preferably 1-40 mg / kg / day. For topical administration, an ointment, for example, may contain 0.1-10% by weight of the pharmaceutical formulation, especially 0.5-5% by weight. If desired, the pharmaceutical preparation containing gemcitabine esters or amides may contain an antioxidant, for example tocopherol, N-methyl-tocophermin, butylated hydrocyanol, ascorbic acid or butylated hydroxytoluene. Combination therapies, ie, in which administration of the gemcitabine ester or amide of this invention can be carried out together with other therapies, for example surgery, radiation, treatment and chemotherapy, are also contemplated. For example, the preferred treatment of brain tumors is likely to be a combination of surgery and treatment with an ester or gemcitabine amide of this invention by systemic or local administration.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:
1. A derivative of gemcitabine that has the formula (I) characterized in that Rl t R2 and R3 are independently selected from hydrogen and saturated and monounsaturated acyl groups C18 and C20, with the proviso that Rlf R2 and R3 can not all be hydrogen.
2. 2. The compound according to claim 1, characterized in that only one of Rl t R2 and R3 is an acyl group.
3. 3. The compound according to claim 2, characterized in that the monoacyl substitution is in the 3'-0 or 5'-O position of the sugar portion.
4. 4. The compound according to claim 3, characterized in that the monoacyl substitution is in the 5'-O position of the sugar portion.
5. 5. The compound according to any preceding claim, characterized in that R., R2 and R3 are selected from oleoyl, elaidoyl, cis-eicosenoyl and trans-eicosenoyl.
6. 6. The ester (5 ') -gemcitabine of elaidic acid.
7. 7. The amide (N4) -gemcitabine of elaidic acid.
8. 8. A pharmaceutical composition, comprising gemcitabine ester or amide according to any preceding claim, and a pharmaceutically acceptable carrier or excipient.
9. 9. An ester or amide of gemcitabine according to any of claims 1-7, characterized in that it is used as an anticancer agent.
10. 10. An ester or amide of gemcitabine according to any of claims 1-7, characterized in that it is used as an antiviral agent
11. 11. The use of a gemcitabine ester or amide according to any of claims 1-7, characterized in that it is used in the manufacture of a pharmaceutical composition having anticancer activity.
12. 12. A process for preparing a gemcitabine derivative, according to claim 1, characterized in that gemcitabine is reacted with a compound of the formula: Fax wherein Fa is an acyl group of a Cs or C20 monounsaturated fatty acid, and X is a leaving group.