Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems

Definitions

• the invention relates to 3-(3-aminobenzylamino)-4-(3-chlorophenylamino)-1 H-pyrazolo[3,4- d]pyrimidine, its salts, solvates and tautomers, and to a process for its preparation, pharmaceutical formulations which comprise 3-(3-aminobenzylamino)-4-(3-chlorophenyl- amino)-1 H-pyrazolo[3,4-d]pyrimidine or its pharmaceutically acceptable salts or solvates, and the use of these derivatives as pharmaceuticals.
• the prefix "lower” designates a radical having up to a maximum of 7, in particular having up to a maximum of 4, and especially having 1 or 2, carbon atoms.
• Salts of the compound of the formula I are, because it has basic properties, acid addition salts with organic or inorganic acids, in particular the pharmaceutically acceptable non-toxic salts.
• suitable inorganic acids are carbonic acid (preferably in the form of carbonates or bicarbonates); hydrohalic acids such as hydrochloric acids, sulfuric acid; or phosphoric acid.
• suitable organic acids are carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2-hydroxybutyric acid, gluconic acid, glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galacta c acid, amino acids such as glutarmic acid, aspartic acid, N-methylglycine, acetylaminoacetic acid, N- acetylasparagine or N-acetylcystein, pyruvic acid, acetoacetic acid, phosphoserine, 2- or 3- glycerophosphoric acid, glucose-6-phosphoric acid, glucose-1 -phosphoric acid, fructose
• the compound of the formula I and intermediates which comprise a pyrazole residue for preparing it may, under certain conditions, for example when they are dissolved in certain solvents, be partly in a tautomeric form in which the hydrogen atom which is normally located on nitrogen N-1 has shifted to another suitable nitrogen atom, for example N-2, N-5 or N-7.
• the invention also relates to these tautomers.
• the compound of the formula i and its pharmaceutically acceptable salts, solvates and tautomers have valuable pharmacological properties.
• they show specific inhibitory effects which are of pharmacological interest. They act primarily as protein tyrosine kinase inhibitors.
• they show a potent inhibition of the tyrosine kinase activity of the receptor for epidermal growth factor (EGF) and of c-erbB2 kinase.
• EGF epidermal growth factor
• c-erbB2 kinase a potent inhibition of the tyrosine kinase activity of the receptor for epidermal growth factor (EGF) and of c-erbB2 kinase.
• EGF-induced activation of the receptor-associated protein tyrosine kinase is in various types of cells a prerequisite for cell division and thus proliferation of the cell population. Multiplication of EGF receptor-specific tyrosine kinase inhibitors thus inhibits multiplication of the cells.
• Analogous statements apply to the other protein kinases mentioned hereinbefore and hereinafter.
• EGF-R-PTK EGF receptor-specific protein tyrosine kinase
• EGF-R ICD recombinant intracellular domain of the EGF receptor
• the compound of the formula I and its salts, solvates and tautomers inhibit the enzyme activity compared with the control without inhibitor by 50% (IC50) for example in a concentration of 0.001 to 0.003 ⁇ M.
• the compound of the formula I and its salts and solvates likewise show in the micromolar range for example also an inhibition of cell growth in EGF-dependent cell lines, for example the epidermoid BALB/c mouse keratinocyte cell line (see Weissmann, B.A., and Aaronson, S.A., Cell 32, 599 (1983)) or the A431 cell line, which are acknowledged to be useful standard sources of EGF-dependent epithelial cells (see Carpenter, G., and Zendegni, J. Anal. Biochem. 153, 279-282 (1985)).
• the inhibitory effect of the compounds of the formula I is measured in a known test method (see Meyer et al., Int. J.
• BALB/MK cells (10,000/microtitre plate well) are transferred into 96-well microtitre plates.
• the test compounds dissolved in DMSO
• concentration series concentrations
• the plates are incubated for three days, during which the control cultures without test substance are able to pass through at least three cell division cycles.
• the growth of the MK cells is measured by means of methylene blue staining: after the incubation, the cells are fixed with glutaraldehyde, washed with water and stained with 0.05% methylene blue.
• IC 50 value in these experiments is reported as the concentration of the particular test compound which results in a cell count which is 50% less than the control without inhibitor.
• the compound of the formula I and its pharmaceutically acceptable salts show inhibitory effects in the micromolar region, for example an IC 50 of about 0.1 ⁇ M.
• the compound of the formula I and its pharmaceutically acceptable salts and solvates also show in vivo an inhibition of the growth of tumour cells, for example as shown by the test described hereinafter:
• the test is based on the inhibition of the growth of the human epidermoid carcinoma A431 (ATCC No. CRL 1555; American Type Culture Collection, Rockville, Maryland, USA; see Santon, J.B., et al., Cancer Research 46, 4701 -4705 (1986) and Ozawa, S., et al., Int. J. Cancer 40, 706-710 (1987)), which is transplanted into female BALB/c nude mice (Bomholtgard, Denmark).
• This carcinoma shows a growth which correlates with the extent of EGF receptor expression.
• tumours which have been grown in vivo and have a volume of about 1 cm 3 are removed surgically from experimental animals under sterile conditions. These tumours are comminuted and suspended in 10 volumes (w/v) of phosphate-buffered saline. The suspension is injected s.c. (0.2 ml/mouse in phosphate-buffered saline) into the left flank of the animals. Alternatively, 1 x 10 6 cells from an in-vitro culture in 0.2 ml of phosphate-buffered saline can be injected. Treatment with the test compound of the formula I or a salt thereof is started 5 or 7 days after the transplantation when the tumours have reached a diameter of 4-5 mm.
• the particular active substance is administered (in various doses in different animal groups) once a day for 15 consecutive days.
• the tumour growth is determined by measuring the diameters of the tumours along three mutually perpendicular axes.
• the tumour volumes are calculated using the known formula p x L x D 2 /6 (see Evans, B.D., et al., Brit. J. Cancer 45, 466-8 (1982)).
• T/C% T/C%
• the protein tyrosine kinases involved in signal transmission mediated by tropic factors for example the abl kinases, such as, in particular v-abl kinase, kinases from the family of src kinases, such as, in particular, c-src kinase, and the serine/threonine kinases, for example the protein kinase C, and cdc2 kinase, all of which play a part in growth regulation and transformation of mammalian cells, including human cells, are inhibited only much more weakly or virtually not at all.
• enzymes such as Flt1 , Flk, c- Met and Tek. As can be easily understood, this is presumably also associated with fewer side effects.
• Another advantage of these compounds is the high cell permeability and the high blood levels which are reached.
• the compound of the formula I and its pharmaceutically acceptable salts which inhibit the tyrosine kinase activity of the receptor for epidermal growth factor (EGF) can therefore be used, for example, for treating benign or malignant tumours, for example those of the lung, of the breast, of the bladder, of the skin or of the intestine. They are able to bring about tumour regression and to prevent tumour metastasis and the growth of micrometastases. They can be used in particular in cases of epidermal hyperproliferation (psoriasis), in the treatment of neoplasms of an epithelial nature, for example carcinomas of the breast, and for leukaemias.
• epidermal hyperproliferation psoriasis
• the compound of the formula I and its pharmaceutically acceptable salts can furthermore be employed for treating disorders of the immune system involving protein tyrosine kinases which are inhibited; this compound of the formula I can also be used to treat disorders of the central or peripheral nervous system as long as signal transmission by these protein tyrosine kinases is involved.
• the present invention generally also relates to the use of the compound of the formula I and its pharmaceutically acceptable salts for inhibiting said protein kinases.
• the compound of the formula I according to the invention and its pharmaceutically acceptable salts, solvates or tautomers can be used either alone or else in combination with other pharmacologically effective substances, for example together with inhibitors of the enzymes of polyamine synthesis, inhibitors of protein kinase C, inhibitors of other tyrosine kinases, cytokines, negative growth regulators, for example TGF- ⁇ or IFN- ⁇ , aromatase inhibitors, antioestrogens and/or cytostatics.
• the compound of the formula I and its salts can be prepared in a manner known per se, for example as exemplified in the example part.
• the preparation process according to the invention comprises a) eliminating the protective group from a compound of the formula II
• Rj is an aminoprotective group
• Suitable aminoprotective groups such as, in particular, tert-butyloxycarbonyl (Boc), and their elimination, are familiar to the skilled person.
• the Boc protective group can be eliminated in an acidic medium, for example by adding 3-normal methanolic hydrochloric acid.
• a suitable reducing agent is, in particular, hydrogen in the presence of a suitable catalyst such as, in particular, Raney nickel.
• Acid addition salts of the compound of the formula I are obtained in a manner known per se, for example by treatment with an acid or a suitable anion exchanger.
• Acid addition salts can be converted in a conventional way into the free compound, for example by treatment with a suitable basic agent.
• Solvates often form "automatically” during working up of the reaction mixture, and hydrates form on standing in air.
• Mixtures of isomers can be resolved in a manner known per se, for example by fractional crystallization, chromatography etc., into the individual isomers.
• the invention also relates to those embodiments of the process which start from a compound which can be obtained as intermediate at any stage of the process, and the missing process steps are carried out, or the process is stopped at any stage, or a starting material forms under the reaction conditions or is used in the form of a reactive derivative or salt.
• the starting materials preferably used are those which in the process result in the compounds described above as particularly valuable.
• the invention also relates to a method for the treatment of warm-blooded animals suffering from an oncosis, wherein an effective tumour-inhibiting amount of a compound of the formula I or of a pharmaceutically acceptable salt or solvate thereof is administered to warm-blooded animals requiring such a treatment.
• the invention additionally relates to the use of a compound of the formula I or of a pharmaceutically acceptable salt or solvate thereof for inhibiting EGF receptor-specific protein tyrosine kinase C in warm-blooded animals or for manufacturing pharmaceutical products for use for the therapeutic treatment of the human or animal body.
• the invention also relates to pharmaceutical products which comprise an effective amount, in particular an amount effective for the prophylaxis or therapy of one of the above- mentioned diseases, of the active substance together with pharmaceutically acceptable carriers which are suitable for topical, enteral, for example oral or rectal, or parenteral administration, and may be inorganic or organic, solid or liquid.
• tablets or gelatin capsules which comprise the active ingredient together with diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol, and/or lubricants, for example diatomaceous earth, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
• diluents for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol
• lubricants for example diatomaceous earth, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
• Tablets may likewise comprise binders, for example magnesium aluminium silicate, starches, such as maize, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcelluiose and/or polyvinylpyrrolidone, and, if required, disintegrants, for example starches, agar, alginic acid or a salt thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, colorants, flavourings and sweeteners.
• binders for example magnesium aluminium silicate, starches, such as maize, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcelluiose and/or polyvinylpyrrolidone, and, if required, disintegrants, for example starches, agar, alginic acid or a salt thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, colorants, flavourings and sweeteners.
• Solutions of this type are preferably isotonic aqueous solutions or suspensions, it being possible for the latter, for example in the case of lyophilized products which comprise the active substance alone or together with a carrier, for example manitol, to be prepared before use.
• the pharmaceutical products may be sterilized and/or comprise adjuncts, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts to regulate the osmotic pressure and/or buffers.
• the present pharmaceutical products which, if required, may comprise further pharmacologically active substances, such as antibiotics, are manufactured in a manner known per se, for example using conventional mixing, granulating, coating, dissolving or lyophilizing processes, and comprise about 1 % to 100%, in particular about 5% to about 90%, of the active substance(s).
• RV rotary evaporator brine: saturated sodium chloride solution
• Example 1 3-(3-Aminobenzylamino)-4-(3-chlorophenylaminoH H-pyrazolor3,4-dlpyrimidine
• the starting material is obtained in the following way:
• Stage 1.1 43.6 ml (400 mmol) of benzylamine are added to a suspension of 68.4 g (400 mmol) of 3,3-bis(methylthio)-2-cyanoacrylonitrile [3,3-bis(methylsulfanyl)-2-cyano- acrylonitrile; Maybridge] in 400 ml of ethyl acetate.
• Stage 1.2 24 ml (0.48 mol) of hydrazine hydrate are added dropwise to a solution of 92 g (0.4 mol) of 3-benzylamino-3-methylthio-2-cyanoacrylonitrile in 400 ml of methanol. During this, the temperature rises to 40°C. It is slowly heated to boiling ( ⁇ MeSH evolution), boiled for 2 h, cooled to RT and evaporated to a residual volume of 200 ml.
• Stage 1.4 60 g (0.47 mol) of 3-chloroaniline are dissolved in 255 ml (0.56 mol) of 2.2 N methanolic HCI. Concentration and stirring of the residue in diethyl ether afford 3- chloroaniline hydrochloride after filtration and drying.
• Stage 1.5 79.2 g (295 mmol) of N'-(3-benzylamino-4-cyano-1 H-pyrazol-5-yl)-N,N-dimethyl- formamidine are suspended in 700 ml of methanol with exclusion of moisture, 60.6 g (369 mmol) of 3-chloroaniline hydrochloride are added, and the mixture is boiled under reflux for 22 h. The resulting yellow reaction solution is cooled to 50°C and poured into 2 litres of ice-water, 200 ml of sat. NaHC ⁇ 3 solution and 1 I of ethyl acetate. The aqueous phase is separated off and extracted twice with ethyl acetate.
• Stage 1.6 Residual water is removed from a suspension of 75.8 g (216 mmol) of 3- benzylamino-4-(3-chlorophenylamino)-1 H-pyrazolo[3,4-d]pyrimidine in 1.5 litre of benzene by distilling off a little solvent. This suspension is then added, with exclusion of moisture, to 84 g of aluminium chloride (Fluka, Buchs/Switzerland) in 500 ml of benzene, and the mixture is heated at 80°C for 2.5 h.
• aluminium chloride Feluka, Buchs/Switzerland
• the reaction mixture is cooled to RT, the supernatant benzene phase is poured into 2 kg of ice-water (a green oily residue remains behind), and the solid which separates out is filtered off with suction and thoroughly washed with water ( ⁇ K-
• the benzene is evaporated off from the filtrate in a rotary evaporator, the remaining aqueous phase is added together with 1 kg of ice to the green oily residue, and the mixture is hydrolysed at 40°C for 2 h.
• the crystalline product is filtered off with suction and washed with water ( ⁇ K2).
• K2 are taken up in 1 litre of methanol, acidified with 4N aqueous HCI and partly evaporated.
• Stage 1.7 4-(3-Chlorophenylamino)-3-(3-nitrobenzylamino)-1 H-pyrazolo[3,4-dlpyhmidine 0.907 g (6 mmol) of 3-nitrobenzaldehyde is added to a solution of 1.043 g (4 mmol) of 3- amino-4-(3-chlorophenylamino)-1 H-pyrazolo[3,4-d]pyrimidine [see Stage 1.6] in 100 ml of methanol, 100 ml of DMEU and 0.48 g (8 mmol) of acetic acid, and the mixture is stirred at RT for 1 h.
• Example 2 3-(3-Aminobenzylamino)-4-(3-chlorophenylamino)-1 H-pyrazolo[3,4-d]pyrimidine
• the title compound is obtained in the following way:
• the starting material is prepared as follows Stage 2 1 3-(N-Boc-am ⁇ no)-benzylam ⁇ ne
• Stage 2 3-[3-(N-Boc-am ⁇ no)benzylam ⁇ no1-2-cvano-3-methylth ⁇ oacrylon ⁇ tr ⁇ le
• Stage 2.3 5-Amino-3-[3-(N-Boc-amino)benzylamino]-4-cyanopyrazole
• Production process The powdered substances mentioned are forced through a screen with a mesh width of 0.6 mm. 0.33 g portions of the mixture are packed into gelatin capsules by a capsule-filling machine.
• the powdered active ingredient is suspended in PEG 400 (polyethylene glycol with M r between about 380 and about 420, Fluka, Switzerland) and Tween 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Ind., Inc., USA, supplied by Fluka, Switzerland), and ground in a wet pulverizer to a particle size of about 1 to 3 mm. 0.43 g portions of the mixture are then packed into soft gelatin capsules by a capsule-filling machine.
• Example 8 In vivo antitumour activity (s.c. xenografts in nude mice) of 3-(3-amino-benzyl- amino)-4-(3-chlorophenylamino)-1 H-pyrazolo[3,4-d]pyrimidine (compound I)
• mice Female Balb/c nu/nu mice (Novartis animal farm, Sisseln, Switzerland) are kept under sterile conditions (10 to 12 mice per cage Type 111) with free access to food and water. Tumours are established after subcutaneous injection of cells (minimum 2 x 10 6 cells in 100 ul PBS or medium) in carrier mice (4-8 mice per cell line). The resulting tumours are serially passaged for a minimum of three consecutive transplantations prior to start of treatment. Tumour fragments (approx. 25 mg) are implanted s.c. into the left flank of animals with a 13-gauge trocar needle under Forene (Abbott, Switzerland) anaesthesia. Treatment is started when the tumour reaches a mean tumour volume of 100 mm 3 .
• Tumour growth is monitored twice and 24 hours after the last treatment by measuring perpendicular diameters.
• Tumour volumes are calculated according to the formula L x D x ⁇ /6 (Ref.: Evans et al, Brit.J. Cancer, 1982; 45: 466-468).
• Antitumour activity is expressed as T/C% (mean increase of tumour volumes of treated animals divided by the mean increase of tumour volumes of control animals multiplied by 100).
• Treatment Applications are given 7 days a week (p.o. or i.v.). The volumes of application are 25 ml/kg (p.o.) and 10 ml/kg (i.v.). Stock solutions of 40 mg/ml of compound I are dissolved in 100% DMSO (MERCK, Darmstadt, Germany) and stirred at room temperature until clear solutions are obtained. Prior to each administration, 10% Tween 80 (FLUKA, Buchs, Switzerland) is added to the stock solution and then diluted 1 : 20 (v/v) with sterile water (p.o. applications) or NaCI 0.9% (i.v. applications). Solutions and dilutions are prepared daily prior to application.
• Tumours Human epidermoid carcinoma A 431 (ATCC: CRL 1555) Start of treatment: day 5 after tumour transplantation Treatment: once daily for 14 consecutive days

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=4232904

Family Applications (1)

Country Status (4)

Cited By (259)

Citations (2)

• 1997
  • 1997-09-25 ID IDP973288A patent/ID18494A/id unknown
  • 1997-09-30 AU AU47069/97A patent/AU4706997A/en not_active Abandoned
  • 1997-09-30 WO PCT/EP1997/005377 patent/WO1998014451A1/en active Application Filing
  • 1997-10-01 ZA ZA9708801A patent/ZA978801B/xx unknown

Patent Citations (2)

Also Published As

Similar Documents

Legal Events