MXPA97005969A - New farnesil inhibitors transfer your preparation and the pharmaceutical compositions that contain them - Google Patents

Abstract

New inhibitors of farnesyl transferase, its preparation and the pharmaceutical compositions containing them in the general formula (I): R1 represents YS-A1 (Y = hydrogen atom, amino acid residue, fatty acid residue, alkyl or alkoxycarbonyl radical, or radical R4 - S - in which R4 represents an alkyl radical having from 1 to 6 carbon atoms optionally substituted by a phenyl radical or radical of general formula (II): in which A1, X1, Y1, R2, R'2, X2, Y2, X, R3, R'3 and R are defined as follows, and A1 is the alkoxy radical having from 1 to 4 carbon atoms optionally substituted in (alpha) of the Group> C (X1) (Y1) by an amino radical, alkylaminoalkanoylamino, alkoxycarbonylamino, X1 and Y1 each represent a hydrogen atom or form together with the carbon atom or with which a group is linked> C = O, R2 represents a straight or branched alkyl radical having 1 to 4 atoms of carbon eventually replaced by a cyclohexyl radical, R'2 represents a hydrogen or alkyl, X2 and Y2 each represent a hydrogen atom or together they form with the carbon atom to which a Group is bound; C = O, R3 represents an alkyl radical having from 1 to 4 carbon atoms optionally substituted by hydroxy alkoxy mercapto, thioalkyl, alkylsulfinyl or alkylsulfonyl where it is understood that, when R3 represents an alkyl radical substituted by a hydroxy radical R3 can form with the carboxy radical in (alpha) a lactone, R'3 represents hydrogen or alkyl, X represents an oxygen or sulfur atom, and R represents a hydrogen atom or an alkyl radical optionally substituted by an optionally substituted phenyl radical. These new products anti-cancer properties

Description

NPKVOS INHIBITORS DB PARNBSI TRANSFKRASA. sp PRE AP ^ T N.? PHARMACEUTICAL COMPOSITIONS QTTg LOS -OW IRHBN.

The present invention relates to new inhibitors of farnesyl transferase of the general formula. its preparation and the pharmaceutical compositions containing them.

The inhibition of farnesyl transferase and, as a consequence of farnesylation of the ras protein, blocks the ability of the mutated ras protein to transform normal cells into cancer cells.

The C-terminal sequence of the ras gene contains the "CAAX" or * 'Cys-AaAl-Aaa2-Xaa "portion in which Aaa represents an aliphatic amino acid and Xaa represents any amino acid.

It is known that tetrapeptides with a "CAAX" sequence can inhibit the farnesylation of ras protein. For example REF: 25083 EP 0618221 presents peptide derivatives of tetrahydro 1,2,3,4-isoquinoline but whose side chain is fixed on the 3-position of the heterocyclic backbone in the application P.C.T. WO 91/16340 and in the application EP 0461869 were described inhibitory peptides of the farnesyl transferase Cys-Aaal-Aaa2-Xaa that are particularly represented more by peptides Cys-Val-Leu-Ser, Cys-Val-Ile-Met and Cys- Val-Val-Met that manifest their inhibitory activity at close concentrations of 10-6 M or 10-7 M.

It was now found, and it is the purpose of the present invention, that the peptides of general formula (I) manifest their inhibitory activity (lC5o) at close concentrations of 10-8M.

In the general formula (I) R1 represents a radical of general formula YS-Al in which y represents a hydrogen atom, or an amino acid residue or a fatty acid residue or an alkyl or alkoxycarbonyl radical, or a radical R4-S in which R4 represents a alkyl radical containing from 1 to 6 carbon atoms optionally substituted by a phenyl radical or a radical of the general formula.

In which Al, XI, Yl, R2, R'2, X2, X, R3, R'3 and R are defined as follows, Y Al represents a straight or branched alkylene radical having from 1 to 4 atoms of carbon optionally substituted in a of group C (XI) (Yl) by an amino radical, alkylamino having from 1 to 4 carbon atoms or alkoxycarbonylamino whose alkyl part contains from 1 to 4 carbon atoms, XI and Yl represent each of the hydrogen atoms or form together with the carbon atom to which they are attached, a group > C = 0, R 2 represents a straight or branched alkyl radical having from 1 to 4 carbon atoms optionally substituted by a cyclohexyl radical, R'2 represents a hydrogen atom or a straight alkyl radical or branched having 1 to 4 carbon atoms, X2 and Y2 each represent a hydrogen atom or form together with the carbon atom, to which a > C = 0, R3 represents a straight or branched alkyl radical having from 1 to 4 carbon atoms optionally substituted by a hydroxyl radical, alkoxy having from 1 to 4 carbon atoms, mercapto, or alkyl, having from 1 to 4 carbon atoms, alkylsufinyl , which has 1 to 4 carbon atoms or alkylsufonyl, having 1 to 4 carbon atoms, of course, when R3 represents an alkyl radical substituted by a hydroxyl radical, R3 can form with the carboxyl radical in a lactone , R'3 represents a hydrogen atom or a straight or branched alkyl radical having from 1 to 6 carbon atoms, X represents an oxygen or sulfur atom, and R represents a hydrogen atom or an alkyl radical optionally substituted by an alkoxy radical having from 1 to 4 carbon atoms, thioalkyl having 1 to 4 carbon atoms, alkylsulfinyl having 1 to 4 carbon atoms, alkylsulfonyl having 1 to 4 carbon atoms, phenyl, phenoxy, thiophenyl, phenylsulfinyl, phenylsulfonyl, alkylamino having 1 to 4 carbon atoms or diacylamino where each alkyl part contains 1 to 4 carbon atoms, or a phenyl radical optionally substituted by one or more identical or different atoms or radicals selected from halogen atoms and alkyl, alkoxy, thioalkyl, or alkenyl radicals having from 1 to 4 carbon atoms. More specifically, R 1 represents a radical of formula YS-Al- in which Y represents a hydrogen atom or a lysine residue or a fatty acid residue having up to 20 carbon atoms and Al represents an ethylene or propylene radical which is optionally substituted by an amino radical, XI and Yl each represent a hydrogen atom or form together with the carbon atom to which a group is attached > C = 0, R2 represents a methyl isopropyl, 1-methyl, propyl, tert-butyl or cyclohexyl radical, R'2 represents a hydrogen atom or a methyl radical X2 and Y2 each represent a hydrogen atom or form together with the carbon atom to which a group is formed > C = 0, R3 represents a methyl or ethyl radical substituted by a hydroxyl, methoxy, mercapto, thiomethyl, methylsulfinyl or methylsulfonyl radical, R'3 represents a hydrogen atom or a methyl radical, X represents an oxygen atom, and R represents a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms which is optionally substituted by an alkoxy radical, or a phenyl radical.

More particularly, Rl represents a radical of formula Y-S-.Al in which Y represents a hydrogen atom and Al represents an ethylene or propylene radical optionally substituted by an amino radical, XI and Yl each represent a hydrogen atom or form together with the carbon atom with which they are bound. group > C0, R2 represents an isopropyl, l-methyl propyl, tert-butyl or methyl cycloexyl radical, R'2 represents a hydrogen atom X2 and Y2 each represent a hydrogen atom or form together with the carbon atom to which a group is linked > C = 0, R3 represents a methyl or ethyl radical substituted by a hydroxyl, methoxy, mercapto or thiomethyl radical, R'3 represents a hydrogen atom, and R represents a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms.

What is particularly interesting are the products of general formula (I) in which R 1 represents a 2-mercapto ethyl radical or 1-amino-2-mercapto ethyl, X 1 and Y 1 each represent a hydrogen atom or form together with the carbon atom with which a group is bound > CO, R 2 represents an isopropyl radical, X 2 and Y 2 each represent an a hydrogen atom or they form together with the carbon atom with which a group is bound > C = 0, R'2 represents a hydrogen atom, R3 represents a 2-thiomethyl-ethyl or 2-methyl sulfinyl-ethyl radical, R'3 represents a hydrogen atom and R represents a hydrogen atom.

The present invention also relates to the stereoisomeric forms of products of general formula (I). The amino acid residues represented by RlC (Xl) (Yl), R 2 CH (NR'2) [C (X 2) (Y 2) and R 3 CH (NR'3) CO-OH preferably have the configuration of the natural amino acids.

The present invention also relates to mineral or organic salts, as well as esters of products of general formula (I) With this invention, the new products of general formula (I) can be obtained by synthesis. In solid phase using a synthesis strategy "9-fluoronimetroxicabonilo (FMOC)". In this case, the thiol groups are protected by tritium or acetamidomethyl groups, the functions aminated by the Boc group (T. butoxycarbonyl) and the acid functions in the form of ester t. butyl, alcohol functions by T. butyl group and the functions amides and imidazole by tritium grouping. The synthesis can be done on a resin confined in a solid phase extraction syringe 3 cm3 in high density polyethylene supplied with Teflon filters. The syringes are placed on a two-way Teflon valve and closed by a plug with single-use high density polyethylene flaps. The agitation of the syringes is carried out on a rotary apparatus for hemolisi tubes. The washing and filtering operations are conducted on a solid phase extraction work station.

The synthesis can be performed on 50μ resin springs. The couplings of the amino acids are made by treating the resin for 250 hours by 250μ springs of the amino acid suitably protected in the presence of 250μ springs of 2- (lH-benzotriazol-1-yl) -1,3,3-tetramethyluronium, Hexafluorophosphate (HBTU) 250μ springs (N-hydroxyl benzotriazole and 750μ springs of diisopropylethylamine in l, 2cm3 of a mixture of N-methyl pyrrolidone-2 (NMP) / dimethylformamide (l / l in volumes) The deprotection of the FMOC grouping is carried out by three successive treatments of the resin twice for one minute and 20 minutes for each 2 cm3 of piperidine in 2% (v / v) solution in the NMP.

For example, the Cys - (NMe) Val- [(R, S) -tetrahydro-1,2,3,4-isoquinoline-1-carbonyl) Met can be prepared in the following manner: 50 μmol of resin Fmoc-Met resin Wang, Wang et al., J. Amer. Chem. Soc. Chem. Soc, 95, 1328 (1975)) to the following treatments: = > deprotection of the FMOC group, = > Washing 5 times with 2 cm3 of NMP, ^ Coupling of acid FMOC - (R, S) - tetrahydro 1,2,3,4 Isoquinoline-1-carboxylic, = > Washing 5 times with 2 cm 3 of NMP, = > Deprotection of the FMOC group, = > Washed 5 times with 2 ctn3 of NMP, = Coupling of FMOC - N - Methylovaline, = > Washing 5 times with 2 cm3 of NMP, = > Deprotection of the FMOC Group, = Wash 5 times with 2 cm3 of NMP, = > Coupling of the FMOC cysteine (S-trityl) = > Washing 5 times with 2 cm3 of NMP, = Deprotection of the FMOC Group, y = > Wash 5 times with 2 cm3 of NMP; At the end of the synthesis, the products are separated by treatment of the resin by 10 cm3 of a mixture of trifluoroacetic acid - Phenol-Ethane-dithiol-thioanisole-Water (40-3-1-2-2 in volumes) for one hour and 30 minutes. minutes The resin is then removed by filtration, the filtrate is concentrated under reduced pressure by means of a centrifugal evaporator (RC 10-10 Jouan) equipped with a finned pump and a trap at -90 ° C for 1.5 hours, the temperature of the evaporation chamber is maintained at 50 ° C. The final volume of this concentrate is about 1 cm3. The product is then precipitated by the addition of 15 cm 3 of a mixture of methyl-tert-butyl ether and petroleum ether (2-1 in volumes) then it is collected by centrifugation. The residue is then solubilized in lcm 3 of precipitated trifluoroacetic acid by the addition of 15 cm 3 of ethyl-tert-butylether and washed with 15 cm 3 of methyl-tert-butylether. The product is then dried under reduced pressure (3.5 KPa). The product is finally purified by high quality liquid chromatography (HPLC) on a C18 100A column (250 x 10 mm, Rad) diluted by an acetonitrile gradient having 0.07% trifluoroacetic acid (in volumes) in the water having 0.07 % trifluoroacetic acid (in volumes) with an expenditure of 6cm3 / min and lyophilized. The product obtained is characterized by a mass spectrum (electroroc? o).

Tetrahydro-1,2,3,4-isoquinoline-, l-carboxylic acid, in racemic form can be prepared by hydrogenation of isoquinoline-1-carboxylic acid under the conditions described by RT. Shuman Coll., J. Med. Chem., 36,314 (1993).

The introduction of the protective group FMOC on an amino acid is effected by the action of the amino acid on the chcoroformiate of 9-fluoronylmethyl (FMOC-Chloride) in the presence of a base.

The inhibitory activity of "Farnesyl transferase" and of Farnesylation of Ras protein can be evidenced in the following test: Farnesyl transferase activity is determined by the amount of (3H) farnesyl transferase from (3H) Farnesylpyrrophosphate [(3H) FPP) on the p21 H-ras protein. The standard reaction mixture is composed, for a final volume of 60μ 1, of 50mM Tris-HCl, MgC125mM, 5mM dithiothreitol, octyl-β-D-glucopyranoside 0.2%, p21 H Ras 200 Picomoles, (3H) FPP (at 61000 dpm / picomol) 4.5 picomoles.

The reaction is initiated by addition of about 5 ng of purified human farnesyl transferase from THP1 cell culture. After incubation for 20 minutes at 37 ° C in microtitre plates having 96 hollows of 1 cm3 per plate (titer píate®, Beckman), the reaction is stopped by the addition of 0.4 cm3 of 0.1% SDS in methanol at 0 ° C. The mixture is then added with 0.4 cm3 of 30% trichloroacetic acid (TCA) in the methanol. The plates are left for an hour on the ice. The content is then precipitated on a Filtermat® glass fiber membrane (Pharmacia) with the filtration unit (Combi tell Harvester® Skatron) and rinsed with 6% trichloroacetic acid in distilled water. The membranes are dried in a microwave oven and then impregnated with meltilex® hot-melt scintillation (Pharmacia) and finally counted in cpm in a ß-Píate® (LKB) counter, each test is repeated 3 times .

The unit of activity is defined by a picomole of (3H) FPP transferred over p21 H-Ras in twenty minutes.

Percentages of inhibition are obtained by comparison of tests with and without inhibitor. After the deduction of the targets, the IC50 are measured from the inhibitions obtained with nine different concentrations using the software Enzfitter® or Grafit®.

The results obtained are mentioned in Table 1, BOARD 1 The new peptides of general formula (I) can be presented in the form of non-toxic and pharmaceutically acceptable salts. These non-toxic salts include salts with mineral acids (hydrochloric, sulfuric, bromide, phosphoric, nitric acids) or with organic acids (acetic, propionic, succinic, maleic, hydroxymelic, benzoic, fumaric, methanesulfonic, trifluoroacetic, or oxalic acids) ) or with the mineral bases (sodium, potassium, lithium, lime) or organic (tertiary amines such as triethylamine piperidine, benzylamine) according to the nature of the amino acids that make up the peptide of general formula (I).

The new peptides according to this invention which inhibit farnesyl transferase and farnesylation of Ras protein, are extraordinary anticancer agents that act both at the level of solid and liquid tumors.

The present invention also relates to pharmaceutical compositions having at least one peptide of general formula (I) in association with one or more pharmaceutically acceptable diluents or auxiliaries which are inert or physiologically active.

These compositions can be administered orally, parenterally or rectally.

The compositions by oral administration comprise tablets, pills, powder or granules. In these compositions the active product according to this invention is mixed with one or more inert diluents such as sucrose, lactose or starch. These compositions may comprise other substances in addition to the diluents. For example, a lubricant such as magnesium stearate.

As liquid compounds for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, having inert diluents such as water or paraffin oil may be used, these compounds may also contain other substances in addition to the in addition to diluents such as wet products, sweeteners or flavorings.

The compounds according to this invention by parenteral administration can be sterile aqueous or non-aqueous solutions, suspensions or emulsions, as a solvent or vehicle, the propylene glycol, a polyethylene glycol, vegetable oils, in particular, olive oil or injectable organic esters, for example, can be used. the ethyl wave. These compounds may also contain adjuvants, in particular wet agents, emulsifiers and dispersants. The sterilization can be done in several ways, for example with the aid of a bacteriological filter, incorporating the compound, sterilizing agents or heating. They can also be prepared in the form of sterile solid compounds that can be dissolved at the time of use in sterile water or other sterile injectable medium.

Compounds by rectal administration are suppositories which may contain in addition to the active product excipients such as cocoa butter.

The compounds according to this invention are particularly useful in human therapy for the treatment of cancers of diverse origin, in human therapy, the doses depend on the sought effect, the duration of treatment and the characteristics of the subject under treatment.

Generally the doses are included in man, between 0, 1 and 2 mg / kg per day intraperitoneally

Claims (5)

REINVTDICATIONS

1. The new peptides dß general formula in which R1 represents a radical of general formula YS-Al in which Y represents a hydrogen atom or an amino acid residue or a fatty acid residue or an alkyl or alkoxycarbonyl radical, or a radical R4-S- in which R1 represents an alkyl radical containing from 1 to 6 carbon atoms optionally substituted by a phenyl radical, or radical of general formula. (II) wherein Al, XI, Yl, R2, R'2, X2, X, R3, R'3 and R are defined as follows, and Al represents a straight or branched alkylene radical having from 1 to 4 atoms of carbon optionally substituted in a of the group > C (X1) (Y1) by an amino radical, alkylamino having from 1 to 4 carbon atoms or carbonylamino alkoxy whose alkyl part contains from 1 to 4 carbon atoms, XI and Yl represent each of the hydrogen atoms or form together with the carbon atom to which a group is attached > C = 0, R 2 represents a straight or branched alkyl radical having from 1 to 4 carbon atoms optionally substituted by a cyclohexyl radical, R'2 represents a hydrogen atom or a straight or branched alkyl radical having from 1 to 6 carbon atoms, X2 and Y2 each represent a hydrogen atom or form together with the carbon atom, to which a group is linked > c = o, R3 represents a straight or branched alkyl radical having from 1 to 4 carbon atoms optionally substituted by a hydroxyl radical, alkoxy having 1 to 4 carbon atoms, mercapto, thioalkyl having 1 to 4 carbon atoms, alkylsufinyl, having 1 to 4 carbon atoms or alkylsufonyl, having 1 to 4 carbon atoms, of course, when R3 represents an alkyl radical substituted by a hydroxyl radical R3 it can form with the carboxyl radical in a lactone, R'3 represents a hydrogen atom or a straight or branched alkyl radical having from 1 to 6 carbon atoms, X represents an oxygen or sulfur atom, and R represents a hydrogen atom or an alkyl radical optionally substituted by an alkoxy radical having from 1 to 4 carbon atoms, thioalkyl having 1 to 4 carbon atoms, alkylsulfinyl having 1 to 4 carbon atoms, alkylsulfonyl having 1 to 4 carbon atoms, phenyl, phenoxy, thiophenyl, phenylsulfinyl, phenylsulfonyl, alkylamino has from 1 to 4 carbon atoms or dialkylamino where each alkyl part contains from 1 to 4 carbon atoms, or a phenyl radical optionally substituted by one or more atoms or radicals, identical or different chosen from the halogen atoms and the alkyl radicals , alkoxy, thioalkyl, or alkenyl having 1 to 4 carbon atoms.

2. New peptides according to claim 1, characterized in that Rl represents a radical of formula YS-Al in which Y represents a hydrogen atom or a lysine residue or a fatty acid residue having up to 20 carbon atoms and Al represents an ethylene or propylene radical which is optionally substituted by a amino radical, XI and Yl each represent a hydrogen atom or form together with the carbon atom to which a group is attached > C = 0, R2 represents an isopropyl, 1-methyl, propyl, tert-butyl or cyclohexylmethyl radical, R'2 represents a hydrogen atom or a methyl radical X2 and Y2 each represent a hydrogen atom or form together with the carbon atom to which a group is formed > c = o. R3 represents a methyl or ethyl radical substituted by a hydroxyl, methoxy, mercapto, thiomethyl, methylsulfinyl or methylsulfonyl radical, R'3 represents a hydrogen atom or a methyl radical, X represents an oxygen atom, and R represents a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms which is optionally substituted by an alkoxy radical, or a phenyl radical.

3. New peptides according to claim 1, characterized in that: R 1 represents a radical of formula Y-S-Al in which Y represents a hydrogen atom and Al represents an ethylene or propylene radical optionally substituted by an amino radical, XI and Y1 each represent a hydrogen atom or form together with the carbon atom with which a group is linked > C0, R2 represents an isopropyl, 1-methyl propyl, tert-butyl or methyl cycloexyl radical, R'2 represents a hydrogen atom X2 and Y2 each represent a hydrogen atom or form together with the carbon atom with which a group is linked to > C = 0, R3 represents a methyl or ethyl radical substituted by a hydroxyl, methoxy, mercapto or thiomethyl radical, R "3 represents a hydrogen atom, and R represents a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms.

4. New peptides according to claim 1, characterized in that R1 represents a mercapto-ethyl or amino-mercapto-2-ethyl radical, XI and Y1 each represent a hydrogen atom or form together with the carbon atom to which a group is linked > Co, R 2 represents an isopropyl radical, X 2 and Y 2 each represent a hydrogen atom or form together with the carbon atom to which a > C = 0, R'2 represents a hydrogen atom, R3 represents a 1-methyl-ethyl or 2-methylsulfinyl-ethyl radical, R'3 represents a hydrogen atom and R represents a hydrogen atom.

5. Pharmaceutical composition characterized in that it contains a sufficient amount of a peptide according to the claims 1 to 4 in association with one or more pharmaceutically inert or physiologically active diluents or adjuvants. SUMMARY OF THE INVENTION New inhibitors of farnesyl transferase, its preparation and pharmaceutical compositions containing them In the general formula (I) R1 represents YS-Al (Y = hydrogen atom, amino acid residue, fatty acid residue, alkyl or alkoxycarbonyl radical, or radical R4-S - in which R4 represents an alkyl radical having from 1 to 6 carbon atoms optionally substituted by a phenyl radical or radical of general formula. wherein Al, XI, Yl, R2, R'2, X2, Y2, X, R3, R'3 and R are defined as follows, and Al radicals having from 1 to 4 carbon atoms eventually replaced in a of the Group > C (X1) (Y1) by an amino, alkylamino, alkanoylamino, alkoxycarbonylamino radical, XI and Y1 each represent a hydrogen atom or form together with the carbon atom with which a group is linked > C = 0, R2 represents a straight or branched alkyl radical having 1 to 4 carbon atoms optionally substituted by a cyclohexyl radical, R'2 represents a hydrogen or alkyl, X2 and Y2 each represent a hydrogen atom or form together with the carbon atom with which a Group is linked to > C = 0, R3 represents an alkyl radical having 1 to 4 carbon atoms optionally substituted by hydroxy alkoxy mercapto, thioalkyl, alkylsulfinyl or alkylsulfonyl where it is understood that, when R3 represents an alkyl radical substituted by a hydroxy radical R3 can form with the carboxy radical in a lactone, R'3 represents hydrogen or alkyl, x represents an oxygen or sulfur atom, and R represents a hydrogen atom or an alkyl radical optionally substituted by an optionally substituted phenyl radical. These new products have anti-cancer properties,