CZ249997A3 - Novel inhibitors of farnesyl transferase, process of their preparation and pharmaceutical compositions containing such inhibitors - Google Patents

Abstract

Novel farnesyl transferase inhibitors of general formula (I), preparation thereof, and pharmaceutical compositions containing same. In general formula (I), R1 is Y-S-A1- (where Y is a hydrogen atom, an amino acid residue, a fatty acid residue, an alkyl or alkoxycarbonyl radical, or a radical R4-S-, where R4 is a C1-6 alkyl radical optionally substituted by a phenyl radical, or a radical of general formula (II), wherein A1, X1, Y1, R2, R'2, X2, Y2, X, R3, R'3 and R are as defined below, and A1 is a C1-4 alkylene radical optionally alpha -substituted in the >C(X1)(Y1) grouping by an amino, alkylamino, alkanoylamino or alkoxycarbonylamino radical); X1 and Y1 are each a hydrogen atom or, taken together with the carbon atom to which they are attached, a >C=O grouping; R2 is a straight or branched C1-4 alkyl radical optionally substituted by a cyclohexyl radical; R'2 is hydrogen or alkyl; X2 and Y2 are each a hydrogen atom or, taken together with the carbon atom to which they are attached, a >C=O grouping; R3 is a C1-4 alkyl radical optionally substituted by hydroxy, alkoxy, mercapto, alkylthio, alkylsulphinyl or alkylsulphonyl, with the proviso that, when R3 is an alkyl radical substituted by a hydroxy radical, R3 may form a lactone with the alpha -carboxy radical; R'3 is hydrogen or alkyl; X is an oxygen or sulphur atom; and R is a hydrogen atom or an optionally substituted alkyl radical or an optionally substituted phenyl radical. These novel products have anticancer properties.

Description

Novel farnesyltransferase inhibitors, processes for their preparation and pharmaceutical compositions containing these inhibitors.

ÓBIN'3! -W9 ^ad (

FIELD ⁹ j θ

BACKGROUND OF THE INVENTION The present invention relates to novel farnesyl transferase inhibitors, to processes for their preparation and to pharmaceutical compositions containing them as active ingredient. I _ϋ , i

BACKGROUND OF THE INVENTION

Inhibition of farnesyl transferase and hence farnesylation of 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 motif CAAAX or Cys-Aaa.

It is known that tetrapeptides with a CAAX sequence can inhibit farnesylation of ras protein. Thus, for example, EP 0 618 221 discloses peptide derivatives of 1,2,3,4-tetrahydroisoquinoline whose side chain is bound at position 3 of the heterocyclic skeleton. In PCT Application WO 91/16340 and EP 0461869 describes peptides inhibiting farnesyl transferase, Cys-Aaa, -Aaa2 ^ ^_ Xaa whose crowds are particularly CysVal-Leu-Ser-CysVal Ile-Met and Cys Val-Val-Met and which exhibit inhibitory activity at concentrations close to 10 or 10 ^-7 M.

SUMMARY OF THE INVENTION

It has now been found, and this is the essence of the invention, that the peptides of formula (I)

COOR (i)

SUBSTITUTE SHEET show inhibitory activity (ΙΟ ^ θ) at concentrations of about 10 ⁸ M.

In the above general formula

R1 is a group of the formula YS-Ap wherein Y is a hydrogen atom, an amino acid or a fatty acid residue or an alkyl or alkoxycarbonyl group or a group R1-Sj in which R1 is an alkyl group having 1 to 6 carbon atoms which is optionally substituted with a phenyl group or a group of formula II

wherein X ₁ , Y ₁ , R ₂ , R ' ₂ , X ₂ , Y ₂ , X, R 1, R' ₃ and R have the following meanings, and A 1 is a straight or branched alkylene group containing 1 to 4 carbon atoms optionally substituted at the alpha position of a C 4 -C 8 (Y p amino group, C 1 -C 4 alkylamino group, C 1 -C 4 alkanoylamino group or C 1 -C 4 alkoxycarbonylamino group,

X | ^ and Y each represents a hydrogen atom or form together with the carbon atom to which they are attached represent C = 2 ^ o _R

R ₂ represents a straight or branched alkyl group containing by one to four carbon atoms which is optionally substituted cyclohexyl,

R ' ₂ represents a hydrogen atom or a straight or branched alkyl group having 1 to 6 carbon atoms,

X ₂ and Y ₂ each represent a hydrogen atom or form together with the atom

R.

the carbon to which they are attached means a straight or branched (C 1 -C 4) alkyl group optionally substituted by hydroxy, (C 1 -C 4) alkoxy, mercapto, (C 1 -C 4) alkylthio, (C 1 -C 4) alkylsulfinyl Having 4 carbon atoms or an alkylsulfonyl group containing from 1 to 4 carbon atoms, provided that when it is an alkyl group substituted with a hydroxy group, R ^ may form with the carboxyl group in the alpha position a lactone, a hydrogen atom or a straight or branched alkyl group containing 1 to 4 6 is an oxygen or sulfur atom and is a hydrogen atom or an alkyl group optionally substituted by an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms. carbon atoms, C 1 -C 4 alkylsulfinyl, C 1 -C 4 alkylsulfonyl, phenyl, phenoxy, phenylthio, phenylsulfinyl, phenylsulfonyl, C 1 -C 4 alkylamino or a dialkylamino group each containing 1 or a phenyl group optionally substituted by one or more of the same or different substituents selected from the group consisting of halogen atoms, (C 1 -C 4) alkyl, (C 1 -C 4) alkoxy, (C 1 -C 4) alkylthio C 4 -C 4 alkanoyl containing from 1 to 4 carbon atoms.

In particular, in formula (I), YSA- is one in which Y is a hydrogen atom, a lysine residue or a fatty acid residue containing up to 20 carbon atoms and A ₁ is an ethylene or propylene group optionally substituted by an amino group,

X ₁ and Y ₁ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group R 7 = O, R 12 represents an isopropyl group, a 1-methylpropyl group, a tert-butyl group or a cyclohexylmethyl group,

R '2 represents a hydrogen atom or a methyl group,

X2 and ^ 2 ^each represent a hydrogen atom or together with the carbon atom to which they are attached form a group 7 ^ C = O,

R 6 represents a methyl or ethyl group which is substituted by a hydroxy, methoxy, mercapto, methylthio, methylsulfinyl or methylsulfonyl group,

R @ 1 represents a hydrogen atom or a methyl group,

X represents an oxygen atom and ---

R represents a hydrogen atom or an alkyl group containing up to 4 carbon atoms optionally substituted by an alkoxy group or a phenyl group.

Especially in said formula I

R1 represents a group of the formula Y-S-A1 - in which Y represents a hydrogen atom and Aj represents an ethylene or propylene group optionally substituted by an amino group,

X ₁ and Y ₁ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group> 0 = 0,

R2 is isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl,

R * 2 represents a hydrogen atom,

X 2 and Y ₂ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group C = O,

R 6 represents a methyl or ethyl group which is substituted by a hydroxy group, a methoxy group, a mercapto group or a methylthio group,

R 'is hydrogen and

R represents a hydrogen atom or an alkyl group containing up to 4 carbon atoms. _.

Particularly interesting peptides of the invention are peptides of the general formula I in which

R ₁ represents a 2-mercaptoethyl group or a 1-amino-2-mercaptoethyl group, X 1 and Y 2 each represent a hydrogen atom or together with the carbon atom to which they are attached form a group C = O, R ₂ represents an isopropyl group, X ₂ and each represents a hydrogen atom or forms together with the carbon atom to which they are attached a group 3; C = O, R ' ₂ represents a hydrogen atom,

R1 is 2-methylthioethyl or 2-methylsulfinylethyl, R1 is hydrogen and R is hydrogen.

The invention also relates to the preparation of stereoisomers of the compounds of formula (I). Amino acid residues RJC (, XJ (YJ, _R, CH (NR _') [C _(X2) (YJ] and _R3 CH (NR ₃₎ -CO-OH · have preferences'

Ί natural amino acid configuration.

The invention also relates to mineral or organic salts and esters of the compounds of formula (I).

According to the invention, these novel products of formula (I) can be obtained in the solid phase using the synthesis of a 9-fluoromethoxycarbonyl derivative (EMOC). In that case the thiol groups are protected by trityl or acetamidoraethyl groups, the amino functions are protected by groups

-6Boc (t-butoxycarbonyl) and acid functions are protected in the form of butyl esters, alcohol functions are protected by t-butyl groups and amide and imidazole functions by trityl groups.

The synthesis can be carried out on a resin which is enclosed in ³ cm ³ solid-density extraction syringes of high density polyethylene. The syringes are fitted with a Teflon filter and a two-way Teflon cap, sealed with a single-use, high-density polyethylene wing plug. The swinging movement of the syringes is provided by a rotating device for hemolysis tubes. Washing and filtration are carried out in a solid-phase extraction station.

The synthesis was performed using 50 pmol resin. Amino Acid Binding - · occurs during. one hour. The "25.0 µmol" amino acids are suitably protected in the presence of 250 µm of 2- (1H-benzotriazole-1 -} - 1,1,3,3-tetramethyluronium, hexafluorophosphate (HBTU), 250 µm of N-hydroxybenzyltriazole and

750 μπιοί diisopropylethylamine in 1,2 ml N-2-methylpyrrolidone (NMP) / dimethylformamide (1/1 v / v) The removal of the EMOC protecting group is carried out by three successive resin adjustments over two minutes followed by 2 ml piperidine in 2 minutes % solution (v / v) in N-2-methylpyrrolidone.

The following example illustrates the invention in more detail without limiting its scope in any way.

DETAILED DESCRIPTION OF THE INVENTION

Cys- (NMe) Val - [(R, S) -1,2,3,4-tetrahydroisoquinoline-1-carbonyl] -Met can be prepared as follows:

pmol Fmoc-Met 'resin [Wang resin,' Wang et al., J. Amer. Chem. Soc., 95, 1328, (1973)] is subjected to the following procedure:

-removal of the FMOC protecting group

-Ί-wash five times with 2 ml of NMP -acid binding

FMOC- (R, S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid

- wash 5 times with 2ml NMP

-removal of the FMOC protecting group

- wash 5 times with 2 ml NMP

-binding of FMOC-N-metbylvaline

- wash 5 times with 2 ml NMP

removal of the FMOC protecting group

- wash 5 times with 2 ml NMP

-binding of FMOC-cysteine (S-trityl)

- wash 5 times with 2 ml of NMP - remove the FMOC protecting group

Wash 5 times 2 ml. NMP. . .. ... ....

The synthesis ends with the isolation of the products. The products are isolated by treating the resin with 10 ml of a mixture of trifluoroacetic acid-phenol-ethanedithiol-thioanisole-water in a volume ratio of 40: 3: 1: 2: 2 in one hour and 30 minutes. The resin is then separated by filtration. The filtrate was concentrated under reduced pressure on a rotary evaporator (RC10-10 Jouan) equipped with a vane pump and a trap (-90 ^E C) over 1.5 hours while the temperature of the vaporization chamber is maintained at 50 ° C. The final volume of the concentrate is approximately 1 ml. The product is then precipitated by the addition of 15 ml of a mixture of methyl tert-butyl ether and petroleum ether (2: 1 by volume), then centrifuged. The pellet is further dissolved in 1 and again precipitated by the addition of 15 ml and washed with a further 15 ml

The product is then dried under reduced purification by high pressure liquid chromatography (HPLC) on a C18 100 Å column (250 x 10 mm,

BioRady; The mixture was yellowed with a gradient of acetonitrile containing Ό.07% trifluoroacetic acid (v / v) in water containing 0.07% trifluoroacetic acid (v / v) at a flow rate of 6 ml / min and then.

ml of trifluoroacetic acid, methyl tert-butyl ether and methyl tert-butyl ether pressure (3.5 kPa); and

-8 is lyophilized. The product obtained is characterized by its mass spectrum.

1,2,3,4-tetrahydro-1-isoquinolinecarboxylic acid, in racemic form, can be prepared by hydrogenation of 1-isoquinolinecarboxylic acid under the conditions described by R.F. Shuman et al., J. Med. Chem., 36, 314 (1993).

The introduction of the FMOC protecting group into the amino acid is accomplished by treating the amino acid with 9-fluoromethylchloroformate (FMOC-chloride) under basic conditions.

Farnesyltransferase inhibitory activity and ras protein farnesylation are described in more detail in the following assay:

Farnesvltransferase activity is determined by the amount transferred. . (¾). farnesylpyrophosphate ( ³ H) FPP) to the p21 protein. The standard reaction mixture consists, for a final volume of 60 ml, of 50 mM Tris-HCl, 5 mM MgCl, 5 mM dithiotreitol, 0.2% octyl-β-D-glucopyranoside, 200 picomol p21, 4.5 picomol (3H) FPP (61000 dpm / picomol).

The reaction is initiated by the addition of about 5 ng of human farnesyltransferase purified from THP1 cell culture. After 20 min incubation at 37 ° C in a 1 ml 96-well microtiter plate (Titer Plate®, Beckman), the reaction was terminated by the addition of 0.4 ml 0.1% SDS in methanol at 0 ° C. The mixture was then added to 0.4 mL of 30% trichloroacetic® (TCA) in methanol. The plates are kept in the freezer for one hour. The precipitate content is then retained on a fiberglass® membrane (Filtermat®, Pharmacia) with a filter unit (Combi Cell Harvester®, Skarton) and rinsed with 6% trichloroacetic acid in distilled water. The membranes are dried in a microwave oven, then saturated with warm air Meltilex® (Pharmacia). Inhibitory activity is calculated in cpm on a β-Plate® (LKB) counter. Each attempt is repeated three times ......

The unit of activity is defined by one picomol (3HJFPP, which is transferred to p21 in 20 minutes).

99 Percent inhibition is obtained by comparing experiments with and without inhibitor after subtracting the blank, IC ₅₀ was measured for inhibition obtained with 9 different concentrations using means known under the trade name Enzfitter ® or Grafit ®.

The results are summarized in Table I.

Table I

Product Inhibitory activity of ic ₅₀ Cys - (N-MeVal - [(R, S) -tetrahydro-1,2,3,4- 2,6 χ ΙΟ ⁸ M isoquinoline-1-carbonyl] -Met - - - -.

The novel peptides (I) may exist in the form of pharmaceutically acceptable non-toxic salts. These non-toxic salts include salts of mineral acids (such as hydrochloric, sulfuric, hydrobromic, phosphoric, nitric) or salts of organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic, oxalic) of mineral bases (sodium hydroxide) , potassium hydroxide, lithium hydroxide, calcium hydroxide) or organic bases (tertiary amines such as triethylamine, piperidine, benzylamine) depending on the nature of the amino acids that make up peptide (I).

The novel peptides of the present invention inhibit farnesyltransferase and farnesylation of ras protein. They are significantly anticancer agents, acting at the level of both solid and liquid tumors.

. The present invention also relates to pharmaceutical compositions comprising at least one peptide of formula (I) in combination with one or more diluents or acceptable

Pharmaceutical excipients which are inert or physiologically active.

The pharmaceutical compositions may be administered orally, parenterally or rectally.

Formulations for oral administration include tablets, pills, powders, or granules. In these compositions, the active ingredient of the invention is admixed with one or more inert fillers such as sucrose, lactose or starch. These compositions may contain other substances as fillers, for example lubricants such as magnesium stearate.

As liquid compositions for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, alcohol-containing syrups containing inert fillers such as water or paraffin oil may be used. ..These means can. also contain other excipients, for example wetting, sweetening or flavoring agents.

The compositions of the patent for parenteral administration may be in the form of sterile water or non-aqueous phase solutions such as suspensions or emulsions. Propylene glycol, polyethylene glycol, an oil plant, particularly olive oil or injectable organic esters such as ethyl oleate can be used as a solvent or vehicle.

These compositions may also contain adjuvants, in particular wetting, emulsifying or dispersing agents. Sterilization can be carried out in a number of ways, for example by means of bacteriological filters, insertion of the sterilizing agent into the preparation or by heating. The agents may also be prepared in the form of solid sterile materials, which may be dissolved immediately in sterile water or other sterile injectable formulations prior to use.

Formulations for rectal administration are presented as suppositories, which may contain other active ingredients, such as a vehicle such as cocoa butter.

The compositions of the invention are particularly useful in human therapy in the treatment of various cancers.

In human therapy, the dosage depends on the effect desired, the duration of use and the individual requirements of the patient for whom the substance is intended.

Generally, the human dose is between 0.1 and 20 mg / kg daily (for intraperitoneal administration).

Claims (5)

PATENTOVÉ Claims CLAIMS 1. Peptides of the general formula I in which: R represents a group of the formula YS-Ap in which Y represents a hydrogen atom or an amino acid or a fatty acid residue or an alkyl or alkoxycarbonyl group or a group R 6 -Sy in which R 6 represents an alkyl group having 1 to 6 carbon atoms which is optionally substituted with a phenyl group or a group of formula II wherein A ₁ , X ₁ Y ₁ , R ₂ , R ₁ , X ₂ , Y ₂ , R ₃ , R 3 ^{, R} 3 ^{and R} have the following meanings; means a straight or branched C1-C4 alkylene group optionally substituted in the alpha position of the C4-C group (xp (Yp by amino, C1-C4 alkylamino, C1-C4 alkanoylamino) or alkoxycarbonylamino, the alkyl radical contains 1 to 4 carbon atoms, X ₁ and Y ₁ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group -C 1 -C 0, 1 * 2 represents a straight or branched alkyl group having 1 to 4 carbon atoms, which is optionally substituted by a cyclohexyl group, R * 2 represents a hydrogen atom or a straight or branched alkyl group having 1 to 6 carbon atoms, X2 and Y2 each represent a hydrogen atom or together with the carbon atom to which they are attached form a group TuC = O, R3 represents a straight or branched (C1-C4) alkyl group optionally substituted by hydroxy, (C1-C4) alkoxy, mercapto, (C1-C4) alkylthio, (C1-C4) alkylsulfinyl or alkylsulfonyl containing from 1 to 4 carbon atoms, provided that when it is an alkyl group substituted with a hydroxy group, R @ 1 may form with the carboxyl group in the alpha position a lactone, R < 1 > represents a hydrogen atom or a straight or branched alkyl group having 1 to 6 carbon atoms, X is oxygen or sulfur; and R represents a hydrogen atom or an alkyl group optionally substituted by a (C 1 -C 4) alkoxy group, a (C 1 -C 4) alkylthio group, a (C 1 -C 4) alkylsulfinyl group, a C 1 -C 4 alkylsulfonyl group, a phenyl group, a phenoxy group , phenylthio, phenylsulfinyl, phenylsulfonyl, (C 1 -C 4) alkylamino or dialkylamino, each C 1 -C 4 alkyl radical, or phenyl optionally substituted by one or more of the same or different substituents selected from the group consisting of halogen atoms, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, and alkanoyl of 1 to 4 carbon atoms. Peptides according to claim 1, in which R | represents a group of the formula Y-S-A-, in which Y represents a hydrogen atom or a lysine residue or a fatty acid residue containing up to 20 carbon atoms and A A represents an ethylene or propylene group optionally substituted by an amino group, X ^ and Y ^ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group 3 ^ 2 = O, R ₂ represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical, R ' ₂ represents a hydrogen atom or a methyl group, X ₂ and Y ₂ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group R1 is methyl or ethyl which is substituted by hydroxy, methoxy, mercapto, methylthio, methylsulfinyl or methylsulfonyl, R ' ₂ represents a hydrogen atom or a methyl group, X represents an oxygen atom and R is hydrogen or alkyl 1-4 carbon atoms optionally substituted by alkoxy or phenyl. Peptides according to claim 1 of the general formula I, in which R 6 represents a group of the formula Y-S-A 6 -, wherein Y represents a hydrogen atom and A 6 represents an ethylene or propylene group, which is optionally substituted with an amino group i • r J r X ^ and Y ^ each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ₂ represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical, R ' ₂ is hydrogen, X ₂ and Y ₂ each represent a hydrogen atom or together with the carbon atom to which they are attached form a group jC = O, R3 represents a methyl or ethyl group which is substituted by a hydroxy, methoxy, mercapto or methylthio group, R 'is hydrogen and R is hydrogen or alkyl. '_________ 1 to 4 carbon atoms. __ Peptides according to claim 1 of the general formula I, in which R 1 represents a 2-mercaptoethyl group or 1-amino-2-mercaptoethyl group, X ₁ and each represents a hydrogen atom or forms together with the carbon atom to which they are attached a group / i C = O, R ₂ represents an isopropyl group, X ₂ and Y ₂ each represents a hydrogen atom or forms, together with the carbon atom to which they are attached, a group j = C, R ' ₂ represents a hydrogen atom, R3 is 2-methylthioethyl or 2-methylsulfinylethyl, R1 is hydrogen and R is hydrogen. A pharmaceutical composition comprising a sufficient amount of a peptide of formula (I) according to any one of claims 1 to 4 in combination with one or more pharmaceutically acceptable diluents or one or more pharmaceutically acceptable excipients which are inert or physiologically active. Represented by: