CA2080582A1

CA2080582A1 - Heterocyclicethenediyl compounds which inhibit egf receptor tyrosine kinase

Info

Publication number: CA2080582A1
Application number: CA002080582A
Authority: CA
Inventors: Alfred P. Spada; Paul E. Persons; Alexander Levitzki; Chaim Gilon; Aviv Gazit
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-04-16
Filing date: 1991-04-16
Publication date: 1991-10-17
Also published as: WO1991016305A1; JPH05507072A; EP0527181A1; EP0527181A4; AU7756891A; AU662480B2; IL97873A0

Abstract

Heteroarylethenediyl or heteroarylethenediyl aryl compounds wherein the heteroaryl group can be mono- or bicyclic heteroaryl and the aryl group can be mono- or bicyclic heteroaryl or bi- or tricyclic carbocyclic, said compound optionally substituted or polysubstituted, with the proviso that the heteroaryl group is not furyl or thienyl when the ethenediyl group has geminal cyano substituents, and pharmaceutical compositions comprising said compounds, and the use thereof for inhibiting cell proliferation in a patient suffering from such disorder.

Description

20~0~82 HETEROCYCLICETHENEDIYL COMPOUNDS WHICH
INHIBIT EGF RECEPTOR TYROSINE KINASE

FIELD OF THE INVENTION
This invention relates to the inhibition of cell proliferation. More specifically, this invention relates to the use of heterocyclicethenediyl compounds in inhibiting cell proliferation, including compounds which are useful protein tyrosine kinase (PTX~ inhibitors.

Normal cellular reproduction is believed to be triggered - by the exposure of the cellular sùbstrate to one or more growth factors, examples of which are insulin, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).
Such growth Pact~rs are typically specific for corresponding growth factor receptors which are imbedded in and which penetrate through the cellular membrane. The initiation of cellular reproduction is believed to occur when a growth factor binds to the corresponding receptor on the external surface of the cellular membrane. This growth factor-receptor binding alters the chemical characteristics of that portion of the receptor which exists within the celI and which functions - as an enzyme to catalyze phosphorylation of either an

2 ~ g ~S ~ 2 PCT/US9l/025c~

intracellular substrate or the receptor itself, the latter being referred to as autophosphorylation. Examples of such phosphorylation enzymes include tyrosine kinases, which catalyze phosphorylation of tyrosine amino acid residues of substrate proteins.

Many diseased states are characterized by the uncontrolled reproduction of cells. These diseased states involve a variety of cell types and include disorders such as leukemia, cancer, psoriasis, atherosclerosis and restenosis injuries. The inhibition of tyrosine kinase is believed to have utility in the control of uncontrolled cellular reproduction, i.e., cellular proliferative disorders.

Initiation of autophosphorylation, i.e., phosphorylation of the growth factor receptor itself, and of the phosphorylation of a ~ost of intracellular substrates are some of the biochemical events which are involved in mitogenesis and cell proliferation. Autophosphorylation of the insulin receptor and phosphorylation of substrate proteins by other receptors are the earliest identifiable biochemical hormonal Z0 responses.

Elimination of the protein tyrosine kinase (PTK) activity of the insulin receptor and of th~ epidermal growth factor (EGF) receptor by site-directed mutagenesis of the cellular genetic material which is responsible for generation of insulin and E~F results in the complete elimination of the receptors' biological activity. This is not particularly desirable because insulin is needed by the body to perform other biological functions which are not related to cell proliferation. Accordingly, compounds which inhiblt thé PTK
portion of the EGF receptor at concentrations less than the concentrations needed to inhibit the PTK portion of the insulin receptor could provide valuable agents for selective treatment of cell proliferation disarders.

WO91/16305 2 ~ PCT/US91/02597 REPORTED DEVELOPMENTS
The use of furanal malononitri:Le and thienylidene malononitrile compounds as tumor growth inhibitors is disclosed in Gal et al., Studies on the Biological Action of Malononitriles. I. The Effect of Substituted Malononitriles on the Growth of Transplanted Tumors in Mice, Cancer Research, ~2:565-72, 1952.

It has been reported that the most potent inhibitors of EGF receptors inhibit EGF-induced proliferation of A431/clone ~0 15 cells with little or no effect on the proliferation of such cells when induced by other growth factors. It has been reported also that erbstatin inhibits the autophosphorylation of the EGF receptor in membranes of A431 cells. Low concentrations of erbstatin are required to inhibit EGF
receptor autophosphorylation, whereas much higher concentra-tions of erbstatin are required to inhibit cyclic adenosine

3',5'-monophosphate (cAMP)-dependent protein kinase.

SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a heteroarylethenPdiyl or a heteroarylethenediyl aryl compound, or a pharmaceutically acceptable salt thereof, exhibiting protein tyrosine kinase inhibition activity, wherein the heteroaryl group can be mono-or bicyclic heteroaryl and the aryl group can be mono- or bicyclic heteroaryl or bi- or tricyclic carbocyclic, said compound optionally substituted or polysubstituted, provided that the heteroaryl ~roup is not furyl or thienyl when the ethenediyl ~roup has geminal cyano substituents.

Another aspect of the present invention relates to pharmaceutical compositions comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of the aforementioned type of compound.

WO91/16305 2 ~ 8 ~ ~ ~ 2 PCT/US91/025;~

Still another aspect of the present invention relates to novel compounds of the aforementioned type.

Compounds described by Formula I below constitute a class of the aforementioned heteroarylethenediyl or heteroaryl-ethenediyl aryl compounds for use in the practice of thepresent invention:

C=C
W R~
wherein:

W is a heteroaryl ring system having an about 5- to about 7-membered monocyclic ring including l or 2 N, O or S
atoms, or an about 8- to about 12-membered bicyclic ring including l to about 4 N, O or S atoms, said ring system optionally-substituted with one to about three R~ groups;

Rl is alkyl, -CN, -CONRR, -CSNRR or -COOR;

R is hydrogen, alkyl or aralkyl;

R2 is -W, -CN, -CONHR5, -CONRR, -COOR, -CSNRR, -CH=C~CN) 2 -C(NH2)=~(CN)2~ .
_co,~, -Co~
S~
~CO ~ ¦ or-an about 9- to ahout 11-membered bicyclic aryl carbocyclic ring system or an about 12- to about 15-membered tricyclic aryl carbocyclic ring system, said ring system optionally substituted with one to about three R4 groups;

- R3 is hydrogen, alkyl, -CN, -CH2CN, -CONRR, -CSNRR, -COOR
or -CH=C(CN)CONH2;

each ~ -is independently alkyl, hydroxy, alkoxy, halo, amino, mono- and di-alkylamino, acetylamino, alkylthio, -CN, -CF3, nitro, -COOR, -CONRR, -CSNRR, -CHO, -C~=CHCOOH, -NHCO(CH2)2COOH or morpholino;

R5 is amino, -CONH2, -(CH2)~-NHCO-C-CH ~ ~3 -(CH) ~ R,7) ~3 0 each R6 is independently hydrogen, alkyl, hydroxy, alkoxy or halo;

each R~ is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;

n is 0 to about 6; and m is 1 to about 7;

with the proviso that when both R~ and R2 are -CN, W is not furyl or thienyl.

WO9l/1~305 PCT/US91/025~`
2 ~ 8 0 3 8 2 Compounds within the scope of the present invention have also a specific affinity toward the substrate site of the tyrosine kinase domain of EGF receptors, inhibit EGF receptor kinase more than they inhibit PDGF receptor kinase and also effectively inhibit EGF-dependent autophosphorylation of the receptor.

DETAILED DESCRIPTION OF_THE INVENTION
As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Alkyl" means a saturated aliphatic hydrocarbon whlch may be either straight- or branch-chained containing from about 1 to about 6 carbon atoms.

"Lower alXyl" means an alkyl group as above, having 1 to about 4 carbon atoms which may be straight- or branch-chained such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

"Alkoxy" means an alkyl-oxy group in which "alkyl" is as previously described. Lower alkoxy groups are preferred.
Exemplary groups include methoxy, ethoxy, n-propoxy, i-propoxy and n-butoxy.

"Aryl" means an unsaturated or partially unsaturated ring system. Preferred aryl groups are pyridyl and indolyl.

"Acyl" means an organic radical derived from an organic acid, a carboxylic acid, by the removal of its acid hydroxyl group. Preferrecl acyl groups are lower alkyl carboxylic acid groups such as acetyl and propionyl. Benzoyl is also preferred.

"Halo" means a halogen. Preferred halogens include chloride, bromide and fluoride.

` WO91/16305 2 ~ 8 0 ~ ~ 2 PCT/US91/02597 Preferred aralkyl groups are benzyl and phenethyl.

It is believed that therapeutically useful PTK inhibiting compounds should be competitive with the substrate of EGF
receptor tyrosine kinase (EGFRK) and not with adenosine triphosphate (ATP)~ The PTX inhibitors guercetin and genistein, which compete with ATP, inhibit other protein kinases and as a result are highly cytotoxic. As a test of selectivity, compounds which inhibit EGFRK better than they inhibit insulin receptor kinase ~IRK) and/or PDGF receptor kinase are of considerable value.

It is theorized that solubility of the compounds of the present invention both in water and in mildly hydrophobic solvents will enhance the probability that they traverse the cell membrane. Various insoluble compounds, however, have exhibited significant EGFRK inhibition in in vitro testing.

A preferred class of compounds useful in the practice of the present invention include those described by Formula I
where:

W is a 5- or 6-membered monocyclic aryl ring including l or 2 N, O or S atoms, or a 9- or l0-membered bicyclic aryl ring including l to 4 N, O or S atoms, said ring optionally substituted with one to about three R4 groups;
.
R~ is -CN, -CONRR, -CSNRR or -COCR;

R is hydrogen, alkyl, or aralkyl;

R2 is -W, -CN, -CONHR5, -CONRR, -COOR, -CSNRR or ~R7)~3 -CO~

R3 is hydrogen;

WO91/16305 2 ~ 3 2 8 PCT/US91/02S~:

each ~ is independently alkyl, hydroxy, alkoxy or halo;

R5 is amino, -CO~H2, s fN ~R~) ~3 -(CH2)~-NHCO-C=CH ~ or ~ ~ R7)~3 each ~ is independently hydrogen or alkyl;

each R7 is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;

n is 0-4; and m is 2-6;

with the proviso that when both R~ and R2 are -CN, W is not furyl or thienyl.

The more preferred compounds of this invention lnclude those of Formula I where:

W is furyl, pyrrolyl, thienyl, thiazolyl, pyridyl, imidazolyl, isoimidazolyl, pyridazinyl, pyrimdinyl, benzofuranyl, indolyl, indolinyl, indolinonyl, benzothienyl, benzothiazolyl, quinolinyl, isoquinolinyl, chromenyl, l,3-benzodioxolyl or 2,3-dihydro-l,4-benzodioxinyl.

The most preferred compounds are described by Formula I
where:

" WO91/16305 2 0 8 0 ~ g 2 PCT/~S91/02597 W is pyridyl, indolyl, imidazolyl, benzothiazolyl, 1,3-benzodioxolyl or 2,3-dihydro-1,4-benzodioxinyl;

R~ is -CN, -CONRR, -CSNRR or -COOR;

is hydrogen, alkyl, or aralkyl;

R2 is -W, -CN, -CONHR5, -CONRR, -COOR, -CSNRR or (R7)~3 -CO~

R3 is hydrogen;

each ~ is independently lower alkyl, hydroxy, lower alkoxy or halo;

-(CH2) -NHCO-C=CH ~ R~)~3 -(CH2) ~ R7)~3 each R~ is independently lower alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;

n is 0-4; and m is 2-6.

Compounds of this invention may be useful in the form of the free base, in the form of salts and as a hydrate. A11 forms are within the scope of the invention. Acid addition W091/16305 2 ~ 8 ~ ~ 8 ~ PCT/~S9t/025~-salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of the base form. The acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base~, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to the animal organism in pharmaceutical doses of the salts, so that the beneficial properties inherent in the free base are not vitiated by side effects ascribable to the anions.
lo Although pharmaceutically acceptable salts of said basic compound are preferred, all acid addition salts are useful as sources of the free base form even if the particular salt per se is desired only as an intermediate product as, for example, when the salt is formed only for purposes of purification and identification, or when it is used as an intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures.

Pharmaceutically acceptable salts within the scope of the invention include those derived from the following acids:
mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like.

~ he correspondin~ acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, methanesulfonate, ethanesulfonats, benæenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.

The acid addition salts of the compounds of this invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by ` WO91/16305 ~ S 2 PCT/US91/02597 evaporating the solution, or by reacting the free base and acid in an organic solvent, in which case the salt separates directly or can be obtained by concentration of the solution.

Compounds useful in the practice of this invention can be prepared by known methods, for example, Xnoevenagel condensation reactions such as those disclosed in ~.s. Patent No. 3,149,148.

Co~pounds of this invention may be prepared by the following reaction sequence:

W-C(R3)0 + R~-CH2-R2 > C=C
W R~

Knoevenagel condensation of a heterocyclic aldehyde of formula W in a polar media with an active methylene compound of the formula R,CH2R2 in the presence of ammonia or amines such as piperidine and raised heat results in the products of this invention. When substitution of the R3 group is desired, the corresponding ketone starting material is used. Reaction temperatures in the range of 25~C to reflux and reaction times vary depending on the materials being used in the condensation.

Compounds of this invention are either comme~cially available, known in the literature or can be made by known procedures.

- Various R, R~, R2, R3, R4, R5, ~ and R7 substituents on the hetero ring or chain can be present in the starting compound or added after formation of the condensation product by methods known in the art for substitution or conversion on one group to another. If the substituents ~hemselves are reactive, then the substituents can themselves be protected according to the techniques known in the art. A variety of protecting groups known in the art, may be employed. Examples ~ J~
WO91/16305 PCT/US91/025a:

of many of these possible groups may be found in "Protective Groups in Organic Synthesis" by T. W. Green, John Wiley and Sons, 1981. For example, nitro groups can be added to the aromatic ring by nitration and the nitro group converted to other groups, such as amino by reduction, and halo by diazotization of the amino group and replacement of the diazo group. Acyl groups can be substituted onto the aryl groups by Friedel Crafts acylation. The acyl groups can then be transformed to the corresponding alkyl groups by various methods, including the Wolff-Kishner reduction and Clemmenson reduction. Amino groups can be alkylated to form mono- and di-alkylamino groups; and mercapto and hydroxy groups can be alkylated to form corresponding ethers. Primary alcohols can be oxidized by oxidizing agents known in the art to form carboxylic acids or aldehydes, and secondary alcohols can be oxidized to form ketones. Thus, substitution or alteration reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product.

Compounds within the scope of this invention exhibit significant activity as protein tyrosine kinase inhibitors and possess therapeutic value as cellular antiproliferative ayents for the treatment of certain conditions including, for example, psoriasis and restenosis injuries. It is expected that the invention will be particularly applicable to the treatment of atherosclerosis. With regard to the treatment of some~conditions, for example, atherosclerosis, certain people may be identified as being at high risk, for example, due to genetic, environmental or historical factors. Compounds within the scope of the present invention can be used in preventin~ or delaying the occurrence or reoccurrence of such conditions or otherwise treating the condition.

Compounds of the present invention can be administered to a mammalian host in a variety of forms adapted to the chosen route of administration, i.e., orally, or parenterally.

WO91/1630~ 2 0 ~ O .~ ~ ~ PCT/US91/02597 Parenteral administration in this respect includes administration by the following routesO intravenous, - intramusc~lar, subcutaneous, intr~ocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation and aerosol and rectal systemic.

The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. SUG~ compositions and preparations should contain at least 0.1~ of active compound. The percentage of the compositions and preparations may, of cour~e, be varied and may conveniently be between Z0 about 2 to about 6% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 1 and 1000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starchJ alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other WO91/1630~ 2 ~ g ~ PCT/US91/025~ l !

materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved aqainst the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of -` WO91/16305 2 0 8 0 S ~ ~ PCT/US91/02597 microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

The therapeutic compounds of this invention may be administered to a mammal alsne or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.

The dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment wiil vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and if necessary, will be increased by small increments until the optimum effect under the circumstances is WO~1/1630S 2 0 ~ 03 8 ~ PCT/US91/025~
, reached. The therapeutic human dosage, based on physiological studies using rats, will generally be from about 0.01 mg to about 100 mg/kg of body weight per day or from about 0.4 mg to about 10 g or and higher although it may ~e administered in several different dosage units from once to several times a day. Oral administration requires higher dosages.

EXAMPLES
Embodiments of the present invention are described in the following non-limiting examples which include a description of pharmacological test procedures believed to correlate to therapeutic activity in humans and other animals. Examples 1-11 below are illustrative of compounds within the scope of the present invention. Examples 1-5 illustrate various unsubstituted heteroaryl malononitriles, i.e., Rl and R2 are each -CN and the other R substituents are all hydrogen.
Example 6 illustrates a compound wherein R~ is -CN,:R2 is -C(NH2~=C(CN)2, R3 is hydrogen and there are no ~a substituents.
Example 7 illustrates a nitro-substituted heteroaryl malononitrile, i.e., R~ and R2 are each -CN, R3 is hydrogen and R4 is nitro. Example 8 illustrates a diheteroarylethenediyl compound, i.e., R~ is -CN, R2 is pyridyl and the other R
substituents are all hydrogen. Example g illustrates a heteroarylethenediyl carbocyclic compound, i.e., R~ is hydrogen, R2 is phenanthryl, R3 is -CN and there are no R~
substituents. Examples 10 and 11 illustrate heteroarylethenediyl aryl compounds, i.e., R~ is -CN,. R2 is 3,4-dihydroxybenzoyl and R3 is -H.
.
Example 1 2-Imidazolidene malononitrile To 0.4 g (4 mmol~ 2-formylimidazole (Aldrich) and 0.5 g (4.5 mmol) malononitrile in 15 ml ethanol was added 30 mg ~-alanine. The reaction was refluxed for one hour, evaporated to dryness and the dark solid obtained was flash-chromatographed on silica gel (elution with CH2Cl2 to 3 WOg1~163~5 2 0 ~ ~ a ~ 2 PCT/~S9l/~25g7 ethanol) to give 0.2g (33% yield) of a yellow solid, m.p.
180C (decomposition~.

Example 2 5-Pvrazolidene malononitrile S (a) To CH2N2 (from 10 g diazald) in 50 ml ether was added 2.5 g (16.5 mmol) propyne acetal. After 3 days of refrigeration, 50 ml ethanol and 10 drops concentrated ~Cl were added and the reaction mixture was stirred for 15 minutes at room temperature, evaporated to dryness and triturated with CCl4 to give 90 mg of a formyl pyrazole, a light yellow solid.
(b) To 80 mg of the pyrazole from step (a) above and 70 mg malononitrile in 20 ml ethanol was added 20 mg ~-alanine and the reaction mixture was refluxed for 20 hours. Water was added and the reaction mixture was extracted with EtAc, evaporated and flash chromatographed to give 30 mg (25~ yield) of product, a yellow solid, m.p. lS4C.

Example 3

4-Imidazol~idene malononitrile 4-Hydroxymethyl imidazole (Aldrich) was oxidized with HNO3 and the reaction mixture was evaporated to dryness to give 0.5 g of a light brown solid, to which, in 10 ml ethanol and 2 ml H20, was added 0.45 g (6.8 mmol) malononitrile and 50 mg ~-alanine. The reaction mixture was refluxed for 1 hour, evaporated to dryness and flash chromatographed. The second fracti~n yielded 20 mg of product as a yellow solid, m.p.
176C.

Exam~le 4 l-Indolinidene malononitrile Indoline, 0.8 g (6.7 mmol) and 0.8 g ~6.7 mmol) ethoxymethylene nialononitrile in 30 ml CH3CN were stirred for 30 minutes at room temperature and the precipitate was filtered off to give 0.78 g (51% yield) of product as a pale yellow solid, m.p. 228C.

wO91/16305 2 ~ 2 PCT/US91/025~^

Exam~le 5

5-Indolidene malononitrile To 150 mg (1 mmol) 5~formyl indole and 80 mg (1.2 mmol) malononitrile in 10 ml ethanol was added 1 drop piperidine.
The reaction mixture was refluxed ~or 2.5 hours, H20 was added, the mixture cooled and filtered to give 140 mg (70~ yield) of product as a yellow solid, m.p. 225 ~C.

Example 6 1,1 3-Tricvano-2-amino-4-f5-indole~butadiene To 0.29 g (2 mmol) 5-formylindole and 0.28 g (2.1 mmol) malononitrile dimer in 10 ml ethanol was added 30 mg ~-alanine. The reaction mixture was refluxed for 4 hours, cooled, filtered, washed with H20 and ethanol and dried to give 0.16 g (31% yield) of product as a yellow-orange solid, m.p.
242C.

Example 7 3,4-Methylenedioxy-6-nitrobenzylidene malononitrile 1 g (5.1 mmol) 3,4-Methylenedioxy-6-nitrobenzaldehyde, 0.4 g S6 mmol) malononitrile and 0.2 g ~-alanine in 30 ml ethanol were stirred 16 hours at room temperature. 50 ml H20 was added. Filtering gave 1 g, (80% yield) of a bright yellow solid, m.p. 104C.

Example 8 Following the procedures of example 7 above, 2-(2-pyridyl~-3-[3,4-(methylenedioxy)phenyl]-2-propenenitrile was prepared, m.p. 153--55C.

Exam~le 9 2-(4-Pyridyl~-3-~9-Dhenanthryl)-2-proeenenitrile To a solution of 0.5 g (2.43 mmol) phenanthrene-9-carboxaldehyde in 40 ml absolute ethanol was added 0.375 g (2.43 mmol) 4-pyridylacetonitrile-~cl and 0.5 ml piperidine, and the reaction mixture was stirred and heated to reflux for 2 hours, at which time a yellow precipitate had formed. The ~ ` WO 91/16305 2 0 ~ O ~ 8 2 PCT/US91/02~97 reaction mixture was heated to reflux for an additional 2 hours, cooled to room temperature, and the solvent was partially evaporated. The mixture was partitioned between CH2Cl2 and H2O, washed with 5% NaHC03, dried over anhydrous Na2SO4, filtered, concentrated to give a yellow solid which was then flash chromatographed on silica gel. The material was loaded onto the column with CH2Cl2 and eluted with EtOAc:hexan~
~ to give 0.328 g (44% yield) of product as an amorphous yellow solid, m.p. 20Ç-208.

Example 10 2-(3l4-Dihydroxybenzoyl)-3-(5-indolYl~-2-propenenitrile 5-Formyl indole (145 mg, 1 mmole), 3,4-dihydroxy-benzoylacetonitrile (177 mg, 1 mmole) and ~-alanine (30 mg) in 30 ml ethanol were refluxed for 3 hours. Water was added and the reaction mixture was extracted with EtOAc. The organic layer was concentrated and the resulting oily residue was purified by chromatography on silica gel to give 86 mg (31%) of the title compound as an orange solid, m.p. 185C.

Example ll 2-~3.4-Dihydroxybenzovl)-3-(3-indolyl)-2-~ropenenitrile 3-Formyl indole (105 mg, 0.7 mmole~, 3,4-dihydroxy-benzoylacetonitrile (130 mg, 0.7 mmole) and B-alanine (20 mg3 in 30 ml ethanol were refluxed for 5 hours. Water was added and the reaction mixture was extracted with EtOAc. The organic layer was concentrated and the resulting oily solid was triturated with CH2Cl2 to give 160 mg (72%) of the title compound as an orange solid, m.p. 228C.

Compounds of this invention are subjected to various biological tests~ the results of which correlate to useful cellular antiproliferative activity. These tests are useful in dPtermining EGF receptor kinase, PDGF receptor kinase and insulin receptor kinase inhibition activities of the compounds disclosed herein.

WO91/16305 2 ~ 8 ~ ~ 8 2 PCT/US91/025~

~GF-Receptor Purification `
~GF-receptor purification is based on the procedure of Yarden and Schlessinger. A431 cells are grown in 80 cm2 -bottles to confluency (2 x 107 cells per bottle). The cells are washed twice with PBS and-harvested with PBS containing l.0 mmol EDTA (l hour at 37C), and centrifuged at 600g for lO
minutes. The cells are solubilized in l ml per 2 x 107 cells of cold solubilization buffer (50 mmol ~epes buffer, p~ 7.6, 1% Triton X-lO0, 150 mmol NaCl, 5 mmol EGTA, 1 mmol PMSF, 50 ~g/ml aprotinin, 25 mmol benzamidine, 5 ~g/ml leupeptic, and - lO ~g/ml soybean trypsin inhi~itor) for 20 minutes at 4C.
After centrifugation at lOOOOOg for 30 minutes, the supernatant is loaded onto a WGA-agarose column (lO0 ~l of packed resin per 2 x 107 cells) and shaken for 2 hours at 4OC.
The unabsorbed material is removed and the resin washed twice with HTN buffer (50 mmol Hepes, pH 7.6, 0.1% Triton X-lO0, 150 mmol NaCl), twice with HTN buffer containing l M NaCl, and twice with HTNG buffer (50 mmol Hepes, pH 7.6, 0.1% Triton X-100, 150 mmol NaCl, and lO~ glycerol). The EGF receptor is eluted ba~tchwise with HTNG buffer containing 0.5 M N-acetyl-D-glucosamine (200 ~l per 2 x 107 cells). The eluted material is stored in aliquots at -70C and diluted before use with TMTNG
buffer (50 mmol Tris-Mes buffer, pH 7.6, O.l~ Triton X-lO0, 150 mmol NaCl, 10% glycerol).

EGFR Kinase Catalyzed Phosphorylation of PolyLGAT! and its Inhibition WGA-purified EGFR (0.25 ~g/assay) is preactivated with EGF (0.85 ~M) in 50 mmol Tris-Mes buffer, pH 7.6 for 20 minutes at 4C. The assay is initiated by addition of a mixture which contains Mg(Ac)2 (60 mmol), [~-32P]ATP (125 ~M, 2- ;
5 ~Ci/assay), poly(GAT) (0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml), and six concentrations of inhibitor in duplicates.
The temperature of the assay is 22C and the production of phosphorylated copolymer is found to be linear up to 20 minutes. The PTK inhibitors tested are solubilized in water or a mixture of ethanol and water such that the final WO91/16305 2 ~ 8 2 Pcr/us9l/o2597 concentration of ethanol does not exceed 4% in the assay. Up to 4% ethanol in the assay has no effect on the EGFR kinase activity. The concentration of EGF in the assay is 300 nM in a final volume of 40 ~ fter 5, lO or 20 minutes, aliquots of 25 ~l are applied onto Whatman 3-mm paper cuttings, which are then soaked in cold 10% TCA cont:aining O.Ol M sodium pyrophosphate. After being washed overnight at 4C, the paper cuttings are dried and counted, measuring 32p Cerenkov radiation. Concentration dependence on poly(GAT) was Michaelian with a Km ~ O.076 + 0.007 mg/ml or 0.069 + O.007 mmol i~ calculated per Glu5Ala3Tyr(GAT) unit. The EGF response for the poly(GAT) phosphorylation is graphed. The Km for ATP
in the assay was found to 2.9 ~M. Compounds of the examples were assayed for their ability to inhibit the EGF receptor tyrosine kinase activity. Table I below summarizes the results of this assay. "IC50" refers to the concentration (micromolar amounts) of inhibitor required to inhibit 50% of polyGAT phosphorylation under standard assay conditions.
PolyGAT concentration was equal to 2.3 Km values.

. .
Table I
Example IC5~l~M~
l 532

6 820 - The results of this assay indicate that the compounds of the present invention are effective in inhibiting substrate phosphorylation catalyzed by EGFRK.

Time_Dependence_oE EGF-Receptor Auto~hos~horYlation WGA-purified EGF receptor from A431 cells (0.5 ~g/assay) is activated with EGF (800 nM) for 20 minutes at 4C. The reaction is initiated by the addition of Mg(Ac)2 (60 mmol), Tris-~es buffer, pH 7.6 (50 mmol), and [32P]ATP (20 ~M, 5 ~Ci/assay). The reaction is conducted at either 4 or l5C and 2~3~2 WO91/16305 PCr/US91/02' terminated by addition of sodium dodecyl sulfate (SDS) sample buffer (10% glycerol, 50 mmol Tris, pH 6.8, 5~ ~-mercapto-ethanol, and 3% (SDS). The samples are run on a 8~ SDS
polyacrylamide gel (SDS-PAGE) (prepared from 30~ acrylamide and 0.8% bis-(acrylamide) and contained 0.375 M Tris, pH 8.8, 0.1% SDS, 0.05~ TEMED, and 0.46% ammonium persulfate). The gel is dried and autoradiography performed with Agfa Curix ~P2 X-ray film. The relevant radioactive bands are cut and counted in the Cerenkov mode. The fast phase of autophosphorylation continues for another lO minutes. The extent of phosphorylation completed in the first lO-s at l5~C
comprises l/3 of the total autophosphorylation signal and probably reflects the phosphorylation of the first site on the receptor. The lO-s interval is therefore chosen for use in subsequent autophosphorylation experiments.

ATP and EGF Dependence of Auto~hosphorylation WGA-purified EGF receptor from A431 cells tO.5 ~g/assay is activated with EGF (0.85 ~M~ for 20 minutes at 4C. The assay is performed at 15C and initiated by addition of Mg(Ac) 2 (60 mmol), Tris-Mes buffer, pH 7.6 (50 mmol), [32P]ATP (carrier free, 5 ~Ci~assay), and increasing concentrations of nonradioactive ATP. The assay is terminated after lO-s by addition of SDS sample buffer. The samples are run on a 6%
SDS polyacrylamide gel. The gel is dried and autoradiographed as described above. The relevant radioactive bands are cut and counted in the Cerenkov mode. the Km for ATP determined in this fashion is found to be 7.2 ~M. With use of the lO-s assay protocol, the EGF concentration dependence of EGFRK
autophosphorylation is determined.

Inhibition of Copoly(Glu4Tyr) Phosphorylation by Insulin-ReceDtor Kinase (InsRK) Rat liver membranes are prepared from the livers of 6-week-old rats as described by Cuatrecasas. WGA-purified insulin receptor is prepared according to Zick et al. WGA-purified rat liver InsRK (1.25 ~g) is preincubated with or without 330 nM insulin in 50 mmol Tris-Mes buffer, pH 7.6, for W091/16305 2 0 $ ~ ~ ~ 2 PCT/USgl/02597 30 minutes at 22C. The assay is performed at 22C and initiated by addition of a mixture which contains Mg(Ac)2 ~60 mmol), NaV03 (40 ~M), [~-32P~ATP (125 ~M, 3-5 ~Ci/assay), and poly(GT) [poly(Glu4Tyr)] at three concentrations: whenever an inhibitor is tested, it is added at the proper concentration.
The final concentration of insulin in the assay is 125 nM.
The total volume of the assay is 40 ~l. After 20 minutes, aliquots of 30 ~l are applied on Whatman 3-mm paper and soaked in cold 10% TCA, containing O.Ol M sodium pyrophosphate.
After being washed overnight, the papers are dried and counted, measuring Cerenkov radiation. The InsRk-catalyzed phosphorylation of poly(GT) obeys Michaelis-Menten kinetics.

Inhibition of EGFR Autophos~horylation A43l cells were grown to confluence on human fibronectin coated tissue culture dishes. After washing 2 times with ice-cold PBS, cells were lysed by the addition of 500 ~l/dish of lysis buffer (50 mmol Hepes, p~ 7.5, 150 mmol NaCl, 1.5 mmol Mg Cl2, l mmol EGTA, lO~ glycerol, 1% triton X-lO0, l mmol PMSF, l mg/ml aprotinin, l mg/ml leupeptin) and incubating 5 minutes at 4C. After EGF stimulation (500 ~g/ml lO minutes at 37C) immunoprecipitation was performed with anti EGF-R (Ab 108) and the autophosphorylation reaction (50 ~l aliquots, 3 ~Ci t~-32P]ATP) sample was carried out in the presence of 2 or lO ~M of compound, for 2 minutes at 4 C. The reaction was stopped by adding hot electrophoresis sample buffer. SDS-PAGE
analysis (7.5% els) was followed by autoradiography and the reaction was quantitated by densitometry scanning of the x-ray films. In order to test the compounds for selective inhibition, the procedure is repeated using PDGF stimulation in place of EGF stimulation. rhe results of this test are summarized in Table II below.

Table II
IC50 (~M) Example EGF PDGF
8 l 20 WO91/1630S 2 ~ 8 ~ ~ ~ 2 PCT/US91/0259-~

The results of this assay show that the compounds of the present inv~ntion inhibit EGF receptor kinase better than they inhibit PDGF receptor Xinase.

Inhibition of Cell Proliferation as Measured by Inhibition of DNA Synthesls~
Cells were seeded at 1 x 105 cells per well in 24-well Costar dishes pre-coated with human fibronectin (by incubating for 30 minutes at room temperature with 10 ~g/0.5 ml/well).
The cells were grown to confluence for 2 days. The medium was changed to DMEM containing 0.5 calf serum for 36-48 hours and the cells were then incubated with EGF (Toyobo, New York, NY) (20 ng/ml) or serum (10% calf serum; and di~Leren~
concentrations of the inhibitory compounds. [3H]thymidine, (NEN, Boston, MA) was added 16-24 hours later at 0.5~Ci/ml for 2 hours. TCA precipitable material was quantitated by scintillation counting fC~.

Cell Culture Cells termed HER 14 and K721A (=DK) were prepared by trans~ecting NlH3T3 cells (clone 2.2) (From C. Fryling, NCI, NIH), which lack endogenous EGF-receptors, with cDNA
constructs of wild-type EGF-receptor or mutant EGF-receptor lacking tyrosine kinase activity (in which Lys 721 at the ATP-binding site was replaced by an Ala residue, respectively).
All cells were grown in ~EM with 10% calf serum (Hyclone, Loganl Utah).

The results obtained by the above experimental methods evidence the useful protein tyrosine kinase inhibition properties of the compounds within the scope of the present invention.

Claims

We claim:

1. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a heteroarylethenediyl or a heteroarylethenediyl aryl compound, or a pharmaceutically acceptable salt thereof, wherein the heteroaryl group can be mono- or bicyclic heteroaryl and the aryl group can be or mono- or bicyclic heteroaryl or bi- or tricyclic carbocyclic, said compound optionally substituted or polysubstituted, with the proviso that the heteroaryl group is not furyl or thienyl when the ethenediyl group has geminal cyano substituents.

2. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a heteroarylethenediyl or a heteroarylethenediyl aryl compound, or a pharmaceutically acceptable salt thereof, wherein the heteroaryl group can be mono- or bicyclic heteroaryl and the aryl group can be or mono- or bicyclic heteroaryl or bi- or tricyclic carbocyclic, said compound optionally substituted or polysubstituted, with the proviso that the heteroaryl group is not furyl or thienyl when the ethenediyl group has geminal cyano substituents.

3. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 2.

4. A compound of the formula wherein:

W is a heteroaryl ring system having an about 5 to about 7-membered monocyclic ring including 1 or 2 N, O or S
atoms, or an about 8- to about 12-membered bicyclic ring including 1 to about 4 N, O or S atoms, said ring system optionally substituted with one to about three R4 groups;

R1 is alkyl, -CN, -CONRR, -CSNRR or -COOR;

R is hydrogen, alkyl or aralkyl;

R2 is -W, -CN, -CONHR5, -CONRR, -COOR, -CSNRR, -CH=C(CN) 2, -C(NH2)=C(CN)2, , , or an about 9- to about 11-membered bicyclic aryl carbocyclic riny system or an about 12- to about 15-membered tricyclic aryl carbocyclic ring system, said ring system optionally substituted with one to about three R4 groups;

R3 is hydrogen, alkyl, -CN, -CH2CN, -CONRR, -CSNRR, -COOR or -CH=C(CN)CONH2;

each R4 is independently alkyl, hydroxy, alkoxy, halo, amino, mono- and di-alkylamino, acetylamino, alkylthio, -CN, -CF3, nitro, -COOR, -CONRR, -CSNRR, -CHO, -CH=CHCOOH, -NHCO(CH2)2COOH or morpholino;

R5 is amino, -CONH2, or ;

each R6 is independently hydrogen, alkyl, hydroxy, alkoxy or halo;

each R7 is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;

n is 0 to about 6; and m is 1 to about 7;

with the proviso that when both R1 and R2 are -CN, W is not furyl or thienyl.

5. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 4, or a pharmaceutically acceptable salt thereof.

WO 91/16305 PCT/US91/025?

6. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 5.

7. A compound according to Claim 4 wherein:

W is a 5- or 6-membered monocyclic aryl ring including 1 or 2 N, O or S atoms, or a 9- or 10-membered bicyclic aryl ring including 1 to 4 N, O or S atoms, said ring optionally substituted with one to about three R4 groups;

R1 is -CN, -CONRR, -CSNRR or -COOR;

R is hydrogen, alkyl or aralkyl;

R2 is -W, -CN, -CONHR5, -CONRR, -COOR, -CSNRR or ;

R3 is hydrogen;

each R4 is independently alkyl, hydroxy, alkoxy or halo;

R5 is amino, -CONH2, or ;

each R6 is independently hydrogen or alkyl;

each R7 is independently alkyl, hydroxy, alkoxy, halo, nitro, carboxy or carbalkoxy;

n is 0-4; and m is 2-6.

8. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 7, or a pharmaceutically acceptable salt thereof.

9. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 8.

10. A compound according to Claim 7 wherein W is furyl, pyrrolyl, thienyl, thiazolyl, pyridyl, imidazolyl, isoimidazolyl, pyridazinyl, pyrimdinyl, benzofuranyl, indolyl, indolinyl, indolinonyl, benzothienyl, benzothiazolyl, quinolinyl, isoquinolinyl, chromenyl, 1,3-benzodioxolyl or 2,3-dihydro-1,4-benzodioxinyl.

11. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 10, or a pharmaceutically acceptable salt thereof.

WO 91/16305 PCT/US91/025?

12. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 11.

13. A compound according to Claim 10 wherein:

W is pyridyl, indolyl, imidazolyl, benzothiazolyl, 1,3- benzodioxolyl or 2,3-dihydro-1,4-benzodioxinyl; and R5 is or .

14. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 13, or a pharmaceutically acceptable salt thereof.

15. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 14.

16. A compound according to Claim 13 wherein:

each R4 is independently hydroxy, lower alkyl, lower alkoxy or halo; and each R7 is independently lower alkyl, halo, alkoxy, hydroxy, nitro, carboxy or carbalkoxy.

17. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 16, or a pharmaceutically acceptable salt thereof.

18. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 17.

19. A compound according to Claim 16 wherein said compound is 2-(4-pyridyl)-3-(9-phenanthryl)-2-propenenitrile.

20. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 19, or a pharmaceutically acceptable salt thereof.

21. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 20.

WO 91/16305 PCT/US91/025?

22. A compound according to Claim 16 wherein said compound is 2-(3,4-dihydroxybenzoyl)-3-(5-indolyl)-2-propenenitrile.

23. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 22, or a pharmaceutically acceptable salt thereof.

24. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 23.

25. A compound according to Claim 16 wherein said compound is 2-(3,4-dihydroxybenzoyl)-3-(3-indolyl)-2-propenenitrile.

26. A pharmaceutical composition for inhibiting cell proliferation in a patient suffering from such disorder comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a compound according to Claim 25, or a pharmaceutically acceptable salt thereof.

27. A method of inhibiting cell proliferation in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 26.

28. A method for the treatment of psoriasis in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 2.

29. A method for the treatment of psoriasis is a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 5.

30. A method for the treatment of atherosclerosis in a patient suffering from such disorder comprising the administration to the patient of a composition according to Claim 2.

31. A method for the treatment of atherosclerosis in a patient suffering from such disorder comprising the administration to the patient of a composition according to

Claim 5.