CA2265630A1 - Use of quinazoline derivatives for the manufacture of a medicament in the treatment of hyperproliferative skin disorders - Google Patents

Abstract

This invention is directed to methods and compositions for treating hyperproliferative skin disorders utilizing a quinazoline derivative as an active ingredient.

Description

USE OF QUTNAZOLINE DERIVATIVES FOR THE MANUFACTURE OF A MEDICAMENT IN THE TREATMENT OFCA 02265630 l999-03- 12W0 98/ 10767. HYPERPROLIFERATIVE SKIN DISORDERS10152025F. 11 E 1 I V .The present invention relates to methods,compositions and formulations for treating skin diseasesand disorders characterized by keratinocytehyperproliferation. In particular, the present inventionrelates to formulations of quinazoline compounds andprocesses for the manufacture of quinazoline derivatives.k f e nv ' nApproximately four and one half million people in theU.S. are afflicted with psoriasis. Psoriasis is a skindisease often confined to localized areas of skin. It istypified by dry, scaly skin, abnormal thickening ofepidermis, and rapid cell turnover in the skin. Psoriasiscan be exacerbated by external factors including sunexposure,blockerviral infections, and corticosteroid or beta-use. Histologically, it is characterized byabnormalities including keratinocyte hyperplasia, abnormaldifferentiation sequenceaffectedtheEsgziaaig: CellEdinburgh, 1983), ,of keratinocytes inepidermis, and accumulation of leukocytes withinEds.,(Churchill Livingstone,et al.,epidermis (Wright and Camplejohn, .pp. 147-295;85:579, 1985).OtherWeinstein,skin disorders characterized by cellseborrheicskinhyperproliferation include actinic keratoses,keratoses and skin cancers such as basal cell carcinoma.Infection with papilloma viruses also causes skincell hyperproliferation (Zur Hausen, int, gev, Exp, E§th.,25:307—326 (1983); Pfister, Rev. Phvs. Biochem.SUBSTITUTE SIIEET (TIME 28)PCT /US97I 161451015202530CA 02265630 l999-03- 12W0 98/ 107672P ma , 99:lll-181 (1984).certain papilloma viruses has been strongly linked to aInfection of cervix bymajority of cervical cancers, the second largest cause ofQQQQELIQncggeng, l:25l—256Cells—DNA Tumor1986).Keratinocyte hyperplasia in psoriasis is linked tocancer deaths in women worldwide4l:l84—187 (1988);(1987); BrokerViruses, New York: Cold Spring Harbor Laboratory,(Parkin, et al.,Durst, et al.,and Botchan, Canceroverproduction of cytokines such as TGFd and interleukin—6(IL-6) and overexpression of epidermal growth factorreceptor (EGF—R) in affected skin (Krueger, et al., Q;Invest. DgrmatQl., 94:l355-1405, 1990). EGF—R is a 180-kDcell—surface receptor whose activity is regulated by bothEGF and TGFd. In psoriasis vulgaris, EGF—R persiststhroughout the epidermis from the basal layers to thestratum corneum. Such persistent EGF—R has been shown tobe biologically active in vivo in nude mice98:296-301, 1992).Suggested treatment for psoriasisof(Nanney, etal., nv . D ,includes directofJ. gellMany topical products currentlyinhibition keratinocyte growth and inhibitionactivated lymphocyte proliferationBigl. ll3:857—865, 1991).available are irritating,(Dvir et al.,messy or simply ineffective.Topical steroids account for 90% of the psoriasis marketin the United States and have many side effects includingformation of striae andcutaneous atrophy, telangiectasia,tachyphylaxis. Epidermal hyperproliferation and angiogenesishallmarksinvention it has been found that quinazoline derivativesareof psoriasis. In the scope of the presentsuch as 4-(3—Bromophenylamino)—6,7—dimethoxyquinazoline,4-(3—Chlorophenylamino)-6,7-dimethoxyquinazoline,SIIBSTITIHE SHEET (fllllf 28)PC17US97fl6l451015202530CA 02265630 l999-03- 12W0 98/ 1076734-(3-Chlorophenylamino)—6—methylquinazoline,4- [3- (tri.flu<)rornetl1yl) phe3nyl.ami_no] -6 , 7 — d i m e t h o x y q u i n a 2 o l i n e ,4-(3-Cyanophenylamino)-6,7—dimethoxyquinazoline,4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline(identified as Al, A2, A3, A4, A5, and A6 in Table 1)the of inhibitinghaveEGF—Rtyrosine kinase activity and skin cell hyperproliferation.properties or reducing theThe administration of an effective amount of an abovementioned compound to a patient suffering from psoriasisor other hyperproliferative skin diseases will be able toinhibit the abnormal proliferation of skin cells, anddecrease cornification, scaling or uneven thickness andother undesirable symptoms of psoriasis. The presentinvention is based on the aforementioned finding and isaccordingly concerned with the novel use of the compoundsin the treatment or prevention of skin diseases anddisorders characterized by hyperproliferation.Therefore, the present invention relates to methodsand compositions for the treatment or prevention ofhyperproliferative skin disorders, including, but notlimited to, psoriasis (e.g. psoriasis vulgaris, psoriasispustulosa, psoriasis erythrodermica, psoriasisarthropathica, parapsoriasis, palmoplantar pustulosis) andskin cancer. In preferred embodiments, a host (e.g. amammal or human) is administered a composition containinga pharmaceutically effective amount of a quinazolinederivative of Formula V described in Figure 4e,Al, A2, A3, A4, A5, A6,salt thereof. The compositions of this invention cure,such asor a pharmaceutically acceptablereduce or prevent keratinocyte hyperproliferation or skinlesions in the host. A preferred drug is highly potentand selective with low toxicity.SIIBSTITIHE SIIEH NILE 23)PCTIUS97/161451015202530CA 02265630 l999-03- 12W0 98/10767 PCT/US97/ 161454In addition to psoriasis, other diseases orpathological conditions characterized byhyperproliferation and/or EGF—Roverexpression andhyperactivity can be treated with the above—describedcompositions. These diseases and pathological conditionsinclude, but are not limited to, keratinocyteproliferation and skin lesions caused or induced byPapilloma virus infection, seborrheic keratoses,acanthosis nigricans, ichthyosis (e.g. ichthyosis vulgarisand congenital ichthyoses), keratodermias, genodermatoseswith pathological cornification disorders (e.g. Darier’sdisease), further lichen ruber planus, pityriasis rubrapilaris, and skin cancers such as basal cell carcinoma,squamous cell carcinoma and melanoma.By “pharmaceutically effective” is meant the abilityto cure, reduce or prevent one or more clinical symptomsof keratinocyte hyperproliferation, including, but notlimited to, cornification, scaling, uneven thickness,inflammation, and rapid cell turnover in the skin.The composition containing a pharmaceuticallyeffective ingredient may be administered topically orsystemically. In a preferred embodiment, it isadministered topically to an affected skin area.In a preferred embodiment, A1 or its pharmaceuticallyacceptable salts are used in the composition. Such acomposition is especially suitable for topical treatmentvis—a—vissystemic treatment of skin conditions because-ofthe following properties of Al: (1) low toxicity, (2)short plasma half-life, and (3) relative high solubilityin nonirritant solvents among anticancer compounds. Lowtoxicity is a desirable feature for a drug used to treata non-life threatening disease. Short plasma half—lifehelps to keep the drug's therapeutic effects localized toFormulations withSIIBSTITIITESIIET (BIILE 23)where it is topically applied.1015202530W0 98/ 10767CA 02265630 l999-03- l2PCTIUS97l16l455nonirritant solvents alleviate the suffering of patientswho have to use the drug repeatedly to receive the desiredtherapeutic effects.In another aspect, this invention features apharmaceutical composition for the treatment of ahyperproliferative skin disorder containing apharmaceutically effective amount of a compound selectedfrom the group consisting of A1, A2, A3, A4, A5, A6 andtheir pharmaceutically acceptable salts; and apharmaceutically acceptable carrier. In a preferredembodiment, the pharmaceutical composition is for topicalapplication to a host. In another preferred embodiment,the compound is selected from the group consisting of A1and its pharmaceutically acceptable salts.The composition may be in a unit dosage form or amultiple use dosage form. In a preferred embodiment, thecomposition is held within a container which includes alabel stating to the effect that the composition isapproved by the FDA in the United States (or an equivalentregulatory agency in a foreign country) for treating ahyperproliferative skin disorder such as psoriasis (e.g.psoriasis vulgaris, psoriasis pustulosa, psoriasiserythrodermica, psoriasis arthropathica, parapsoriasis,palmoplantar pustulosis), skin lesions caused by Papillomavirus infection, seborrheic keratoses, acanthosisnigricans, ichthyosis (e.g. ichthyosis vulgaris andcongenital ichthyoses), keratodermias, genodermatoses withpathological cornification disorders (e.g. Darier’sdisease), further lichen ruber planus, pityriasis rubrapilaris, or a skin cancer such as basal cell carcinoma,squamous cell carcinoma or melanoma. Such a containerprovides a therapeutically effective amount of the activeingredient to be administered to a host.SIIBSTITIIT E SHEET (fllllf 26)1015202530WO 98/10767CA 02265630 l999-03- l2PCT/US97/161456In yet another aspect, this invention features amethod of making a composition for the treatment of ahyperproliferative skindisorder by providing apharmaceutically effective amount of a compound selectedfrom the group consisting of Al, A2, A3, A4, A5, A6 andtheir pharmaceutically acceptable salts, and admixing the.compound with a pharmaceuticallyacceptable carrier.Preferably, the composition is further packaged into acontainer in a unit dosage or a multiple use dosage.This invention also features a method of preparing acompound for treating a hyperproliferative skin disorderby providing a plurality of compounds of Formula V,testing these compounds’ ability to specifically inhibitEGF—R tyrosine kinase activity using the assays deseribedand disclosed in this application (such as those inExamples 1-8), and selecting those with activity in therange of Al, A2, A3, A4, A5, and A6. A pharmaceuticallyeffective amount of such compounds is further packaged ina container with a pharmaceutically acceptable carrier.Other features and advantages of the invention willbe apparent from the following detailed description of theinvention, and from the claims.Brief Description of the DrawingsFigures 1(a), 1(b), l(C). l(d); 2(3); 2(b), 2(C) and2(d) are general formulae of compounds that can beprepared with a process of this invention. C1 in Figuresl(b) and 2(b) may be replaced by another displaceablegroup which includes, but is not limited to, halogen,alkoxy, aryloxy and sulphonyloxy groups. Preferably, thedisplaceable group is chloro, bromo, nmthoxy, phenoxy,methanesulphonyloxy or toluene—p—sulphonyloxy.Figures 3a, 3b and 3c are flow charts showing thegeneral process for preparing compounds of the formulae inSIIBSTIIIJTE SHEET (IIIILE 28)1015202530CA 02265630 l999-03- 12W0 98/ 107677Figures 1 and 2. Ethanol is used as solvent only as anexample.Figure 4a shows the structure of a compound offormula I.Figure 4b shows the structure of a compound offormula II.Figure 4c shows the structure of a compound offormula III.Figure 4d shows the structure of a compound offormula IV.Figure 4e shows the structure of a compound offormula V.D n e mI ui v iv f ’ Inv n ' t —‘c e t’ H ' a ‘v:;Jsin__DJ..a=&.sssThis invention concerns treating or preventingpsoriasis and other conditions characterized bykeratinocyte hyperproliferation by inhibiting or reducingEGF—R activity with quinazoline derivatives.with(PLC)Psoriatic epidermis is associated alteredprotein tyrosine kinase, phospholipase C(PKC)and proteinkinase C activities, all of which are changed bychronic EGF—R activation. Growth factors, cytokines andtheir receptors are involved with the disease process ofpsoriasis (Krueger, et al., gé_;ggg§;;_Qg;matgl;, 94:l355—1405, 1990). Among them, EGF—R plays a crucial role inthe hyperproliferation of keratinocytes in psoriaticlesions.EGF—R is overexpressed ill psoriatic keratinocytes(Nanney, et al., £i_lnM§§Li_D§Lm§LQli, 85=260'255, 1986).The normal basilar distribution of EGF receptors inepidermal keratinocytes is markedly altered in psoriasisSIJBSTITIIT E SIIET (RIILE 28)PCT/US97/161451015202530CA 02265630 l999-03- 12W0 98/10757 PCT/US97/161458vulgaris where they are also observed in the upperkeratinocyte compartment (Nanney et al., Q, invest.Qermatgl. 83:385—393, 1984; Green et al., Q. Invest.Dermatgl. 85:239—245, 1985). In a study on benignepidermal dermatoses, EGF receptor expression throughoutthe epidermis returned to a basal layer distribution whenthe lesion resolved (Ellis317:l582-1587, 1987).et al., N. Enal. J. Med.Psoriatic keratinocytes persistently secrete TGFQ due(Gottlieb,et al.,to the overexpression of the TGFd geneJ. . Med., l67:670—675, 1988; Elder,243:8l1-814, 1989; et al.,5:714-727, 1991).autocrine stimulation of the keratinocyte EGF—R (Grossman,et al., Prgg, uatl Agad §ci gsa, 86:6367—6371, 1989).IGF—1 receptor is also overexpressed in the psoriaticet al., J. Inv . l., 96:419-et al., QL_flxg__MegL, 175:l08l—1090,Activation by IGF—1 stimulates the synthesis ofet al.,Scien e,Vassar, Genes and Develon.,TGFd overexpression causes enhancedepidermis424, 1991;1992).EGF-R and thus amplifies the effect of the EGF-R/TGFQThe that(Krane,Krane,autocrine loop. finding transgenic miceoverexpressing TGFd in their skin develop lesionset al., §§Q§§_§QQ_Q§1§lQQ;, 5:714-727, 1991)crucial role of EGF-R in the pathogenesis of the disease.An EGF-R(Vassar,supports thethree functionalhas domains: anextracellular ligand binding domain, a transmembranedomain and a cytoplasmic domain capable of phosphorylatingtyrosine residues. Ligand binding to the extracellularligand binding domain of membrane-bound EGF—R induces theformation of receptor dimers and allosteric changes thatactivate the intracellular kinase domain to result in thephosphorylation (autophosphorylation and/or transphosphor—ylation) residues. Theof tyrosine phospho—tyrosineresidues of the cytoplasmic domains of EGF—R then interactSIIBSTITIITE SIIEET (HIM 26)1015202530W0 98/ 10767CA 02265630 l999-03- l29with a host of cytoplasmic signaling molecules to activatesignal transduction pathwaysgall 6l:203-212, 1990).cells, the tyrosine kinase activity of EGF-R results in an(Ullrich and Schlessinger,During mitogenic activation ofincreased activity of phospholipase C (PLC). Theactivation of EGF—R also results in the activation ofprotein kinase C (PKC),increased calciunl uptake, andhydrolysis of membrane lipids to yield diacylglycerols(DAG) (IP3).In psoriasis, hyperproliferation of keratinocytes is(Elder,and inositol-triphosphatedriven mainly by EGF-R and its ligands et al.,figigngg, 243:670-675, 1989). Some EGF—R blockers arrestthe growth of psoriatic keratinocytes and are consideredfor clinical use (Ben-Bassat, et al., Egper, Qermatgl,,4(2):82-84, 1995). Tyrosine kinase blockers of thetyrphostin family have been shown to block theproliferation of psoriatic keratinocytes grown in culture(Dvir, et al., Q+_§ell_§igl;, 113:857-865, 1991; Ben-Bassat, et al., §z;h___Qg;ma;g;+, 4:82-88, 1995).Tyrphostins inhibit EGF-R autophosphorylation andEGF-dependent tyrosine phosphorylation of intracellulartarget proteins in keratinocytes.Skin cancers have also been associated with theexpression of EGF-R ligands and anti-EGF-R antibodies havebeen shown to inhibit the growth of a skin cancer cellline expressing EGF—R. (See Hagedorn, M. and T.Baukrecht, ;*_flau;kr 65(6):575—577, 1990; Kawamoto, T., etal. J. aigl. ghem. 259(l2):776l—7766, 1984).The present application shows that quinazolinederivatives of this invention,able toi.e., Al, A2, A3, A4, A5and A6, are inhibit EGF—stimulated EGF—Rphosphorylation. These compounds also are capable ofinhibiting EGF—mediated skin cell growth in vitro andpsoriatic skin cell proliferation in vitro.SIIBSTHIHE SIIEH (HIM 23)Specifically,PCTIUS97/161451015202530W0 98/ 10767CA 02265630 l999-03- l2PCTIUS97ll614510Al potently inhibited ligand—inducedautophosphorylation of the EGF receptor, and downstreamsignal transduction events, including DNA replication andcell cycle progression. The compound is specific for theEGF receptor, since it displayed little or no activityagainst unrelated receptor tyrosine kinases such as thereceptors for platelet~derived growth factor orinsulin-like growth factor—l. Al was shown to block EGFreceptor—dependent growth of tumor cells and fibroblastsengineered to overexpress EGF receptor. At micromolarconcentrations, Al was shown to inhibit the proliferationof keratinocytes isolated from psoriatic lesions. In skinpenetration studies, radiolabelled compound penetratedhuman cadaverskin, reaching biologically effectiveconcentrations in the epidermis within a 24-hour period.' f C mThe chemical structures of Al, A2, A3, A4, A6 and A5II. ' i ' n rare shown below in Table 1. Methods for preparing thesePatent 5,457,105 issuedOctober lO, 1995 and PCT publication WO 95/03283 publishedcompounds can be found in U.S.February 2, 1995, the totality of which is incorporated byreference herein.In an example, Al was prepared essentially asdescribed in Barker, AJ. European Patent Application 0 566226 Al, October 20, 1993; Med.Qhgm‘ 4:l203—l207,2-amino—4,5—dimethoxybenzoate was treated with formamideat 180°C.and Gazit et al., i1996. Briefly, methylThe reaction was cooled and diluted with water.The precipitate was collected by filtration, washed withwater and dried to give 6,7—dimethoXyquinazolone, whichwas treated with thionyl chloride and dimethylformamide atconcentrated, and stirred with sodium bicarbonateTheSIIBST IT IITE SIIEH (HIILE 28)reflux,solution. resulting solid was collected and1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97I16l45llcrystallized from hexane to give4-chloro—6,7—dimethoxyquinazoline, which was refluxed with3—bromoaniline in ethanol, cooled, treated with 1 normalNaOH and the resulting solid collected by filtration toyield(A1).4-(3—bromophenylamino)-6,7—dimethoxyquinazolineII. The above identified compounds may be used to treatskin diseases or pathological conditions in mammals,especially in humans.The hyperproliferative diseases which cause abnormalscaling and cornification of the skin include all forms ofpsoriasis, e.g. psoriasis vulgaris, psoriasis pustulosa,psoriasis erythrodermica, psoriasis arthropathica,parapsoriasis, palmoplantar pustulosis, all forms ofichthyoses, e.g. ichthyosis vulgaris and congenitalichthyoses, keratodermias of all types, e.g., palmoplantarkeratodermia, other genodermatoses with pathologicalcornification disorders, e.g. Darier’s disease, furtherlichen ruber planus and pityriasis rubra pilaris.In addition to psoriasis, other diseases orpathological conditions characterized byhyperproliferation and/or EGF—R overexpression andhyperactivity can be treated with the above-describedcompositions. These diseases and pathological conditionsinclude, but are not limited to, keratinocyteproliferation and skin lesions caused or induced byPapilloma virus infection, seborrheic keratoses,acanthosis nigricans, ichthyosis (e.g. ichthyosis vulgarisand congenital ichthyoses), keratodermias, genodermatoseswith pathological cornification disorders (e.g. Darier’sdisease), further lichen ruber planus, pityriasis rubraSIIBSTITIITE SIIEH (lllllf 25)1015202530W0 98/10767CA 02265630 l999-03- 1212pilaris, and skin cancers such as basal cell carcinoma,squamous cell carcinoma and melanoma.V. Ioxicitv and Efficacv of Ouinazoline CompoundsToxicity and therapeutic efficacy of the aboveidentified compounds can be determined by standardpharmaceutical procedures in cell cultures or experimentalanimals. A measure of the effectiveness and cell toxicitythethe dose required toof a compound can be obtained by determiningLD5o/IC50. IQ0 ,achieve 50% EGF-R tyrosine kinase activity inhibition, cantherapeutic index:be measured using standard techniques thoseof thepopulation, can be measured by standard techniques, suchJ. Immggngl .by measuring the amount of LDHsuch asdescribed herein. LDw, the dose lethal to 50%as using an MTT assay as described by Mossman,Methods 65:55-63 (1983),released as described by Korzeniewski and Callewaert, Q‘I u eth 64:3l3 (l983)and Decker andLohmann-Matthes, J, Immunol. Methods 1l5:6l (1988), or bymeasuring the lethal dose in animal models. Anothermeasure is the ratio LDW/EDw. §D is the dosetherapeutically effective in 50% of the population.Compounds which exhibit large therapeutic indices arepreferred. The therapeutic index should be greater than2, preferably at least 10, more preferably at least 50.For any compound used in the method of the invention,the therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dosecan be formulated in animal models to achieve acirculating plasma concentration range that includes theICW as determined in cell culture.Plasma half-life and biodistribution of the drug andmetabolites in plasma and major organs can be determinedto facilitate the selection of drugs most appropriate forSUBSTITUTE SIIEET (|||llE 28)PCT/US97ll6l451015202530W0 98/ 10767CA 02265630 l999-03- l2PCTIUS97/1614513the inhibition of a disorder. Such measurements can becarried out, for example, using HPLC analysis. Compoundsthat show potent inhibitory activity in the screeningassays, but have poor pharmacokinetic characteristics, canbe optimized by altering their chemical structure andretesting. In this regard, compounds displaying goodpharmacokinetic characteristics can be used as models forthe synthesis of other compounds.Toxicity studies can also be carried out by measuringthe blood cell composition. For example, toxicity studiescan be carried out as follows: (1) the compound isadministered to mice (an untreated control mouse shouldalso be used); (2) blood samples are periodically obtainedvia the tail vein from one mouse in each treatment group;and 3) the samples are analyzed for red and white bloodcells for indication of toxicity.The data obtained from these cell culture assays andanimal studies can in; used ix) formulating a range ofdosage for use in humans. The dosage of such compoundslies preferably within a range of circulatingconcentrations that include the EDW with little or notoxicity. The dosage may vary within this range dependingupon the dosage form employed and the route ofadministration utilized. ggLL;_3ng_ggl;_§ultu;e. All tumor cell lines wereobtained from the American Type Culture Collection(Rockville, MD), unless otherwise specified. NIH3T3 mousefibroblasts overexpressing the EGF receptor, IGF—lreceptor, insulin receptor, PDGF—b receptor, or a chimeraof the EGF receptor extracellular domain fused to theHer-2 cytoplasmic domain were engineered using retroviralThese cells will be referred to as 3T3—EGFR,SW81 ITIITE SIIEET (Bl|lE 28)vectors.1015202530CA 02265630 l999-03- 12WO 98/10767 PC'I‘lUS97Il6145143T3—IGFlR, 3T3-IR, 3T3-PDGF-bR, and 3T3-EGFR/Her-2,respectively. All cell culture media and supplements werepurchased from Gibco Life Technologies (Grand Island, NY)unless otherwise specified. All cells were grown in ahumid atmosphere of 90-95% air and 5—lO% CO2 at 37°C, Celllines were maintained under standard conditions in culturemedia recommended by the ATCC, unless otherwise specified.3T3-EGFR, 3T3-PDGF—bR, and 3T3-EGFR/Her—2maintained in DMEM containing 10% calf serum (CS) and 2 mMGLN.cells were3T3—IGFlR and 3T3-IR cells were maintained in DMEMcontaining 10% FBS and 2 nmd GLN. Keratinocytes wereobtained from small biopsy specimens of split—thicknessskin from patients with psoriasis and from healthy controldonors. The biopsy specimens were treated to yield apopulation of cells enriched for keratinocytes, which weremaintained in keratinocyte growth mediuni as described(Ben-Bassat et al., §gp;_Qe;maLglé 4:82-88, 1995).In Vitro functional studies. The effect of Al onreceptor phosphorylation, DNA replication, cell cycleprogression, and cell growth was studied. Cell linesexamined in receptor phosphorylation ELISA were 3T3—EGFR,3T3—EGFR/Her-2, 3T3-PDGF-bR, 3T3—IGFlR, and 3T3-IR. Foranalysis of DNA replication (measured as incorporation ofBrdU) and cell cycle progression, 3T3—EGFR and 3T3—PDGF—bRcell lines were used. Cell growth studies were carriedout on A431 (EGF—R+), BT474 (Her-2+), and C6 (PDGF—bR+)cell lines. The effect of Al on the growth of humanpsoriatic keratinocytes was determined as previouslyExp. Qermatgl. 4:82-88,described (Ben—Bassat et al.,1995).§g;Q_£ggg;;a;;gg. In vitro skin penetration studieswere conducted using human cadaver skin. The vehicleconsisted of aSIIBSTITIITE SIIEET (MILE 29)formulation petrolatum based topical1015202530W0 98/ 10767CA 02265630 l999-03- 1215ointment containing 5.0% mineral oil, 3.0%1.5% benzyl alcohol and 2.5% oleic acid.(0.5%, 1.0%, 2.0%,were evaluated during each study. Theglycerylmonostearate,Six replicates of each Al concentrationand 4.0% drug)formulations were spiked with radiolabeled ”C—Al to obtaina specific radioactivity of 25 uci/g. Radiolabeled “C—Alwas provided by SynPep Corp, Alameda, California5.89(specific#O20196CLO01). Theformulation was applied (16.9 mg/cu?) to human cadaver skin(1.77 cm2activity mCi/mmol, lotsurface area and approximately 200 um splitthickness)mounted on a Franz static dffusion chamber. Thechambers were filled with 4% BSA isotonic saline solution(6-10 mLtemperaturevolume) andof 37°C by areservoir equilibrated to acirculating pump. Percentpenetration through the skin was determined by measuringcumulative radioactivity in the reservoir medium at theend of 24 hours or at regular intervals during the 24 hourperiod. Tissue distribution of the drug in the stratumcorneum, epidermis and dermis was determined at the end of24 hours. The stratunx corneun1 wasseparated by tapestripping with cellophane tape until “glistening”. Thedermis and epidermis were separated by microwave techniqueas described (Kumar et al., 2harma;§uLigal_Be§l 6:740-741,1989; Bronaugh, “Preparation of biological membranes”, In: , pp 61-66- Ed-Kempanien and William G. Reifenrath, CRC Press,Barbara1990).Example 1:E . .Specific Inhibition of EGF—R Tvrosine KinaseE§E_R flhgle gel] Kjnafig AfigaxNIH3T3 clone C7 engineered to over—express human EGF—R and the human glioblastoma line U1242 that expressesPDGFR—beta were used for cellular kinase assays.SIIBSTITIITE SIIEH (NILE 28)PCT /US97ll6l4510152025W0 98/ 10767CA 02265630 l999-03- 1216The activity of the above identified compounds ininhibiting EGF—stimulated EGF-R phosphorylation wasmeasured in an ELISA assay.EGF—R kinase activity (EGF-R-3T3 assay)cells was measured as described below:in wholeA. Coat ELISA plates (Corning, 96 well, Cat. #25805—96)with 05-101 antibody at 0.5 pg per well in PBS, 150 Mlfinal volume/well, and store overnight at 4"C. Coatedplates are good for up to 10 days when stored at 4"C.On day of use, remove coating buffer and replace withblocking buffer (5% Carnation Instant NonFat Dry Milk inPBS).(about 23“C to 25"C)remove blocking buffer and wash plate 4 times with TBSTbuffer.Incubate the plate, shaking, at room temperaturefor 30 minutes. Just prior to use,B. Seeding CellsEGF-R/C7 cell line was used for this assay.Choose dishes having 80-90% confluence for theexperiment. Trypsinize cells and stop reaction by adding10% CS DMEM medium. Suspend cells in DMEM medium (10%calf serum (CS) DMEM medium)rpm, and once at room temperature for 5 minutes.and centrifuge once at 1000Resuspend cells in seeding medium (DMEM, 0.5% bovineserum), and count the cells using trypan blue. ViabilitySeed cells in DMEM medium (0.5%bovine serum) at a density of 10,000 cells per well, 100above 90% is acceptable.Ml per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO2at 37“C for about 40 hours.SIIBSTIWTE SHEET (ME 26)PCT/US97/1614551015202530CA 02265630 l999-03- 12W0 98/1076717C. Check seeded cells for contamination using an(10 mg/ml in DMSO)then transfer 5 pl to a test well for1:200 DMSOControl wells receive DMSO alone.Incubate in 5% CO2at 37‘C for one hour.inverted microscope.1:10 in DMEM medium,Dilute drug stocka final drug dilution ofand a finalconcentration of 1%.Prepare EGF ligand: dilute stock EGF in DMEM so thatupon transfer of 10 ul dilute EGF (1:12 dilution),25 nMfinal concentration is attained.Prepare fresh HNTG'sufficient for 100 Ml per well;and place on ice.HNTG*: 10 mlHNTG stock (5x) 2.0 mlmilli-Q H20 7.3 mlEDTA, (100 mM, pH 7.0) 0.5 mlNa3VOL (0.5 M) 0.1 mlNa,POL (0.2 M) 0.1 mlAfter two hours of incubation with a drug, addprepared EGF ligand to cells, 10 #1 per well, to yield afinal concentration of 25 nM. Control wells receive DMEMalone. Incubate and shake at IOOHI temperature for 5minutes.Remove drug, EGF, and DMEM.Transfer HNTG’to cells,Wash cells twice withPBS. 100 Ml per well.Place onice for 5 minutes. Meanwhile, remove blocking buffer fromother ELISA plate and wash with TBST as described above.With a fitted to amicropipettor, scrape cells from plate and homogenize cellpipette tip securelymaterial by repeatedly aspirating and dispensing the HNTG’lysis buffer. Transfer lysate to a coated, blocked,washed ELISA plate.andIncubate and shake atIOOITItemperature for one hour.SIIISTIWTE SIIEET (ME 28)PCT /US97ll6l451015202530CA 02265630 1999-03-12WO 98110767 PCTlUS97ll6l4518Remove lysate and wash 4 times with TBST. Transferfreshly diluted anti—phosphotyrosine (anti-Ptyr) antibodyto ELISA plate at 100 ul per well. Incubate shaking atroom temperature for 30 minutes in the presence of theanti-Ptyr antiserum (1:3000 dilution in TBST).Remove the anti-Ptyr antibody and wash 4 times withTBST. Transfer the freshly diluted TAGO 30 anti—rabbitIgG antibody (anti—rabbit IgG antibody: l:3000 dilution inTBST) to the ELISA plate at 100 #1 per well. Incubateshaking at room temperature for 30 minutes.Remove detection antibody and wash 4 times with TBST.freshly prepared ABTS/H53100 pl per well.20 minutes. ABTS/Hzozsolutionz 1.2 #1 30% H;)2in 10 ml ABTSTransfer solution to ELISAplate, Incubate at room temperature forstock.Stop reaction by adding 50 ul 5N &fiO4(optional), anddetermine O.D. at 410 nm.The maximal phosphotyrosine signal is determined bysubtracting the value of the negative controls from theThe ofphosphotyrosine content for extract—containing wells ispositive controls. percent inhibitionthen calculated after subtraction of the negativecontrols.D- MaLsLial§_i_B§£g§nL§1) EGF Ligand: stock concentration = 16.5 uM; EGF 201,TOYOBO, Co., Ltd. Japan.2) 05-101 (UBI) (a monoclonal antibody recognizing anEGF—R extracellular domain).3) Anti—Phosphotyrosine antibody (polyclonal) (madeaccording to Fendley et al., Qang§;_3g§ga;gh 50:l550—l558,1990).4) TAGO antibody: Goat anti—rabbit lgG horse radishperoxidase conjugate, TAGO, Inc., CA.SIIBSIIWIE SIIEH (ME 26)Burlingame,1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97/16145195) TBST buffer:Tris—HCl, pH 7.2, 50 nMNaCl, 150 mM,Triton X-100 0.1%6) HNTG 5X stock:HEPES 0.1 MNacl 0.75 MGlycerol 50%Triton X-100 1.0%7) ABTS stock:Citric Acid 100 rnMNa2HPO, 250 mMHCl, conc. 4.0 pHABTS‘ 0.5 mg/ml*(2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid).Keep solution in dark at 4"C until use.8) Stock reagents of:EDTA 100 mM; pH 7.0Na3VO, 0.5 MNa,PQ 0.2 MTable 2 shows the activities of the quinazolinederivatives in the cellular EGF-R kinase assay and theligand dependent cellular proliferation assay.Specifically, it shows the effectiveness of Al, A2, A3,A4, A6 and A5 in inhibiting EGF-R tyrosine kinase activityand curtailing EGF stimulated cell proliferation and theselectivity of the compounds. Table 2 shows ICSO (uM) ofthe above identified compounds. IC50 is the dose requiredto achieve 50% inhibition. In the order of decreasingactivity, these compounds are Al, A4, A2, A3, A5 and A6.To examine the effects of Al on EGF~mediated receptorautophosphorylation, NIH 3T3 cells engineered to expressEGF receptors (3T3-EGFR) were pretreated with titratedSIIBST ITIIT E SHEET (|llllE 28)1015202530CA 02265630 l999-03- 12W0 98/10767 PCT/US97l16l4520doses of Al prior to the addition of ligand. ELISAanalysis showed that EGF—stimulated receptorphosphorylation was decreased in a dose—dependent manner.The average ICW of six determinations was 13 nM.To examine the effect of Al on other receptortyrosine kinases (RTK), NIH 3T3 cells engineered tooverexpress various RTK were exposed to theircorresponding ligands after pretreatment with Al, and theextent of ligand—stimulated receptor phosphorylation wasdetermined by ELISA.As shown in Table 10, Al inhibitedreceptor phosphorylation of the EGF receptor and a closerelative, Her-2. However, other RTKs such as theplatelet-derived growth factor receptor (PDGF-bR), theinsulin-like growth factor receptor (IGF~lR) and theinsulin receptor (IR) were not inhibited at concentrationsup to 100 pM.In Table 10, NIH 3T3 cells engineered to overexpressvarious RTKs were treated with corresponding ligand for 5to 10 min following a 2 hr pretreatment with Al. Receptorphosphorylation was measured by ELISA. EGF—R/Her—2denotes a chimeric receptor consisting of theextracellular (ligand—binding) domain of EGFR, and thetransmembrane and cytoplasmic domains of Her—2.Example 2: Inhibition «of NIH 3T3 Cells OverexpressingF-R uThe efficacy of the above identified compounds ininhibiting or reducing EGF—R stimulated proliferation ismeasured by the 3T3 cell growth assay. The 3T3 growthassay was carried out as follows:EGF—RC7R) and NIH 3T3C7 cellsSUBSTITUTE SHEET (HIIEE 28)(NIH 3T3 C7 cells engineered to express EGF~(as the control) were used for this1015202530W0 98/ 10767CA 02265630 l999-03- 12PCT /US97ll614521assay. NIH3T3C7 cells were seeded at 2500 cells/well, 10Ml/well in 10% CS + 2 mM Glutamine/DMEM, in a 96 wellplate; EGF—RC7 cells were seeded at 6000 cells/well, 100ul/well in 2% FBS + 2 mM Glutamine/DMEM, in a 96 wellplate. Cells were incubated at 37°C, 5% Cozovernight toallow for cell attachment to the plate.A quinazoline compound was added to the cells at day2. The compound was prepared in the appropriate growthmedium (10% CS -+ 2 mM glutamine in DMEM for" NIH3T3C7cells; 2% FBS+2 mM Glutamine in DMEM for EGF-RC7 cells) ina 96 well plate, and serially diluted. A total of 100pl/well medium of the diluted compounds was added into thecells. The total volume of each well was 200 pl.After the cells were treated with the compound for 4days, the cells were washed with PBS and fixed with 200ul/well ice—cold 10% TCA for one hour at 0-5°C.Remove TCA and rinse wells 5 times with deionizedwater. Dry plates upside down with paper towels.cells with 0.4% SRB at 100 Ml/well for 10 minutes.StainPour off SRB and rinse plate 5 times with 1% aceticacid. Dry plate completely.Solubilize the dye with 10 mM Tris-base at 100Ml/well for 10 minutes on a shaker.Read the plate at dual wavelengths at 570 nm and 630nm on Dynatech Elisa plate reader.To determine whether the above identified compounds’inhibitory effect on EGF—R C7 cells is selective, theexperiment described above was modified, replacing EGF—RC7 cells with human glioblastoma line Ul242 that expressesPDGFR—beta.The results of the assays described in Examples 1 and2 are shown in Table 2. Example 1 demonstrates that thecompounds of the invention are highly potent inhibitors ofEGF-R enzymatic activity. Example 2 demonstrates that theSUBSTITUTE SIIEET (RIILE 26)1015202530W0 98/10767CA 02265630 l999-03- 1222compounds of the inventionare alsohighly potentinhibitors of EGF—R mediated cellular proliferation andthat they are highly selective as well. The most potentand most selective compound is Al.Materials and Reagents(1) Dulbecco's Modified Eagle Medium (D-MEM), Gibco511965-050;(2) Calf serum, Gibco 16170-O29;(3) Trypsin-EDTA, Gibco 25200-056;(4) Fetal Bovine Serum Certified, Gibco 16000-028;(5) Dulbecco' 5 Phosphate—Buffered Saline (D—PBS), 10Gibco 14190-O29;(6) Sulforhodamine B (SRB), Sigma 5-9012 0.4% SRB in 1%acetic acid;(7) 10 mM Tris-base, Fisher BP152-5;(8) 10% TCA, Trichroloacetic acid, Fisher A322—500;(9) 96-well flat bottom plate (sterile), Corning08—757—155;(10) 100 ml reagent reservoir 9 (sterile), MatrixTechnologies Corporation, 8086;(11) Sterile pipet tips, Fisher 2l—l97—8E;(12) 50 ml sterile TBST tubes, Fisher 05-539-6.E e - T icit SThe each of the theinvention was evaluated by determining the LDN and LDWtoxicity of compounds of(the dose lethal to 10% or 50% of a population)BALB/Cin mice.Briefly, female mice (5 per group)injected with a single dose of compound 1P in 50ul DMSO.wereSurvival was measured at seven days. The results arepresented in Table 3 below and demonstrated that thecompounds of the invention are relatively non-toxic.SIIBSTITIITE SIIEH (Hill! 23)PCT /US97/1614510152025CA 02265630 l999-03- 12“'0 93’1°7‘7 PCTIUS97/1614523Multiple dose toxicity studies were also conducted.Al in DMSO was administered 1P to Balb/c nude mice atdoses ranging from 5 to 30 mg/kg/day for 17, 20 or 30days. The results are shown below:Exp Dose Duration %Nbruflity1 30 17 O2 5 20 03 10 20 04 30 20 O3 30 33 25Up to 12.5% mortality can periodically be observed withprolonged treatment (33 days) with vehicle control alone.Example 4: Skin Penetration and Absorption StudiesThreetotal)formulation categories (nine formulationsconsisting of Petrolatum Ointments, EmollientCreams and Polyethylene Glycol Ointments were developed(Tables 6 and 7). Thedrug concentration was 2.0% for all formulations and time1 and 24 hours.for the skin penetration studiespoints were drawn at 0, There was animmediate turnaround of these results which indicated thatthe petrolatum Ointments and the emollient creams showedapproximately ten fold increase in penetration as comparedto the PEG ointment with the percents listed in Tables 7and 8.Plasma levels of Zn. were measured jJ1 nu/nu mace.Approximately 60 mg of a 2% (w/w/ointment was applied tothe backs of four female mice using a stainless steelspatula. The dose was spread evenly over the back surfaceof each mouse.SIIBSTITIITE SIIEET (RULE 28)1015202530W0 98/ 10767CA 02265630 l999-03- l2PCTIUS97/1614524Plasma was prepared from blood samples taken 30 or 60min. following application and(2 mice per time point)examined by HPLC..50 mg of the 2%manner .In a second experiment,(w/V)Blood samples were taken at 15 and 30 min and atl, 2.25 and 30 hours.increased grooming behaviorapproximatedointment was applied in the sameThe mice exhibited no discomfort orfor the(<2ug/ml)detected at 30 min. or later time points.duration of theexperiments. amounts of A1 wereOnly traceThe lower limitof quantitation for the HPLC assay is l.0ug/ml.Based on the above describedstudies, a(see Table 9).topicalformulation of Al is preparedIn another example, skin penetration studies werecarried out using a1.0,petrolatum—based2.0 and 4.0% of Al.formulationcontaining 0.5, The results aresummarized in Table 12. These data show that the amount ofAl recovered in the epidermis increased with theconcentration applied to the skin and did not reachsaturation at 4.0% Al. Furthermore, 0.5% A1 appliedrtopically to human cadaver skin resulted in aconcentration in the epidermis after 24 hours ranging from141-355 yM.inhibition of psoriatic keratinocyte proliferationThis is 10 to 25 fold more than the Ic” for(byextrapolation of the growth inhibition curve, the IC” wasThus,estimated to be 14 MM on day 5). biologicallyrelevant concentrations of the drug penetrated the skinand reached the target tissue (epidermis) within 24 hoursof application.Calculations to determine micromolar were madeassuming that 100 microns of the skin sectiontotal)(200 micronsrepresented epidermis. The area of the cadaverskin used was 1.77 cmz. The calculated volume using thisassumption is 17.7 ML. Molar concentration was calculatedfrom the microgram recovery in the epidermis.SIIBSTIIIIIE 8IlEEH|llIlE 23)1015202530W0 98/ 10767CA 02265630 l999-03- 1225Example 5: Inhibition of EGF-driven DNA replicationAutophosphorylation of receptor tyrosine kinases suchas the EGF-R initiates a signaling cascade that results innuclear changes in the cell, including DNA replication andentry into the S~phase of the cell cycle. Therefore, theeffect of Al on ligand—induced DNA replication wasstudied. 3T3—EGFR cells and 3T3-PDGF—bR cells werestimulated with the corresponding ligand in the presenceof titrated doses of Al. After 20 hours, DNA replicationwas determined by measuring the(BrdU).incorporation ofbromodeoxyuridine EGF-driven DNA replication in3T3-EGFR cells was significantly inhibited by Al in adose-dependent manner, with an ICW of 30 nM.PDGF—driven DNAIn contrast,3T3—PDGF-bRinhibited only by much higher concentrations of Al10 uM).replication in cells was(lcso =Example 6: A1 inhibits EGF—driven cell cvcle progressionAl was examined for its ability to inhibit3T3-EGFR and3T3—PDGF—bR cells were incubated with drug and ligand (EGFand PDGF,receptor-stimulated cell cycle progression.respectively) for 20 hours. The percentage ofcells in various phases of the cell cycle was determinedby propidium iodide staining and fluorescence activatedcell sorting (FACS) analysis. Ligand stimulation of eachcell line resulted in an increase in the percentage ofcells in the (4% in resting 52% inPDGF—stimulated, 72% in EGF—stimulated). Similar to theS-phase cells,results seen in receptor phosphorylation and DNA synthesisthe addition of A1 inhibited cellprogression much more potently in the EGF—driven cellsthan in the PDGF-driven cells (ICW = 34 nM [EGF], 4.1 uM[PDGF]). These data demonstrate that the inhibition ofSIIBSTITIHE SIIEH (ME 23)assays, cyclePCT/US97ll6l451015202530W0 98/10767CA 02265630 l999-03- 1226EGF—R autophosphorylation by Al results in inhibition ofdownstream effects, suggesting that Al might specificallyinhibit EGF-driven cellular proliferation.Example 7: A1 inhibits cellular proliferation driven bvthe fififlagfigr family Q: RTKsSince EGF receptor signaling and cell cycleprogression were inhibited by Al, the effect of thecompound on cellular proliferation was also examined.Cells expressing various receptor tyrosine kinases wereseeded overnight in 10% FBS and Al was added in titrateddoses the next day. After 4 days, cell density wasmeasured by SRB staining. Icw values were determined byregression analysis. For these experiments, cell lineswhose growth is dependent on naturally-expressed RTK wereexamined. Cellular proliferation was measured bysulforhodamine B (SRB) staining after a 4 day incubationin the presence of titrated doses of Al, and Icw valueswere calculated. The data in Table 11 clearly show thatAl preferentially inhibited the growth of cell linesdriven by the EGF-R/Her family of RTK, but did not inhibitPDGF-bR—dependent cell growth.E - 1 ’ ' ' ow h f ri ic)$§_r.aLin.Q.<l.‘=LSKeratinocytes were obtained from psoriasis patientsafter informed consent, and the effect of Al on the growthof the keratinocytes was examined as described byBen-Bassat et al., E£2i__D§£m§LQli_ 4:82-88, 1995. Alinhibited the growth of psoriatic keratinocytes, with anICW of < 1.0 uM on days 5 through 12 of culture. Theseresults indicate that blocking EGF-R signaling issufficient to inhibit the proliferation of psoriatickeratinocytes.SIIBSTITIIT E SIIEET (llllli 28)PCT/US97/161451015202530W0 98/ 10767CA 02265630 l999-03- 1227V. Formulations and AdministrationThe compounds of the present invention can beadministered. to a host alone, or in ea pharmaceuticalcomposition comprising the active compound and a carrieror excipient.The compounds also can be prepared aspharmaceuticallyacceptable salts.Examples ofpharmaceutically acceptable salts include acid additionsalts such as those containing hydrochloride, sulfate,phosphate, sulfamate, acetate, citrate, lactate, tartrate,methane sulfonate, ethane sulfonate, benzene sulfonate, p-toluene sulfonate, cyclohexylsulfamate and quinatePCT/US92/03736incorporated by reference herein).(e.g.those disclosed in and PCT/GB94/01544,Such salts can bederived using acids such as hydrochloric acid, sulfuricacid, phosphoric acid, sulfamic acid, acetic acid, citricacid, lactic acid, tartaric acid, malonic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonicacid, p—toluenesulfonic acid, cyclohexylsulfamic acid, andquinic acid. Pharmaceuticallyacceptable salts alsoinclude alkali metal salts such as sodium or potassiumsalts, alkaline earth metal salts such as calcium ormagnesium salts, ammonium salts or salts with an organicbase which afford a physiologically-acceptable cation suchas salts with methylamine, dimethylamine, trimethylamine,piperidine, morpholine or tris-(2-hydroxyethyl)amine.Pharmaceutically acceptable salts can be prepared bystandard techniques. For example, the free base form ofthe compound is first dissolved in a suitable solvent suchas an aqueous or aqueous—alcohol solution containing theacid. The salt is thenappropriate isolated byevaporating the solution. In another example, the salt isprepared by reacting the free base and acid in an organicsolvent.SIIBSTHIHE SHEET (HIM 28)PCT/US97/161451015202530W0 98/ 10767CA 02265630 l999-03- l2PCTIUS97Il6l4528In accordance with this invention the aforementionedquinazoline derivatives can be administered to a subjectfor reducing or inhibiting keratinocyte proliferation.The compounds are useful as a prophylaxis or means fortreating disorders such as psoriasis. The pharmaceuticalcompositions of the invention contain the compounds inassociation with a compatible pharmaceutically acceptablecarrier material.Techniques for formulation and administration may befound in , 18th ed-,Mack Publishing Co., Easton, PA (1990).carrier material can be utilized.Any conventionalThe carrier materialcan be an organic or inorganic carrier material suitablefor topical, enteral, percutaneous or parenteraladministration. Carriers or excipients can be used tofacilitate administration of the compound, for example, toincrease the solubility of the compound. Suitablecarriers and excipients include, but are not limited to,water, ethanol, polysorbate—80, triacetin, gelatin, gumarabic, lactose, starch, magnesium stearate, talc,vegetable oils, polyalkyleneglycols, petroleum jelly,benzyl alcohol, polyethylene glycols (e.g. PEG—3OO andPEG-400), propylene carbonate, propylene glycol,Transcutol, Petrolatum, vegetable oils, mineral oil,stearyl alcohol, Laureth-4, calcium: carbonate, calciumphosphate, various sugars or types of starch, cellulosederivatives and mixtures thereof. Furthermore, thepharmaceutical preparations may contain otherpharmaceutically active agents. Additional additives suchas flavoring agents, preservatives, stabilizers,emulsifying agents, buffers and the like may be added inaccordance withaccepted practices of pharmaceuticalcompounding.SUBSTITUTE SIIEH (fllllf 23)1015202530W0 98/ 10767CA 02265630 l999-03- 1229Table 4 lists the excipients used for solubilitytesting of Al. The amount of compound for each test wasbetween 1 to 15 mg/mL unless otherwise indicated. Visualsolution cflarity was recorded immediately and 24 Hrs.after rocking at room temperature in a type I glasscontainer. Table 5 shows the solubility of Al in moreexcipients.The pharmaceutical preparations can be made up in any(a)capsules,(b) asolutions,(c)administration such as sterile solutions,(d)administrations such as solutions, suspensions, ointments,conventional form including: a solid form for oraladministration such as tablets,and the like;pills, powders,granules, liquid form for oraladministration such as syrups, suspensions,elixirs and the like; preparations for parenteralsuspensions oremulsions; and preparations for topicalcreams, gels, micronized powders, aerosols, aqueous gels,Thepharmaceutical preparations may be sterilized and/or mayPetrolatum ointments, PEG ointments and the like.Contain adjuvants such as preservatives, stabilizers,wetting agents, emulsifiers, salts for varying the osmoticpressure and/or buffers.the thepreferably prepared asFor topical administration to skinaforementioned compounds areointments, tinctures, creams, gels,.solutions, lotions,sprays, suspensions, shampoos, hair soaps,the like.for application to the scalp or skin can be utilized inperfumes andIn fact, any conventional composition utilizedaccordance with this invention. Preferred formulationsinclude gels, lotions and creams. The pharmaceuticalpreparation for topical administration to the skin can beprepared by mixing the aforementioned active ingredient(i.e., a pharmaceutically effective amount of a compound)SIIISTITIITE SHEET (HIILE 23)PCT/US97/ 161 451015202530WO 98/10767CA 02265630 l999-03- l2PCT/US97ll6l4530with non—toxic, therapeutically inert, solid or liquidcarriers customarily used in such preparations.In preferred embodiments, these preparations containat least about 0.0005 percent by weight, of the activeingredient based upon the total weight of the composition.The active ingredient, the compound, may be used intopical compositions in amounts significantly exceeding 10percent. It is preferred that these preparations containabout 0.01 to 10 percent by weight of the activeingredient based upon the total weight of the composition.It is also preferred that these preparations areapplied once or twice daily to the skin. Thesepreparations can be applied according to the need of thepatient. In carrying out this invention, the activeingredient can be applied in an aqueous solution or analcohol solution such as ethyl alcohol.In preparing the topical preparations described aboveadditives such as preservatives, thickeners, perfumes andthe like conventional in the art of pharmaceuticalcompounding can be used. In addition, conventionalantioxidants or mixtures of conventional antioxidants canbe incorporated into the topical preparations containingthe aforementioned active agent. Among the conventionalantioxidants which can be utilized in these preparationsare included N—methyl-d-tocopherolamine,butylatedtocopherols,hydroxyanisole, butylatedhydroxytoluene,ethoxyquin and the like. Cream-base pharmaceuticalformulations containing the active agent, used inaccordance with this invention, are composed of aqueousemulsions containing a fatty acid alcohol, semi—solidpetroleum hydrocarbon, 1,2-ethyleneglycol and anemulsifying agent.Ointment formulations containing the active agent inaccordance with this invention comprise admixtures of aSUBSTITUTE SIIEET (RULE 28)1015202530W0 98/10767CA 02265630 l999-03- l2PCT/US97/1614531semi-solid petroleum hydrocarbon with a solvent dispersionof the active material. Cream compositions containing theactive ingredient for use in this invention preferablycomprise emulsions formed from a water phase of ahumectant, a viscosity stabilizer and water, an oil phaseof fatty acid alcohol, a semisolid petroleum hydrocarbonand an emulsifying agent and a phase containing the activeagent dispersed in an aqueous stabilizer—buffer solution.Stabilizers may be added to the topical preparation. Anyconventional stabilizer can be utilized in accordance withthis invention.In the oil phase, fatty acid alcoholcomponents function as a stabilizer. These fatty acidalcohol components are derived from the reduction of along—chain saturated fatty acid at least about 14 carbonatoms. Also, conventional perfumes and lotions generallyutilized in topical preparation for the hair can beutilized in accordance with this invention. Furthermore,if desired, conventional emulsifying agents can beutilized in the topical preparations of this invention.Parenteral forms can bedosage infusions orinjectable solutions. Such dosage forms can be injected,e.g., intravenously,These preparations can also contain other medicinallysubcutaneously or intramuscularly.active substances. In preferred embodiments, a dailydosage of from about 0.01 mg to about 2 mg per Kg of bodyutilized inweight is parenteral formulations. Infurther preferred embodiments, a daily dosage of fromabout 0.025 mg to about 0.5 mg per kg of body weight ofthe patient is utilized. Additional additives such asflavoring agents, preservatives, stabilizers, emulsifyingagents, buffers and the like may be added in accordancewith accepted practices of pharmaceutical compounding.A preferred oral dosage form comprises capsules ofhard orsoft gelatin methylcellulose or ofSIIBSTIIIITE SHEET (fllllf 28)another1015202530WO 98110767CA 02265630 l999-03- l2PCT/US97ll6l4532suitable material easily dissolved in the digestive tract.The enteral dosages contemplated in accordance with thepresent invention will vary in accordance with the needsof the individual patient as determined by the prescribingphysician. In preferred embodiments a daily dosage offrom about 0.01 mg. to about 2 mg per Kg of body weight isutilized. In further preferred embodiments a daily dosageof from about 0.025 mg to about 0.5 mg per kg of bodyweight of the patient is utilized. This dosage may beadministered according to any dosage schedule determinedby the physician in accordance with the requirements ofthe patient.It is likewise within the purview of the presentthesubstances enumerated herein in any desired amount forinvention to incorporate therapeutically activeenteral administration within the oral unit dosage form.It is preferred, however, to formulate preparationscontaining the active substance of the present inventionin such a manner that each dose contains from about 0.05mg to about 100 mg,10 mg of theparticularly from about 0.1 mg toactive substance withfillersabout suitabletherapeutically inert and diluents. It isespecially preferred to incorporate such a dosage intosoft gelatin capsules and tablets.A. The present invention is concerned with novelformulations of quinazoline,quinazoline derivatives, andpharmaceutically acceptable salts thereof which aresuitable for topical application for treatment ofpsoriasis and other skin diseases.Topical formulations include ointments, tinctures,creams, gels, solutions, lotions, sprays, suspensions,hair soaps,SIIBSTIIIITE SHEET (fllllf 28)shampoos, perfumes and so on. It is an object1015202530W0 98/10767CA 02265630 l999-03- l2PCTIUS97/1614533of the present invention to provide a vehicle forquinazoline, quinazoline derivatives, and pharmaceuticallyacceptable salts thereof. Such vehicle possesses one ormore of the following characteristics:1. Non-irritating or low-irritating, and non-allergenicor low—allergenic to skin.2. High capacity in storing pharmaceutically effectiveamount of quinazoline, quinazoline derivatives, andpharmaceutically acceptable salts thereof.3. Physically and chemically stable at ambienttemperature.4. Efficient drug release and delivery, good skinpenetration.In that regard, this invention generally features atopical formulation containing a pharmaceuticallyeffective amount of quinazoline, a quinazoline derivative,or a pharmaceutically acceptable salt thereof dispersed ina nonpolar hydrocarbon mixture composed of compounds madeof carbon and hydrogen, and an excipient which incombination with the nonpolar hydrocarbon mixture enhancesthe penetration of quinazoline or its derivative throughthe skin. Preferably, the nonpolar hydrocarbon mixture isa petrolatun1 ointment, including, but not limited to,white petrolatum USP ointment. Other nonpolar hydrocarbonmixtures include, but are not limited to, mineral oil USP,light mineral oil NF, paraffin NF, synthetic paraffin NF,squalane NF, microcrystalline was NF, hexane, isohexane,heptane, decane, decene, decyne, octadecane, benzene,toluene, naphthalene, polyethylene, polypropylene, andpolystyrene.The concentration of quinazoline or its derivative inthe topical formulation is preferably from about 0.01% toabout 10.0% w/w, more preferably from about 0.1% to aboutSIIBSTITIHI SIIET (Rl|lE 28)1015202530W0 98I10767CA 02265630 l999-03- l2PCT/U S97! 16145344.0%about 1.0% w/w.w/w, and even more preferably from about 0.25% toQuinazoline or its derivative in theformulation may be micronized to facilitate suspension intopicalthe ointment, dispersion and/or skin absorption. Variousknown methods of particle size reduction may be utilizedto reduce the size of the quinazoline particles.Preferably, the majority of the particles have a maximumthan about 50and more preferably 90% or more of the particlesdimension (i.e., diameter) of no moremicrons,have a maximum dimension of no more than about 20 microns.thequinazoline or its derivative that reachesAn excipient which increasespercentage ofdermis andepidermis (as measured by % penetrated to reservoir,dermis, epidermis and stratum corneum (R + D + E + SC)) isselected for the topical formulation of this invention.A method for selecting excipients is described in Example2 in the Detailed Description of the PreferredEmbodiments. Preferably, the excipient increases %penetrattion (R + D + E + SC) by more than fifty percent.Examples of skin penetration enhancer include, but are notlimited to, low molecular weight alcohol such as benzylalcohol, NF, and unsaturated fatty acid such as oleicacid, NF, or unsaturated fatty alcohol. The unsaturatedfatty acid or unsaturated fatty alcohol preferably has atleast one double bond and no fewer than twelve carbons.Other excipients disclosed or described in Eergutaneous . CRC Press (E. W.Maibach ed. 1995, ISBN 0-8493-2605-2)penetration of drugs can be used in the formulations ofSmith and H. I.as enhancing skinthis invention as well, especially those described inChapter 9.1 —— “Fatty Acids as Skin Permeation Enhancers”.The content of the book isentire incorporated bySIIBSTITIITE SIIEH (RIJLE 26)1015202530CA 02265630 l999-03- 12W098/10767PCT /US97/1614535reference herein. Excipients may be used alone or incombination with each other in the topical formulation.Benzyl alcohol may be used in a concentration fromabout 0.050% to about 5.0% w/w,about 3.0%1.0% w/w.preferably no more thanw/w, and more preferably no more than aboutOleic acid may be used in a concentration from about0.050% to about 10.0% w/w, preferably no more than about5.0% w/w, and more preferably no more than about 2.5% w/w.An antioxidant may be used in combination with oleicacid to reduce oxidation damage. Candidate antioxidantsinclude, but are not limited to, butylated hydroxytoluene(BHT), NF, butylated hydroxyanisole (BHA), ascorbic acid(Vitamin C), hydroquinone,ascorbyl palmitate, acetylcysteine, N-methyl-d-tocopherolamine,, ethoxyquin,nordihydroguaiaretic acid (NDGA), sodium bisulfite, propylgallate and a—tocopherol (Vitamin E). They may be used ina concentration from about 0.001% to about 1.0% w/w. Inthe case of BHT, preferably it is used in a concentrationfrom about 0.0005% to about 0.1% w/w, and more preferablyno more than 0.01% w/w.Inert ingredients which stabilize the petrolatumsuspension may be added, including, but not limited to, anemollient stabilizer(e.g. mineral oil)emulsifier (e.g. glyceryl monostearate).and an nonionicOptionally, minor amounts of dyes, perfumes,sunscreens, or other agents which are commonly used inadded.such topically active medicaments as theanti—inflammatory corticosteroids and antimicrobials maytopical pharmaceutical compositions may beFurthermore,also be incorporated.Preferably, Al or a pharmaceutically acceptablethereof is used in the composition. Such a composition isespecially suitable for topical treatmentSIIBSTIHHE SHEET (RULE 23)vis—a-visW0 98/107671015202530CA 02265630 l999-03- l2PCT/US97/ 1614536systemic treatment of skin conditions because of Al's lowtoxicity and short plasma half—life. Low toxicity is adesirable feature for a drug used to treat a non—lifethreatening disease. Short plasma half—life helps to keepthe drug's therapeutic effects localized to where it istopically applied.The formulation may be in a unit dosage form or amultiple use dosage form. In a preferred embodiment, aformulation of this invention is held within a containerwhich includes a label stating to the effect that thecomposition is approved by the FDA in the United States(or an equivalent regulatory agency in a foreign country)for treating a hyperproliferative skin disorder such aspsoriasis (e.g. psoriasis vulgaris, psoriasis pustulosa,psoriasis erythrodermica, psoriasis arthropathica,parapsoriasis, palmoplantar pustulosis), skin lesionscaused by Papilloma virus infection, seborrheic keratoses,acanthosis nigricans, ichthyosis (e.g. ichthyosis vulgarisand congenital ichthyoses),keratodermias, genodermatoseswith pathological cornification disorders (e.g. Darier'sdisease), further lichen ruber planus, pityriasis rubrapilaris, or a skin cancer such as basal cell carcinoma,squamous cell carcinoma or melanoma. Such a containerprovides a therapeutically effective amount of the activeingredient to be administered to a host.In another aspect, this invention features a methodof administering quinazoline, a quinazoline derivative, ora pharmaceutically acceptable salt thereof topically byapplying a topical formulation described above.Generally, disease—inflicted skin is applied with atopical formulation containing a pharmaceuticallyeffective amount of quinazoline, a quinazoline derivative,or a pharmaceutically acceptable salt thereof dispersed ina nonpolar hydrocarbon mixture, and an excipient which inSUBSTITUTE SIIEET (fllllf 28)1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97/1614537combination with the nonpolar hydrocarbon mixture enhancesthe penetration of quinazoline or its salt through skin.Preferably, the nonpolarhydrocarbon mixture is apetrolatum ointment or mineral oil.Em Ointment formulations containing the active agentcomprise admixtures of a semi—solid petroleum hydrocarbonwith a dispersion of the active material in an inertliquid ingredient.Cream—base pharmaceutical formulations are composedof oil—in~water emulsions containing a wide variety ofexcipients, including, but not limited to, fatty alcohol,semi—solid petroleum hydrocarbon, propylene glycol or 1,2-propanediol. Cream compositions containing the activeingredient preferably comprise emulsions formed from a"water" phase containing one or more humectants, aviscosity stabilizer, a preservative and water, an "oil"phase containing one or more fatty alcohols, a semisolidpetroleum hydrocarbon, and one or more emulsifying agents.Stabilizers may be added to the topical preparation.The petrolatum formulations initially developed areshown in Table 13. These formulations were put on a threemonth stability screening protocol as described in Example9. The petrolatum formulations were also sent for inVitro skin penetration studies with human cadaver skin;six replicates were run for each formulation. A summaryof the test protocol is given in Example 10 and a summaryof the skin penetration results is given in Table 13.The drugformulations tested.concentration was equal in all thePetrolatum ointments and emollientcreams containing dispersed drug substance showsubstantially better drug delivery in terms of microgramsdelivered and percent of dose delivered than PEG ointmentsSIIBSTIIIIII SIIEEI (RIILE 26)W0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/1614538containing dissolved drug substance(Creams and PEGointments are not shown in Table 13). In comparing thetwo petrolatum ointments and the three creams, they areall equal considering test error. The petrolatumointments containing suspended drug showed total skinpenetration (R+D+E+SC) of about 4% and 6%. Formulation Fldelivered 47 micrograms to R+D+E+SC, at an efficiency of7.9%. .Formulation F2 also gave good penetration at 34micrograms (5.6%) delivered to (R+D+E+SC). The lack of apenetration enhancer in this formula probably accounts forits lower delivery compared to F1.£mm A second series of five petrolatum formulations forskin penetration evaluation are shown in Table 14. Thisgroup consists of the formulation from Series I showing(F1)ointments which were variations of F1 designed for im-the highest penetration and four new petrolatumproved penetration. Four new cream formulations were alsoprepared which were variations of the best cream fromSeries I designed for improved penetrationTable 14).(not shown inThe mineral oil used in the petrolatum ointment(Light Mineral Oil,The USP grade has aallformulations was changed from NF gradeNF) to USP grade (Mineral Oil, USP).higher viscosity. The USP grade was used insubsequent work.Because oleic acid is susceptible to oxidation due toits C=C bond, USP)0.2% alpha tocopherol (Vitamin E,(see Table 14).wasadded to several formulations However, itwas found that this high level of tocopherol caused adarkening of the color of the formulated product after twoa lower level ofSIIBSTHIIII SIIET (MILE 26)weeks at 40°C. In the alternative,1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97ll6l4539tocopherol or approximately 10 ppm (0.00l%) BHT is addedto the petrolatum ointment formulation to help preventpossible oleic acid oxidation.The penetration results of thein Table 14.gavepetrolatumformulations are shownThe petrolatumbetterDue to thelarge errors present in this type of test, a general ruleis that aointments (except F6) substantiallypenetration than the creams in this series.significant difference between twoexistsformulations if there is at least a 2-fold differencebetween results. In this case, the average penetrationdifference between the four best ointments and the fourcreams is a factor of about 3-fold.Among petrolatum formulations, formulation Fl againgave the best penetration results of all the formulationstested, delivering 44 micrograms ‘to (R+D+E+SC),at anefficiency of 7.4%.Formulation F5, containing 2.5% oleic acid and 1.5%benzyl alcohol, also gave very good penetration at 36(6.1%) delivered to (R+D+E+SC).micrograms This resultcan be considered equal to F1. Because oleic acid is apotential irritant, this formulation is not as irritant asFl.Formulations F3 and F4 were modifications of F1containing additional excipients (benzyl alcohol andlaureth 4) which have relatively high drug solubility.These formulations did not perform as well as Fl; thereason may be that addition of a good solvent to asuspended drug formulation favors the drug remaining inthe formulation rather than moving toward and through theskin.Formulation F6, containing 5% propylene glycol (PG,a penetration enhancer), gave poor penetration results.This may be because PG is not miscible with petrolatum andSIIBSTIWTE SHEET (lllilf 28)CA 02265630 l999-03- 12WO 98/10767 PCT/US97/ 16145101520253040forms a two-phase system. In this case, the PG,shows very low drug solubility, may have interfered withwhichthe penetration enhancing properties of the oleic acid.Overall, the creams did not perform as well in thisstudy (maximum 2% penetration) as they did in Series I,while the petroleum ointments continued to show excellentpenetration (up to 6%).Therefore, petrolatum was chosenas thepreferred vehicle, and another penetrationexperiment was performed to optimize the formulations.Fo m ' r"The third series of eight petrolatum formulations forThisseries was designed to study the effects of varying theskin penetration evaluation is shown in Table 15.drug concentration, and also for optimization of theconcentrations of the excipients benzyl alcohol and oleicacid. The penetration results are shown in Table 15.One cream formulation was prepared as well, whichcontains (%):Al 2.00WHITE PETROLATUM, USP 5.00BENZYL ALCOHOL, NF 2.00STEARYL ALCOHOL, NF 10.00OCTYLDODECANOL, NF 5.00BRIJ72l 2.00BRIJ72 2.40PROPYLENE GLYCOL, USP 5.00METHYLPARABEN, NF 0.20HYDROXYETHYL CELLULOSE, NF 0.30PURIFIED WATER, USP 66.10The penetration result of the cream formulation is3.6% (R+D+E+SC).SUBSTITUTE SIIEET (ME 23)WO 981107671015202530CA 02265630 l999-03- l2PCT/US97/1614541Overall, the cream did not perform as well in thisstudy as it did in Series I, while the petrolatum ointmentcontinued to show good drug penetration into the skinlayers. .All the petrolatum ointments at. 2% drug gavesubstantially better penetration than the cream at 2%drug, a conclusion drawn also from Series II and confirmedin Series III.The petrolatum ointment control sample in this test,F10, was identical to F5 in Series II except that theSeries II formula also contained 0.2% tocopherol, whichshould not have substantially affected penetration. Theresults for F10 in this test are very close to those ofF5: only a 2 nucrogram difference in total reservoir +dermis + epidermis + stratum corneum (R+D+E+SC)penetration.Among petrolatum ointments, the drug concentrationdose study gave essentially a linear response over theof 0.5 to 4.0% A1 R+D+E+SCpenetration to the nearest 5 micrograms gives an exactlyrange (rounding off thelinear result of 80, 40, 20 and 10 Inicrograms). Thedouble themicrogram penetration of the best of the 2% drug formulas.formula at 4% aboutdrug therefore gaveThis would allow drug concentration to be varied to meeta given target delivery, with good predictability.F11, the effect ofvarying the oleic acid concentration from 2.5 to 1.25 toComparing F10, and F12 gives0% in the same base formula. Looking at the R+D+E+SCpenetration implies that 2.5% is equal to 1.25%, and theseare probably better than 0%. The 0% formula still gavefair penetration probably because the benzyl alcohol wasmaintained at 1.5%.F13, the effect ofvarying the benzyl alcohol concentration from 1.5 to 0.75Comparing F10, and F14 givesto 0% in the same base formula. The results of thisSIIISTHIITE SHEET (lllllf 29)W0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/1614542comparison show that 0.75% and 0% are equal, while 1.50%may be slightly worse. In any case, the differences arerelatively small, similar to the oleic acid dose study.This may imply a synergistic effect between the oleic acidand the benzyl alcohol. Since F12 gave the lowestpenetration results of the 2% oleic acid isthanointments,probably more important to penetration benzylalcohol.Overall, most of the 2% ointmentresults arerelatively close. F11 and F13 gave the highest R+D+E+SCpenetration. The physical stability results showed thatthe latter might possess slightly better stability.Because the drug substance is present as a solidsuspension in the formulated ointment, it was micronizedfor the formulations to be used in toxicology studies andclinical studies. Micronization produces smallerparticles and increases the total drug substance surfacearea and dispersion of the suspension in the ointment;these characteristics may result in more efficient skinpenetration of the drug substance. Drug micronizationcontributes to better characterization and definition ofdrug substance particle size for future bioequivalenceneeds. The drug micronizing process also improves thecosmetic elegance of the formulation as an emollient byeliminating palpable detection of solids in the ointment.The micronized drug substance was tested by HPLCwhich indicated that the micronization did not reduce thedrug's purity.F1,F1 and F13 are stable at ambient temperature for atF11 and F13 were screened for stability withHPLC.least three months. F11 is stable at ambient temperaturefor at least two months.An additional exemplary petrolatunl formulation isdescribed in Table 16.SIIBSTITIHE SIIEH (NILE 29)1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97ll6l4543All the excipients in the selected formulation arecompendial. A manufacturing process is described inExample 11.Ez§mple_2;_ Phvsical Stability Screeningmfhm:A 5 to 15 gram sample is placed in a 20 ml glassscintillation vial and capped with a urea screw caphaving a PE cone liner.fitggggg ggnditigns:50°C (45 to 55°C)40°C (37 to 42°C)30°C (27 to 32°C)Ambient Room Temperature(2 to 8°C)(-10 to -30%Freeze—thaw cycling (1 week at freezer followed by 1(15 to 30°C)RefrigeratorFreezerweek at ambient per cycle)Eygiuagjgg limes:1 week (optional), 2 weeks (optional), 1 month, 2months & 3 months.The 50°C sample is typically terminated at 1 month.Samples at any test station may be terminated basedon gross instability or other pertinent reason. :Evaluation is by physical examination for generalappearance, color,odor, consistency/viscosity,separation/syneresis, etc.Example 10: In—vitro Skin Penetration Studv Protocol§sf;u2=with a 4% BSA isotonic buffered saline solutionFranz static diffusion chambers are filled(6-10 mLreservoir volume) and equilibrated to a temperature of 37°CSIISTITIITE SIIEET (IIIILE 23)1015202530WO 98/10767CA 02265630 l999-03- l2PCT/US97/1614544by a circulating water pump. Excised human cadaver skin(approximately 200 micron thickness)is placed onto eachchamber. After sealing with an O—ring,skin1.77A total of 54 diffusion cells are used in the study.the exposedsurface area has a diameter of 15 mm and the area iscm?Nine different formulations are tested; each formulationis done in six replicates. Skin from the abdominal area(dorsal) from a single human donor is used for the study.Depending upon the experimental design,theskin can also betape-stripped to"glistening" point (removal ofstratum corneum) before starting the experiment.Iggt Formulations:Dow Dermatologics,Test formulations provided byInc., containing non-radioactive drugsubstance at the formulated concentration, are spiked with“C or 3H—radiolabeled drug substance and mixed thoroughlywith a this is donespatula; immediately prior toapplication to the skin chamber. The target for specificSpikedformulations are tested for uniform specific activity byactivity is about 0.050 uCi/mg test formulation.liquid scintillation counting.t u i n A ' ‘ n: Test formulations areapplied to the skin surface with a Gilson positivedisplacement pipet. The amount applied is 17 mg/cmz;therefore, about 30 mg of test formulation is applied toeach cell.1;mg_ggig;§: A sample of 1.0 mL is taken with aGilson pipet from the reservoir under the skin sample atthe following time points: 0, 1, and 24 hours. The sampleis placed in a vial containing Ecoscint scintillationfluor. At the O and 1 hour time points the 1.0 mL volumeThe O and 1hour time points are used to assess the integrity of thethe 1highis replaced with the BSA saline solution.If any holes or other defects exist,willskin sample.showSUBSTITUTE SHEET (RIILE 28)hour reservoir sample unusuallyCA 02265630 l999-03- 12W0 98/10767 PCTIUS97/1614545radioactivity. In such cases that cell is removed fromthe study. It is common that several cells are omitted1015202530from the data calculations for each study. The endpointcan be varied from 3-24 hours.be added to the test protocol,W d aAdditional time points canat additional cost.At theexperiment, the skin is removed from the cell.of theTo recoverendexcess formulation from the skin surface, the skin isfirst washed with three 1.0 mL volumes of 2% Oleth-20 inwater; The skin is thenthis is retained for counting.gently wiped with three separate cotton gauzes which aresaved and Counted.i C t t e ' t‘ s:withThe skin is tape-stripped cellophane tape until "glistening"(approximately 22 strips). The first two strips thatremove the excess drug adhering to the outer surface of(SC)counts are included in total recovery but excluded from SCthe stratum corneum are counted separately. Thesecompartment recovery. Four groups each consisting of fiveconsecutive tape strips are placed in a scintillation vialcontaining Scintilene. Dermis and epidermis are separatedby xnicrowave technique and placed in vials containingReadyProt cocktail for tissue digestion. All samples arecounted in a Beckman LSC counter and corrected forquenching. Recovery in the reservoir, washes, gauzewipes, and the respective skin compartments (SC,epidermis, and dermis) is calculated by determining thepercent of the total counts applied. Total radioactiverecovery is typically 70—lOO%.Example 11:(1)Mineral Oil.Process for Making 1% and 4% Al OintmentIn a clean vessel, weigh. micronized .Al andUsing a stainless steel spatula, spatulatethe mixture until a homogeneous paste is achieved.SIIBSTITIITE 8|lEHfl|l|.E23)W0 98/ 107671015202530CA 02265630 l999-03- 12PCT /US97/1614546(2)and Glycerol Monostearate.until melted.(3)Hydroxytoluene to Step 2.In a separate vessel, weigh White PetrolatumHeat to approximately 75°CAfter mixing, add Oleic Acid and ButylatedMix for approximately 5 minutesuntil homogeneous.(4) With continuous mixing, add Benzyl Alcohol toStep 3. Mix for 5 minutes.With continuous mixing, add Step 1 to Step 4. Mixfor approximately 20 minutes to ensure Homogeneity.Begin cooling process using a water bath, withcontinuous mixing and side scraping. Continue mixing andcooling until temperature is below 30°C.VI. synthesis gf Quinazglige DerivativesEuropean Patent Application Publication No.226 Al (October 12, 1993) A. J.,by reference herein in its entirety,O 566by Barker, incorporateddescribes processesfor the nmnufacture of certain quinazoline derivatives(e.g., pp 18-20 of EP 0 566 226 Al).conventional protocolsHowever,synthesis for quinazolinederivatives have had relatively low yields and requirereactions at high temperatures, the use of column’ chromatography and a 2—propanol solvent.This invention features new and improved processesfor the preparation of quinazolone, chloroquinazoline, 4-arylamino quinazoline, and their derivatives. Much betteryields of the intermediate molecules and the final productare achieved with the processes of this invention thanwith conventional methods. The reaction to preparequinazolone is conducted at a lower and more convenienttemperature. In the isolation and purification ofcholoroquinazoline, column chromatography is not required.The final free base form of the 4-arylamino quinazoline isSIIBSTITIJTE SIIEH (lllllf 23)1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97/1614547obtained in high purity from. the reaction mixture bysimply adding an alkaline solution.In addition to quinazolone, other substitutedmonocyclic, heterocyclic or polycyclic fusedbicyclic,ring compounds and their salts containing a pyrimidonering (see Figure l and below) can be prepared with theprocess of this invention from their B—amino acrylic acidprecursors which contain a B—aminoacrylic acidfunctionality such as acid, ester or —OC(O)—.Thus, thisin one aspect, invention features aprocess of preparing a substituted monocyclic, bicyclic,heterocyclic or polycyclic fused ring compound containinga pyrimidone ring by reacting a precursor which has a B-amino acrylic acid functionality with formamidine or aformamidine salt to fuse thepyrimidone ring. Inpreferred embodiments, the reaction is conducted with a(e.g., ethanol).The reaction is conducted at a temperature of no less thanformamidine salt in refluxing alcohol20°C (preferably no less than 60°C, and more preferably noless than 80°C)than 120°C,and no more than 150°C (preferably no moreand more preferably no more than 100°C). Thefused ring compound is quinazolone or a quinazolonederivative and the precursor is an aminobenzoate or aminobenzoic acid. Specific compounds which can be prepared bythis process are described in the detailed description ofthe invention.In another aspect, this invention features a processof preparing a 4—halogen-pyrimidine (e.g.chloroquinazoline) by reacting a pyrimidone (e.g.quinazolone or a quinazolone derivative) with ahalogenating agent (e.g. chlorinating agent) and isolatingor purifying the end product, 4—halogen—pyrimidine (e.g.chloroquinazoline), by precipitation, crystallization orsublimation. In preferred embodiments,SIIBSTIIIITE SIIET (lllllf 20)the pyrimidoneW0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97l16l4548reacts with the halogenating agent in the presence of acatalyst such as dimethylformamide which promotes theformation of the 4—halogen-pyrimidine. The halogenatingagent includes, but is not limited to,thionyl chloride,phosphorus oxychloride and oxalyl chloride. This processmay be modified by replacing the halogenating agent withanother displacement agent to prepare a quinazoline whosedisplaceable group .is alkoxy,thearyloxy or sulphonyloxy.For example, displaceable group may be methoxy,phenoxy, methanesulphonyloxy or toluene—p—sulphonyloxy.'In yet another aspect, this invention features aprocess for preparing a 4-arylaminopyrimidinehydrochloride salt by dissolving a substitued aniline insolution ethanol),an alcohol (e.g.,chloropyrimidine to the alcohol solution to react with theadding asubstituted aniline, and isolating the end product, 4-arylaminopyrimidine hydrochloride salt. In a preferredembodiment, the 4—arylaminopyrimidine hydrochloride saltis a 4-arylamino quinazoline hydrochloride salt and theChloropyrimidine is chloroquinazoline or achloroquinazoline derivative.In addition, this invention features a process forpreparing a free base 4-arylaminopyrimidine directly bydissolving a substitued aniline in an alcohol solutionethanol),(e.g., adding a chloropyrimidine to the alcoholsolution to react with the substituted aniline, adding analkaline solution to the alcohol solution, and isolatingthe end product, 4-arylaminopyrimidine. In a preferredembodiment, the 4-arylaminopyrimidine is a 4-arylaminoquinazoline and the chloropyrimidine is chloroquinazolineor a chloroquinazoline derivative.Furthermore, this invention features a process forconverting an isolated 4—arylaminopyrimidine hydrochloridesalt to its free base form by mixing it with an alkalineSUBSTITUTE SIIEET (RULE 29)W0 98/107671015202530CA 02265630 l999-03- l2PCT/US97/ 1614549solution and isolating the end product, 4-arylaminopyrimidine (e.g. 4—arylamino quinazoline).E E. . . E IAs used hereinafter, an “alkyl”saturated aliphatic hydrocarbon, including straight-chain,group refers to abranched-chain, and cyclic alkyl groups.Preferably, thealkyl group has 1 to 12 carbons. More preferably, it isa lower alkyl of from 1 to 7 carbons, more preferably 1 tosuch e+g*,4 carbons, as, methyl, ethyl, n-propyl,iso—propyl, n~buty1, sec—butyl, iso—butyl, tert-butyl, and2—methylpentyl. The alkyl group may be substituted orunsubstituted. When substituted the substituted group(s)is preferably hydroxyl, cyano, alkoxy, =0, =5, N02, N(CHQ2,amino, or SH.An “alkoxy”“alkyl” isgroup refers to an “-O~alkyl”described above,iso—propoxy,tert—butoxy,group,defined as such aswheremethoxy, ethoxy, n—propoxy, n—butoxy,sec-butoxy, iso—butoxy, trifluoromethoxy,3—hydroxyhexyloxy, 2-carboxypropyloxy, 2-fluoroethoxy,carboxymethoxy and cyanobutyloxy and the like.An “alkenyl” group refers to an unsaturatedhydrocarbon group containing at least one carbon-carbondouble bond, including straight—chain, branched-chain, andcyclic groups. Preferably, the alkenyl group has 1 to 12carbons. More preferably it is a lower alkenyl of from 2to 7 carbons, more preferably 2 to 4 carbons. The alkenylgroup may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferablyhydroxyl, cyano, alkoxy, =0, =S, Nos, halogen, N(Cg 5,amino, or SH. An “alkynyl” group refers to an unsaturatedhydrocarbon group containing at least one carbon-carbontriple bond, including straight—chain, branched-chain, andcyclic groups.Preferably, the alkynyl group has 2 to 12SIIBSTITIHI SIIEH MILE 23)1015202530W0 98/ 10767CA 02265630 l999-03- l2PCT/US97/1614550carbons. More preferably, it is lower alkynyl of from 2to 7 carbons, more preferably 2 to 4 carbons. The alkynylgroup may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferablyhydroxyl, cyano, alkoxy, =0, =S, Nos, N(CHg2, amino or SH.A "heterocycle" denotes a chain of carbon and atleast one non-carbon atoms which together form one or morearomatic or non—aromatic rings having preferrably betweenabout 5-14 atoms, such as, e‘g+, furyl, thienyl,imidazolyl, indolyl, pyridinyl, thiadiazolyl, thiazolyl,piperazinyl, dibenzfuranyl, dibenzthienyl. These ringsmay be optionally substituted with one or more functionalgroups which are attached commonly to such rings, such as,gig‘, hydroxyl, bromo, fluoro,thio, alkylthio,heteroaryl, carboxyl, oxo, alkoxycarbonyl, alkyl, alkenyl,chloro, iodo, mercapto orcyano, cyanoamido, heterocycle, aryl,nitro, amino, alkoxyl, amido, and the like to form rings§&g+Ichlorothiazol-4-yl,such as, 2-aminothiazol-4-yl, 2—amino—5—2~amino-thiadiazol-4—yl,2,3-dioxopiperazinyl, 4—alkylpiperazinyl,2-iodo—3—dibenzfuranyl and 3—hydroxy—4-dibenzthienyl andthe like.An “aryl” group refers to an aromatic group whichhas at least one ring having a conjugated pi electronsystem and includes carbocyclic aryl, heterocyclic aryland biaryl groups, all of which may be optionallysubstituted. Preferably, the aryl is a substituted orunsubstituted phenyl or pyridyl. Preferred arylsubstituent(s) are halogen, trihalomethyl, hydroxyl, SH,OH, N02, amine, thioether, cyano, alkoxy, alkyl, and aminogroups.An “alkylaryl” group refers to an alkyl as describedabove covalently joined to an aryl group as describedabove.Preferably, the alkyl is a lower alkyl.SIIBSTITIIIE SIIEH (NILE 26)W0 98/ 107671015202530CA 02265630 l999-03- 12PCT /US97/1614551“Carbocyclic aryl” groups are groups wherein thering atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted.“Heterocyclic aryl” groups are groups having from 1to 3 heteroatoms as ring atoms in the aromatic ring andthe remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen.The heterocyclic aryl groups of this invention include,but are not limited to, furanyl, thienyl, pyridyl,pyrrolyl, N—lower alkyl pyrrolo, pyrimidyl, pyrazinyl,imidazolyl and the like, all optionally substituted.An "aryloxy" denotes -OAr, where Ar is an aryl groupas defined above.An "aralkyl" denotes -RAr, where R is alkyl and Aris aryl, both as defined above.refers to anAn “amide" —c (0) —NH—R,either alkyl, aryl, alkylaryl or hydrogen.where R isAn “amine” refers to a -N(R”)R”’, where R" and R”’,alkyl,alkylaryl, provided that R” and R”’ are not both hydrogen.is independently either hydrogen, aryl, orAn "amino" denotes the group NRR', where R and R‘may independently be alkyl, aryl or acyl as defined above,or hydrogen.A "cyanoamido" refers to the group —NH—CEN.§emr_al__ErLtmu.La_§Compounds of the formulas as depicted in Figures la-ld, 2a—2d, and their hydrochloride salts can be preparedby the process of this invention.In Figures la, lb, 1c and 1d:X = 0, 1 or 2.R1 istrifluoromethyl, C1-C6 alkoxy, Cl-C6 alkyl, nitro, cyano,either hydrogen, hydroxy, halogen,or amino.SIIBSTIIIITE SHEET (NE 26)W0 98/ 1076710152025CA 02265630 l999-03- l2PCTIUS97/1614552R2 and R3 are independently either hydrogen, C1-C6alkyl, Cl-C6 alkyl, C1-C6 (C1-C3alkenedioxy). nitro, halogen, C1-C6 alkoxycarbonyl, cyano,alkoxy, cycloor amido.M and N are independently either single or doublebonds. If M and N are single bonds then the bonds betweenatoms B and C can be a double bond.Each of A, B, C and D is independently carbon or aheteroatom such as N, O, or S to give bicycles including,but not limited to, quinazoline derivatives.With both M and N double bondsFor example: N C C N pteridineC N N C pyrazino[2,3d]pyrimidineN C C C pyridino[2,3-d]pyrimidineC N C C pyridino[3,4-dlpyrimidineWith both M and N single bonds and the bond betweenB and y a double bond 1 ,4-dithia—1 ,4-dihydroquinazolme1 ,4—dithia-1.2.3,4-tetrahydroquinazolineWith both M and N single bonds C C Q 1 ,4-d|oxolo[2,3-d]pyrimidine 2b, 2c and 2d:With M a single bond and N a double bondIn Figures 2a,ring svstemthien[2,3-b]pyrimidineSUBSTITUTE SITEET (NILE 23)CA 02265630 l999-03- 12W0 98/10767 PCTIUS97ll6l4553O C C furan[2,3—b]pyrimidineN C C pyrro[2,3—b]pyrimidineC C C cyclopenteno[2,3-b]pyrimidineS N C isothiazo[4,5—d]pyrimidine5 S C N thiazo[4,5-d]pyrimidineO N C isoxazo[4,5—d]pyrimidineO C N oxazo[4,5-d]pyrimidine1015202530With M a double bond and N a single bondB C Ring svstemthien[2,3—b]pyrimidinefuran[2,3—b]pyrimidinepyrro[2,3-b]pyrimidinecyclopenten[3,4—b]pyrimidineisothiazo[4,5-d]pyrimidinethiazo[4,5—d]pyrimidineOUJUJOZOUJisoxazo[4,5—d]pyrimidineZOZOOOOODHOZOZOOOOO oxazo[4,5-d]pyrimidineThe process may also be employed for preparingsubstituted mono—, bi- and tricyclic fused ring compoundscontaining a pyrimidone ring by starting with any compoundwith a B—amino acrylic acid functionality. For example,the following tricyclic compounds can be prepared by thismethod: naptho[l,2-d]pyrimidine, naptho[2,3-d]pyrimidine,quinolino[2,3-d]pyrimidine, and quinolino[3,4—d]pyrimidine.Quinazoline derivatives of formula V in Figure 4eand their pharmaceutically—acceptable salts and theirprecursors of formulae II and III may be prepared by theprocesses of this invention.II, III and V, m is l,is independently selected from theIn formulas I,Each R12 or 3 and nis 1 or 2.group consisting of hydroxy, amino, carboxy, carbamoyl,ureido, (l-6C)alkoxycarbonyl, N>(1-6C)alkylcarbamoyl, N,N>di-[(l-6C)alkyl]carbamoyl, hydroxyamino,SIIBSTITIWE SIIEH (lllllf 28)(l-6C)alkoxyami-W0 98/107671015202530no,(1-6C)alkoxy,morpholino,fluoromethyl), hydroxy—(l—6C)alkyl,(1—6C)alkoxy'(1-6C)alkyl,(l-6C)alkoxycarbonyl-(1—6C)alkyl,6C)alkyl,(1-3C)alkylenedioxy,[(1-6C)alkyl]amino,piperazin—l—yl,(l—6C)alkylthio,6C)alkylsulphonyl, halogen-(l—6C)alkylCA 02265630 l999-03- 1254(2—6C)alkanoyloxyamino, trifluoromethoxy,pyrrolidin—l-yl,N—(l-6C)alkylcarbamoyl—(1—6C)alkyl,6C)alkyl]carbamoyl-(1-6C)alkyl,6C)alkylamino-(l-6C)alkyl,PCT /U S97! 16] 45(1-6C)alkyl,(1-6C)alkylamin0,piperidino,4-(1-6C)alkylpiperazin—1—y1,(1-6C)alkylsulphinyl,(1-(other than tri-(2-6C)alkanoyloxy—(l—carboxy—(1-6C)alkyl,carbamoyl—(l-6C)alkyl,N,N-di-[(l-amino-(1—6C)alkyl,di—[(1—6C)alkyl]amino—(l—(1-6C)alkyl, piperidino—(1-6C)alkyl, morpholino-(l—6C)alkyl,piperazin—1-yl-(1-6C)alkyl,(1-6C)alkyl,6C)alkylamino—(1(1-6C)alkyl,6C)alkylthio-(l—6C)alkyl,6C)alkyl,6C)alkoxy,6C)alkoxy,koxy,6C)alkoxy,6C)alkoxy,—6C)alkyl,phenoxy—(1-6C)alkyl,phenylthio-(l-6C)alkyl,hydroxy-(2-6C)alkoxy,(2—6C)alkanoyloxy,hydroxy-(2-6C)alkoxy-(l~6C)alkyl,6C)alkoxy-(2—6C)alkoxy—(l—6C)alkyl,4-(1-6C)alkylpiperazin—l-yi-(1-hydroxy-(2-(1-6C)alkoxy-(2-6C)alkylamino-(l—6C)alkylthio—(1—6C)alkyl,hydroxy—(2—(1—6C)alkoxy-(2—6C)alkylthio—(1-anilino—(l—6C)alkyl,cyano—(1—6C)alkyl, halogen—(2—(2-6C)alkanoyloxy—(2-(1-6C)alkoxy-(2—6C)alkoxy,(1-6C)alkoxycarbonyl—(l—6C)alkoxy,carboxy-(l—6C)al-carbamoyl-(1-N=(1—6C)alkylcarbamoyl—(1—6C)alkoxy,[(1—6C)alkyl}carbamoyl-(l—6C)alkoxy,(l-6C)alkylamino-(2—6C)alkoxy,N,N-di-amino-(2-6C)alkoxy,di-[(1-6C)alkyl]amino-(2-hydroxy—(2—6C)alkanoyloxy,(1-6C)alkoXy-(2—6C)alkanoyloxy, phenyl—(1—6C)alkoxy,phenoxy—(2—6C)alkoxy, anilino—(2—6C)alkoxy, phenylthio-(2-6C)alkoxy,6C)alkoxy,piperidino-(2-6C)alkoxy,morpholino-(2—piperazin—l—yl—(2-6C)alkoxy,6C)alkylpiperazin—l-yl-(2—6C)alkoxy,6C)alkylamino,4-(1-halogen—(2—hydroxy-(2-6C)alkylamino,SIIBSTITIHE SIIEH (BM 28)(2-di-W0 98/ 107671015202530CA 02265630 l999-03- l2PCTIUS97/16145556C)alkanoyloxy—(2-6C)alkylamino, (l-6C)alkoxy-(2-(1-carbamoyl—(l—6C)alkylamino, carboxy—(1-6C)alkylamino,6C)alkoxycarbonyl~(l—6C)alkylamino,6C)alkylamino, NF(1-6C)alkylcarbamoyl—(l—6C)alkylamino,N,N>di—[(1—6C)alkyl]carbamoyl—(1-6C)alkylamino, amino-(2-6C)alkylamino, (1-6C)alkylamino-(2—6C)alkylamino, di—[(l—6C)alkyl]amino—(2-6C)alkylamino, phenyl-(l-6C)alkylamino,phenoxy—(2-6C)alkylamino, aniline-(2—6C)alkylamino,(1-(l—6C)alkylsulphonylamino,phenylthio—(2—6C)alkylamino, (2-6C)alkanoylamino,6C)alkoxycarbonylamino,benzamido,benzenesulphonamido, 3-phenylureido, 2-oxopyrrolidin-1-yl, 2,5~dioxopyrrolidin—l—yl, halogen-(2-H-carboxy—(2—(1—6C)alkoxycarbonyl—(2—N%l—N,N-di-[(1-6C)alkanoylamino, hydroxy—(2—6C)alkanoylamino,6C)alkoxy-(2—6C)alkanoylamino,6C)alkanoylamino,6C)alkanoylamino, carbamoyl-(2—6C)alkanoylamino,6C)alkylcarbamoyl-(2—6C)alkanoylamino,6C)alkyl]carbamoyl—(2—6C)alkanoylamino, amino—(2~6C)alkanoylamino, (1-6C)alkylamino—(2—6C)alkanoylamino anddi-[(1-6C)alkyl]amino—(2—6C)alkanoylamino, and whereinsaid benzamido or benzenesulphonamido substituent or anyanilino, phenoxy or phenyl group in a R1 substituent may(l-6C)alkyl or (1-each Rzof formulatheconsisting of hydrogen, hydroxy, halogen, trifluoromethyl,(1-6C)alkyl, (l—6C)alkoXy, cyclo[(1—(l-6C)alkylamino, di-[(l—6C)alkyl]amino,(1—6C)alkylsulphinyl (1-Z of formula IV is a cflsplaceableoptionally bear one or two halogen,6C)alkoxy substituents. In addition,IV or V is independently selected fromgroupamino, nitro, cyano,3C)alkenedioxy],(1-6C)alkylthio,6C)alkylsulphonyl.andgroup which includes, but is not limited to, halogen,alkoxy, aryloxy and sulphonyloxy groups. Preferably, Z isselected from the group consisting of chloro, bromo,SIIBSTITIITE-SIIEH (MILE 25) hW0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/X614556methoxy, phenoxy, methanesulphonyloxy and toluene-p-sulphonyloxy groups.In a preferred embodiment, each R1 is independentlyselected fronx the group consisting of hydroxy,amino,ureido, methoxycarbonyl, ethoxycarbonyl, hydroxyamino,trifluoromethoxy, methyl, ethyl methoxy, ethoxy, propoxy,isopropoxy, butoxy, methylenedioxy, ethylenedioxy,methylamino, ethylamino, dimethylamino, diethylamino,piperidino, morpholino, mehtylthio, ethylthio,bromomethyl, dibromomethyl, methoxymethyl,piperidinomethyl, morpholinomethyl, piperazin-1-ylmethyl,methoxyethoxymethyl, methylthiomethyl, 2-hydroxyethylthiomethyl, anilinomethyl, phenylthiomethyl,cyanomethyl, 2—bromoethoxy, 2-hydroxyethoxy, 3-hydroxypropoxy, 2—methoxyethoxy, 2—ethoxyethoxy, 3-methoxypropoxy, 3-ethoxypropoxy, methoxycarbonylmethoxy,ethoxycarbonylmethoxy, carbamoylmethoxy, 2-dimethylaminoethoxy, 2-diethylaminoethoxy, 2-methoxyacetoxy, benzyloxy, 2—anilinoethoxy, 2-peperidinoethoxy, 2—morpholinoethoxy, 2—(piperazin—1—yl)ethoxy, 2-hydroxyethylamino, 3—hydroxypropylamino, 2-methoxyethylamino, 2—ethoxyethylamino, 3-methoxypropylamino, 3-ethoxypropylamino, 2-dimethylaminoethylamino, 2—diethylaminoethylamino, 3-dimethylaminopropylaminno, 3—diethylaminopropylamino,acetamido, propionamido, benzamido, 3—phenylureido, 2-chloroacetamido, 2—oxopyrrolidin—l-yl, 2-hydroxyacetamido,2—methoxyacetamido and 2-ethoxyacetamido. In addition,each Rzis independently selected from the group consistingof hydrogen, fluoro, chloro, bromo, trifluoromethyl,nitro, cyano, methyl and ethyl.In a further preferred embodiment, (RHm is selected6,7-6—trifluoromethoxy,SIIBSIITIITE SHEET (RIILE 26)from the group consisting of 6—hydroxy, 7—hydroxy,dihydroxy, 6—amino, 7-amino, 6—ureido,W0 98/ 107671015202530CA 02265630 l999-03- 12PCT /US97/16145576—methyl, 6,7-dimethyl,dimethoxy, 6,7—diethoxy, 6—hydroxy—7—methoxy, 7—hydroxy—6-6-methoxy, 7—methoxy, 6,7-methoxy, 6-amino—7—methoxy, 6—amino—7—methylthio, 5—amino-6,7—dimethoxy, 6-methoxy-7-isopropoxy, 6,7—methylenedioxy,6,7-ethylenedioxy, 6-methylamino, 7-methylamino, 6—dimethyiamino, 6—amino-7—methylamino, 6-methoxymethyl, 6-bromomethyl, 6-(2—methoxyethoxymethyl), 6—cyanomethyl, 6-methylthiomethyl, 6-phenylthiomethyl, 7—(2-hydroxyethoxy)—6-methoxy, 6,7-di—(2-hydroxyethoxy), 6-(2-bromoethoxy), 6-(2—methoxyethoxy), 6—methoxy—7-(2—methoxyethoxy), 6,7—di-(2-methoxyethoxy), 7-(2—bromoethoxy)-6-methoxy, 7—benzyloxy—6-methoxy, 6-(2-methoxyethylamino), 6-acetamido,6—benzamido, 6—(2-chloroacetamido), 6—(2—methoxyacetamido)and 7-(2—methoxyacetamido). In addition, (Rfln is selectedfrom. the group consisting of hydrogen, 4'~fluoro, 3'-chloro, 3'-bromo, 3',4‘-dichloro, 4'-fluoro—3'—chloro, 3‘-trifluoromethyl, 4‘—fluoro—3'—trifluoromethyl, 3'—nitro,3'—nitro—4‘—chloro, 3'-nitro-4'—flouro and 3'—methylgroups.&mm A quinazoline derivative as depicted in Figures 1and 2, or a pharmaceutically acceptable salt thereof, canbe prepared by the processes shown in Figures 3a, 3b and3c.§1;.aL2_lAn aspect of this invention is the improved yieldand low reaction temperature in the first stepaccomplished by the use of formamidine or a formamidinesalt instead of formamide. For example, the commerciallyavailable 6,7—dimethoxyanthranilic acid methyl ester iscondensed with formamidine acetate by refluxing in ethanolto give 6,7~dimethoxyquinazolone in greater than 90%SIIBSHTIHE SIIEET (lllllf 28)W0 98/ 107671015202530CA 02265630 l999-03- l2PCTlUS97ll6l4558yield. Similarly, 6,7-dimethoxyanthranilic acid is alsocondensed with formamidine to give >80% yield. Thisreaction is not restricted to unsubstituted formamidinesbut may employ substituted formamidines to give 3-substituted quinazolones.This step converts a substituted ortho-aminobenzoicester or a substituted isatoic anhydride or hydrochlorideto give a quinazolone. The reaction is preferably carriedout by heating in the presence of an inert solvent such asmethanol, ethanol, isopropanol, a chlorinated solvent suchas dichloromethane or chloroform, an ether such astetrahydrofuran or dioxane, an amide such asdimethylformamide or dimethylacetamide, water or any otherinert solvent. The reaction is heated to between 20°C andthe boiling point of the solvent,between 20°C and 120°C.cooling the reaction to ambient temperature or below andpreferably heating toThe quinazolone is obtained byfiltering the solid product, and, optionally,recrystallizing.Alternatively, condensing with formamide at atemperature of about 170° C gives a yield of 75%, which isnot as high as with a formamidine in ethanol and at a lessconvenient temperature.Products which are not quinazoline or quinazolinederivatives but which contain heteroatom substituents inthe benzene ring of the quinazoline and/or are saturatedin this ring can also be prepared by this method. InFigures 3a—3c, Y is alkoxy or hydroxy.S_ts.L2The compound prepared in Step 1 is reacted with anof thionyl chloride with a such asexcess catalystdimethylformamide in the absence of solvent or in thepresence of a trapping agent such as toluene, benzene, orSUBSTITUTE SIIET (HIILE 28)W0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/1614559xylene to remove free chlorine. Alternatively, anotherchlorinating agent can be used such asphosphorusoxychloride or oxalyl chloride. The crude product isisolated by evaporating the solvent and/or adding a co-solvent to precipitate it. The crude product, either asthe solid or in solution in an inert solvent such as anyof the solvents of Step 1, is washed with dilute aqueousbase, such as sodium carbonate or bicarbonate and isolatedby evaporation or precipitation with an inert co—solventsuch as toluene, benzene or hexane. The washed product issatisfactory for conversion to the quinazoline in Step 3or can be further purified by crystallization orsublimation.For example, 6,7-dimethoxy—4-quinazolone can bereacted with thionyl chloride and dimethylformamide toform 4-chloro-6,7-dimethoxyquinazoline, which is purifiedby precitation and washing or sublimation. Such processrequires no solvent and produces4-chloro-6,7—dimethoXyquinazoline of high purity.Compared to conventional methods, this process has a muchbetter yield (about 89% versus about 27%) and eliminatesthe chromatography step.31:29.;The chloroquinazoline of Step 2 is reacted with asubstituted aniline present in slight excess in theabsence of solvent or in an inert solvent, or with anequivalent of aniline and a suitable base such aspyridine, triethylamine, ethyl morpholine, or any otherunreactive base or an inorganic base such as sodium orpotasium carbonate or bicarbonate. The inert solvent canbe methanol, ethanol, isopropanol, a chlorinated solventsuch as dichloromethane or chloroform, an ether such astetrahydrofruan, 1,2-dimethoxyethane or dioxane, an amideSIIBSTITIIIE SIIEH (|l||lE 26)W0 98/ 1076710152025CA 02265630 l999-03- 12PCT /US97/1614560such as dimethylformamide ordimethylacetamide, ordimethylsulfoxide. The reaction is carried out between20°C and the boiling point of the solvent, preferablybetween 20°C and 150°C. The solid product quinazolinehydrochloride salt is isolated bycooling and/orconcentrating and filtering.For example, 4—chloro-6,7—dimethoxyquinazoline isreacted with 3-chloroaniline to form4-(3—chlorophenylamino)-6,7—dimethoxyquinazoline. In thisinvention, denatured alcohol was used as solvent insteadof 2—propanol used in conventional methods. Applicantfound that 4-chloro-6,7—dimethoxyquinazoline does notreact with the alcoholic solvent as long as the aniline isalready present in the mixture.§1;ep__iAn alkaline solution can be added directly to thereaction mixture of Step 3, stirred, and the free baseisolated in Step 3 instead of the hydrochloride salt.This is simpler than conventional methods in which anadditional step is required to obtain the free basequinazoline from its hydrochloride salt.Alternatively, the quinazoline hydrochloride saltprepared in Step 3 is converted into its free base bytreatment of the salt alone, or suspended in an inertorganic solvent as described above, with a suitableorganic or inorganic base as described above. The productis isolated by filtration with or without concentrating.The following six compounds of formula V wereprepared with the process of this invention.SIIBSTITIITESIIEET (|l|l|.E 23)CA 02265630 l999-03- 12W0 98/10767 PCT/US97/1614561§:,ss1_rI12.Le_a R’ R’S1 6,7-dimethoxy 3-chloroS2 6,7~dimethoxy 3—bromoS3 6,7-dimethoxy 3—trifluoromethyl5 S4 6,7-dimethoxy 3-cyanoS5 6-methyl 3-chloroS6 6-methyl 3—trifluoromethylQsamL2.T&_.1..2. :4-(3—Chloroohenvlamino)-6.7—dimethoxvcuinazoline10 Step 1660 g of methyl 2-amino-4,5-dimethoxybenzoate, 660 g offormamidine acetate, and 2.46 kg of ethanol were stirredand refluxed in a 22 L flask for 10 hours. Heating wasdiscontinued and the reaction was allowed to cool. The15 product was collected by vacuum filtration and washed withwarm ethanol. The product was dried at 55°C under vacuumto give 609 g (about 95%) of 6,7—dimethoxy—4-quinazolone.Alternatively, formamide (73.7lg) and 82.80 g ofmethyl 2—amino—4,5—dimethoxybenzoate were stirred and20 heated at 170°C in a 250 ml flask maintained in a nitrogenatmosphere for 5.7 hours. Heating was discontinued and thereaction was allowed to cool. The mixture was dilutedwith 150 ml of water and the solids collected by vacuumfiltration and washed three times with 50 ml of water.25 The product was dried at 60°C under a vacuum of 0.1 mm Hgto give 59.0 g (about 73%) of 6,7—dimethoxy—4—quinazolone.§l:ep._2Thionyl chloride (256.8 9): 59.0 g of6,7—dimethoxy—4-quinazolone, and 5 ml of dimethylformamide30 were stirred and heated at 93°C for 9.3 hours. Heating wasdiscontinued, the mixture cooled to room temperature,SUBSTITUTE SIIEH (RIM 23)W0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/1614562transferred to an evaporating flask using 100 ml ofmethylene chloride, and evaporated to dryness at 50°C. Theresulting solid was stirred with 400 ml of saturatedsodium bicarbonate, collected by vacuum filtration, andwashed repeatedly with water (400 ml total).was dried at 50 - 60°C under a vacuum of 0.1 mm Hg to give63.7 g (~1OO % yield) of4-chloro—6,7—dimethoxyquinazoline.The productcrudeThe crude was dissolved in 825 ml of boiling hexane,hot filtered and allowed to crystallize. The solids werecollected, washed with 80 ml of hexane and air dried togive 66 g of product. The crude solid was sublimed at150°C under a vacuum of < 0.1 mm Hg to give, in three55.7 g (about 89 % yield)(180.8°C softens) 185.0 —l86.5°C.was consistent with the expected structure.crops, of an off—white solid, mpThe proton NMR spectrumTLC (5 %Rf 0.30.-dimethoxyquinazolinemethanol in methylene chloride, silica gel):4-chloro—6,7can be prepared with the method described in Step 2 ofAlternatively,Example 13 below..S1;§2S__3__ansi_4_To a 500 ml reaction flask was added 18.09 g of3-chloroaniline, 150 ml of denatured alcohol and then 13.0g of 4-chloro-6,7—dimethoxyquinazoline. The reaction washeated to 80 i 5°C and stirred vigorously for one hour.The reaction was allowed to cool to room temperature too f4-(3—chlorophenylamino)—6,7—dimethoxyquinazolineg i V e a p r e c i p i t a t ehydrochloride salt. The flask was then cooled to O — 4°Cwith stirring.One Normal sodium hydroxide (58 ml,was added and the reaction stirred at O -one equivalent)4°C for 30The product was collected by vacuum filtration,SIIBSTITIIIE SHEET (HIILE 23)minutes.W0 98/ 107671015202530CA 02265630 l999-03- l2PCT/US97/1614563washing twice with 20 ml portions of ethanol/water 2/1(v/v). The product was dried under a vacuum of 0.1 mm Hgat ambient temperature to give 16.7 g of4-(3—chlorophenylamino)-6,7—dimethoxyquinazoline (about84% yield). M.P. 178 — 181°C.In order to prepare the hydrochloride salt of4-(3-chlorophenylamino)-6,7-dimethoxyquinazoline, thereaction was conducted as above but omitting the additionof l N sodium hydroxide to the cooled reaction mixture.Instead, the cooled reaction mixture was stirred for 30at o — 4°C,and washed twiceminutes the product collected by vacuumfiltration, with 20 ml portions ofethanol.Hg at to4-(3—chlorophenylamino)-6,7—dimethoxyquinazolineThe product was dried under a vacuum of 0.1 mmambient temperature givehydrochloride salt.Example 1 324-(3-Bromophenvlamino)-6.7—dimethoxvduinazolineStep 12.500 kg of 2—amino—4,5—dimethoxybenzoate and 1.48kg of formamidine acetate in 9.300 kg of ethanol werefor 10 (at thinchromatography showed completion of the reaction).refluxed hours which timelayerThemixture was cooled to room temperature and the crudeproduct isolated by vacuum filtration. The crude productwas resuspended in 5.75 kg of ethanol and stirred at 40°C for 30 minutes. The mixture was cooled to roomtemperature, the product isolated by vacuum filtration anddried under vacuum at 55° C until the loss on drying was<1%. The 2.351 kg (96.8%) of 6,7-dimethoxyquinazolone at a purity of 99.48% by HPLCyield wasrelative area %.SUBSTITUTE STTEET (TTTTLE 28)W0 98/ 107671015202530CA 02265630 l999-03- l2PCTIUS97/1614564§_te.1L2.1.18 kg of 6,7 -dimethoxyquinazolone, 5.13 kg ofand 0.094 kg ofdimethylformamide were refluxed for 6 hours.thionyl chloride, 0.53 kg of toluene,Thin layerThemixture was cooled to room temperature and then 6.82 kg ofchromatography showed completion of the reaction.toluene was added. The mixture was stirred for 30 minutesand the crude solid product colleted by vacuum filtration.The solid was suspended in 26.2 kg of methylene chlorideand agitated at room temperature for about 1 hour with8.24 kg of aqueous solution containing 0.37 kg of sodiumcarbonate. The organic layer was separated, dried with0.59 kg of anhydrous sodium sulfate, filtered to removethe sodium sulfate, and concentrated at £400 C to about 25%of its original weight.To the thick slurry of product in methylene chloridewas added 11.15 kg of toluene. The mixture was againconcentrated until its temperature reached about 800 C.The mixture was cooled to room temperature and the solidproduct was collected by vacuum filtration and dried undervacuum at 550 C to give 0.884 kg of 4-chloro-6,7 —dimethoxyquinazoline (68.7% yield), 98.87 % pure by HPLCarea %. The second batch of 4-chloro-6,7dimethoxyquinazoline was manufactured by the same processas the first batch to give 1.088 kg of 4-chloro-6,7 —dimethoxyquinazoline (85.7% 99.32%yield), pure by HPLCarea %._S_t_e_p__3_To a solution of 0.734 kg of 3-bromoaniline in 6.9added 0.874 kg of 4-chloro-6,7 —This mixture was refluxed for aboutkg of ethanol wasdimethoxyquinazoline.2 hours at which time thin layer chromatography showedcompletion of the reaction. The mixture was cooled toSIIBSTITIHE SIIEEI (RULE 2|!)W0 98/1076710152025CA 02265630 l999-03- l2PCTIUS97/1614565room temperature and the solid product isolated by vacuumfiltration. The product was washed by suspending in 6.1Thesolidproduct again collected by vacuum filtration to give 1.855kg of ethanol and refluxing for about 30 minutes.mixture was cooled to room temperature and thekg of 4—(3—bromophenol) amino—6,7(93.7%)Batch of—dimethoxyquinazoline98.37% pure by HPLC4—(3—bromophenol)hydrochloride sale yield),area %. amino-6,7 —dimethoxyquinazoline hydrochloride was manufactured by thesame process as the first batch to give 2.610 kg of crude4—(3—bromophenol) amino—6.7(lO4.6% yield),~dimethoxyquinazolinehydrochloride 99.04% pure by HPLC area %.Srix 94.400 kg of crude 4—(3—bromophenol amino—6,7 —dimethoxyquinazoline hydrochloride and 8.5 kg of watercontaining 0.34 kg of sodium hydroxide was stirred in13.33 kg of ethanol at room: temperature for about 90minutes. The solid product was isolated by vacuumfiltration, washed by resuspending in 13.6 kg of water andagain collected by vacuum filtration. The solid product13.6 kg of ethyland collected bywas washed again by suspending inacetate, refluxed for about one hour,vacuum filtration. Finally the product was washed bysuspending in 13.6 kg of water,at 450 C,stirred about 30 minutescooled to room temperature, and collected byvacuum filtration. Drying under vacuum at 55° C gave 2.689kg of 4—(3—bromophenol)(83% yield), 99.5%containing 4.57 % water.amino—6,7 ~dimethoxyquinazolinepure by HPLC relative area % andSIIBSTITIITE SHEET (NILE 29)W0 98/107671015202530CA 02265630 l999-03- l2PCT/US97/ 1614566Ex e 4:6.7—Dimethoxv-4—[3-(trifluoromethvl)phenvlaminolduinazol11.112This compound was prepared from the4-chloro-6,7-dimethoxyquinazoline of Example 12 and3—(trifluoromethyl) aniline by the method of Example 12. :4- — o la i — 7- ' e h x iThis compound was prepared from the4—chloro—6,7-dimethoxyquinazoline of Example 12 and3-cyanoaniline by the method of Example 12.Ex le4-{§—gQ1g:Qpnenylamino)-6-methylggjgazolige2—Amino—5-methyl benzoic and formamide werecondensed as in Example 12 to give 6-methyl-4-quinazolonein about 70% yield. 6—Methyl—4—quinazolone waschlorinated mith thionyl chloride as in Example 12 to give4-chloro-6—methylquinazoline in 84%by4-(3-Chlorophenylamino)-6-methylquinazoline was preparedaboutyield andp u r i f i e d s u b l i m a t i o nfrom 4-chloro-6-methylquinazoline and 3—chloroaniline asin Example 12 in about 60% yield. :- -4- 3- ' om en 1 in ui zol'neThis compound was prepared from the4-chloro—6—methylquinazoline of Example 13.All publications referenced are incorporated byreference herein, including the nucleic acid sequences andamino acid sequences listed in each publication. All thecompounds disclosed and referred to in the publicationsmentioned above are incorporated by reference herein,SIIBSTIHHE SHEET (RIM 29)CA 02265630 l999-03- 12W0 98/10767 PCT/US97/1614567including those compounds disclosed and referred to inarticles cited by the publications mentioned above.Other embodiments of this invention are disclosed inthe following claims.5 I3ble_l; Structures of Ouinazoline Compounds.H\N x1X2<::;ZTX3 N_C_c>r_np_ca.md X1 X2 213A1 Br methoxy methoxyA2 Cl methoxy methoxy10' A3 C1 methyl HA4 CF3 methoxy methoxyA5 CN methoxy methoxyA6 CF3 methyl HSUBSTITUTE SHEET (RULE 28)CA 02265630 1999-03-12W0 98/ 10767 PCTIUS97/1614568Table 2: Inhibition of EGF-R Tvrosine KinaseActivitvand Cellular Growth bv Ouinazoline derivativesCompound EGFR Kinase 3T3-EGFR 3T3-PDGFR PDGFR/EGFRIC50 (MM) ICSO (MM) ICSO (uM) IC50/ICSOA4 <0.006 0.4 28 705 free baseA1 0.0009 0.075 15 200free baseA2 <0.015 0.11 18 163free base10 A6 0.9 6.0 30 5free baseA3 0.058 2.0 10 5free baseA5 0.2 1.5 >100 6715 free baseSUBSTITUTE SIIEH (ME 28)CA 02265630 l999-03- 12W0 98/1076‘! PCT/US97/1614569Table 3: Ioxicitv,of Ouinazoline Comoounds (mg/kg)Toxicity* Plasma Concentration**(mg/kg) (ug/mL)LD“, LDSO 5 min 15 min 60 minA3 LD20=4OO >400 7.87;+_0.98 3.43_-tO.82 1.34;!-_0.llA2 >200 >200 l1.5i4.0 6.5il.3 3.7_4;0.5no deaths no deathsA6 >400 >400 4.45_tl.38 2.0liO.53 O.54_t0.2no deaths no deaths5 A1 75 ~300 17.9_+;4.3 6.3-_+_3.7 2.01-_O.9A4 LD,o=40O LD.,o=400 l3.31l.5 l2.73_r_3.0 3.9il.5A5 >153 >153 0.95j_O.42 0.65_+_0.l9 O.l3;+_0.06no deaths no deaths* Single dose, IP, in BALB/c, female mice.** Single dose (50 mg/kg), IP, in BALB/c, nu/nu, female10 mice.SUBSTITUTE SIIEET (RIILE 28’)CA 02265630 l999-03- 12WO 98/10767 PCT/US97/1614570Table 4. EXCIPIENT SCREENING FOR A1Excipient Excipient [Drug] SolubilityRatio (mg/mL) After 24 Hrs.Distilled H20 Insoluble1.0N HCI Insoluble/Yellow5 Cone. NaOH discolorationInsolubleEthanol Partially SolublePolysorbate-80 10 Slightly SolubleEtOH/Poly-80 50:50 10 Slightly SolubleBenzyl Alcohol Soluble10 Triacetin Partially SolubleOleic Acid Insoluble/Yellow/Cloudylsopropyl Myristate lnsolublelcloudyPEG-300 SolublePropylene Glycol Slightly15 Propylene SolublelClearIParticulatesCarbonate SolubleI8OC/10 min.SIIBSTHIIIE SIIEET (NILE 23)CA 02265630 l999-03- 12W0 98/ 107671015202571PCT/US97/16145Table 5: SOLUBILITY OF A1 IN PURE SOLVENTSSoluble at Not Soluble atCompendial Excipients (% w/w) (% w/w)Benzyl Alcohol, NF 3.60 4.77Betizyl Berizoate, USP 0,35Dehydrated Alcohol, USP 0.76 0.98lsopropyl Alcohol, 99%, usp _ 1.25Octyldodecanol, NF 0.68Oleic Acid, NF 1.00Polyethylene Glycol 300, USP 1.00 2.10Polyethylene Glycol 400, USP 1.76 2.44Polysorbate 20, NF 0.99Propylene Carbonate, NF 0.98 1.46Propylene Glycol, USP 0.59Triacetin, USP 0.96Non-Compendial ExcipientsCapryliclcapric Triglyceride 0.84(Miglyol 812)Diethyl Sebacate not compatible (yellow)Diisopropyl Adipate 0.66Dimethyl isosorbide 0.97Ethoxydiglycol (Transcutol) 2.41 3.74Finsolve TN (Finetex) 1.27Hexylene Glycol 0.98 2.05lsotearyl Alcohol 0.99Laureth 4 4.10 -SIIBSTITIITE SIIEH llllllf 23)CA 02265630 1999-03-12W0 98/1076772PCT /US97ll6l45Table 6: A1 FORMULATIONS FOR SKIN PENETRATION STUDIESFormulation Drug FormCategoryMajorExcipients1. Aqueous Gels Solution52. Petrolatum Solution3. PEG Ointments Solution104. Creams Suspended15Water, PEG 400,Transcutol, propyleneglycol, benzyl alcoholPetrolatum, Ointmentpropylene carbonatePEG 400, benzyl yalcohol, Laureth—4,TranscutolAqueous phase: propyleneglycol, water,surfactants, Oil phase:mineral oil, stearylalcohol.Drug Concentration: 2% w/w in all the formulations (except aqueousgels)SUBSTITUTE SIIEH (MILE 23)10CA 02265630 l999-03- 12W0 98/ 10767 PCT /US97/1614573Table 7: SUMMARY RESULTS A1 PENETRATION STUDYPolyethylene Glycol OintmentsSG-94A SG-89A SG-90A SG-91AExcipients A B C DPolyethylene Glycol 400, NF 71.0 64.0 64.0 58.5Benzyl Alcohol, NF 3.0 ***** ***** 2.0Laureth 4 amen-* um mu-** 2_5White Petmlatum’ USP *.m ...m H... umPropylene Carbonate, NF ***** ***** 10.0 *****Propylene Glycol, USP ***** 10.0 ***** 10.0Polyethylene Glycol 3350, NF 24.0 24.0 24.0 25.0A1 2.0 2.0 2.0 2.0 §pg Penetrated (R+D+E)* 1.8 1.6 1.9 3.2pg Penetrated (R+D+E+SC)** 3.0 2.7 2.9 6.5Percent of Dose (R+D+E) 0.3 0.3 0.3 0.5SIIBSTITIHE SHEET (NILE 23)CA 02265630 l999-03- 12W0 98/ 1076710ExcipientsWhite Petrolatum, USPBenzyl Alcohol, NFLt. Mineral Oil, NFWhite Wax, NFPropylene Carbonate, NFWhite Wax, NFGlyceryl Monostearate, NFA1Oleic Acid, NFPenetration Resgltg,ug Penetrated (R+D+E)*pg Penetrated (R+D+E+SC)**Percent of Dose (R+D+E)74PCT/US97/16145Petrolatum OintmentsSG—92AE83.51.5_5.0***************3.02.05.034.447.25.7SIIBSTITIHI SHEET (|l|llE 23)SG-93AF82.5**********5.07.55.03.02.0*****21.333.63.610152025CAW0 98/10767ExcipientsStearyl Alcohol, USPCetyl Alcohol, USPWhite Petrolatum, USPOctyldodecanol, NFSorbitan Monostearate, NFPolyoxyl 40 Stearate, NFBrij 721Brij 72Purified Water, USPPropylene Glycol, USPBenzyl Alcohol, NFEthoxydiglycol [Transcutol]Methylparaben, USP10% NaOH SolutionCarbopol 980, NFHydroxyethyl Cellulose250HHXA1Eenetrgtion Resultspg Penetrated (R+D+E)*pg Penetrated(R+D+E+SC)**Percent of Dose (R+D+E02265630 1999-03-12PCT/US97/161457 5fi_r_r_1o[Ii§n; CreamsSG-98A SG-98B SG-99AG H I10.0 1 0.0 6.0m... .m. 055.0 5.0 *****5.0 5.0 5.0......... m... H)...m ...m 4_02.0 2.0 *****2.4 2.4 *****58.1 66.1 60.85.0 5.0 20.0...m 2.0 m...1 OD mu ......*0.2 0.2 0.2*~u»** um 02..m_* ..m 030.3 0.3 *****2.0 2.0 2.016.5 20.7 14.822.7 27. 1 21 .0) 2.8 3.4 2.5*R+D+E = Reservoir + Dermis + Epidermis**R+D+E+SC = Reservoir+ Dermis = Epidermis + Stratum CorneumSIIIISITIIITE SIIET (|llllE 29)W0 98/ 10767CATable 8.76SKIN PENETRATION STUDIES02265630 1999-03-12PCT /US97l16l45FormulationCategoryNo. ofFormulationDrug Form(2% w/w)MajorExcipientsPercent SkinPenetrat1on*PetrolatumOintment2SuspendedWhitePetrolatum(84%),acid,oleicpropylenecarb., benzylalcohol3.6 - 5.7CreamsSuspendedWater (60%),surfactant,alcoholpropylenecarb.,mineral oil2.5 ~ 3.4PolyethyleneGlycolOintmentDissolvedPEG —400(S8 — 71%)benzylalcohol,laureth—4,transcutol0.3 - 0.5SUBSTITUTE SHEET (RIM 28)1015WO 98/10767CA 02265630 l999-03- l2PCTIUSQ7/1614577Table 9: A1 TOPICAL FORMULATIONFORMULATION PERCENTDESIGNATION CONC. (W/W)A1 1.0 or 4.0%Mineral Oil, NF 5.00%Glyceryl Monostearate, NF 3.00%Benzyl Alcohol, NF 0.75%Oleic Acid, NF 2.50%Butylated Hydroxytoluene 0.001%White Petrolatum, USP QS to 100%Table 10: Selectivity of A1I RECEPTOR I LIGAND I ICE (MM) IIEGF-R EGF 0.02EGF-R/Her-2 EGF 0.60PDGF-bR PDGF >100IGF-IR IGF-1 >100IR Insulin >100SIIBSTITIITE SIIET (||lllE 28)W0 98/1076710CA02265630 1999-03-12 PCT/US97/1614578Table 11: Effect of Al on cell growthCELL LINE RTK IC (uM)A431 EGF-R 1.6BT474 Her-2 0.9C6 PDGF-bR >1001e tui'ecveof1i eieiPercent Epidermis peg Epidermis uMA1 Recovery Concentration _0.5 1.6 :t 0.7 248 :1: 1071.0 2.3:t1.0 355115]2.0 4.5:+:1.0 712i 1574.0 16.3 :1: 9.4 2561 :1: 1469SIIBSTITIITE SIIEH (RULE 20)CA 02265630 l999-03- 12W0 98/10767 PCT/US97/1614579Table 13: TOPICAL FORMULATION SERIES IFORMULATION COMPONENTS F1 F2A1 2.0 2.0White Petrolatum, USP 83.5 82.55 Benzyl Alcohol, NF 1.5 N/ALight Mineral Oil, NF 5.0 N/APropylene Carbonate N/A 7.5White Wax, NF N/A 5.0Glyceryl Monostearate 3.0 3.010 Oleic Acid, NF 5.0 N/Apg Penetrated (R+D+E+Sc) 47 34% Penetrated (R+D+E+Sc) 7.9 5.6SIIBSTHIITE SHEET (RULE 23)CA 02265630 l999-03- 12W0 98/ 10767 PCT/US97/1614580Table 14: TOPICAL FORMULATION SERIES IIFORMULATION F1 F3 F4 F5 F6COMPONENTSA1 2.0 2.0 2.0 2.0 2.05 White Petrolatum, 83.5 81.8 80.3 85.8 78.3USPBenzyl Alcohol, NF l.5 3.0 l.5 1.5 1.5Mineral Oil, USP 5.0 5.0 5.0 5.0 5.0Propylene Glycol, N/A N/A N/A N/A 5.010 USPLaureth 4 N/A N/A 3.0 N/A N/AGlyceryl 3.0 3.0 3.0 3.0 3.0MonostearateOleic Acid, NF 5.0 5.0 5.0 2.5 5.015 Vitamin E, USP N/A 0.2 0.2 0.2 0.2pg Penetrated 44 28 22 36 9(R+D+E+Sc)% Penetrated 7.4 4.6 3.6 6.1 1.4(R+D+E+Sc)SIIBSTITIHE SHEET (MILE 2|!)CA 02265630 l999-03- l2PCT/US97/16145W0 98/10767815m+m+o+E“Tm m.n m.m ma m.m m. >6 » omoo mo ucmoumm.1, 6m+m+a$:mm 3 am 2 mm NH am am omumfimcmm mimuaflmmm counumnumawmoo.m oo.N cod oo.N oo.m omio 8... co; Emmomom.~ om.N . mm; om.N omzm om.N om.N mz .aHU¢ Bmqooo.m 86 oo.m oo.m oo.m oo.m oo.m oo.m miammemozoz dcmmufiwoo.m 85 oo.m oo.m oo.m oo.m oo.m oo.m mmo :30 flwmuzaz mic om; om; om; omg omg om; mz iomooi dfizmm3.3 2.3 omam 3.5 8.3 8.5 ooém 8.5 mmo .zP:EoE.mm mix:Em mam Nah Sm 3m 3 mm Pmmazflfizmo znafiomaumHHH muwumm cowumasfiom HMOMQOB "ma mannaOHSIIBSTITIITE SHEET (RULE 23)CA 02265630 l999-03- 12W0 98/107157 PCT/US97ll614582Table 16: EXEMPLARY TOPICAL FORMULATIONFORMULAT ION PERCENTDESIGNATION CONC . (W/W)Al 0.25 or 1.0%5 Mi 5.00%Glyceryl Monostearate, NF 3.00%Benzyl Alcohol, NF 0 to 1.50%Oleic Acid, NF 2.50%Butylated Hydroxytoluene, NF 0.001%10 White Petrolatum, USP QS to 100%SUBSTITUTE SHEET (|T||lE 23)

Claims (44)

1. A method for treating a hyperproliferative skin disorder wherein said hyperproliferative skin disorder is not cancer in a host, comprising the step of administering to said host a composition containing a pharmaceutically effective amount of a compound selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanophenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof.

2. The method according to claim 1 wherein said composition is administered to said host by topical application.

3. The method according to claim 1 wherein said hyperproliferative skin disorder is selected from the group consisting of psoriasis, and hyperproliferation caused by papilloma virus infection.

4. The method according to claim 1 wherein said compound is 4-(3-Bromophenylamino)-6,7-dimethoxy-quinazoline or a pharmaceutically acceptable salt thereof.

5. The method according to claim 1 wherein said composition comprises a pharmaceutically acceptable carrier.

6. A method for reducing keratinocyte proliferation in a host wherein said keratinocyte proliferation is not cancer, comprising the step of administering to said host a composition containing a pharmaceutically effective amount of a compound selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanophenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof.

7. A method for reducing skin lesions caused by Papilloma virus infection in a host wherein said papilloma virus infection is not cancer, comprising the step of administering to said host a composition containing a pharmaceutically effective amount of a compound selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanophenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof.

8. A method for treating psoriasis in a host, comprising the step of administering to said host a composition containing a pharmaceutically effective amount of a compound selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanophenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof.

9. The use of a compound selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanophenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof; in the manufacture of a medicament for the treatment of a hyperproliferative skin disorder wherein said hyperproliferative skin disorder is not cancer.

10. The use of claim 9, wherein said medicament is for topical application to a host.

11. The use of claim 9 wherein said compound consists of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline or a pharmaceutically acceptable salt thereof.

12. A composition for topical application, comprising:
(a) a nonpolar hydrocarbon mixture;
(b) a pharmaceutically effective amount of a compound selected from the group consisting of quinazoline, a quinazoline derivative, and a pharmaceutically acceptable salt thereof; and (c) an excipient; wherein said compound is dispersed in said nonpolar hydrocarbon mixture, and said excipient in combination with said nonpolar hydrocarbon mixture enhance the ability of said compound to penetrate through the skin.

13. The composition of claim 12, wherein said non polar hydrocarbon mixture is selected from the group consisting of a petrolatum ointment and mineral oil.

14. The composition of claim 12, wherein said excipient is oleic acid or benzyl alcohol.

15. The composition of claim 14, wherein the concentration of said oleic acid is from about 0.05% to about 5.0% by weight.

16. The composition of claim 12, further comprising an effective concentration of an antioxidant.

17. The composition of claim 16, wherein said antioxidant is selected from the group consisting of butylated hydroxyanisole, butylated hydroxytoluene, .alpha.-tocoprerol, ascorbic acid, and propyl gallate.

18 The composition of claim 16, wherein said antioxidant is butylated hydroxytoluene with a concentration of from about 0.0001% to about 0.01% by weight.

19. The composition of claim 14, wherein the concentration of said benzyl alcohol is from about 0.05 to about 3.0% by weight.

20. The composition of claim 12, wherein said compound is dispersed in said nonpolar hydrocarbon mixture in particles and more than half of said particles have a diameter of no more than about 50 microns.

21. The composition of claim 12, wherein said compound is selected from the group consisting of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6,7-dimethoxyquinazoline, 4-(3-Chlorophenylamino)-6-methylquinazoline, 4-[3-(trifluoromethyl)phenylamino]-6,7-dimethoxyquinazoline, 4-(3-Cyanopnenylamino)-6,7-dimethoxyquinazoline, 4-[3-(trlfiuoromethyl)phenylamino]-6-methylquinazoline, and a pharmaceutically acceptable salt thereof.

22. The composition of claim 21, wherein said compound is 4-(3-Bromophenylamino)-6,7-dimethoxyauinazoline or a pharmaceutically acceptable salt thereof.

23. The composition of claim 22, wherein the concentration of said compound is from about 0.1% to about 4.0% by weight.

24. A petrolatum ointment for topical treatment of psoriasis, comprising:
from about 0.1% to about 4.0% by weight of 4-(3-Bromophenylamino)-6,7-dimethoxyquinazoline or a pharmaceutically acceptable salt thereof, from about 1.0% to about 5.0% by weight of oleic acic, and rcm abour 0.0001% to about 0.01% by weight of butylated hydroxytoluene.

25. A process for preparing a substituted monocyclic, heterocyclic or polycyclic fused ring compound containing a pyrimidone ring, comprising the step of reacting a .beta.-amino acrylic acid precursor of said compound with formamidine or a formamidine salt under conditions suitable to fuse said pyrimidone ring.

26. The process of claim 25, wherein said precursor is reacted with formamidine or a formamidine salt in refluxing ethanol.

27. The process of claim 25, wherein said fused ring compound containing a pyrimidone ring is quinazolone or a quinazolone derivative, and said precursor is 2-aminobenzoate or 2-amino benzoic acid.

28. The process of claim 27, wherein the reaction is conaucted at a temperature of no more than 150°C.

29. The process of claim 27, wherein the reaction is conducted at a temperature of no more than 120°C.

30. The process of claim 27, wherein the reaction is conducted at a temperature of no more than 100°C.

31. The process of claim 27, wherein said formamidine salt is formamidine acetate.

32. The process of claim 25, wherein said fused ring compound containing a pyrimidone ring has the structure of formula II and said precursor has the structure of formula I, wherein Y represents hydroxy or an alkoxy group, m is 1, 2 or 3 and each R1 is independently selected from the group consisting of hydroxy, amine, carboxy, carbamoyl, ureido, (1-6C)alkoxycarbonyl, N-(1-6C)alkylcarbamoyl, N,N-di-[(1-6C)alkyl]carbamoyl, hydroxyamino, (1-6C)alkoxyamino, (2-6C)alkanoyloxyamino, trifluoromethoxy, (1-6C)alkyl, (1-6C)alkoxy, (1-3C)alkylenedioxy, (1-6C)alkylamino, di-[(1-6C)alkyl]amino, pyrrolidin-1-yl, piperidino, morpholino, piperazin-1-yl, 4-(1-6C)alkylpiperazin-1-yl, (1-6C)alkylthio, (1-6C)alkylsulphinyl, (1-6C)alkylsulphonyl, halogen-(1-6C)alkyl (other than trifluoromethyl), hydroxy-(1-6C)alkyl, (2-6C)alkanoyloxy-(1-6C)alkyl, (1-6C)alkoxy-(1-6C)alkyl, carboxy-(1-6C)alkyl, (1-6C)alkoxycarbonyl-(1-6C)alkyl, carbamoyl-(1-6C)alkyl, N-(1-5C)alkylcarbamoyl-(1-6C)alkyl, N,N-di-[(1-6C)alkyl]carbamoyl-(1-6C)alkyl, amino-(1-6C)alkyl, (1-6C)alkylamino-(1-6C)alkyl, di-[(1-6C)alkyl]amino-(1-6C)alkyl, piperidino-(1-6C)alkyl, morpholino-(1-6C)alkyl, piperazin-l-yl-(1-6C)alkyl, 4-(1-6C)alkylpiperazin-1-yl-(1-6C)alkyl, hydroxy-(2-6C)alkoxy-(1-6C)alkyl, (1-6C)alkoxy-(2-6C)alkoxy-(1-6C)alkyl, hydroxy-(2-6C)alkylamino-(1-6C)alkyl, (1-6C)alkoxy-(2-6C)alkylamino-(1-6C)alkyl, (1-6C)alkylthio-(1-6C)alkyl, hydroxy-(2-6C)alkylthio-(1-6C)alkyl, (1-6C)alkoxy-(2-6C)alkylthio-(1-6C)alkyl, phenoxy-(1-6C)alkyl, anilino-(1-6C)alkyl, phenylthio-(1-6C)alkyl, cyano-(1-6C)alkyl, halogen-(2-6C)alkoxy, hydroxy-(2-6C)alkoxy, (2-6C)alkanoyloxy-(2-6C)alkoxy, (1-6C)alkoxy-(2-6C)alkoxy, carboxy-(1-6C)alkoXy, (1-6C)alkoxycarbonyl-(1-6C)alkoxy, carbamoyl-(1-6C)alkoxy, N-(1-6C)alkylcarbamoyl-(1-6C)al-koxy, N,N-di-[(1-6C)alkyl]carbamoyl-(1-6C)alkoxy, amino-(2-6C)alkoxy, (1-6C)alkylamino-(2-6C)alkoxy, di-[(1-6C)alkyl]amino-(2-6C)alkoxy, (2-6C)alkanoyloxy, hydroxy-(2-6C)alkanoyloxy, (1-6C)alkoxy-(2-6C)alkanoyloxy, phenyl-(1-6C)alkoxy, phenoxy-(2-6C)alkoxy, anilino-(2-6C)alkoxy, phenylthio-(2-6C)alkoxy, piperidino-(2-6C)alkoxy, morpholino-(2-6C)alkoxy, piperazin-1-yl-(2-6C)alkoxy, 4-(1-6C)alkylpiperazin-l-yl-(2-6C)alkoxy, halogen-(2-6C)alkylamino, hydroxy-(2-6C)alkylamino, (2-6C)alkanoyloxy-(2-6C)alkylamino, (1-6C)alkoxy-(2-6C)alkylamino, carboxy-(1-6C)alkylamino, (1-6C)alkoxycarbonyl-(1-6C)alkylamino, carbamoyl-(1-6C)alkylamino, N-(1-6C)alkylcarbamoyl-(1-6C)alkylamino, N,N-di-{(1-6C)alkyl]carbamoyl-(1-6C)alkylamino, amino-(2-6C)alkylamino, (1-6C)alkylamino-(2-6C)alkylamino, di-[(1-6C)alkyi]amino-(2-6C)alkylamino, phenyl-(1-6C)alkylamino, phenoxy-(2-6C)alkylamino, anilino-(2-6C)alkylamino, phenylthio-(2-6C)alkylamino, (2-6C)alkanoylamino, (1-6C)alkoxycarbonylamino, (1-6C)alkylsulphonylamino, benzamido, benzenesulphonamido, 3-phenylureido, 2-oxopyrrolidin-1-yl, 2,5-dioxopyrrolidin-l-yl, halogen-(2-6C)alkanoylamino, hydroxy-(2-6C)alkanoylamino, (1-6C)alkoxy-(2-6C)alkanoylamino, carboxy-(2-6C)alkanoylamino, (1-6C)alkoxycarbonyl-(2-6C)alkanoylamino, carbamoyl-(2-6C)alkanoylamino, N-(1-6C)alkylcarbamoyl-(2-6C)alkanoylamino, N,N-di-[(1-6C)alkyl]carbamoyl-(2-6C)alkanoylamino, amino-(2-6C)alkanoylamino, (1-6C)alkylamino-(2-6C)alkanoylamino and di-[(1-6C)alkyl]amino-(2-6C)alkanoylamino, and wherein said benzamido or benzenesulphonamido substituent or any anilino, phenoxy or phenyl group in a R1 substituent may optionally bear one or two halogen, (1-6C)alkyl or (1-6C)alkoxy substituents.

33. The process of claim 32, wherein (R1)m is selected from the group consisting of 6-hydroxy, 7-hydroxy, 6,7-dihydroxy, 6-amino, 7-amino, 6-ureido, 6-trifluoromethoxy, 6-methyl, 6,7-dimethyl, 6-methoxy, 7-methoxy, 6,7-dimethoxy, 6,7-diethoxy, 6-hydroxy-7-methoxy, 7-hydroxy-6-methoxy, 6-amino-7-methoxy, 6-amino-7-methylthio, 5-amino-6,7-dimethoxy, 6-methoxy-7-isopropoxy, 6,7-methylenedioxy, 6,7-ethylenedioxy, 6-methylamino, 7-methylamino, 6-dimethyiamino, 6-amino-7-methylamino, 6-methoxymethyl, 6-bromomethyl, 6-(2-methoxyethoxymethyl), 6-cyanomethyl, 6-methylthiomethyl, 6-phenylthiomethyl, 7-(2-hydroxyethoxy)-6-methoxy, 6,7-di-(2-hydroxyethoxy), 6-(2-bromoethoxy), 6-(2-methoxyethoxy), 6-methoxy-7-(2-methoxyethoxy), 6,7-di-(2-methoxyethoxy), 7-(2-bromoethoxy)-6-methoxy, 7-benzyloxy-6-methoxy, 6-(2-methoxyethylamino), 6-acetamido, 6-benzamido, 6-(2-chloroacetamido), 6-(2-methoxyacetamido) and 7-(2-methoxyacetamido).

34. A process for preparing a 4-halogen-pyrimidine, comprising the steps of reacting pyrimidone or a pyrimidone derivative with a halogenating agent under conditions suitable for production of said 4-halogen-pyrimidine and isolating or purifying the end product by precipitation, crystallization or sublimation.

35. The process of claim 34, wherein said 4-halogen-pyrimidine is chloroquinazoline or a chloroquinazoline derivative, said pyrimidone derivative is quinazolone or a quinazolone derivative, and said halogenating agent is a chlorinating agent.

36. The process of claim 35, wherein said pyrimidone derivative is reacted with said chlorinating agent in the presence of a catalyst which promotes the formation of said chloroquinazoline or chloroquinazoline derivative and a trapping agent which removes free chlorine from the reaction.

37. The process of claim 36, wherein said catalyst is dimethylformamide and said trapping agent is toluene.

38. The process of claim 35, wherein said chlorinating agent is selected from the group consisting of thionyl chloride, phosphorus oxychloride and oxalyl chloride.

39. A process for preparing a 4-arylaminopyrimidine hydrochloride salt, comprising the steps of dissolving a substituted aniline in an ethanol solution, adding to said solution a chloropyrimidine under conditions which cause reaction with said substituted aniline, and isolating the resulting 4-arylaminopyrimidine hydrochloride salt from said solution.

40. The process of claim 39, wherein said 4-arylaminopyrimidine hydrochloride salt is a 4-arylamino quinazoline hydrochloride salt and said chloropyrimidine is chloroquinazoline or a chloroquinazoline derivative.

41. A process for preparing a 4-arylaminopyrimidine, comprising the steps of dissolving a substituted aniline in ethanol, adding to said solution a chloropyrimidine to react with said substituted aniline, adding an alkaline solution to said ethanol solution, and isolating said 4-arylaminopyrimidine.

42. The process of claim 41, wherein said 4-arylaminopyrimidine is a 4-arylamino quinazoline and said chloropyrimidine is chloroquinazoline or a chloroquinazoline derivative.

43. A process for preparing a 4-arylaminopyrimidine, comprising the steps of dissolving a substituted aniline in an ethanol solution, adding to said solution a chloropyrimidine under conditions which cause reaction with said substituted aniline, isolating the resulting 4-arylaminopyrimidine hydrochloride salt from said solution, dissolving said 4-arylamino quinazoline hydrochloride salt in an alkaline solution, and isolating the resulting 4-arylaminopyrimidine.

44. The process of claim 43, wherein said 4-arylaminopyrimidine is a 4-arylamino quinazoline, said 4-arylaminopyrimidine hydrochloride salt is a 4-arylamino quinazoline hydrochloride salt, and said chloropyrimidine is chloroquinazoline or a chloroquinazoline derivative.