MXPA96002680A - Treatment of disorders related to platelet derived growth factor, such commons, using growth receiver inhibitors derived from plate - Google Patents

Abstract

The present invention relates to compounds that can inhibit the activity of the platelet-derived growth factor receptor (PDGF-R), preferably said compounds also inhibit the activity of other members of the PDGF-R superfamily and are selective for the members. of the PDGF-R superfamily, the PDGF-R superfamily includes PDGF-R and Flt and KDR kinases related to PDGF-R, the preferred compounds are active on cell cultures to produce the activity of the PDGF-R and preferably one or more kinases related to PDGF-Rs, an example of a preferred compound, A10 (see formulas of group 1) and its ability to inhibit the growth of tumor cells in vivo is described, using the present application as a guide, others can be obtained compounds capable of inhibiting PDGF-R and preferably Flt and / or KDR, said compounds are preferably used to treat patients suffering from characterized cell proliferation disorders s for inappropriate PDGF-R activity

Description

TRIFICIENT PE RELEVANT DISORDERS WITH FRCTQR PE REFERRED REGIMENT? E PLROUETRS. SUCH AS CANCER, USING INHIBITORS OF GROWTH RECEIVER DERIVED FROM PLATELETS CRMPQ PE INVENTION The present invention relates to methods and compositions for treating disorders of cell proliferation characterized by a growth factor receptor activity derived from inappropriate platelets (PDGF-R).

SQLICITUP RELRCIQNRPfi The present application is a part of the Hirth et al., Entitled "TREATMENT OF PLATELET DERI ED GROWTH FACTOR RELRTED DISORDERS SUCH AS CANCERS" (TREATMENT OF DISORDERS RELATED TO GROWTH FACTOR DERIVED FROM PLATELETS, SUCH AS WITH CANCERES), E.U.A. Series No. 08 / 179,570, ^ posted on January 7, 1994, whose full content - including the drawings is incorporated herein by reference.

NTEQEPENTES PE Lfl INVENTION The platelet-derived growth factor receptor (PDTF-R) is a thryocin kinase of a transnsrnembrane receptor. The binding of ligands to the receptor results in the trimerization of two receptors that lead neraly to inter-cellular phosphorylation of each receptor, commonly known as α-tophosphorylation or transphosphorylation, and activation of the receptor complex. PDGF, which is a ligand for PDGF-R, is a dirneric protein having two polypeptide chains linked by disulfide bridges. Each polypeptide is either a chain A polypeptide or an E chain polypeptide. Therefore, PDGF can have either two A chains, two B chains or one A chain and one B chain. PDGF-R consists of two isomers s. and ß "The - receptors containing a and (3 have been associated with mitogen activity, whereas only the receptor having β has been associated with chemotaxis and actin reorganization (Heldin, CH, EMBO Journal 11: 4251-4259, 1992). with Píate and others, Laboratory Investigation 4: 529-534, 1992: PDGF is a potent growth factor for rnesenquirnatosas and neuroectodermal cells. The endothelial cells fc2 ns -'- cl0 considered as cells that do not correspond to PDGF, 'but a recent study has shown that PDGF may have a role in angiogenesis during the development of the placenta. In addition, PDGFR-b has been shown to be expressed in endothelial cells in inflammatory tissue and glial tumors. This suggests that PDGF may play a role in vascular functions under pathological conditions. CSe omit citations.] Helding, previously cited, describes the relationship of PDGF and its receptor, and describes the function of PDGF in the 4 > cancer, observing that certain cancers do not produce PDGF and have central .ecrosis. Heldin states: The adverse effects of PDGF in certain diseases as described above, make PDGF antagonists highly desirable. Recently, some approaches have been followed to develop such antagonists. Antibodies against PEDGF have been shown to be useful in inhibiting both autocrine stimulation in cells transformed in SSV and the atherosclerotic process that occurs after 'endothelialization of the carotid arteries of rats. In addition, a soluble form of the PDGF receptor has been shown to bind to PDGF and inactivate it, and therefore could potentially be useful in inhibiting the action of PDGF i vivo. Another approach would be to design or find agents that compete in an antagonistic manner with PDGF for receptor binding. In order to identify peptides that interfere with PDGF binding, peptides erivated from the B chain sequence were selected. The peptide was found to inhibit PDGF binding and autophosphorylation of alpha and beta receptors. However, the peptide also showed some cellular toxicity and further development will be necessary before the peptide antagonists become useful for in vivo studies. Low molecular weight compounds have been described that interfere with receptor binding, e.g., suramin; however, surarnin is not specific enough to be used clinically as a PDGF agonist. Recently it has been found that another low molecular weight compound, neomycin, at high concentrations, inhibits the binding of PDGF-BB to the α- receptor, but not a binding to the β receptor. This compound, therefore, represents an antagonist that distinguishes between the two types of receptors; however, its low power makes it unsuitable for use in vivo. It is hoped that the experiments with surarnin and neomycin will help the future design of more powerful and specific PDGF receptor antagonists. The design of antagonistic Chemis would be greatly facilitated by the elucidation of the three-dimensional structure of the PDGF receptor complex. The antagonistic activity of PDGF could also be achieved by the inhibition of PDGF receptor directing. By way of hypothesis, the rnonomeric PDGF might not induce the dimerization of the receptor and therefore could have antagonistic activity. Since the reduction of PDGF results in loss of linkage of the receptor, disulfide bonds between chains are tried in order to debris and thus avoid ligand dimerization. This turns out to be very difficult to the high density of system residues in PDGF. The approach that ultimately succeeded included the partial reduction of the PDGF molecule using a concentration of lithium reitol that reduced only the interchain disulfide bonds, and did not affect the interchain bonds. By this procedure, the second and fourth system residues of the N-terminal group were found to be the two links between chains in PDGF. Analysis of a B-chain mutant of PDGF in which these two system residues had resolved to serine residues, revealed that it retained receptor binding activity. Is it a receptor antagonist ?. The answer is no; in fact, monomeric PDGF induced both dimerization and "autophosphorylation of the receptor. This result may indicate that PDGF-induced receptor dimerization was only a matter of bridge formation between two receptor molecules: dimerization may also involve a ligand-induced conformational change of the extracellular domains of receptors that promote protein interactions. receiver to receiver. One possible way to achieve an antagonist effect, which is currently being explored, is to combine a chain of wild type PDGF with a mutated chain that does not bind to the PDGF receptor but that can actively prevent the dimerization of lops receptors. Spada A.P., et al., Entitled "Bis Mono- and Bicyclic Aryl and Heteroaryl Cornpounds Which Inhibit EGF and / or PDGF Receptor Tyrosine Kinase," PCT / US92 / 03736, mentions the use of certain bis and mono bicyclic aryl compounds. According to Spada: According to the present invention there is provided a method for inhibiting the proliferation of normal cells in a patient suffering from a disorder characterized by said proliferation comprising administering to a patient an effective inhibitory amount of an EGF receptor and / or PDGF an aryl and / or heteroaryl and / or bicyclic heteroaryl compound showing inhibition of tyrosine kinase protein, wherein each aryl and / or heteroaryl group is a ring system containing 0-4 heterogeneous atoms, said compound being optionally substituted or polysubstituted.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to compounds that They can inhibit the activity of the platelet-derived growth factor receptor (PDGF-R), preferably said compounds also inhibit the activity of other members of the PDGF-R superfarnil and are selective for members of the PDGF-R superfarnil. The PDGF-R superfamily includes PDGF-R and PDGF-R the kinases related to Fit, and KDR.

Preferred compounds are active in cell cultures to reduce the activity of PDGF-R and preferably one or more active compounds. Their ability to inhibit the growth of turnoral and living cells is described below. Using the present application as a guide, other compounds capable of inhibiting PDGF-R and preferably Fit and / or KDR can be obtained. Such compounds are preferably used to treat patients suffering from cell proliferation disorders characterized by inappropriate PDGF-R activity. The proliferation of unwanted cells can result from the inappropriate PDGF-R activity that runs in different types of cells including cancer cells, cells that surround cancer cells (stomach cells), endothelial and smooth muscle cells. For example, an increase in PDGF-R activity of endothelial cells surrounding cancer cells can induce an increase in tumor vascularization, thus facilitating the growth of cancer cells. Therefore, inappropriate PDGF-R activity can contribute to a disorder of cell proliferation in different ways such as by increasing the production of growth factors, causing the aberrant growth of a cell, and increasing the formation and dispersion of blood vessels in solid tumors thus supporting tumor growth. Other members of the PDGF-R superfarminal are also involved in the support of the turnoral growth. The The members of the PDGF-R superfamily have a kinase domain that contains at least 45% frequency similarity to the kinase domain of PDGF-R a or β. Vascular endothelial growth factor (VEGF) activates at least the tyrosine kinase receptors; Flk-1 or its human counterpart, KDR, and Flt-1. These two receptors are expressed in endothelial cells and appear to be important in angiogenesis. Píate et al., Nature 359: 845-848, 1992; Shweiki, et al., Nature 359: 845, 1992; llauer et al., cell 72: 835-846, 1993; Píate et al., Cancer Res-, 53: 5822-5827, 1993; Waltenberger et al., Journal of Biological Chemistry 43: 26988-26995, 1994. Vascularization is essential for solid tumor growth and is thought to be regulated by tumor cell factors that have quiniotactic and / or mitogenic endothelial cell effects. PDGF-R KDR and Flt-I are all involved in the formation of blood vessels and the dissemination and feeding of solid tumors. By inhibiting both PDGF-R and one or more related Wrosine kinase activities, the growth of aberrant cells and the limitation of such growth can be inhibited. Many examples of compounds (formulas of group 1) belonging to the important groups (chemical structures of groups 1-10) are described. Those skilled in the art can obtain other compounds, to inhibit PDGF-R and preferably Fit and / or KDR, which have equivalent or greater activity in those receptor tyrosine kinases using the present compounds belonging to important groups (chemical structures of groups 1-10) for equivalent activity. Using normal tests, the site of action of any of the compounds described below can be determined, as well as other active compounds in the same site. The methods and compositions are designed to inhibit the proliferation of unwanted cells by altering the activity of PDGF-R, and preferably also alternating the activity of Fit and / or KDR. Without being limited by any theory, the inhibition of unwanted cell proliferation can lead to altered activity of PDGF-R (e.g., inhibiting tyrosine phosphorylation, PDGF-R, inhibiting binding of substrate protein or adapter to receptor , or inhibiting other signaling events downstream), thus inhibiting the activity of PDGF.-R. However, unless otherwise specified, the use of all and claimed compositions are not limited to this particular theory. Therefore, a first aspect of the present invention includes a method for treating a patient suffering from a cell proliferation disorder characterized by inappropriate PDGF-R activity. The method involves the step of administering to the patient a therapeutically effective amount of a composition comprising a compound of formula II-Aj, or the active compound formed of this type is placed under physiological conditions (i.e., the active structural entity of a pro - drug described above) administration of a particular compound is achieved by providing a particular compound to the patient or by providing a prodrug of the compound to a patient forming the particular compound in vivo "cell proliferation disorders" refers to to disorders where the unwanted proliferation of JÍÉulas of one or more subsets of cells in a multicellular orgamsrno occurs resulting in damage "eg discomfort or loss of hope due to the multicellular organism. Cells can occur in different types of animals and in humans. n of cells including cancers, disorders of proliferation of blood vessels and fibrotic disorders. "Inappropriate PDGF-R activity" refers to 1) that of PDGF-R in cells that do not normally express PDGF-R; 2) expression of PEDGF by cells that do not normally express PDGF; 3) increased PDGF-R expression that leads to unwanted cell proliferation; 4) increased PDGF expression that leads to the proliferation of unwanted cells; or 5) mutations that lead to the constitutive activation of PDGF-R. The existence of inappropriate or abnormal PDGF and PDGF-R levels or activities thereof is determined by methods well known in the art. The compositions can be used to treat a cell proliferation disorder by administering a therapeutically effective amount of the composition to a patient (i.e., a human or animal having a cell proliferation disorder). The compositions can also be used in in vitro studies the mechanism of action of PDGF-R or PDGF itself. A "therapeutically effective amount", in reference to the treatment of a cancer, refers to an amount sufficient to carry out one or more of the following results: reduce the size of the cancer, inhibit the metastasis of the cancer, inhibit the treatment of the cancer, retain cancer growth, alleviate discomfort due to cancer, or prolong the life of an affected patient poicancer. A "therapeutically effective amount", in "Non-specificity to the treatment of a cell proliferation disorder other than cancer refers to an amount sufficient to produce one or more of the following results: inhibit the growth of the cells that produce the disorder, alleviate the discomfort due to the disorder or prolong the life of a patient suffering from said disorder. "Significant inhibition" of the tyrosine kinase activity of a receptor refers to a less than or equal to 75 μM using one or more of the tests described in the "Examples" above. cited. Preferably, the compound can inhibit the activity of PDGF-R with a Clase less than or equal to 50μM, most preferably less than or equal to 1μM, most preferably still less than 1μM. The lower ICSAs are preferred because the CIso provides an indication as to the in vivo effectiveness of the compound. Other compounds known in the art, such as the half-life, biodistribution and toxicity of the compound should also | be used for therapeutic uses. Such factors may allow a "" compound or a lower Cl-sa to have a higher efficacy than a compound having a higher value. Selective inhibition of PDGF-R superfarnil is achieved by significantly inhibiting the activity of PDGF-R, while having an insignificant effect (ie, a CISo for tyrosine phosphorylation greater than 100 μM or EGF-R. another member of the superfarmilia d'GF-R is significantly inhibited.Preferably, the compound is either A10, All, A12, Al3 BIO, Bll, B12, B13, B14, B15, B16, B17, B18, B19, CIO CU, C13, Dll, D12, D13, D14, D15, D16, D17, D18, D20 E10 , Eli, E12, E13, E14, E15, E16, FIO, Fll, F12, G10 Gil, G12, G13, G14, G15, G16, G17, Glf G19, G20, G21, G22, G23, G24, G25, G27 , G28, G29, G30, H12, 110, Jll, PlO, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25? or the active drug of said compounds, or pharmaceutically acceptable salts thereof. The compound is preferably used in a pharmaceutical composition formed by mixing one of the above compounds and a physiologically acceptable carrier. A physiologically acceptable carrier is a formulation to which it is compounded may be added to solver or otherwise facilitate the administration of the compound. Examples of physiologically acceptable vehicles include water, saline, physiologically regulated solution in their pH, cyclodextrins and PBTEsDdW. Hydrophobic compounds such as AlO are preferably administered using a carrier such as PBTE: D5W. An important factor in the choice of a suitable physiologically acceptable vehicle is to choose a vehicle in which the compound remains active or the combination of the vehicle and the compound produces a J active play. The compound can also be administered in a continuous manner using a slow release formulation or pump to maintain a constant or variable drug level in a patient. • Another aspect of the present invention includes a method of treating a patient suffering from a cell proliferation disorder characterized by the activity of Inappropriate PDGF-R using AlO, A12, or Bll. The method involves a therapeutically amount. Another aspect of the present invention describes a method for treating a patient suffering from a cancer characterized by inappropriate PDGF-R activity using combination therapy. The combination therapy is carried out using one or more agents described herein in conjunction with normal anticancer agents. The method is carried out by administering to a cancer patient an effective therapeutic amount of a composition comprising an agent capable of significantly inhibiting the activity of PDGF-R and a cytotoxic agent. Preferably, the cytotoxic agent is VP-16 or cisplatin. Most preferably, the cytotoxic agent is cisplatin and the cancer is lung cancer. Another aspect of the present invention includes a method for treating a patient having a cell proliferation disorder characterized by inappropriate PDGF-R activity using mutated PDGF-R, or nucleic acid that purifies ift PDGF-R mutated. The "mutated" PDGF-R refers to PDGF-R wherein one or more anirno acids are missing or altered.

As illustrated below, a nucleic acid encoding a Mutated PDGF-R (ie, truncated one) lacking a kinase domain can inhibit turnoral growth in vivo. He Mutated PDGF-R can be administered as a protein, or recominant nucleic acid that encodes the protein and expresses the protein within a cell. In other aspects, the invention includes "novel" compositions that include one of the preferred compounds described herein and the PBTE vehicle: D5W wherein the preferred compound is soluble in PBTE: D5W; and the novel compounds BIO, B12 CIO, CU, E10, Eli, E12, E13, E14, E15, E16, FIO, Fll, F12, G21, G22, Hll, H12, H13 and H14 Other characteristics and advantages of the invention will be evident from the following description of the preferred embodiments thereof, and from the indications.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. NIH3T3 cells overexpressing the human PDGF-b (A) receptor or the human EGF (B) receptor were treated with AlO as indicated. The percentage of cells in the S phase of the cell cycle was determined by flow cytometry. Figure 2. In two separate experiments, 06 cells (1 x 10 cells in 4μL) were implanted in the brain of BALB / c, nu / nu mice. AlO was administered IP in 100 μL of PBTE: D5W at the indicated doses on each day beginning one day after implantation. V = vehicle control, n = 8 to 12 (Expt # 1) or 5 (Expt # 2) animals per group. + P < 0.00001; * • * P < 0.002; * • * - * • P < 0.02 compared to the vehicle control. Figure 3. In two separate experiments, C6 cells (5 x 10"* cells in 20 μL CExpt # 13 or 5 μL CExpt # 23) were implanted in the brain of rats atirnicae.AlO was administered IP in 500 μL of PBTE at the doses indicated daily beginning one day after implantation V = vehicle control, n = 7 to 8 animals per group. * P = 0.0002; * • * P = 0.0003 compared to the vehicle control.

PESCRIPCIQN PETflLLflPfl PE LR INVENTION The present invention includes methods and compositions for treating cell proliferation disorders characterized by inappropriate PDGF-R activity. The present application demonstrates the ability of compounds capable of significantly inhibiting the activity of Flt-1 and / or PDGF-R, and provides examples of such compounds useful for treating a proliferation disorder, such as cancer. The compounds described herein can be used in the treatment of other proliferation conditions associated with the inappropriate expression of PDGF-R, for example, disorders of blood vessel proliferation and fibrotic disorders characterized by PDGF-R activity. Using the present disclosure as a guide, those skilled in the art can readily determine which of the compounds described herein are useful for treating a particular proliferative disease. A single objective site, the presence of activity of "Inappropriate PDGF-R, for a large number of disorders of many * objectives proposed in the art together with compounds capable of inhibiting the activity of PDGF-R and preferably the activity of Fit and / or KDR are described in the present application. Preferably, the activity of PDGF-R together with KDR and / or Fit activities are inhibited by a single compound such as AlO. Combinations of compounds or types of treatments can also be used to guide different tyrosine kinases related to PDGF-R. Examples of such combinations include the use of compounds inhibiting PDGF-, R activity together with a KDR inhibitor compound, and the use of PDGF-R nucleic acid to inhibit the production of PDGF-R together with a KDR inhibitor compound. . The compounds (also known heretofore as "drugs") useful in this invention belong at least to eight different groups. The preferred compounds of these groups, and in others as indefinite groups, which generally have shown significant inhibition of pyre receptor activity PDGF are shown in formulas of groups 1-11. Although the generic formulas are presented, those skilled in the art will recognize that those compounds useful in the invention can be determined by selecting methods described in present and known in the art. The ability of AlO, truncated versions of a PDGF-R compound or other compounds to inhibit tumor growth in animals illustrates the effectiveness and efficacy of these? CFomposites. These animal studies support the effectiveness of the compounds demonstrating that the compounds can be effective in animals despite several problems that are inherently associated with the use of compounds in animals to treat a particular condition. The inherent problems include the animal that is integrated by a ?! =; heterogeneous cell population, various pharmacological considerations such as bioavailability of the compound, half-life of the compound and range of the compound. These inherent often prevent a compound from exerting a physiological effect. Below are examples that illustrate the ability of several compounds to inhibit phosphorylation of PDGF-R. Examples are also provided illustrating the ability of the compound named AlO (see formulas A10-A13) to inhibit cancers in vivo. Rather, using the present disclosure as a guide, one skilled in the art can use the preferred methods and compositions to obtain compounds Additional hibi ores and facing other cell proliferation disorders characterized by an activity of PDGF-R inappropriate.

I. Superfamily PDGF-R fl. PDGF-R activity The binding of ligands to PDGF-R induces the formation of dimeric receptors and allosteric changes that activate the intracellular kinase domains, and results in transphosphorylation and / or autophosphorylation of the receptor or tyrosine residues. Phosphorylation of the receptor stimulates a physical association of the activated receptor with target molecules. Some of the target molecules are in turn phosphorylated, which transmits the signal to the cytoplasm. Other target molecules are not phosphorylated, but they help in signal transmission by acting as cutting molecules or for secondary signal transducing proteins. Secondary signal transducing molecules generated by activated receptors result in a cascade of signals that regulate cell functions such as cell division (See, FfV M.3 and others.) P otein Science 2: 1785-1797. 1993). Therefore, an increase in the activity of PDGF-R is characterized by an increase in one or more of the activities that may occur under ligand binding of PDGF-? 1) phosphorylation or autophosphorylation of PDGF-R, 2) phosphorylation of a substrate of PDGF-R (e.g., PL 3-kinase, RasGAP, PLCg, see Frv above supra), 3) activation of an adapter molecule, and 4) increased cell division. These activities can be measured using techniques described below and known in the art. For example, the autophosphorylation of PDGF-R can be determined as described in the examples given below using an antibody t-phosphotyrosine, and increased cell division can be performed as described below by measuring the incorporation of aH- tynidin in DNA Preferably, the increase in PDGF-R activity is associated with an increased amount of phosphorylated PDGF-R and DNA synthesis.

, Kinases related to PP6F-R * / The kinases related to PDGF-R Flt-1 and KDR can be activated by VEGF. VEGF is a rnonodimeric glycoprotein with structural homology to PDGF. Four different splice variants of VEGF have been isolated. Rosenthal, i O ros. Growth Fae ón ?, 4; 53-52, 1990; Qonn V rtrps. prpq, N + .lr Rca == - (USR?, 87s3.323-I327., 19907 HowK, others,? J,. Endocrinol .. 5: 1806-1814.1991, two are segregated forms and two remain associated with cells. has shown to be?> bregulated by hypoxia and acts specifically on endothelial cells, Piette et al., Nature, 359: 845-848, 1992: Sh? eike., et al., Nature 359: 843-845, 1992. KDR activity. is characterized by an increase in one or more of the activities that may occur under VEGF ligand binding: 1) phosphorylation or autophosphorylation of KDR, 2) phosphorylation of a KDR substrate, 3) activation of an adapter molecule, and 4) Implied cell division »The activity of Flt-1 is characterized by an increase in one or more of the activities that can occur- under the binding of the VEGF ligand: 1) phosphorylation or autophosphorylation of Flt-1, 2) phosphorylation of a Flt substrate -1, 3) activation of an adapter molecule, and 4) increased cell division. ii, CQ? PUESTQS PREFFRXPOS n Compounds from groups 1 to 11 are shown at the beginning of each paragraph A to I and in K.

R. COMPUESTOS PEL CRUPO I The compounds of group 1 have the following where R ^, & > "RX, R" 2, and &"X are independently selected from the group consisting of hydrogen, halogen, triahalogenomethyl, and N02, preferably R ^ and Rs are independently CF3, O? O hydrogen, and R's, R 'X, and R 'X pn hydrogen, and R3 is selected from the group consisting of hydrogen, carboxy, alkoxy or carboalkoxy, preferably hydrogen, carboxyl or methyl Examples of compounds of group 1 are listed in Table I and are shown in the formulas , All, A12 and A13.

GROUP 1 A11 A13 TABLE I «Fc It is believed that these compounds act as drugs because the ring is cleaved in vivo to produce active B. GROUP COMPOUNDS 2 The group compounds have the following wherein R ^ and Rs are independently halogen, hydrogen, trihalogenomethyl, or NO ^, preferably R ^ is CF3 and Rs is H; R & is either aryl, alkyl, alkenyl or alkynyl; R_s. is alkyl or one of the substituents of the compounds listed in Table II. Examples of the compounds of group 2 are listed in table II and are shown in formulas B10-B19 B19 QUPPRQ II V5rn position ^ BIO NOs H CHS Bll CF3 H CH: B12 CFa H 4-Fluorophenyl B13 CFS H cyclohexyl B14 CFa H 2,2,3,3-tetra- rnet i 1 cyclopropyl BIS CF-H pentafl orophenyl • ML TB316 CF, 3-phenoxy phenyl B17 CF, H benzyl B18 CF, H 2 -methylpropyl B19 CF, H diphenylmethyl O. COMPOUNDS OF GROUP 3 The compounds of group 3 have the following NH? wherein R-r, RX, and RT are independently halogen, OH, hydrogen, alkoxy, SH, NHS, or C (CH3) 3, preferably RX, R = and R is independently selected from H, OH and C (CH3) 3; most preferably R-r and R are OH; R > is aryl or hydrogen, preferably hydrogen or phenyl. Examples E compounds of group 3 are listed in table III and are shown in formulas CIO, Cll and C13. GROUP 3 C13 TABLE III D. COMPOUNDS OF GROUP 4 The compounds of the group have the following where 1D is either = S, = 0, SH, OH, or MHS; and Ra.a- R SH, OH, NH2, or dihydroxyphenyl; or R o and R a. taken together are aryl, preferably 3-amino-4-cyano-5-pyrazole or l-phenyl-3-arnino-4-cyano-5-pyrazole; and R 22 is hydrogen, aryl, alkyl, alkenyl or alkynyl, preferably hydrogen, - (CH ^) sC - a (CHS) X (CH3) a - Examples of compounds of group 4 are listed in table IV and are shown in the formulas Dll - D18, D20.

CUflPRQ IV E. COMPOSED OF GROUP 5 The compounds of group 5 have the following wherein R ^ a, Rx ^, R s, R < s, R -r, and Rβ are independently hydrogen, halogen, alkoxy, OH, < arnino, alkylamino or SH; preferably hydrogen or OH. Examples of the compounds of group 5 are listed in when V and are shown in formulas E10-E16.

CUflPRQ V F. COMPOUNDS OF GROUP 6 The compounds of group 6 have the following chemical structure: where R -5 > is aryl, alkyl, alkenyl or alkynyl, preferably 2- (3,4-dihydroxyphenyl) ethyl; R 5 is an alkyl, preferably ethylenehydroxy; or R - = > and Rao are together aryl, preferably a ring of rnorpholine having a substituent = CH- (mono or dihydroxyphenyl).

Examples of compounds of group 6 are set forth in table VI and are shown in formulas FIO, Fll and F12.

F12 TABLE VI G. COMPOUNDS OF GROUP 7 ^ rT ** The compounds of group 7 have the following chemical formula: wherein b is an optional pi bond, * Y and Z are independently carbon or nitrogen; Rs > and R22 are independently hydrogen, halogen, OH, SH, NHs, Os, alkyl, alkenyl, alkynyl, alkoxy, benzoyl, COOH, or carbalkoxy, preferably OH, NOs, CHs, rnetoxy, benzolyl or COOH; or RsX and Rss > together they form an ar-ornatic ring to give an aryl, preferably phenyl; R23 is hydrogen, halogen, = 0, OH, SH, NHS, alkoxy, COOH, aryl, preferably a substituted or unbranched anilino, a substituted or unsubstituted phenyl, hydrogen, COOH, = 0, alkoxy, or rnetoxy, provided that if - ^ is = O b is present as a link; R2 is hydrogen, or aryl, preferably a substituted or unsubstituted anilino, phenyl or 2-thienyl; and R2s is hydrogen, halogen, = S, or 0 =, where if »is = 0 or = S, b is present as a bond; provided that if b is not bond, the adjacent nitrogen optionally has a substituent selected from that consisting of hydrogen, alkyleneamino, alkylenenarinoalkyl and alkylenenocyano. Examples of compounds of group 7 are set forth in table VII and are shown in the formula G10-G25, G27-G30.

TABLE VII H. GROUP COMPOUNDS 8 The compounds of group 8 have the following chemical structure: wherein R26 and RHB are independently alkyl, aryl, alkenyl or alkynyl; and RST is aryl. Examples of compounds of group 8 are set forth in table 8 and are shown in formulas H10-H14.

- ^^ F GROUP 8 TABLE VIII COMPOUNDS PE GROUP 9 The compounds of group 9 have the following chemical structure: wherein R30 is either alkyl, alkenyl, alkynyl, preferably CH3; R3X is aryl, preferably phenyl; And Ras is either O or S. An example of a compound of group 8 is 110, shown in formula 110. 3. COMPOUNDS OF GROUP 10 The compounds of group 10 have the following chemical structure: wherein R3s is alkyl or aryl; R3A, Ras, and ao are independently halogen, OH,? &Idrogen, alkoxy, SH, NH = or C (CHa) 3, preferably R3 ^., Ss, and s * are independently selected from H, OH and C (CH3 )3. Examples of compounds of group 10 are 310 and 311, shown in formulas 310 and 311.

GROUP 10 J10 K, COMPOUNDS OF GROUP 11 Examples of compounds of group 11 are shown in the formulas PlO, P12-P25. The compounds of group 11 are identified by means of a "P". * w ^ ' P25 L- CHEMICAL NOMENCLATURE XJr 'The definitions of some of the chemical groups mentioned in the application are described below: An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight chain, branched chain and cyclic alkyl groups. Preferably, the alkyl group has from 1 to 12 carbon atoms, most preferably from 3 to 9 carbon atoms. The alkyl group can be substituted or unsubstituted. When substituted, the substituted group is preferably hydroxyl, cyano, alkoxy, = 0, = S, NOs or N (CH3) s, arnino, SH, or aryl. An "alkenyl" group refers to a hydrocarbon group containing at least one carbon-carbon double bond, including straight chain, branched chain and cyclic groups. Preferably, the alkenyl group has from 1 to 12 carbon atoms, most preferably from 3 to 9 carbon atoms. The alkenyl group can be substituted or not • ^^ substituted. When substituted, the substituted group is N (CH3) s, arnino, SH or aryl. An "alkynyl" group refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight chain, branched chain and cyclic groups. Preferably, the alkynyl group has from 1 to 12 carbon atoms, most preferably from 3 to 9 carbon atoms. The alkynyl group can be substituted or unsubstituted. When substituted, the substituted group is preferably hydroxyl, cyano, alkoxy, = 0, = S, NOS or N (CH3) s, arnino, SH, or aryl. An "alkoxy" group refers to a group "-O-alkyl" wherein "alkyl" is defined as described above. An "aryl" refers to an aromatic group having at least one ring that has an electron and conjugate system and includes carbocyclic aryl, heterocyclic aryl, and biaryl groups, all of which may be substituted. Preferred substituents of aryl groups are hydroxyl, cyano, alkoxy, alkyl, alkenyl, alkynyl, arnino and aryl. The carbocyclic aryl groups are groups in which the ring atoms in the aromatic ring are carbon atoms.

The carbon atoms are optionally substituted. Carbicyclic aryl groups include rnonocicly carbocyclic aryl groups and optionally substituted naphthyl groups. The heterocyclic aryl groups are groups that have If ^ * - from 1 to 3 heterogeneous atoms as ring atoms in the aromatic ring and the rest of the ring atoms are carbon atoms. Suitable heterogeneous atoms include oxygen, sulfur and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrirnidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. A "carbalkoxy" group refers to a COOX group, wherein "X" is a lower alkyl group. * Q The term "lower" referred to herein in connection with organic radicals or organic compounds respectively defines the same up to and including 7, preferably up to and including 4, and advantageously one or two carbon atoms. These groups can be straight or branched chain II Proliferative cell disorders The compositions and methods described are designed to inhibit cell proliferation diseases by inhibiting the activity of PDGFR. As described above, proliferation disorders result in unwanted cell proliferation of one or more subsets of cells in a multicellular organism - resulting in organ damage. Inappropriate PDGF activity can stimulate cell proliferation disorders. Two ways in which inappropriate PDGF or PDGF-R activity can • * stimulate the unwanted cell proliferation of a particular type of cells by directly stimulating growth of the particular cell, or by increasing the vascularization of a particular area, such as turnorral tissue, thus facilitating tissue growth. The use of the present invention is facilitated by first identifying whether the cell proliferation disorder is driven by PDGF. Once such disorders are identified, patients suffering from such disorders # can be identified by analysis of their symptoms or by well-known procedures by doctors. Such patients can be treated as described herein. The determination of any cell proliferation disorder that is driven by PDGF-R can be carried out by first determining the level of PDGF-R activity that occurs in the cell or in a particular body site. For example, in the case of cancer cells, -1 level of one or more activities of PDGF-R is compared for cancers not driven by PDGF-R (eg R431 cells as described below) and cancers driven by PDGF-R (eg, glioblastoma cells T98G as described below). If cancer cells have a higher level of PDGF-R activity than cancers not driven by PDGF-R, preferably equal to or greater than the PDGF-R-driven cancers, then can be used for treatment of PDGF-R described. cell proliferation disorders that arise due to unwanted proliferation of non-cancerous cells, the level of PDGF-R activity is prepared at the level that occurs in the general population (eg, the average level occurs in the general population of people or animals excluding those persons or animals suffering from a cell proliferation disorder). If the disorder unwanted cell proliferation is characterized by a level If PDGF-R is superior to that occurring in the general population, then the disorder can be considered for treatment using the PDGF-R inhibitors described. Cell proliferation disorders include cancers, blood vessel proliferation disorders and fibrotic disorders. These disorders are not necessarily independent. For example, fibrotic disorders can be related or overlap with blood vessel disorders. For example, atherosclerosis (characterized J ßor a blood vessel disorder) results in the abnormal formation of fibrous tissue. A cancer cell refers to several types of malignant neoplasms, most of which can invade surrounding tissues, and can spread by metastasis to different sites, as defined by Stedman's Medical Dictionary Stedman's Medical Dictionary 25a. edition (Hensyl ed. 1990). Examples of cancers that can be treated with of prostate, lung cancers, Kaposi's sarcoma, and skin cancers, which may have inappropriate PDGF-R activity. These types of cancers can also be characterized. For example, intraaxial cancers include glioblastorna multiforme, anaplastic atrocytoma, astrocytoma, ependirnorna, oligodendroglioma, eduloblast a, meningliorna, sarcoma, hernangioblast a and pineal parenquirnal. The formation and dissemination of blood vessels, or vasculogenesis and angiogenesis respectively, play important roles in a variety of physiological procedures such as embryonic development, wound healing and organ regeneration. They also play a role in the development of cancer. Blood vessel proliferation disorders refer to angiogenic and vasculogenic disorders generally resulting in the abnormal proliferation of blood vessels. Examples of He said disorders include restenosis, retinopathy and atherosclerosis. Advanced atherosclerosis lesions result from an excessive inflammatory proliferative response to a damage to the endothelium and smooth muscle of the arterial wall (Ross R., Nature 362: 801-809 (1993).). Part of the response seems to be mediated by secretion of PDGF-BB, and the activation of PDGF-R in endothelial and smooth muscle cells. Both the migration of cells and the proliferation of cells played a role in the formation of atherosclerotic lesions. Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include liver cirrhosis and mesanglial cell proliferation disorders. Liver cirrhosis is characterized by the increase in constituents of the extracellular matrix resulting in the formation of a hepatic scar. Liver cirrhosis can produce diseases such as cirrhosis of the liver. An increased extracellular matrix that results in a hepatic scar may also be caused by viral infection such as hepatitis. Lipocytes seem to play an important role in liver cirrhosis. Inappropriate PDGF-R activity can stimulate the proliferation of lipocytes. The proliferation disorders of rnesanglial cells refer to disorders produced by the abnormal proliferation of rnesanglial cells. Slangular proliferation disorders include several human renal diseases, such as glornerulonephritis, diabetic nephropathy, malignant nephrosclerosis, syndromes of thrombotic disease, rejection of transplant and glornerulopathies. PDGF has been implicated in the maintenance of mesanglial cell proliferation (Floege, 3. et al., Kidney International 43S: 47-54 (1993).) As noted above, other rheumatization diseases of this type can be identified by from Fté.cnicas normal, and by determining the effectiveness of the action of the compounds described. ft Ovarian cancer One aspect of the invention relates to the treatment of ovarian cancer. Epithelial ovarian cancer accounts for almost 90% of all ovarian tumors and remains a highly lethal malignancy. Approximately 19,000 new cases of ovarian cancer are diagnosed in the United States each year, and 12,000 of these women will die of cancer (Rodriguez et al., in DeVita, Hellrnan, Rosenberg (eds) Biologic Therapy of Cancer, 3B Lippincott, 1991) . The treatment of advanced ovarian cancer generally includes cyto-productive surgery followed by combination chemotherapy with alkylating agents such as cisplatin and cyclophosphamide. However, the long-term survival of patients with advanced ovarian cancer is extremely deficient, on a scale of 10% to 20%, mainly due to the incidence of etastatic tumors through the peritoneal cavity and in some non-lymph nodes. Furthermore, chemotherapy with cisplatin carries a potential for renal toxicity and progressive neuropathy. The invention reveals a pathological relationship between the expression of the PDGF receptor and ovarian epithelial cancer, and provides compositions and methods for inhibiting inappropriate PDGF-R activity in epithelial ovarian cancer cells to inhibit the proliferation of the disease. Methods of treating ovarian cancer comprise administering a composition that inhibits inappropriate PDGF-R activity in ovarian carcinoma cells, in supporting stromal cells (i.e., the framework over which a tumor or a rnetastatic lesion grows, including but not limited to connective tissue and vascular endothelial cells), and / or in associated vascular fibricothelial cells. Ovarian cancers susceptible to treatment with the compounds described herein include ovarian epithelial carcinoma, ovarian tumor metastasis, and other ovarian cells expressing PDGF receptors. As described below, compositions that inhibit the activity of PDGF-R also inhibit the proliferation of ovarian cancer cells in vitro and inhibit the growth of ovarian tumors in vivo. Very specifically, the use of a of the invention, RIO, results in the almost complete inhibition of ovarian tumor growth in mice to which human ovarian cancer cells are xenotransplanted, without significant cytotoxicity or mortality, thus providing a drastic therapeutic effect. Accordingly, as an example of the method of the invention, RIO is administered to a patient who has been diagnosed with ovarian cancer through any route of any appropriate pharmaceutical carrier that brings AlO into contact with positive ovarian cancer cells. for PDGF receptor and / or surrounding estrone cells. In view of the localized spread of ovarian cancer throughout the peritoneal cavity, a preferred method of administration, particularly in advanced cases, is by intravenous or intraperitoneal injection of a non-toxic pharmaceutical formulation of AlO.

The preparation and use of effective compositions for treating ovarian cancers are described in detail in the following and by way of example, mentioned above. In addition to the compositions specifically described herein, the invention provides for the identification of other compositions which, due to their inhibitory effect on the activity of PDGF-R, may be useful for inhibiting the proliferation of ovarian neoplasms. Possible compositions can be identified by their ability to inhibit autophosphorylation of the PDGF receptor by using any suitable test, such as ELISR tests of inhibition of autophosphorylation and vitro inhibition of tyrosine kinase. Possible compositions can be evaluated for therapeutic efficacy by testing their ability to inhibit the growth of ovarian cancer cells, and ideally by testing the inhibition of xenografted tumors in vivo. The procedures described in the aforementioned examples, or similar procedures, can be employed by introducing said tests.

B Glioma Another aspect of the invention relates to the treatment of primary intraaxial brain tumors of the gliorna family, including but not limited to astrocytomas and glioblastomas. Glioblastoma multiforme is the most common and malignant tumor of astrocytic origin in adult Ernes and represents more than half of all primary brain tumors (see, for example, Cecil Texbook of Medicine, Uyngaarden,? Rnith, Bennett (eds.) WB Saunders, 1992, p 2220). The gliornas have the common property of directing the invasive wrapping of brain tissue, and they are fundamentally malignant, and are fatally fatal. Patients with glioblastoma have an average survival time of less than one year even when they are treated aggressively Bte a combination of surgery, chemotherapy and radiotherapy, unfortunately, successful surgery is extremely rare in view of the difficulty or inability to define microscopic limits of a glioma within normal brain tissue. Similarly, chemotherapy with alkylating agents has been achieved with very little success and no more than 10% of patients with gliorna respond significantly. Radiation therapy has shown some value in the control of the growth of gliona, but often results in The substantial neurological process. Therapy with interferon-ß, in combination with radiotherapy and chemotherapy, has had some success (DeVita, Helinan, Rosenberg (eds) Biologic Therapy of Cancer, 3B Lippincott, 1991). The invention discloses a pathological relationship between the expression of PDGF receptor and glioma, and provides compositions and methods for inhibiting the activity of PDGF in gliorna cells to inhibit the proliferation of the disease. All of the glyoxin treatment comprises administering a composition that inhibits the activity of PDGF-R expressed in glioma cells and / or in nearby vascular endothelial cells. In particular, most of the compositions specifically described herein are highly active to inhibit autophosphorylation of PDGF receptor in human gliorna cells in vitro. Some of these compositions inhibit the growth of cultured gliorna cells, and one of these, RIO, also inhibits the growth of several glioma cultures. Furthermore, AlO strongly suppresses the growth of grafted xenografted gliornas in mice; In some animals, the turnoral growth was inhibited by more than 95% in relation to untreated controls. Accordingly, as an example of the method of the invention, AlO is administered to a patient with gliorna via any route of administration and in any suitable pharmaceutical carrier which results in carrying AlO in contact with positive glyogen cells to the PDG receptor. as well as nearby vascular endothelial cells, which typically proliferate in high-grade glycines. Intravenous and intraarterial routes may be preferred routes of administration. In addition, the newly developed icrocatheter technology can be particularly effective in delivering the compositions of the invention directly to the glioma site, thereby achieving immediate localized contact with the cancer and nearby endothelial cells and thus possibly reducing the potentiality associated with the assortment. intraarterial more di tanto. The preparation and use of therapeutically effective compositions for the treatment of glyorines is described in detail in the following sections and by way of examples, mentioned below. In addition to the compositions herein described in specific form, the invention provides identification of other compositions which, due to their inhibitory effect on the activity of the PDG receptor, may be useful for inhibiting the proliferation of several intraaxial tumors. Possible compositions can be identified by their ability to inhibit the activity of the PDGF receptor using any suitable test, such as the ELISA tests of inhibition of chitosis autophosphorylation and the inhibition of tyrosine kinase. Possible compositions can be evaluated for therapeutic efficacy by inhibiting the growth of gliorna cells and ideally by testing the inhibition of xenografted tumors in vivo.

III fllQ The present invention describes various compositions that can be used to inhibit the activity of PDGF-R and thus inhibit cell proliferation disorders. The use of RIO to inhibit the growth of tumors in animals demonstrates the ability of these compositions to function in vivo despite several pharmacological considerations that prevent the composition from exerting its effect. Said inhibition in vivo is illustrated in the examples described below.

AlO is also known as Leflunornide, HUIA 485, and 4- tri-luorome il-anili a of 5-methyl-xylo-4-carboxylic acid. Several publications have described different possible uses of AlO. According to the abstracts of Kommerer F-3 and others, patents of E.U.fl. No. 4,284,786 (1981) and Kórnrnerer F-3 and others, "US Ph.D. No. 4,351,841 (1982), AlO" has antirheumatic, antiphlogistic, antipyretic and analgesic option, and can be used for the treatment of multiple sclerosis. "According to Talrnadge 3.E. and Twardzik DR floents and Actions 355 : 135-141 (1991) "it was suggested that the mechanisms of activity of Lefluno ide may be the inhibition of a specific kinase of the cytokine." Robertson SM and Lang LS, European Patent Application 0 413 329 A2 (published in 1991 ), linked with 5-methylisoxazole-4-carboxylic acids which irabarcan Leflunomide, asserts that: The present invention is directed to methods for treating ocular diseases with immune aetiology through the use of 5-methyl-isoxazole-4 Carboxylic and hydroxyethylidene cyanoacetic acid anilide derivatives In addition, the compounds are useful for treating ocular manifestation associated with systemic diseases with immune aetiology. It has a suppressant, mild and moderate antiallergic effect and is useful for the treatment of eye diseases such as uveitis (including reurnatoid nodules), retinitis, allergy (vernal ceratoconjunctivitis and allergic or giant papillary conjunctivitis) and dry eyes ). Additionally, the compounds are useful for prolonging corneal or other ocular tissue graft survival and are useful as adjuncts to surgery in patients who are atopic or in an altered condition. "? The summary by Barlett RR et al, entitled" Isoxazole-4-Carboxamides and Hydroxyalkylidene-Cyanoacetamides, Drugs Containing These Compounds and Use of Such Drugs ", asserts: Isoxazole-4-carboxaraids derivatives and hydroxyalkylidene-nitrate derivatives are suitable for the treatment of cancer diseases These compounds can be prepared by methods of the prior art.

? Silos are new, and are also suitable for the treatment of rheumatic diseases. Bartlett R.R. and others., ftgents and Actions 32: 10-21 (1991) states that "C13 efluno ide has been shown to be very effective in preventing and curing various autoimmune diseases in animals." Barlett also states that: ..., it can be shown that the tyrosine phosphorylation of the RR-SRC peptide substrate and the autophosphorylation of the epidermal growth factor receptor (EGF) were #hibited, in a dose-dependent manner, by Leflunomide. Matter et al., FEBS 334: 151-154 (Nov. 1993) (not admitted to be the prior art) describes the use of the active metabolite of Leflunomide to inhibit the growth of EGF-dependent cells, including A431 cells. Matter also states: Platelet-derived growth factor-dependent tyrosine phosphorylation was also inhibited by R77 1725 in intact cells at concentrations similar to EGF-dependent described in Figure 3 (no data shown). Studies on a composition of the invention, AlO, described more fully and by way of example, mentioned below, the establishment of its potency against cancer cells of brain, lung, prostate, ovary, skin and colon, characterized by inappropriate PDGF-R activity rather than EGF activity. As illustrated in the feeles described below, RIO inhibits the activity of PDGF-R while having little, if any, effect on the EGF receptor or HER2 phosphorylation. In addition, although AlO inhibited the growth of tumors characterized by appropriate PDGF-R activity, AlO did not significantly inhibit the growth of xenotransparent cells by expressing an EGF receptor (epidermoid cells R431).

These data are surprising because of the results described by Bartlett and others, mentioned above, Agents and Actions in which Leflunomide was shown to inhibit EGF receptor autophosphorylation induced by EGF and cell proliferation, and Matter et al., Mentioned above, wherein the active metabolite of Leflonornide inhibited the growth of A431 cells. The present disclosure demonstrates the ability of AlO to inhibit inappropriate PDGF-R activity and unwanted cell proliferation in vivo. such co occurs in cancers characterized by PDGF-R activity. As illustrated in the examples that are illustrated below, RIO can be used to selectively inactivate inappropriate PDGF-R activity. A compound is considered to effect phosphorylation if its ability to inhibit the phosphorylation of a receptor (e.g., as described below) is less than its cytotoxic effect (e.g., DLso as described below).

The inhibition of phosphorylation of different receptor such as PDGF-R receptor, EGF-R or HER-2 depends on conditions such as the concentration of the drug. By "selective inhibition" it is understood that a compound can be used at a particular concentration to inhibit the phosphorylation of PDGF-R and have little effect, if any, on phosphorylation of the EGF-R and / or HER receptor. 2 at the same concentration. Preferably, the compound, like AlO, can inhibit PDGF-F while having little, if any, effect on phosphorylation of EGF.R and / or HER-2 HER-2. By "little effect, if any" on the activity of EGF-R, or HER-2, it is understood that the activity of the receptor is affected not more than S%, most preferably not more than 20%, most preferably still «R not more than 10%, in a particular receiver. Tyrosine kinases are important in many biological procedures including cell growth, differentiation, aggregation, chemotaxis, cytokine release, and concentration in the muscle. Many of these cases are mediated through different tyrosine kinase factors. In addition, different tyrosine kinase receptors may be important for a biological function in different types of cells. By developing effective inhibitors of PDGF-R, the possible toxic effect of the compound is reduced. The compounds described herein vary in their ability to selectively inhibit PDGF-R. For example, D14, G12, G13 and G14 inhibits phosphorylation of PDGF-R but does not affect EGF or Her-2 phosphorylation while CIO affects EGF-R, PDGF-R, and the phosphorylation of Her-2.

IV. PDGF-R mutated Cell proliferation disorders characterized by inappropriate PDGF-R activity can also be inhibited by using a mutated PDGF-R. Ueno H. , et al., Science 252: 844-252 (1991), describes nucleic acid encoding truncated PDGF-R to inhibit the phosphorylation of PDGF-R in vitro. According to Ueno: When the truncated receptors are expressed in excess compared to the wild-type receptors, the stimulation by PDGF of receptor autophosphorylation, association of phosphatidylinositol-3 kinase with the receptor, and calcium mobilization were blocked. Ueno did not demonstrate that the inhibition of PDGF-R by a mutated protein could inhibit the proliferation of unwanted cells. Said cell proliferation inhibition in viyo V \ o desired is illustrated in the examples described below using nucleic acid encoding PDGF-R having a stop codon just upstream of the first tyrosine kinase domain. The nucleic acid is used to introduce the truncated protein into a cell. For example, the nucleic acid encoding the truncated PDGF-R is placed in a retroviral vector using normal recombinant DNA techniques. The vector then affects a cell where its nucleic acid is finally ~ ras to the protein that produces a PDGF-R. Other means for introducing the ruined protein into a cell include preparing the mutated protein in vitro and introducing the protein and the cell with a vector, such as a liposome. The mutant PDGF-R must be constructed in a manner that interferes with the intermolecular phosphorylation that occurs between the directed receptor. This can be achieved by various means such as 1) truncation of PDGF-R, preferably to eliminate a tyrosine kinase domain, and most preferably.to eliminate both tyrosine kinase domains; 2) mutations that inhibit the catalytic capacity of the catalytic domain of PDGF-R such as a mutation of lysine 602 to agrinin which prevents ATP binding. Of these methods, mutation of tyrosine residues is preferred and truncation of the receptor is highly preferred. The use of nucleic acid encoding truncated PDGF-R to inhibit tumor growth in animals demonstrates the ability of such truncated receptors to function in vivo to various pharmacological considerations that would be expected to prevent the composition from exerting its effect. Therefore, the present disclosure demonstrates that the use of nucleic acid encoding the truncated PDGF receptor is not limited to the inhibition of PDGF-R * in cell culture. Rather, the nucleic acid encoding PDGF-R can be used to inhibit inappropriate PDGF-R activity in animal cells, thereby inhibiting the growth of tumors in animal cells, and -deploying it in other disorders related to PDGF-R.

V. Administration of preferred compounds The compounds of this invention can be administered to a patient alone, or in a pharmaceutical composition comprising the active compound and a carrier or excipient. The compounds can be prepared as pharmaceutically acceptable salts (ie, non-toxic salts which do not cause the compound to exert its effect). The pharmaceutically acceptable salts can be acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfarnate, acetate, citrate, lactate, tartrate, methanesulphonate, ethanesulfanate, benzenesulfonate, p-toluensulphonate, cyclohexylsulfarnate and cinate (cf. .gr., cited above PCT / US92 / 03736). Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfasane acid, acetic acid, citric acid, lactic acid, tartaric acid, rhenalonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid. , cyclohexylsulfanic acid and chemical acid. The pharmaceutically acceptable salts can be prepared by normal techniques. For example, the free base form of the compound is first dissolved in a suitable solvent such as an aqueous or non-aqueous solution of alcohol, which contains the appropriate acid. The salt is then isolated by evaporation of the solution. In another example, the salt is prepared by reacting the free base and the acid in an organic solvent. Vehicles or excipients can be used to facilitate administration of the compound, for example, to increase the solubility of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, platinum, vegetable oils, polyethylene glycols and physiologically compatible solvents. The compositions or pharmaceutical compositions can be administered by different routes including intravenous, intraperitoneal, subcutaneous and intramuscular, oral, topical or transmucosal. Some of the preferred compounds, such as AlO and Bll, are hydrophobic and therefore not very soluble in water. The effective dose of RIO can be obtained using AlO in combination with PBTE: D5W. PBTE consists of a solution of 3% benzylic Slcohol w / v polysolvate 80, at 8% w / v, and 65% w / v polyethylene glycol (MW = 300 daltons) in absolute ethanol. PBTE: D5U consists of PBTE diluted 1: 1 in a 5% dextrose solution in water. The solubility of AlO in PBTE is about 60 rng / rnl and the solubility of AlO in PBTE: D5W is about 5 mg / ml. The solubility of the other compounds described herein can be obtained using standard techniques. In addition, the active drug itself (e.g., Bll) can be administered in an oral formulation. Another way to overcome the problem of hydrophobic character includes the use of frequent small daily doses more than a few large daily doses. For example, the composition can be administered at short time intervals, preferably, the composition can be administered using a pump to control the time interval or achieve continuous administration. Suitable pumps are commercially available (eg, the ALZETR pump sold by the .za Corporation, and the BARD ambulatory PCR pump sold by Bard MedSyste). Alternatively, prodrugs having increased solubility can be used. The prodrugs can be cleaved into the active drug under physiological conditions. For example, Patterson et al., 3. Med. Che. 35: 507-510 (1992) describes R12 (3-carboxy-5-methyl-N-C4- (trifluorornethyl) -phenylH-4-isoxazolecarboxarnide), which, like AlO, can act as a pro-drug for Bll. The appropriate dose depends on several factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient. For the treatment of cancers the expected daily dose of AlO is between 1 to 2000 mg / day, preferably from 1 to 250 mg / day and most preferably from 10 to 150 rng / day. Drugs can be given less frequently, as long as plasma levels of the active portion are [sufficient to maintain therapeutic effectiveness. One factor that can influence the dose of the drug is body weight. The drugs should be administered at doses ranging from 0.02 to 25 mg / kg / day, preferably from 0.02 to 15 mg / kg / day, most preferably from 0.2 to 15 mg / kg / day, alternatively, drugs can be administered at 0.5 at 1200 rng / m2 / day, preferably from 0.5 to 150 mg / rc / day, most preferably 5 to 100 mg / m ^ / day. The average level in the plasma should be 50 to 5000 g / rnl, preferably 50 to ng / rnl, and most preferably 100 to 500 mg / rnl. Levels in the plasma can be reduced if the effective pharmacological concentrations of the drug are achieved at the site of interest.

VI Administration of mutated PDGF-R The PDGF-R mutants can be administered as a nucleic acid expressing the protein, using formal techniques some of which are described below. The delivery vehicles include liposomes and other pharmaceutical compositions. The nucleic acid encoding a mutated PDGF-R can also be introduced into a cell using standard techniques such as a paired retroviral and ionic molecule. In those cases where the technique is carried out ex vivo. The cell is then applied to a patient. The administration of protein is facilitated using a carrier or excipient as described above. The specific assortment route of any agent selected depends on the use of the agent (these considerations can also be applied for the administration of the preferred compounds). Generally, a specific assortment program for each agent focuses on the intake of the agent with respect to the intracellular location, followed by the demonstration of efficacy, alternatively, the assortment of these same cells and a protected organ of a Intake studies include valuation ingestion tests, eg, nucleic acid or cell protein intake, regardless of the assortment vehicle or assortment strategy. Said tests also determine the intracellular localization of the agent after ingestion, finally establishing the requirements for the maintenance of stable state concentrations within the cell compartment containing the target sequence (nucleus and / or cytoplasm). Efficacy and cytotoxicity can The toxicity not only includes viability of the cells but also function of the cell. In general, the doses of the mutated protein and nucleic acid is as described above for the preferred compounds. The drug assortment vehicles are effective for both systemic and topical administration. They can be designed to serve as a slow-release reservoir or to deliver their contents directly to the cell or target. A The use of direct drug assortment vehicles is that multiple molecules are taken by ingestion. These vehicles increase the average circulation life of drugs that would otherwise be rapidly eliminated from the bloodstream. Some examples of such specialized drug assortment vehicles that fall into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive microspheres. Pumps can also be used for this purpose. r Of this category of assortment systems, liposomes are "preferred." Liposomes increase intracellular stability, increase the efficiency of intake and improve biological activity Liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to those lipids They have an internal water space to trap water-soluble compounds and vary in size from 0.05 to several microns in diameter.Antibodies can be attached to liposomes to target particular cells. PDGF-R and the preferred compounds is advantageous since it allows localized concentration at the site of administration with minimal systolic adsorption.This simplifies the strategy of assortment of the agent to the site of the disease and reduces the degree of toxicological characterization. of applied material is much less than that required for other as? Lyias of administration. The effective assortment requires that the nucleic acid enter the cell membrane of the cytoplasm of the cells characterized by inappropriate PDGF-R activity and express the protein. The agents can also be administered via the systemic route. Systemic absorption refers to the accumulation of drugs in the bloodstream followed by distribution throughout the body. The routes of administration that lead to systemic absorption include: intravenous, aubcutaneous, tntraperitoneal, intranasal, intrathecal and ophthalmic. Each of these routes of administration exposes the drug to accessible pathological tissue. Subcutaneous administration drains into a localized lymph node that proceeds through the lymphatic network into the circulation. The route of entry into circulation has been shown to be a function of molecular weight or size.

Below, examples are provided to illustrate different aspects and embodiments of the present invention. These examples are not intended to limit the invention described in any way. Rather, they illustrate the methodology by which drugs having the described formula can be easily identified by a routine procedure to ensure that they have the desired activity. That is, those of the formula claimed here can be determined those with the most appropriate activity before being administered to an animal or human being. Other compounds may also be selected to determine the degree of suitability for use in methods of this invention. A description of some of the procedures used in the following examples is described in the appendices given below. The use of AlO in the different procedures is mentioned, however, the compounds 7 different AlO were tested by these procedures replacing RIO with the tested compound.

APPENDIX 1 1. Cell lines Cell lines were purchased from the RTCC at least as specified. U124Q and U1242 cells were obtained from Dr. 3oseph Schlessinger (Ne? York University) and SF763 and SF767 cells were obtained from Dr. Michael Berens (Barrow Neurological Institute). SF767T, SF763%, U118T, and SKOV3T are sublines of the SF767, SF763, U118 and SKOV3 cells, respectively. They were derived from the implantation of SC progenitor cells in mice BALB / c, nu / nu. Tumors that displayed desirable growth characteristics were excised and finely ground in a sterile Pet i box. Two to five rnl of appropriate media were added to the suspension and the tumor pieces were mechanically separated. The resulting suspension was placed in tissue culture flasks and fed with the appropriate culture medium supplemented with 100 units per ml of penicillin G-sodium and 100 μg / μL of streptomycin sulfate (Gibco, Grand Island, NY) . The medium was changed every two to three days. After three to five steps, the antibiotic supplements were removed and the cells were kept in a He gave antibiotics. - and NTH3T3 mouse fibroblasts overexpressing the DGF receptor, Flk-1 receptor, IGF-1 or PDGF-b receptor were genetically engineered using retroviral vectors. The MCF7 / HER2 cells were derived by overexpressing the HER2 gene using retroviral constructs on a background of MCG7. 2. Crops < All cell culture media, glutamine and fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells were cultured in a humid atmosphere of 90-95% air and 5-10% COs at 37 ° C. All cell lines were routinely subcultured twice a week and were negative for microplasma co or determined by the Mycotect method (Gibco). The C6 cells were maintained in Harn FIO supplemented with 5% fetal bovine serum (FBS) and 2 mM glutamine (GLN). T98G cells were cultured in MEM with 10% FBS, 2 mM GLN, 1 rnM sodium pyruvate (NaPyr) and non-essential amino acids (NEAA). SK0V3T cells were cultured in DMEM, 10% FBS and 2 mM GLN. NIH3T3 mouse fibrolasts engineered to overexpress Flk-1 or the EGF-receptor were maintained in DMEM containing 10% fetal serum (CS GLN at 2 M. Engineered NIH3T3 cells - * Or genetic to overexpress the IGF-1 or the insulin receptor were kept in DMEM that contained FG? at 10% and GLN at 2 rnM. HL60 cells were maintained in RPMI 1640 with 10% FBS and 2 nM GLN. Cells 547D and BT474 were maintained in RPMI 1640 with 10% FBS, GMS-G and 2 mM GLN. DU 145 cells were cultured in DMEM F12 with 10% FBS and 2 mM GLN. Cells A172, R431, U118MG and RRG were cultured in DMEM with 10% FBS and 2 nM GLN. Cells L1210 ^ >e cultured in DMEM with 10% horse serum and 2 nM GLN. C1300 cells were cultured in DMEM with 10% heat inactivated FBS, 2 mM GLN and 50 mM β-mercaptoethanol. Cells 598G, 7138MG, 787MG, 7373MG, U1240, U1242, Calu-3, Calu-6, SF767, SF767T, SF763, SF763T, SK-N-MC and SK-N-SH were cultured in MEM with 10% FBS, ERA, NaPyr ai M and GLN. 2 mM. MDA MB 361 and MDR MB 468 cells were cultured in L15 with 10% FBS and GLN. at 2 mM. PC-3 cells were cultured in F12 from Hflrn with FBS at 'km% and GLN at 2 tm ~ ñ543 cells were cultured in F12 from HRM with 10% FBS and 2 mM GLN. ZR75-30 cells were cultured in RPMI 1640 with 10% FBS, GLN at 2 rali and NaPyr at 1 rnM. Cells MCF7, MCF7 / HER2, R375, BT549, 9L, C81-61, ZR 75-1 and K562 were cultured in RPMI 1650 with 10% FBS and 2 mM GLN. Ovcar3 cells were cultured in RPMI 1640 with 20% FBS, 2 nM GLN and 10 rnG / rnL insulin. DIB and T27A cells were cultured in RPMI 1640 with 10% heat inactivated FBS, 2 M GLN and 50 M rercaptoethanol; 7TD1 cells were grown on the same < sdio supplemented with 50 units / mL of IL-6 Coló cells "Recombinant murine% 320DM, WEHI-164.14, and HBL100 where grown in RPMI 1640 with 10% heat inactivated FBS and 2 mM GLN." SKBR3 cells were grown in McCoy's 5A with 15% FBS and 2 M GLN. PA-1 cells were cultured in MEM with 10% heat inactivated FBS, 2 M GLN and NEAA Neuro 2R cells were cultured in MEM with 10% heat-inactivated FBS, 2 nMN GLN, NERR, NaPyr and β-mercaptoethanol at 50 nm.

APPENDIX 2 1. Phosphorylation of the receptor Inhibition of receptor tyrosine kinase activity by AlO was studied by methods of Western Blot and ELISA. For the Western Blott test, the tests were plated on two rnL of growth medium in 6-well Petri dishes (500,000 cells per well) and allowed to settle overnight. The medium was replaced by 2 rnl # of MCDB 105 (UCSF Cell Culture Facility) supplemented with 1% FBS. The plates were then incubated overnight at 37 ° C, ambient C0a. To test the effects of compounds under ligand-mediated receptor autophosphorylation, the cells were exposed to AlO or DMSO for 1 hour at 37 ° C prior to stimulation of receptor tyrosine kinase activity with ligand. After an incubation for 7 minutes at room temperature with ligand, the lacquers were placed on ice and washed three times with a '* ml of PBS cooled with ice plus orthovanadate at 1 mM. Lysis was achieved by pipetting cells in 0.5 rnl pH buffer containing Tris at 50 rnM, pH 7.4, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 M sodium vanadate (Na3VOA), pyrophosphate at 10 rnM, PMSF at 1 mM, 10 rng / rnL for aprotinin, 10 rng / rnL for leupeptin. An aliquot of 300 microliters of each lysate was added immediately to 100 μL 4x of Laemrnli sample buffer (Tris at 0.2 M, pH 6.9, 20% glycerol, 7% SDS, * £ DTfl at 5 M, β 5% mercaptoethanol) containing phosphatase inhibitors, Na3VO. at 2 rnM and pyrophosphate at 10 mM. Samples were boiled for 5 minutes, frozen in ice-cold ethanol and stored at -80 ° C. Proteins were redissolved by SDS-PAGE (Bio-Rad Minif) rotate II) and transferred to nitrocellulose membrane (Schleicher S Schuell) at 120 volts for 1 hour at room temperature in pH buffer containing 25 mM Tris, pH 8.3, 20% methanol, 0.2 M glycine, SDS at Q.1%. The integrity of the * Protein transfer to membrane was determined by staining with 1% Ponceau S in 5% acetic acid for 5 minutes. After destaining in several rinses of distilled water, the membrane was soaked overnight in a pH regulator blocker containing 5% milk in saline regulated at its pH with Tris, 0.05% Tween 20. Phosphotyrosine was detected by incubating the membrane (1 hour at room temperature) with an anti-phosphotorosine antiserum diluted in pH-regulator to 1: 3000. For C6 cells, the content of PDGF-b receptor in the lysates was confirmed on sample days in duplicate by Western Blot using a specific antibody to PDGF-b receptor (UBI). The antibodies were visualized using ECL reagent from Amersham and by exposure to Fuji RX film. For ELISfi tests, the cells were grown for 80-90% confluence in growth medium and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After incubation during * overnight in a medium containing 0.5% serum, the cells were changed to serum-free medium and treated with test compound for two hours in an incubator with 5% C02 at 37 ° C. Cells were then stimulated with ligand for 5-10 minutes followed by lysis with HNTG (Hepes at 20 mM, NaCl at 150 rnM, glycerol at 10%, EDTA at 5 rnM, Na3V0 ^ at 5 mM, Triton X-100 at 0.2% and NaPyr to 2 M). Cell lysates (0.5 mg / well in J ^? PBS) were transferred to pre-coated ELISA plates ^^^ with antibody specific to the receptor and which had been blocked with 5% milk in TBST (Tris at 50 mM-HCl, pH 7.2, NaCl at 150 rnM and Triton X-100 at 0.1%) at room temperature for 30 minutes . The lysates were incubated with shaking for one hour at room temperature. Plates were washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. The anti-phosphotyrosine antibody excess was removed with TB? T plate four times. Goat anti-rabbit IgG antibody was added to the ELISR plate for 30 minutes at room temperature followed by rinsing with TBST four times more. ABTS (citric acid at 100 M, NasHPO ^ at 250 rnM and 0.5 rng / rnL of acid 2, 2-azino-bis (3-etilbenciazolin-6-eulfónico) plus H 02 (1.2 rnL of Hs02 at 30% at 10 rnL of ABTS) was added to the ELISA plates to begin color development.The absorbance at 410 nrn with reference wavelength of 630 nrn was recorded about 10 to 30 minutes after the of ABTS. Cell lines used in ELISA tests included U1242 (PDGF-b receptor), HL-60 cells (GMCSF receptor / 3AK-2), or NIH3T3 cells overexpressed the EGF receptor, F1K-1 receptor, IGF-1 or the insulin receptor. The IC '0 values were estimated by comparing the inhibition of tyrosine phosphorylation by drug in the absence or presence of appropriate ligand. .2 »SYNTHESIS APN The effects of AlO on the PDGF-dependent RDN synthesis were determined by measuring the incorporation of H-tirnidine in the RDN of the cells. The conditions for the test were essentially those described by Pollack. and Q rQS. i. Neurosurg .. 73: 106-112. 1990. with some modifications. The T98G cells in the log phase growth were transferred to 96-well dishes at 20,000 cells in 0 μL of growth medium containing serum. After an overnight binding period, the monolayers were washed twice with 200μL of MCDB 105 medium and the cells were cultured from serum free MCDB 105 medium for 24 hours. The medium in the wells was replaced by a fresh medium alone (MCDB 105 plus 5 μg / rnL insulin), the medium containing PDGF-BB alone, or the medium containing PDGF-BB in combination with various concentrations of RIO. The plates were incubated in COa for approximately 18 hours. H-tirnidine (flrnsersharn, 5 Ci / mmol) was added to each well to produce a final concentration of 5μCi / rnL and the plates were returned to a 37aC incubator. After 4 hours the medium was removed, the plates were placed on ice, and washed twice with 200μL of PBS cooled by ice in each well. The radioactivity incorporated in the RDN was separated from 3H-thyrnidine not incorporated by precipitation with lOOμL of TCa cooled with ice for 10 min on ice. After washing with TCR, cooled with ice, the precipitate was solubilized (1% SDS in 100 L of Tris base at 10 mM) and transferred to liquid scintillation counting flasks. 6 L of combination (Ready -Safe; Beckrnan) were added and the radioactivity was quantified in a liquid scintillation counter L6000SC. 3 »Cell cycle analysis ^ NIH3T3 mouse fibroblasts overexpressing the human PDGF-b receptor were seeded in DMEM added with 10% CS and 2 M GLN. The cells were cultured to approximately 80% confluence and then treated overnight in a serum-free medium (DMEM, 2 mM GLN, 2 nMR NRR, 2 nM NaPyr, and 2 M HEPES). The cells were incubated for 20 hours in the presence of PDGF-BB 100 ng / nL and with various concentrations of AlO (0.1, 1, 10, 25, or 100 M). The a cells were then harvested, stained and analyzed by flow cytometry for the DNA content. 4. Growth tests The RIO was tested for growth inhibition of anchor-dependent tumor cells using the colorimetric test described by Skehan. and others. 3. Nati. Cancer Inst .. 82: 1107-1112. 1990. The test measures the protein content of acid-bound cells using the counterionic bond of sulforhoda ina B (SRB, Sig a) dye. The RIO was solubilized in DMSO (Sigma, cell culture grade) and diluted in an appropriate growth medium at two times the desired final test concentration. In tests using C6 cells, RIO (100 μL) was added to 96-well plates containing bound cell monolayers (2000 cells / well in 100 μL). For other cell lines, the cells (2000 / well in 100 μL) were introduced into the well; immediately after supplying the drug solutions. After 4 days (37 ° C, 5% C0) the rnonolayers were washed 3 times with PBS and fixed with 200μL of 10% TCA cooled with ice (Fisher Scientific), and kept at 4 ° C for 60 minutes. The TCA was removed and the fixed monolayers were washed 5 times with tap water and allowed to dry completely at room temperature on absorbent paper. The cell protein was stained for 10 minutes with 100 μL 0.4% 'fl-S'RB dissolved in 1% acetic acid. After 5 washes with tap water, the dye was solubilized in Tris base at 10 rnM (100 μL per well) and the absorbance reading at 560 nrn on a plate reader Dynatech model MR50Q0. The growth inhibition data are expressed as a percentage of the absorbance detected in the control wells that were treated with 0.4% alone. The DMSO controls were different from the cells that grew in regular growth medium. The IC50 values were determined using a 4-parameter curve fitting function. For the anchor-independent tumor cell growth test, cells (3000 to 5000 per dish) suspended in 0.4% agarose in an eneay medium (DMEM containing 10% FCS) with and without AlO were plated on plates of 35rnrn coated with a base layer of solved agarose (0.8% agarose). After an incubation of two to three weeks at 37 ° C, larger colonies of 50 μm were harvested using an Ornnicon 3800 Tumor Colony counter.

APPENDIX 3 1 »Growth Testing for Cell Lines T unite! For most of the turnoral cell line, the inhibition of cell growth by AlO was determined using an SRB test as described in FIG.

^^ T index 2. For cells K562, DIB, L1210, 7TD1, T27A, and Colo320 DM was used an MTT test to determine cell growth. (Hansen et al., 3. Im unol. 119 = 203-210. 1989) Briefly, 5GrnL of growth medium containing various concentrations of AlO and 50 mL of cell suspension (2,000 cells) was added to each well of a 96-well plate. The cells were incubated at 37 ° C for 4 days in a humidified 7% C0S atmosphere. At the end point, 15 rnL of MTT (5 rnG / rnL in PBS, Sigma) plates were incubated at 37 ° C for 4 hours followed by the addition of 100 mL of solubilization solution (20% w / v SDS in 50% N, N-dirnethylforrnarnide, pH 4.7) to each well. The plates were incubated overnight in a sealed container with a humidified atmosphere. The absorbance was determined at a wavelength of 570 nrn with a reference wavelength of 630 nm using an ELISA plate reader. "2, Growth Test for Primary Tumors The effect of fllO on primary tumor growth was examined by Oncotech, Inc. (Invine, CR). Viable tumors were placed in the medium by the pathologist and the reference institution immediately after surgery, and sent to Oncotech by overnight delivery. As soon as the tumor was received, one portion was fixed in formalin for sectioning and the rest cut from the necrotic tissues, connector and adipose. All the managements of the tumor were executed aseptically. The remaining tumor was placed in a Petri dish containing 5 L of the medium (RPMI 1640 added with 10% FBS, 100 IU / L of penicillin, 100 mg / mL of streptomycin and L-glutamine) and mechanically separated with scissors in pieces of 2 m or smaller. The resulting pastes were mixed with the medium containing 0.003% aßj ^ D ase (2650 Kunitz units / mL) and 0.14% collagenase type 1 (enzymes from Sigma Chemical Co., St. Louis Mo.), placed in flasks of 50 mL with shaking, and incubated for 90 minutes at 37 ° C in a humidified atmosphere 5% C0S a portion of the cell suspension was used for the preparation of cytospin slides and examined after staining with eosin and hernatoxylin from the tissue sections by a pathologist to confirm the diagnosis, and to determine tumor cell count and viability. After the enzymatic dispersion in a suspension # of individual cells, the tumor cells were filtered through the nylon sieve, washed in the medium, suspended in soft agarose (0.12%) and deposited in plates to apr-oxirnadarnente 20,000 cells per well on an agarose layer (0.4% ) in 24-well plates. Cells were incubated under normal culture conditions for 5 days in the presence or absence of RIO. The cells received by application 3H-ti idina (flrnersharn, 5 rnCi per well) for the last 48 hours of the culture period. After the appropriate labeling period, the tissue culture plates were heated to 60 ° C to melt the agarose, the cells were harvested with a micro-harvester over glass fiber filters and the radioactivity was determined. Inhibition (PCI) was determined using the formula PCT = 1 - (treatment group CPM control group II CPM). The determinations of the proliferation of the control group were executed in quadruplicate, while the proliferation of the treatment group was determined in triplicate.

AP NDICE 4 no ales Female atirnian mice (BRLB / c, nu / nu), BRLB / c mice, Wistar rats and Fisher 344 ratae were obtained from Sirnonsen Laboratories (Gilroy, CR). The female fl / 3 mice were obtained from Jackson Laboratory (Bar Harbor, ME). The DR rats were obtained from B &K Universal, Inc. (Frernont, CA). The typical R / Nu rats, the DBR / 2N mice, and the mice BALB / c were obtained from Harlan Sprague Dawley (Indianapolis, iKi). Female C57BL / 6 mice were obtained from Taconic (Gerrnantown, NY). All the animals were kept under clean environment conditions in microisolation cages with dry Alfa bed. They received food for sterile rodents and water Ib- a libituin- 2. Subcutaneous Xenograft Model 0 The cell lines were grown in appropriate medium (see appendix 1). Cells were harvested at or near confluence with 0.05% Trypsm-EDTA and pelleted at 450 x g dur-ante 10 minutes. The pellets were suspended again in PB? sterile or medium (without FBS) at a particular concentration and the cells were implanted in the posterior side of the mice. Turnoral growth was measured at 3 to 6 weeks using vernier calipers. The tumor volumes were calculated as a product of the #gitude X width X the height unless otherwise indicated. The P values were calculated using the student's t-test. The AlO in 50-100 μL of excipient (DMSO, PBTE, PBTE6C: D5W, "or PBTE: D5W) was delivered by IP injection at different concentrations. 3. Xenoinierto Intracerebral model For mouse Cl model, rat C6 glioma cells were harvested and suspended in sterile PBS at a concentration of 2.5 x 10 7 cells / rnL and placed on ice.The cells were implanted in BALB / c mice, nu / nu as follows: the frontoparietal scalp of the mice was shaved with animal shearers if necessary before cleaning with isopos with 70% ethanol.The animals were anesthetized with isofluorane and the needle was inserted through the skull into the hemisphere left of -Free. The cells were supplied from hermetically sealed syringes to the GaT Hámilton using 1.27 caliber 30 caliber needles equipped with sleeves that allowed only 3rnrn penetration. A repeat dispenser was used for an accurate delivery of 4 μL of cell suspension. The animals were monitored daily for welfare and were sacrificed when they had had a weight loss of approximately 40% and / or showed neurological symptoms.

For the Rat Cl model, the rats (Wistar, Sprague ßyley, Fisher 344, or atic R / Nu, approximately 200g) were anesthetized by an IP injection of 100 rnG / kg Ketaset (cetanin hydrochloride; Aveco, Fort Doge, Iowa) and 5 rng / kg Rornpun (xylazine, 2% solution, Bayer Germany). After the start of the anesthesia, the scalp was shaved and the animal was oriented in a stereotaxic apparatus (Stoelting, Wood Dale, ID) The skin at the site of the incision was cleaned 3 times with alternating cleanings of 70% ethanol and Povidone-iodine 10% An average incision 1.0 - 1.5 crn was § | b "cha on the scalp using a sterile surgical blade The skin was slightly separated and spread out to the sides to expose the sutures on the surface of the skull A dental burr (Stoelting, Wood Dale, ID was used to make a small hole (1-2 rnrn diameter) in the skull approximately 1 anterior rnrn and 2 rnm lateral to the bregma.The cell suspension was removed in a Harnilton syringe of 50 μrnL equipped with a standard 23 or 25 gauge bevel needle. Oriented at the orifice at the level of the arachnoid and descended until the tip of the needle was at a depth of 3 m inside the brain structure, where the cellular euepeneion was injected slowly. Deepuée that the cells were injected, the needle was left in the hole for 1-2 minutes to allow complete supply of the cells.The skull was cleaned and the skin was closed with two to three sutures.The animals were observed for Recovery from surgery and anesthesia. Along the Next, the animals were observed at least twice each day for the development of symptoms associated with intracerebral tumor progression. The animals exhibited advanced symptoms (stooping, loss of balance, dehydration, loss of appetite, loss of coordination, termination of cleaning activities, and / or significant weight loss) were humanely sacrificed and the organs and tissues of interest were resected.

I. Intraoperitoneal model The cell lines were cultured in the appropriate medium as described in Appendix 1. The cells were harvested and washed in sterile PBS medium without FBS, resuspended at an appropriate concentration, and injected into the IP cavity of the cell. the mice of the appropriate strain. Before implantation of the 7TD1 cells, the C57BL / 6 patons were initiated by IP injection of 0.5 rnL Pristane. SKOV3T cells were implanted in atirnicotic mice without Pristane initiation. The mice were observed daily for the occurrence of ascites formation. The individual animals were sacrificed when they presented a weight gain of 40% or when the IP tumor burden began to cause undue stress and pain to the animal.

. Immunohistochemistry # ~ X - - - 5 μm frozen tissue sections fixed with acetone from untreated xenografted tumors derived from human, rat, or murine turnoral cells were analyzed by immunohistochemistry using highly specific receptor antibodies. In summary, the non-specific binding sites were blocked with 10% normal goat serum before the application of the primary antibody. The appropriate antibody concentrations were used | > To obtain the desired sensitivity and specificity (receptor- of PDGF-b, rabbit anti-human, 1: 400 and anti-aton Flk-1, 5.5 μg / rnL of rabbit.) The sections of tissue known to contain the protein of interest served as positive controls. The appropriate negative controls of normal rabbit IgG and rabbit antipollo IgG of the same protein concentration and type as the primary antibody were used.The detection method was an indirect three-way method consisting of the binding of primary antibody to a secondary labeling. biotin (goat anti-rabbit IgG 1: 500) followed by horseradish peroxidase conjugated with streptavidin The croAgen / substrate used was 0.05% diarninobenzidine / 0.03% Hs0s The sections of tissue were counterstained with hernatoxylin, dehydrated-through grades ascending ethanol, cleaned as a substitute for xylene, and covered with Per ount for microscopic evaluation. A + 10 +++ was used to identify the general intensity of the expression with + = low, ++ = moderate, and +++ = high intensity. The specific staining reaction was observed as a turnoral T cell or vascular endothelial cell V or both.

APFNDICF, 5 l. In Vitro Immunological Tests • At the indicated times, the animals were sacrificed, and the vessels were removed aseptically and placed in sterile medium. The vasoe were processed into individual cell suspensions by grinding between sterile frozen glass slides. - After a simple wash to remove the tissue debris, the spleen cells were resuspended at a regular pH of "Hypotonic ammonium chloride to lyse erythrocytes. The lymphocytes were washed and resuspended at the appropriate concentrations in a complete medium, consisting of RPMI plus 10% heat-inactivated FBS, 2 mM glutamine, 50 μM β-rnercaptoethanol, and penicillin-streptomycin. Lymphocyte responses were examined in the following tests in accordance with accepted procedures (Curretn Protocols in Irn unoloov, Coligan, 3.E., Kruisbeek, AM Marguliee, DH, Shevach, EM? Trober, W. (Eds.) 3ohn Law and Sons, Inc. 1992. the. Nitrogen responses The T-cell mitogen, Confl, and the T cell-independent B cell mitogen, LPS, were added to 96-well round-bottom plates at the indicated concentrations. The lymphocytes from normal animals with vehicle doses and with drug doses were added to a final concentration of 2.5 x 10s / well. The cultures were usually established in triplicate or quadruplicate. The plates were incubated at 37 ° C in a humidified atmosphere containing 5% C03 for the indicated times. The supernatants (approximately 100 μL) were carefully removed from the wells and stored at -80 ° C for the lymphosin and immunoglobulin tests. To measure the '' oliferation of the lymphocytes 1 μCi of 3H-thymidine was added to each well, and the plates were incubated for 6 hours. The cultures were harvested on glass fiber filters, and the incorporated radioactivity was quantified by liquid scintillation counting (Betaplate, Wallac). l.b Combined Lymphocyte responses jjh Lymphocytes from animals with vehicle doses and normal drug doses were depoeited in 2.5 x 10s /? ozo 96-well round bottom plates. The stimulator cells were then added to the same cell concentrations. The stimulator cells consisted of syngeneic or allogeneic lymphocytes that had been treated with 50 μg / mL of rnitomycin-C for 30 minutes before the test. The plates were incubated for 3 to 4 days, at which time the supernatants were harvested and the cultures were treated with vibration as described for the RNA tests. lc Lymphocyte Tests The supernatants from RNA and MLR cultures were tested for the content of IL-2 and IL-6 by the ability to support the growth of factor-dependent cell lines. HT-2 cells (lOVpozo, IL-2-dependent) and 7TD1 cells (2xl03 / well, IL-6 dependent) were deposited in 96-well round bottom plates, in 60 μL / well. The supernatants were added in 50 μL / well, and the plates were incubated overnight (HT-2) or for 4 days (7TD1). Cell proliferation was determined in the colorometric test (Appendix 3"1),. ld Immunoglobulin ELISA # Flat-bottom 96-well EIA plates were coated with goat anti-mouse Ig antibodies (Southern Biotechnology) overnight at 4 ° C. The plates were blocked by the addition of PBS + 1% BSA. After washing with PBS, the supernatants from the rnurin mitogen and the MLR cultures were added and incubated at room temperature for one hour. The plates were washed with PBS, then goat anti-mouse Ig antibodies labeled with HRP were added and incubated at room temperature for 1 hour. Lae placae were washed and developed by the addition of the substrate (ABTS).

APFNDTCF ñ HPLC test At specific times after the treatment of the mice or rats with AlO, blood was collected in heparinized tubes by terminal cardiac puncture. The plasma was prepared and frozen in liquid nitrogen. The tissues and organs were resected and immediately frozen in liquid nitrogen. After the addition of an internal standard, the plasma samples were acidified with HCl and extracted with acetonitrile. The acetonitrile fraction was evaporated to dryness in a vacuum centrifuge with heating and to dissolve in methanol. The tissue and organ samples were homogenized according to the weight ratio of 1: 5 (p: v) in 50 rnM Tris-HCl, pH 7.4, at 20,000 rev / rnin, using a tissue homogenizer (Brinkmann Polytroin Model PT3000). After the addition of an internal standard, the furnace was acidified with HCl and then extracted with acetonitrile. The acetonitrile fraction was then extracted with an equal volume of diethyl ether. The ether fraction was evaporated to dryness in a vacuum centrifuge with encouragement and redissolved in methanol. * Samples for HPLC tests were injected onto a column of Hewlett Packard Hypersil cartridge 5-μrn C18 (100 x 4.6rnm). The mobile phase was methanol: 35 rnM KH2PO «(pH 4.5) 55:45 containing 4 rnM triethylamine. The flow velocity was 1. 2 rnl / rnin. The compounds were monitored by UV absorption at 254 nrn using a Hewlett-Packard diode array detector (HP Model 1090). The concentrations of The tissue and tissue were determined from normal curves using the peak area for quantification. Normal plasma and tissue curves were prepared from plasma and tissue furnaces obtained from non-drug rats, injected with known amounts of RIO and Bll. The results were corrected for the recovery of the internal standard. The internal standard used was 4-trifluoromethyl-anilide of 5-methyl-pyrazole-4-carboxylic acid.

APPENDIX 7 # 1. Effects of RIO on body weight Atirnic mice (BALB / c, nu / nu, females, from 4 to 5 Di 'weeks of age) received IP administration of AlO (20 ng / kg / day) each day in 100 μl PBTE: D5W (1: 1, v: v) for 101 days. The vehicle control animals received IP administration of 100 μL PBTE: D5W (1: 1, v: v) daily for 101 days, and untreated control animals did not receive treatments. There were 8 animals in each group. The. weights on day 0 (one day before the administration of drugs) and twice a week until the end of the experiment. The percentage change was calculated as an average weight in each determination compared to the average weight on day Q. 2. Determination of "DL," Groups of 5 to 10 atirin mice (BRLB / c. Nu / nu, 'females, or BALB / c mice (males and females) were treated with IP RIO in 50 μL PBTE, 100 μL PBTE, or 50 μL DMSO In one experiment, groups of five female BALB / c mice received IP administration of Decadron (dexarnethamine sodium phosphate by injection, 1.5 mg / kg) once a day for 7 days before the administration of a dose individual RIO IP In a further experiment, groups of five female BALB / c mice received IP injection of the Ei &antin "- (phenytoin-sodium by injection, 20 rng / kg) once a day for 7 days prior to administration of an individual dose of AlO. All animals were observed for 14 days after the last dose was administered.The DLso was calculated from a plot of the percentage of mortality against the dose (log M) using a logistic equation of Four parameters with anti Ig antibodies i- goat mouse (Southern Biotechnology) overnight at 4 ° C. The plates were blocked by the addition of PBS + '% BSA. After washing with PBS, the supernatants were extracted from the murine nitrogen and the MLR cultures were added and incubated at room temperature for 1 hour.The plates were washed with PB ?, then the antibodies were added, Ig anti- tón de gobra labeled with HRP and incubated at room temperature for 1 hour.The plates were washed and developed by adding the substrate (RBTS).

E1EMPLO 1: PDGF AUTOFOSFORILATION INHIBITION THROUGH ALO This example illustrates the ability of RIO to inhibit PDGF-R autophosphorylation of rat C6 glioma cells. Rat CS glioma cells (5 X 1 QS) were plated on MCDB105 medium containing 5% FCS in a 6-well plate and incubated for 24 hours at 37 ° C. The cells were then placed in medium with 1% FC? for another 24 hours. The cells were treated with AlO at 50, 100, rnM for one hour at 37 ° C. The cells were then treated with 20 ng / ml of PDGF-BB for 10 minutes at 37 ° C. The cells were lysed in 50 M Tris-HCl (pH 7.4) containing 2 rnM EDTA, 10% glycerol, 1% NP-40, orthovanadate of a * IrnM, 10 rnM pyrophosphate, 1 mM PMSF, 10 rng / rnl of aprotinin and 10 rng / rnl de .leupeptin. The proteins were then separated by SD-polyacrylamide gel electrophoresis (PAGE). Proteins containing phosphorylated tyrosine were identified by Western blotting with an antibody of # antifosfotiroeiná. The level of phosphorylated tyrosine was determined by quantifying the amount of bound antifoefotiroeiná. The quantification was carried out by integration of peak area using a Molecular Dinamics Computing Densitometer (Model 300S), and Image Ouant v3.0 software (Molecule Dynamics). The data were expressed as relative peak intensity (phosphorylation of a receptor ^^ divided by the total amount of phosphorylated tyrosine). "* 'PDGF-BB stimulated the autophosphorylation of PDGF-R, while AlO inhibited such stimulation.The increasing concentrations of AlO resulted in phosphorylation of the stimulated PDGF receptor.AlO at a concentration of 200 M reduced the PDGF-R phosphorylation below that which occurs in the absence of PDGF-BB stimulation.

ArTrip O 2: SELFTIVE INHIBITION OF FLUTOPHOSFORILLATION PPGF-R VIA A1Q RIO inhibits the autophosphorylation of PDGF-FT in human T98G glioblastoma cells, while it has little effect on the autophosphorylation of the EGF receptor. The T98G cells were plated on MCDB105 medium containing 2% FBS and incubated for 24 hours at 37 ° C. The media was aspirated and then re-placed with CDB105 and the cells were treated for one hour with 200, # 500 or 1,000 rnM AlO. The cells were treated with different concentrations of RIO (0, 200, 500 and 1000 M) and in the presence or absence of the ligand. The cells were then treated with ligand for 10 minutes (20 ng / ml PDGF-BB or 50 ng / ml EGF). The cells were used and the phosphorylated receptor level was quantified as desed in example 1. RIO inhibited the autophosphorylation of PDGF-R by PDGF, although it had little effect on the ability of EGF to stimulate EGF autophosphorylation. -R.

E3FMPLO 3: INHTBICTOIM DF I QS PHOSPHORILATION PDGF-R THROUGH VARIOUS COMPOUNDS This example illustrates the ability of several compounds to inhibit phosphorylation of the stimulated PDGF receptor. U1242 MG cells were plated in 96 well plates at a concentration of 5 x 10 cells / well in cultured medium containing 0.5% FBS.The cells were incubated for 24 hours.The cells were then treated with a compound particular for 2 hours followed by the addition of 100 ng / ml PDGF-BB and incubation for 10 minutes.The cells were used in 0.2 M Hepes, 0.15 M NaCl, 10% glycerol V / V, 0.04% Triton X-100 , 5 rnM EDTR, 5 mM N * vanadate and 2 nM NaCl pyrophosphate. * cellularee were added after an ELISR plate coated with an anti-PDGF receptor antibody (Genzyme). ELISR plates were coated at 0.5 rnG antibody / well in 150 rnl PBS for 18 hours at 4 ° C before the addition of lysate. The lysate was incubated in the coated plates for 1 hour and then washed 4 times in TBST (35 M Tris-HCl pH 7.0, 0.15 11 NaCl, 0.1% Triton X-100). The antiofsphotyrosine antibody (100 ml in PBS) was added and the mixture was * incubated for 30 minutes at room temperature. The wells were then washed four times in TBST, a conjugated secondary antibody for POD (TRGO) was added to each well, and the treated wells were incubated for 30 minutes at room temperature. The wells were then washed four or four times in TBST, ABTS / HsOs solution was added to each well and the wells were incubated for 2 minutes. Abeorbencia was measured at 410 nm. "The cytotoxicity of each drug was also determined. The cells were deposited in wells as described above. Following incubation with drug, cell survival was measured by an MTT test as described by Moss an 3. Im unol. Methods 55: 55-53 (1983), or by measuring the amount of LDH released (Korzeniewski and Callewaert, 3. Immunol. Methods 64: 313 (1983); Decke r and Lohrnann-Matthes, 3. Irnrnunol. Methods 115: 61 (1988). W The results are shown in table IX. The CIaa values (ie, the dose required to obtain a 50% inhibition) were determined using the ELISR sieving test. The LDso values (ie, the dose resulting in "u" 50% toxicity) were determined using an MTT or LDH test. The CI values for inhibiting phosphorylation of PDGF receptor stimulated in U1242 cells varied from 0.4 to jjJ? SQO nM. As seen in Table IX, most of the compounds tested inhibited the phosphorylation of the stimulated PDGF-receptor. In all cases the inhibition of receptor phosphorylation was not due to nonspecific effects on cell viability as shown by the higher DLsa. Therefore, these drugs are good candidates for compounds that can be used to treat cell proliferation diseases by inhibiting the activity of PDGF-R. G13 and G14 had the lowest CISD although ######################################################################### TABLE IX PROGRESSION OF THE CICLQCE U AR This example illustrates the ability of AlO to inhibit the synthesis of stimulated PDGF-BB DNA and the progression of the cell cycle. The effect of AlO on DNA synthesis in the T98G cell in the absence or presence of PDGF-BB was determined by measuring the incorporation of H-thymidine into the DNA. the percentage of cells in the S of the cell cycle was determined by flow cytometry. The cells were cultured as described in the appendices above. The test conditions were essentially those described by Pollack et al., 3.

Neurosurg. 73: 106-112 (1990) with some modifications. Cells (rat C6 or human T98G) in the log phase growth were transferred to 96 well plates at 2 x 10 ** cells ifl? Pn 200 rnl of medium MCDB 105 containing 2% FBS. After a 1 * nighttime binding period the medium was changed to a serum free test medium (MCDB 105 with 5 mg / ml insulin) and the cells were incubated for 18-24 hours. The RDN synthesis studies were initiated by the addition of 50 ng / ml of PDGF-BB alone or in combination with various concentrations of AlO. The effect on the incorporation of basal 3H ~ tirnidine was determined in the absence of PDGF. The plates were incubated at 37 ° C for approximately 18 hours. AH-thyrnidine (A nersham, 5 Ci / mnol) was added to each well to produce a final concentration of 5 rnCi / rnl, the plates were returned to the incubator at 37 ° C, after 4 hours the medium was removed and the plates were placed on ice. Each well was then washed twice with 200 rnl of PBS cooled with ice. The radioactivity incorporated into the RDN was separated from unincorporated H-thymidine by precipitation with 100 rnl of TCR cooled with ice for 10 minutes. After two washes «TCR lion cooled with ice, the precipitate was solubilized (1% SDS in 100 rnl of base Tris at 20 M) and transferred to containers of liquid scintillation counting. They added 6 l of a combination (Ready Safe, Beckman) and the radioactivity was quantified in a Beckrnan liquid scintillation counter model LS6000 SC. RIO decreased the synthesis of RDN PDG stimulated in both cell types, although a greater effect was observed in Ig.the human R98G glioblastoma cells than in the rat C6 gliorna cells. To confirm these results, the effect of the RIO on the PDGF input stimulated in the S phase of the cell cycle was examined. The NIH3T3 cells engineered to overexpress the human PDFG-b receptor were restricted in their growth (serum was removed) followed by treatment with PDGF ligand in the presence or absence of AlO. The cells were analyzed for the content of RDN by flow cytometry. The results of this test are summarized in figure 3. Treatment with PDTF resulted in a marked increase in the residence of the cells in the S phase (62% 9 in relation to the cells not treated with PDGF (11%). However, the cells treated with RIO showed a dose-dependent decrease in the number of cells progressing towards the S phase of the cell cycle in response to PDGF, indicating that the stimulated rnitosie PDGF was blocked by RIO. confirm with the results of a si-mi -lar in which RIO at a concentration of 100 rnM was not able to inhibit mitosis stimulated in cells EGF NIH3T3 that overexpress the human EGF receptor (figure 3).

These results confirm the selectivity of the RIO for PDGF-mediated signaling.

EPLAM 5: TNHIBTTION OF IR ACTIVITY OF DIFFERENT RECEIVER SINASES The ability of different compounds to inhibit the different receptor tyrosine synthases was tested. The test results are shown in table X.

TABLE X M 0 fifteen twenty B F3EI1PUQ 5; EFFICACY XN VITRQ 30 The efficacy of RIO as a growth inhibitor determined on tumor cell lines, and primary tumors isolated from patients. The effects of the RIO on the tumor cell lines were determined by exposing the cells to a scale of drug concentrations and quantifying the cell density after four days using the procedures described in Appendix 3. Table 11 provides the results of tests of different cell lines. .'ái F TABLE XI TC = a values for RIO ranged from 0.8 μM to The effects of RIO on the in vitro growth of isolated primary turnoral cells from six patients with multi-formations of glioblastoma (GBM) and six patients with ovarian carcinoma were tested. Samples were obtained from newly diagnosed and previously untreated patients evaluated with or described in example 13, appendix 3. A positive correlation between inhibition of tumor growth by RIO and PDGF-R expression was observed for both types of tumor. As the concentration of RIO increased (from 0 to 400 mM), the growth of the tumor cell decreased for both types of tumors. Growth inhibition was dose dependent for both tumor types and varied among turnoral cells. CIsa values ranged from 39 μM to 198 μM for GMB tumors and from 20 rnM to 140 mM for ovarian tumors.

AXIS? PLQ 7: IN VITRO EFFECTIVENESS STUDIES USING This example summarizes several experiments that illustrate the RIO's ability to "inhibit the growth" of different tumors in vivo. The first series of experiments focuses on the effects of different formulations and treatment regimens. The second series of experiments addresses the effects of AlO on a variety of different tumors.

DIFFERENT FORMULATIONS AND TREATMENT REGIMES C6 and SKOV-3 (T) cells grew in culture, as described in "cell growth" above, and were implanted in the posterior side of a female Balb / c nu / nu to 3 mouse x 10 cells (for C6 experiments), or 1 x 10"7 cells (for SKOV-3 experiments) in 100 rnl of PBS on day 0. Human glioblastoma cells U87MG, U118MG or U373MG (obtained from RTCC) , or R4312 were also implanted in athymic mice, mice implanted with inores, and mice not implanted received by intraperitoneal injection RIO or Bll in a volume of 50 ml of DMSO, 100 rnl PBTE: D5W, or 100 rnl PBTE starting on day 1 or as otherwise indicated, the tumors were measured using vernier calipers and the volume of the tumor was calculated as a product of tumor length, width and height.In one set of experiments the mice were implanted with R431, cells of rat C6 gliorna, SKOV-3 ovarian purnor cells (T) and treated with RIO 15 mg / kg / day (DMSO). Tumor growth progresses logarithmically in untreated loe and DMSO controls. In contrast, tumor growth progressed only slightly (ie, more than 90% inhibition in "tumor growth after 20 days compared" with control) in the treated animals.

AlO implanted with cells of rat C6 gliornas or ovarian turnoral cells 5KOV-3 (T). The RIO has little effect (ee < say no more than "25%) on the R431 tumor growth." Mouse tumor growth is implanted with rat gliorna cells C5 was inhibited with 15 mg / kg / day of Bll (DMSO) to the same extent as implanted mice treated with mg / kg / day of RIO (DMSO). In another set of experiments the mice implanted with C5 gliorna cells were treated with RIO. Table 12 summarizes the ability of the RIO to inhibit rat C6 gliorna cells in atirin mice using different treatment regimens. The percent inhibition is related to the size of the tumor from animals treated with RIO, divided by the size of the tumor from the animals tr-linked with the vehicle control. The different treatment regimens resulted in 51% inhibition up to more than 95%. TABLE XII * In another set of experiments the effects of different AlO dose regimens were examined on C6 glioma cells. The RIO was administered IP at different doses different regimes starting with a day after antacid. The total dose of RIO ad ini trated to the animals was compared with the percentage of inhibition. These studies showed that the highest doses of RIO administered had less efficacy against tumor equivalent to that observed with lower doses administered daily, provided that the total dose administered was equal. For example, 95% inhibition of tumor growth could be obtained by administering RIO to mice at 20 Fmg / kg every day, 40 mg / kg every two days or 80 mg / kg every four days. C6 cells (3 x 10 * cells) were implanted SC into the posterior flank of BRLB / c, nu / nu mice. In another set of experiments, the effect of different doses of RIO on giioblastoma was determined. Table XIII presents data that "illustrate RIO's ability to inhibit glioblastoma cells in vivo.

TABLE XIII ^ / » Percent inhibition refers to the size of the tumor in animals tri- linked to the size of the turnor in untreated animals. Table XIV compares the efficiency of different RIO formulations in vivo (mice implanted with C6 cells). The formulations PBTE, PBTE: D5W and DMSO shown are equivalent to * iri inhibition "live of turnor growth.

CURRRO XIV The effects of RIO on animal mortality, using the formulations DMSO, PBTE, or PBTE: D5W is presented in table XV (mice implanted with C6 cells). The PBTE: D5W formulations significantly reduced the mortality rate compared to the DMSO formulations and the PBTE formulations.

TABLE XV Wl - INHIBITION OF DIFFERENT TYPES OF TUMOR THROUGH FULL This section describes "experiments that compare the ability of the RIO to inhibit the growth of different types of tumors." In a set of experiments, the ability of the RIO to inhibit turnoral ovarian, melanoma, cell lines, Prostate, lung and breast established as xenoingertosis SC were examined using the procedures described in the appendices.In a second set of experiments the effects of the RIO on the growth of murine leukemia cell lines in a synthetic model were tested using the procedures described in the previous appendices Table XVI summarizes the results of the studies using xenoingertos SC.R doeis that vary from 12 to 20 RIO effectively inhibited the growth of (human ovary), PA-l (human ovary), A375 (human rnelanoma) ), PC-3 (human prostate), Calu-6 (human lung), and DIB and L1210 (murine leukemia). However, AlO did not significantly inhibit the growth of R549 (human lung), MCF7 (human rnarna) and xenoingertos R431 (human epidermoid).

TABLE XVI Table XIV. The turnoral cells were implanted within the indicated strains of mice. The treatment with a vehicle or RIO began one day after the implant, with the following exceptions: + = day 4, ¥ = day 9, £ = day 15, = day 29. f = day 8. * D1B and L1210 are lines of murine tumor cell, C6 and 9L are rat turnor cell lines; all others are human turnoral cell lines. * • - * The data are presented as percentage of tumor growth inhibition on the indicated day as compared to vehicle control; n = 8 to 10 mouse / group ~ ptf J ^ r except DIB and L1210 where n = 4 mice / group. * - ** The P values were calculated using the student's t-test; NS- not significant. "^ In the second set of experiments, it was found that AlO significantly increases the survival of animals suffering from SK0V3T IP xenoingertos and mice suffering from 7DT1 iosteps In one experiment, SKOV3T cells (2x10 ^ cells) were implanted in the IP cavity of BRLB / c nu / nu mice The RIO was administered IP in 50 μM DMSO at 15 mg / kg / day for 21 days starting one day after implantation and mice were monitored for survival (8 animals per treatment and control group) All animals in the control group after 27 days, while animals treated with RIO died at 28 days and 40% of the animals treated with RIO survived after 32 days.

In another experiment, 7TD1 cells (B-cell hybridoma) were implanted IP in syngeneic inununocornpetent C57BL / 6 mice and the animals were treated for 30 days with 15 mg / kg / day of RIO (8 animals per treatment and control group). The RIO was administered IP in 50 μM DMSO at 15 mg / kg / day for 30 days starting one day after implantation. The dosage was terminated on day 30, and the surviving mice were observed for an additional 50 days. All animals in the control group died by day 9, while 3 of the 8 animals in the group treated with RIO survived until after day 80.

EXAMPLE 8 INHIBITION OF ACTIVATED CHARACTERIZATION OF PDGF-R INAPPROPRIATE This example illustrates the ability of AlO to inhibit cancers characterized by inappropriate PDGF-R activity while having little or no effect on tumors that are not characterized by PDGF activity. R. The expression of PDGF-R was measured qualitatively using a ~ Western blot. SRB tests determined cell growth by determining the total cell protein using sulforhodamine-B (Skehan, T et al., 3. Nati, Cancer Inet 82: 1107-1112 (1990). SAA) were carried out by seeding the cells to a colony density on a medium of semi-solid agar on an agar base layer after two to three weeks in which the size and number of the colonies was quantified with a counter of Ornincon 3800 ™ automated tumor foci.

SRE and SAR were expressed as CIso values in rnM. In vivo inhibition was determined in xenografted atirin mice. The results are shown in table XVII.

TABLE XVII As observed in Table XVII, growth inhibition is strongest on cells expressing high levels of PDGF-R, establishing a clear link between receptor activity and proliferation of cancer cells.

E3EMPL0 9: INHIBITION IN LIVE AFTER IMPLEMENTATION USING ALQ This example describes the effects of AlO when administered to mice for a number of days after the implantation of the cancer. In one experiment, the SCID mice containing the PC-3 prostate cell line were treated with RIO starting 15 days after the turnor transplant. The weekly treatment comprised 12 mg / kg / day of RIO (PBTE: D5W) for five days and two days without treatment. They were treated for three weeks. In another experiment, the mice containing the R375 rnelanoin were treated in the same manner as those implanted with PC-3 except that 15 mg / kg / day of RIO was used and the treatment started 9 days after tumor implantation. In both cases, tumor growth was inhibited by RIO. The growth of turnor PC-3 was inhibited approximately 50% after three weeks. Growth of rnelanar R375 was inhibited approximately 40% after three weeks. Therefore, this example further illustrates the utility of the RIO to inhibit turnor growth by showing its ability to inhibit the turnor that has been growing in a host before treatment (example 7 where the -tumor treatment starts one day after the transplant).

EXAMPLE 10: EFFECT OF ALO ON GROWTH OF INTRACEREBRAL TUMOR The effect of RIO on turnor growth in brain tumor models was examined in a series of experiments. In two separate AlO expirers, the atomic mice were administered immediately after the intracerebral (Cl) implantation of C6 cells, the tiernp > or average survival of R10-treated anirnalee was significantly increased compared to controls. The results of this Experiments are shown in Figure 4. In another experiment, in which RIO was administered to athymic mice following the CI implantation of U87MG cells, the average survival of the animals treated with RIO was 55 days cornp> prada with an average survival of 60 days in the control animals an increase that was not significant (P = U.15) .The efficacy of RIO in typhic jfc rats was also proven.As shown in figure 5, RIO has an effect Positive but significant positive on the "survival of athytic rats followed the implantation Cl of" turnoral cells C6 E1EMPLO 11 = INMONOLOGY STUDIES The effects of RIO on various parameters of normal immune function were examined, including the proliferation of lymphocytes, the generation of cytotoxic effector cells, the production of lymphokine, and the secretion of uni-globulin. These studies involved the in vivo treatment of rats and mice with AlO followed by in vitro analysis of immune function. A detailed summary of the methods used in immunological studies is described in Appendix 5.

EFFECTS OF THE IN VIVO ADMINISTRATION OF RIO ON THE FUNCTION IMMUNE OF NORMAL MICE BALB / c mice not afflicted were treated with 15 mg / kg / day of R10___o or the vehicle (PBTE: D5W) for 7 and 21 days. The animals were sacrificed and their spleen cells were tested in vitro for the responses to Confl (mitogen T-cell), LPS (a B-cell heterogeneous independent of T cells), and alloantigens (spleen cells C3H / He3) horn are described in Appendix 5. The content IL-2, IL-6, Ig and cell proliferation were measured after 40 hours - Sjk (mitogen) or 72 hor-as (alloantigens). The results are summarized in table XVIII.

TABLE XVIII Effects of AlO on normal immune function in mice Table XVIII. BALB / c female mice were treated with AlO for 7 or 21 days. Spleen cells were removed and stimulated in vitro with mitogens or alloantigens. Cell proliferation, cytokine production, and Ig production were tested as described in Appendix 5. The results are presented as a comparison between the drug treated animals and the animals treated with vehicle (PBTE: D5W). 0 = no change; + / - = Pligero increase in some animals; + / - = moderate increase in all animals; ++ = strong increase in all animals. Two parameters appeared to be affected by the RIO treatment: the production of induced RIS IL-6 was higher in the group treated with RIO than in the controls on day 7, although this difference was not apparent on day 21. Ig production Induced LPS was slightly higher in the RIO group than in the controls followed by 7 days of treatment, so this difference was not apparent in 3 or 4 mice tr-linked with drug for 21 days. These data indicate that the effects of RIO on IL-6 production and Ig production by splenocytes were transient even with continued administration of RIO.

EfectPS of the administration in V o of AlO on the immune function of mice during a primary immunization The effect of AlO on animals that respond to a Active primary immunization was also examined. BALB / c mice were immunized SC with 50μg of lapano hernocyanin (KLH) suspended in complete Freund's adjuvant (CFA); the control mice were administered an emulsion of saline regulated in their PH with phosphate (PBS) in CFA. Starting on day 1, the mice were divided into 3 treatment groups: untreated, vehicle only (PBTE: D5W), or RIO at 20 rng / Kg day. On day 14, all mice were «Jfc-tsacrificadoe, and eue spleen cells were tested. In addition, the immune responses to KLH and tetanus toxoid were measured as described in Appendix 5. The production of cytokine (IL-2 and IL-6) by the splenocytes was not significantly affected by the vehicle alone or the drug as summarized in Table XIX However, the AlO treatment was associated with a slight inhibition of the proliferative responses of the spleen cells after resternulation with the immunization antigen (KLH) intro, (Table XIX). Sirnilary effects were observed when the spleen cells were stimulated with LPS or allogeneic cells (quadr-or XIX).

Table XIX Effects of RIO on Normal Immune Function in Mice that Resp > onden to a Primary Immunization Table XIX. The BRLB / c mice were immunized with KLH, then divided into three treatment groups; the dosage starts on day 1 and continues until day 14 after immunization. Spleen cells from mice from untreated, vehicle-treated or RIO-treated mice, as well as non-innate (normal) control mice, were cultured with the indicated monitogens or alloantigens. Cell proliferation, cytokine production and immunoglobulin production were tested as described in Appendix 5. The results are presented as a comparison of the responses of the animals treated with drug to those of the controls treated with vehicle. ^ ß! BTE: D5W). 0 = no change; - = slight inumption in all animals.

Effects of the administration of AlO in vivo on the immune function of mice having the tumor BALB / c mice received? C implant of syngeneic WEHI 154.13 fibrosarcorcor cells on day 0. On day 1, the mice were divided into three treatment groups: untreated, daily treatment with vehicles only •? V (PBTE: D5W), or daily treatment with AlO at 20 mg / kg day. On day 17 the animals were sacrificed and their blood vessels were removed. Spleen cells were used in vitro for responses to the alloantigenes and rnitógenoe. The RIO treatment did not affect the proliferation, cytokine secretion, or Ig of the spleen cells stimulated with alloantigen or rnitogens as summarized in Table XX. Spleen cells were also tested for cytotoxic T-cell lymphocytes. "Esp >eci of WEHI-164.13 (CTL) in the culture of combined lymphocyte tumor cell (MLTC). Spleen cells from animals that have the turnor- without treatment but not a vigorous CTL response (44% specific lysis at E: T = 50: 1). This response was almost completely inhibited in animals receiving the vehicle alone (13% specific lysis). The vehicle has an ethanol content of -15% and ethanol is known for the transient effect of CTL generation and activity. (Walia, et al., Proc. Soc, Exp. Biol. Med., 192: 177-200, 1989.) Treatment with AlO increased the CTL response (78% specific lysis) compared to animals treated with vehicle; The CTL response of the spleen cells of the RIO-treated mice was also higher than that of the non-trialated control animals. Thus, it seems that in this inrnunocornpetent mouse model, the RIO dosage exceeds the CTL generation effect.

-F Table XX Effects of RIO on Normal Immune Function in Brass Having a Primary Tumor. '1 •' Table XX. Mice that have tumor were treated with vehicle or RIO, or left untreated, for 17 days after implantation. Mice were sacrificed and lae spleen cells were cultured with the indicated mitogens, alloantigens, or WEUN-164.13 turnoral cells treated with rnitocin-C. The p-cell proliferation, cytokine production, inrnunoglobulin production and CTL activity were tested as described in the Appendix . The results are presented as a comparison of the responses of animals treated with drug to those of the controls (PBTE: D5W). 0 = no change; ++ = strong increase in all animals; NT = not tested.

Effects of in vivo administration of AlO spfrre the function 'I'IIH' II I 'I' I? Wistar rats received C6 cell implants on day 0. Starting "on day 1, and daily thereafter, the rats received IP vehicle doses (PBTE: D5W) or AlO and 8.4 mg / kg / day (equivalent to 20). rng / kg / day of AlO in mice) The control group was not treated after the implantation of the turnor On day 21, the animals were sacrificed and the animals removed for in vitro tests. were contraindicated ConR, LPS, and alloantigene (DR rat spleen cells) as in the murine studies .. Of the parameters measured, only cell proliferation- in the syngeneic combined lymphocyte response (MLR) appeared to be affected by the treatment RIO as summarized in Table 21. The syngenic MLR of untreated animals treated with vehicle or having the tumor was much higher than those of the animals without the turnor.This proliferative response was reduced by one in 4 animals that have the j3? rnor treated with RIO to a level similar to controls that do not have the tumor. The allogeneic response was not reduced. The spleen cells of Wistar rats were also tested for the generation of C6-specific CTL in the MLTC and the results were summarized in Table XXI. The cells of non-trialated animals that have the tumor raised a vigorous CTL response (35% specific lysis to ET = 100: 1), while the cells from control rats that do not have the turnor presented a response of (13% lysis specific to Similar to studies in BRLB / c mice, it appeared that the vehicle inhibited the generation of CTL in a 7-day culture.) In contrast to murine studies, however, RIO does not exceed effect of vehicle inhibition in inrnunocomp> etentee rats.

Table XXI Effects of the RI on the normal immune solution in rats that have a primary tumor.

Table XXI. The Wistar rats received SC implants from C6 cells, followed by the daily vehicle treatment or RIO; The untreated animalee that have the turnor and the animals that do not have the normal tumor were included in the study. On day 21, the animals were sacrificed, and their spleen cells were stimulated with the indicated mitogens (ConR, LPS), syngeneic (Syn) or alloantigenic (Ryl), or C6 turnor cells treated with rnithornnicin C. Cell proliferation, cytosine production, and CTL activity were (stolen as described in Appendix 5. The results were presented as a comparison of the response of animals treated with drugs to those of the controls treated with the vehicles (PBTE : D5W) 0 = no change; +/- = decrease in some drug-treated animals to levels detected in normal animals (no tumors, no drug treatment), NT = unproven The results of these inrunological studies indicate that RIO does not affect adverea lae responses Immunocompetent or immunocompetent rodent immune cells during or after in vivo dosing In addition, the efficacy of the drug when tested against lines of murine turnoral cells in host immunocompetent hosts indicates that RIO does not impair antitumor immunity. .

Eiemolo 12: Pharmacology of RO Several pharmacological properties of AlO have been studied in mice and rats including their metabolism and half-life in plasma and brain tissue.A detailed description of the methods used in the study of AlO pharmacology can be found in appendix 6. In addition, the RIO pharmacokinetic profile was studied in conjunction with formal toxicity studies of the compound in rats and monkeys.

RIO metabolism The RIO isoxazole ring supports an intramolecular reaction to 2-cyano-3-hydroxy-N-C4- (trifluoromethyl) phenyl] -2-butenarnide (Bll). The metabolism of RIO and Bll was investigated ex vivo in heparinized plasma from humans or rats. The conversion of RIO to Bll was monitored using reverse phase HPLC as described in Appendix 6. R 25 ° C (room temperature) RIO supported complete conversion to Bll for 21 hours in human plasma. A similar experiment was performed using rat plasma with incubation at 37 ° C; RIO was completely converted to Bll after an incubation of 2 hours. The conversion of RIO and Bll does not occur in the inactivated plasma, by heat (15 min at 60 ° C) from humans or rats. Additionally, both Bll and AlO together were incubated with fresh human plasma and the concentrations were increased. No AlO was detected after 21 hours; only Bll was present. The rnetabolite Bll appears to be equipotent to AlO in the in vitro growth tests as well as equipotent in inhibiting the synthesis of stimulated RDG PDGF and the stimulated PDGF cell cycle progression. Bll is also equipotent in inhibiting the growth of C6 tumors in vivo. These data suggest that Bll is an active metabolite of the RIO. The use of RIO to treat cell proliferation diseases is preferred because it is more appropriate than Bll for the formulation as an IV solution.

Pharmacokinetics of fll) v Bll i vivo The furanacokinetics of RIO and its metabolite (Bll) was investigated in vivo, after intraperitoneal administration (IP) of RIO to mice and rats. When the atomic mice (BRLB / C, nu / nu, females) received RIO (120 rng / rnE, 40 rng / kg), the drug was converted to Bll at 3 hours after the dose, so the concentration of the parent compound was below of the limits of detection in the systérmico circulation. The Bll was detected in the plasma for more than 48 hours and had a t-1/2 of 16 hours and 3 hours. The area under the curve of the concentration time (RUC) for Bll was calculated to be the rate of release that was calculated to be 2773 μg.h / rnL assuming 100% bioavailability and a mean entry time that is zero . The volume of distribution was calculated to be (VD). in the brains of those mice, 6.7 rnL a Bl "l of 14 hours and a t / 3 of 3 hours was exhibited.

Table XXII. Female nude mice (BRLB / cm nu / nu, four group) were treated IP as indicated. BRLB / c mice (female, four per group) were treated IP with 120 rnG / rn22 (40 rng / kg) RIO. Wistar rats (females, one per time point) were treated IP with 560 rng / rn12 (80 rng / kg) RIO. The plasma and brain samples were prepared and analyzed by HPC as described in Appendix 6. All results are calculated by the internal normalization method. MT = not tested, ND = not detected. The pharmacodynamic profile was also determined in atirn mice for the RIO dose calculated to be the DL3.0 Intraperitoneal administration of RIO and 225 mg / rns (75 rng / kg) resulted in Bll detection for more than 48 hours (the last point of time analyzed) with a t ^ / s of 15.5 hours and Trnsi? 3 hours (table XXII). The RUC was calculated to be 5660 μg hr / rnL. The CL was calculated to be 0.27 rnL / hr. assuming 100% bioavailability and a mean entry time that is 0. The RV was calculated to be 6.4 Ml. RIO DLa_o's pharmacoci ethical profile (75 mg / kg) was 'also studied in mice after 4 IP treatments given once every 7 days. The Bll was detected in the plasma for 48 hours after the last dose and had a Ta- ^ s of 16.9 hr and Tmax of 3 hr (Table XXII). The RUC for Bll was calculated at 5012 μg.hr / L. The CL was calculated to be 0.3 mL / hr, assuming 100% bioavailability and an average entry time that is zero.

The RV was calculated to be 8.2 rnL. The pharmacokinetic profile of AlO was determined in BALB / c mice. BALB / c mice received a single dose of AL (120 rng / rn ^, 40 rng / kg). The RIO has been completely converted to Bll and was not detectable for 1 hour after administration of the drug. Bll was detected in the plasma for more than 48 hours (the last point of time analyzed) and had a t / O of 14.9 hr- and Tms? > < of 3 hr as is summarized in table XXII. The RUC was calculated to be 444 μg.hr / rnL. The CL was calculated to be 0.18 rnL / hr, assuming f100% bioavailability and an average entry time that is 0. The RV was calculated as 3.1 rnL. In the brains of those mice, Bll exhibited a t 1/2 of 2 hours and a Tm; a < of 1 hour (table XXII). When the rats (Wistar females) received an individual dose of RIO (560 mg / m2, 80 rng / kg) the drug was converted to Bll in two 2 hours after dosing, the first time point analyzed. However, RIO was detectable haeta 8 hours after the dose. As summarized in table 22, AlO had a t / 2 of < 2 hr and Tma? of < 2 hr. Bll was detected by rnae 24 hours after the dose and had an X / > of 6 hours Tm¡a > < - The RUC was calculated to be 6417 μg / rnL. The CL was calculated to be 0.26 L / hr, assuming 100% bioavailability and a mean entry time that is 0. The RV was calculated to be 5.3 rnL. In the brains of those mice, Bll, a? 6 hours and one Tma) < of 6 hours although RIO exhibited a tn./: of 20 hor-as and a Tr of 7 hours (Table XXII).

# Plasma levels in vivo The in vivo plasma levels of Bll were determined in atiric mice after > of a single dose or repeated daily doses of RIO and the results are summarized in Table XXII. Three hours later (at Tma <) of an individual administration of RIO (20 mg / kg), the Bll concentration was determined to be 51.2 ± 7.3 μg / rnl (scale 38.8 - 55.2 ^ μg / rnL) while RIO was not detected. At steady-state levels of Bll in the plasma, atirnic mice were also determined after repeated administration (20 mg / kg / day of RIV at intervals of 24 hours (8 administrations).

The maximum stable state level of Bll in the plasma was 79.8 +. 11-2 μg / rnL (scale 65.2 - 94.6 μg / rnL), and the minimum steady state or threshold level was 33.6 +. 7.3 μg / mL (scale of 23.4 - 45.8 μg / mL). The level of Bll in the plasma after obtaining the stable state (maximum stable state level) was ^ K 64% higher than after an individual dose (Table XXII). ^ Table XXII Levels of Bll in the plasma Table XXII. Levels of Bll in plasma were determined after individual or multiple daily administrations of IOP at 20 or 40 mg / kg / day in atirnicos mice. All data are the average of 4 animals. The concentration of Bll in the plasma a, Tma.? it was also determined after a simple administration of RIO at 40 rng / kg. The concentration of Bll was 110.9 +. 15.3 μgS / rnL (scale 93.5 - 131.3 μg / ml) while RIO was not detected. The steady state levels of Bll in the plasma were also in athymic mice after the repeated (40 mg / kg) of RIO at 24-hour intervals (6 administrations). The stable state level of maximum Bll in the plasma "was 161.1 +. 27.8 μg / rnL (scale 119.9 - 204.1 μg / mL) and the minimum level of steady state or umbilical level of 38.2 + 2. 7 μg / rnl (scale 13.5 - 66.9 μg / rnL) The level of Bll in the plasma after reaching the steady state was 69% higher than after an individual dose (table XXII). That data shows that Bll accumulates in the plasma J ^ - after the multiple daily administrations of RÍO. After the plasma levels, the RIO and Bll concentrations were determined in brain tissue after administration of RIO to rats and mice. The results are summarized in table XXIV. The levels of Bll and RIO in the brain differed in both species and strains. The highest level of Bll in the brain was detected in atirnic mice, followed by the BRLB / c mice, and the Wistar rats respectively, fl lae 2 and 4 hours after the doses, there were 11 and 6.5 times, more Bll in the mice. brains of atirnic mice compared with Wistar rats. At 2 hours after dosing, there were 2 times more RIO in the brains of atirnic mice compared to Wistar rats; At 4 hours after the dose approximately equivalent amounts of fllQ were detected in the brains of atiric mice and Wistar rats.

TABLE XXIV Table XXIV. The levels of Bll and RIO in brain tissue were determined as described in Appendix 6. Determined at 2.5 hours after the dose. n = 4 animals. These pharmacological studies show that RIO is metabolized to Bll in both rats and mice. In addition, AlO and Bll can be detected in both plasma and brain tissue. However, there seems to be a difference in the farrnacokinetics and the distribution of AlO and Bll in plasma and ^ jB > The brain between the two species examined.

Example 13: Preliminary studies using QA Preliminary toxicology studies of RIO included testing the effect of RIO on blood cells, body weight, DLso-determinations. To determine the effect of potential adjunctive medications on LSo. of cutó also the combination of experiments. A detailed description of the methods used can be found in Appendix 7. The pharmacology and toxicology studies illustrate that RIO can be administered to animals under conditions that have little, if any, adverse effect on animals, particularly when The PBTE formulations are used: D5W. Other suitable formulations can be obtained by someone skilled in the art using this application as a guide.

Effect of AlO on blood cells Many cancer therapeutics are cytotoxic in nature and have profound effects on blood cells that result in cytopenia. The effects of RIO on the number of red blood cells, white blood cells and the percentage of lymphocytes against polymorphonuclear cells were also examined. RIO at 15 mg / kg / day does not appear to affect blood differentials over a 21-day period for the drug in DMSO, PBTE or PBTE: D5W (1: 1). The drug supplied in PBTE: D5W at 20 or 25 mg / kg / day did not affect the number of RBCs, "WBCs or the percentage of lymphocytes: neutrophils." However, the drug supplied in PBTE alone at 30 mg / kg / day showed a slight decrease in WBCs after 2 to 3 weeks of treatment. The animals given 40 mg / kg / day showed anemia and leucopemia after several weeks of treatment when they were given RIO in PBTE alone. No effects on blood cells were observed when RIO was administered in PBTE: D5W even after 100 days of treatment.

Effects of AlO on body weight The effect of daily administration of AlO (20 rng / kg / day) on body weight was examined over a period of 100 days. AlO has effects on weight gain compared to controls. However, after several weeks, the animals gained weight on a scale similar to that of untreated or vehicle treated animals. During the treatment period, no mortality was observed. In addition there was no effect on blood differentials and there were no observable effects in most of the histopathology of the organs, including heart, liver, lung, kidneys, spleen, long bone, stomach, rnesenteric lymph node, and the large and small intestine and the pancreas.

Determination of DL "" The lethal dose of AlO piara 50% of the animals (BL-sa) was determined for the atirnicos mice (BALB / c, nu / nu, females) and the BALB / c mice (males and females) using a number of dosing regimens. As shown in Table XXV, the RIO DL.3a ranged from 83-145 mg / kg. The effects of Dilantin < F (phenytoin-sodium for injection), an anticonvulsant agent, and DecadronCF ° (dexamethasone sodium phosphate for injection), ^, an anti-inflammatory agent, on DLso of AlQ (Table XIV). The RIO following the pretreatment of animals with DecadronCF or DilantinCR? it was 94 and 144 mg / kg, respectively.

TABLE XXV Determination of AlO in mice k? Cuadr-o XXV. DecadronC F ° a 1 was administered. 5 mg / kg / day 7 days before the administration of an individual dose of RIO. Dilantin < F was administered at 20 ng / kg / day for 7 days before the administration of a single dose of RIO. The DLrao was calculated from a graph of percentage of mortality against the dose (log M) using a logistic equation of four parameters. f = female, rn = male, n = 5 animals per group.

This example illustrates the use of the nucleic encoding of PDGF-β receptor to inhibit turnoral growth in vivo. The C6 rat gliorna cells were infected with retroviruses carrying a mutant gene for the human PDGF-b receptor. Seven selected G418 clones were screened for expression of the truncated receptor by Western blotting with an antibody that recognizes the extracellular aFβ domain of the human-receptor but has no cross-reaction with the wild-type rat receptor. The clones, HiMut.l and H? Mut.2, express high levels of a protein with the predicted molecular weight for the receptor that lacks the majority of the intracellular region. Several clones expressed low levels of the truncated receptor; LoMut.l was selected for additional experiments. Hirnut.l Express PDGF-R 8.3 times higher than LoMut.l. Himut.2 express PDGF-R 9.4 times higher than LoMut.l. The isolation and characterization of the clones that the mutant receptors were carried out as described below. Cell culture. All culture medium, fetal bovine serum (FBS) and chemical agents were purchased from Gibco BRL. CS rat glioma cells were grown in a Harn / F-IQ medium supplemented with 5% fetal bovine serum and 2 nM glutamine. The COS cell were cultured in 10% fetal bovine serum and 2 nM glutamine in a Dulbecco's modified Eagle's medium (DMEM) # Expression of the mutant PDGF-b receptor. A stop codon was introduced by site-directed mutagenesis into the gene from the human PDGF-b receptor directly upstream from the first tyrosine kinase domain. The mutant gene was cloned into a vector under the control of the long terminal repeat of rnurin sarcoma virus (Muller, R.D., et al., Mol. Cell. Biol. 11: 1785-1792, 1991). 4000 g of each of these vectors and a B ^ vector- containing the genes required for retrovoiral virus packaging (Muller, supra) were co-transfected into COS cells (2 x 10 cells / dO rnm plates) by calcium phosphate precipitation ( Chen, C. fl., And H. Okayarna, Biotech 6: 632-638, 1988). The cells were washed with PBS and performed the next day and the conditioned medium was harvested on days 4-5 after transfection. C6 cells (103 cells / 60 nrn plaque) were infected with dilutions of conditioned medium containing 6 μg / ml Polybrene (Sigma). Two days later, the cells were placed within the selection with 800 mg / rnl G418 (Gibco) and the colonies collected when they could be distinguished. The vector control cells were prepared by the same method although with a vector lacking a gene under the LTR. Co-inrnunoprecip > tip > or wild and truncated receptors. Vector control cells and cells expressing high levels of the mutant PDGF-b receptor (HiMut.l) were each seeded with 3 x 10 cells / well on 6-well plates. The next day, the medium was changed by 0.5 rnl 3% FBS in -cys -rnet DMEM (ICN) containing 100 mCi / rnl Tran 3 S-label tag (ICN). The cells were incubated at 37 ° C for 16 hours. They were washed twice are pH-regulator binding (0.1% BSR, .1.0 ing / rnl CaCl_a.2Hs »0, 10 rng / l MgS0? .7Hsa0, 10 mg / ml, aprotonin and 0.2. RnM PMSF in PBS ), and 0.5 rnl of binding pH regulator or • 0.5 rnl 20 ng / ml PDGF-BB (Collaborative Researc Inc.) in regular binding pH that was added to each well. The cells were incubated at 4 ° C for 4 hours, washed 2 times with PBS and used with 0.5 ml 1% Triton X-100 in HNTG (20 rnM HEPES (pH 7.5), 150 rnM NaCl, Triton X-10Q, 10% glycerol, 10 mg / ml each of aprotonin, leupeptin and pepstatin, and 0.2 rnM PMSF). PDGF-BB was included in the lysis buffer of cells that had been treated with PDGF. The lysates were rotated at 100,000xg for 30 minutes at 4 ° C. The supernatants were in new tubes and prewashed with Protein A-agarose (Vector laboratories). SDS was added to a final concentration of 0. 1 to 2 of the samples treated with PDGF- for each cell line. Duplicate samples were inrnunoprecipitated already with an antibody recognizing the C-terminus of the eilvestre rat receptor (UBI anti-PDGF-b receptor) or the human mutant receptor (anti-PDGF-b receptor of Genzi a). He I G anti-rabbit mouse was used co or an anti cue rp > or secondary for samples incubated with the anti-receptor? MfTr Genzirna. The complexes were precipitated with Protein A-agaroea and washed 5 vecee with 0.1% Triton X-100 in HNTG. The proteins were separated by electrophoresis of SDS-polyacrylaminating gel on 7.5% gels under reducing conditions. The gels were fixed, treated with Arnpli fy (Arnersharn) and exposed to X-ray film for 3 days.

Western blottino. Each cell line was plated in multiple wells at 5 x 10 cells / wells on 6-well plates. The next day they were fed with 1% FBS in MCDB 105 (UCSF Cell Culture Facility) and incubated for 24 hours in a 0% CO2 environment. PDGF-Rfl O BB (Collaborative Researc Inc.) was added to a well of each clone at the desired final concentration. After incubation for 7 minutes at room temperature, the cells were washed with PBS and lisadae with 50 rnM Trie-HCl (pH 7.4), 1% nonidet P-40, 10% glycerol, 2mM EDTR, 10 M and sodium pyrophosphate, 10 each of aprotinin and leopéptin, 1 rnM PMSF and 1 rnM sodium orthovanadate. Equal volumes of each lysate were operated on 7.5% multiple SDS polyacrylamide gels and transferred to cellulose (Sceicher 8 ¿Schuell). The membranes were blocked with 5% instant skim milk in Tris buffered saline at its pH 0.05% Tween-20 (TBST-T). Duplicate membranes were incubated with each polyclonal antiphosphotyrosine 1: 3000 or anti-PDGF-b receptor (UBI) 1: 000 in regular of p > H blocking. The second antibody was Wt anti-rabbit IgG, goat conjugated with horseradish peroxidase (Sigma 1: 000. To detect the truncated receptor, a monoclonal antibody against the extracellular domain of the human PDGF-b receptor (Genzyrne) diluted 1: 500 was the one used The secondary antibody used was rabbit anti-mouse IgG conjugated with peroxidase (ICN) 1: 000. ECL (R ersharn) was used for detection on all dyes. Molecular Devices Cornputing Ether Density The levels pb phosphorylation salts were subtracted from each point.

Adherent growth of celUla e? Lines To determine the growth densities, each cell line was seeded with 10 cells / well over 5 24-well plates with samples triplicated in FBS medium Harn / F-10. The media was changed every 3 days. Every two days, the cells on a plate were trypsinized and counted on a counter • ipulter. To determine the cloning efficiencies 100 cells from each cell line were plated on plates of 10 crn in 1% or 5% FBS in Harn / F-10 medium. The media was changed every 3 days for approximately 12 days. The colonies were fixed, stained with methylene blue and registered.

Cell line anchoring independent growth ^ (soft agar test). A base layer is made in 8% plates (FMC BioProducts), 1% FBS 2rnM glutamine, lmM sodium pyruvate, 10 mM HEPES and non-essential amino acids. Cells were suspended in 0.4% agarose containing the other ingredients noted above and the desired concentration of PDGF-BB (Collaborative Research Labs).

The suspension was plated on the base layer with 3000 cells / plate. They were incubated for 2 weeks in a humic chamber with 5% C0S. The colonies were '? registered visually or with an Omincon 3800 ™ automated tumor colony counter.

Growth of cell lines in mice without Hair.

The cells were exp > andidas in rolling bottles, trypsinized and resuspended in PBS. They were counted and the volume adjusted to 3x10"'" cell / rnl. For each cell line, 4 to 8 mice without atirnico hair (Sirnonsen Labs) were injected subcutaneously with 100 ml (3x10 * cells). HE A (ordered the tumor volumes with calibrators twice a week for 18 to 21 days.

Immunohistochemical staining of tumor sections. The tumors were resected from the mice and frozen in OCT (Miles Lab). Sections of 5 ml thick were cut and fixed with acetone. Lae sections were blocked with 10% normal goat serum before incubation with 20 rng / rnl biotinylated antihuman PDGF-b receptor (? X genzirna antibody, biotinylated by Molecular Prober). Streptavidin conjugated with peroxidase (Caltag) 1: 100 and diaminobenzidine (Sigrna) with H202 were used for detection. For a negative control, a biotinylated monoclonal antibody for a p > Unrelated rotein was used in the same protein concentration as the anti-PDGF-b receptor. The anti-dye was Harris hernatoxylin (Rnatech). Using the above-described retroviral expression system fr, a truncated PDGF-b receptor was introduced into the rat C6 glioma cells. In resistant G418 clones expressing the mutant receptor, the tyrosine phosphorylation induced by the wild-type PDGF-BB receptor was significantly reduced. In addition, these cells grew to lower densities and formed smaller colonies in the culture and in soft agar than the progenitor C6 cells. Cells that express the truncated receptor were significantly impaired in their ability to grow in hairless mice. After 21 days, the volumes of the Tumors of HiMut.l and HiMut.2 were only 12-16% of the size of the tumors of the progenitor cells. The tumors derived from C6 progenitor cells and vector control cells were essentially identical indicating that the G418 selection of the vector control cells did not affect their ability to grow in hairless mice. Tumors derived from HiMut.l gave a very dark immunological staining in at least 10% of the cells. The tumors' derived from HiMut.2 were stained in 45-85%. The p > Resistance of the truncated PDGF-b receptor was also confirmed by Western blotting of the turnoral sections used. Therefore, the truncated PDGF-b receptor was expressed in vivo up to 21 days and you or it must be present in at least 10% of the cells that were to be inhibited. These studies demonstrate the uselessness of the dominant negative mutants of PDGF-R to inhibit the growth of apa tumors characterized by inappropriate PDGF-R activity in vivo.

Example - 15 Cell culture v effects j or vitro and other compounds This example illustrates the effect of characterized compounds different from RIO and Bll, on the turnoral growth in cell cultures or in vivo. PDGF-R, C6 SRB activity and in vivo efficacy were measured using the Vrocedures described in the previous appendices. The results are shown in Table XXVI and XXVII. Those results are preliminary data. One skilled in the art can improve the efficiency of different compounds using factors known in the art such as different dosing regimens.

?, F TABLE XXVI w effected in Western test f PICTURES XXVII c- W = Done in Western test Example 15: Combination therapy. XA Studies were conducted to observe the effect on tumor growth when PDGF-R inhibitors are used in combination with known cytotoxic drugs currently used to treat cancer. In separate experiments, CRLU-5 cells and MCF-7 / Her2 cells were implanted subcutaneously in hairless mice. In one experiment the r-atons were then treated with AlO alone, cisplatin alone or a combination of cisplatin and AlQ. AlO was given intraperitoneally twice a week at 5 rng / kg. Isplatin was given in a wave intraperitoneal dose of 5 # rng / kg on day 2. In mice implanted with MCF-7 / Her2 cells, the combination of AlO and cisplatin was better for the inhibition of tumor growth than cisplatin alone or without treatment, but did not improve the turnor suppression compared to AlO alone. However, in mice implanted with CALU-6 cells the combination of AlO and cisplatin resulted in a significant suppression of turnor growth compared to AlO alone, or cisplatin alone or no treatment. In a second experiment with cells CALU-6, mice were treated with AlQ, cisplatin or VP-16 alone (10 mg / kg on days 4, 7 and 10) or the combination of cisplatin and VP-16 or AlO, cisplatin and VP-16. The combination of AlO, cisplatin and VP-16 was better- suppressing tumor growth than either the solee drug or the combination of cieplatin and VP-16.

Example 17: Chemical synthesis. Some of the compounds of this invention can be prepared using procedures known in the literature starting with cornp > well-known intermediaries or intermediaries. The quinoxalines were prepared either 1) by reacting a 1,2-aromatic diamine with a ketoaldehyde or a diketone, or 2) an exchange reaction of a bis-thiose icarbozone and a 1,2-diamine in the presence In the following preparations the aromatic diarrnin was obtained commercially or prepared as described in the example In the examples where the reaction solvent "is not ~ eep > ecificádo, the reaction was carried out in ethanol-acetic acid. Quinoxalines synthesized using this solvent were isolated as comp > away from addition of acid with a molecule of acetic acid, based on elemental analysis. The reactions in ethanol alone, followed by ¡¡-e ap > solvent prayer and recrystallization gave a product • • Clearer and higher performance.

Compounds of group 1 am RIO "may be prepared as in European patent application No. 0 013 376 Al. Alternatively, RIO may be p <or> as follows: gj Step 1: Preparation of acetoacetic acid (4- ri fluorornetyl) aniline. A mixture of 4-tri fluorornetyllaniline (16.1 g, 0.1 mol), 2, 2,6-trimethyl-4H-1, 3-dioxin-4-one (purity 95%, 14.97 g, 0.1 mol), and xylene (20 rnl) was heated to reflux for 30 minutes in a bath preheated to 150 ° C. The resulting dark solution was cooled to room temperature to crystallize the product.

The crystals were filtered and collected. More material was obtained from the mother liquors (the remaining solution after initial crystallization and filtration). The yield of the aniline was 17.75 g (72%), the aniline had a melting point of 153-154 ° C.

Step 2: Preparation of (2-ethoxyrnethylenacetoaceti 1 - (4-trifluoromet 1) aniline). Rcetoacetyl- (4-trifluorornethyl) aniline (14.11 g., 57.6 mmol), triethoxy ethane (9.43 g, 63.4 mmol), and acetic anhydride (16.30 mmol, 173 mmol) were mixed together and heated at reflux for 90 minutes. The resulting dark solution was evaporated to dryness, the residue was resuspended in benzene / isooctane and the product was crystallized, more material was obtained from the mother liquors, the yield of the pure product was "11.93 g (72%), mp 120. -122 ° C.

Step 3: Preparation of RIO 2-ethoxyrnethylenacetoacetyl- (-trifluoromethyl) aniline ßP'O.Ol g, 10.4 rnrnol) in ethanol (6 rnl) was slowly added to an ice-cold solution of hydroxylamine hydrochloride (0.77 g, 11.0 mmol) in 2M NaOH (5.5 nmol). The mixture was refluxed for 1 hour, cooled to room temperature and evaporated to dryness. The residue was resuspended and distributed between ethyl acetate and water. The cap > The organic was separated, extracted with water, dried with sodium sulfate and the solvent was evaporated. The residue was resuspended in toluene and crystallized to yield a solid residue (2.45g, 87%) that had a melting point of 166-167 ° C. auL The preparation of the 5-rnethyl-isoxazole-4-carboxylic acid (3-tri-fluorodyl) -anilide was carried out in 3 steps: Preparation of (3-tri f1 uoromethyl-1) -ani-ida acid acetoacetic acid? "" "0 In a 25-milliliter round bottom flask equipped with a Claisen distillation head and magnetic stirrer, 4 g (18.6 rnM) of ct, ct, a, -trifluoro-p-toluidine, 3.71 g (24.8 g) were combined. M, 1 equiv.), 95%, of 2, 2, 6-trirnethyl-4H-l, 3-dioxin-4-one, 112 ml of diethanolarnin and 12.4 ml of xylene The 5 ternp of the mixture it was increased to 110 ° C, and the mixture was stirred at this temperature for 6 hours while the acetone was distilled out of the system.The progression of the reaction was monitored by TLC (Merck Kieselgel 60 Fss., eluent: petroleum ether plate. f action 90-110 ° C): acetone, 2: 1; 0 with 5% PMR in EtOH After 6 hours the xylene was removed by distillation at "20 mmHg and the residue was purified with medium pressure liquid chromatography ( 2 atrn) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-% | O: 110 ° C): acetone, 2: 1, co or eluent. The fractions of the product at R = 0.3 were collected using the TCC system used for the reaction monitoring, and after removal of the solvent 3.82 g of the (3-trifluoromethyl) -anilide of acetoacetic acid were isolated. Melting point: 91-92 ° C.

^ -H-NMR (p> prn, acetone-d6) flrH 7.35-7.87 4H (rn) NH 9.41 1H (s) CH? 3.62 2H (s) XL Pr portion of (2-acetyl-3-ethoxy-N- (Tri f1uoromethyl) pheny1-2-oropenamide were combined in a dry flask (120 ° C, 30 rnin) of 50 rnl, round bottom, equipped with magnetic stirrer, thermometer, rubber stopper, and Argon balloon in a T tap, 1.0 g (4.1 rnM) of acetoacetic acid (3-trifluoromethyl) -anilide, 0.85 g (5.7 rnM) of triethyl ether - orthophoric, 1.37 g of acetic anhydride and 450 rng of zinc chloride The mixture was stirred in an argon atmosphere at 60 ° C for 30 minutes The progress of the reaction was monitored by TLC (Merck Kieselgel 60 FaS4 plate. petroleum ether (90-110 ° C fraction), acetone, 5: 1 If the reaction was not completed after 30 minutes, 0.85 g (5.7 rnM) of orthophoric triethyl ester was added, and the reaction mixture gradually became brown After the reaction was completed, the reaction mixture was & amp; yornetide to empty; the residue was dissolved in ethyl acetate which was washed with water, the organic phase was dried with magnesium sulfate and then filtered and the ethyl acetate was extracted by vacuum distillation. The 1.16 g of crude product was purified with medium pressure liquid chromatography (2 atrn) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-110 ° C): acetone, 5: 1, as eluent. The product fractions of R ^ - 0.231 were collected using the CCF system used to monitor the reaction and after removing the solvent, 0.45 g of 2-acetyl-3-ethoxy-NC (trifluoromethyl) phenyl-2-arope were isolated. > enarnida. Melting point: 100.5 ° C. ^ H-NMR (pprn, acetone-d6) RRH 7.29-8.00 4H (rn) NH 11.25 1H (s) CH = 8.46 ÍH (s) EtCH3 4.37 2H (s) EtCH3 1.45 3H (s) AcMe 2.48 3H (s) ) c) Preparation of 5-methyl-l-isoxazole-4-carboxylic acid (3-trifluorornetyl) -anilide. In a 25-milliliter round-bottomed flask equipped with a magnetic stirrer and ter-rnórnetro, 0.11 g (1.58 rnM) of hydroxylamine hydrochloride was dissolved in 0. 5 rnl of water and to this solution were added 64 rng (1.6 mM) of sodium hydroxide ** ^) n 0.5 rnl of water. In this solution, 2.2 nrn of rnetanol and 0.44 (1.5 nnM) of 2-acetyl-3-ethoxy-N- (trifluoromethyl) "phenyl-2-pro? Arnide were added at room temperature." The progress of the reaction was monitored pC or CCF (Merck Kieselgel 60 F-254 plate, eluent: petroleum ether (fraction 90-110 ° C): acetone, 4: 1, visualization with 5% PMR in EtOH. After completing the reaction, the mixture of The reaction was concentrated in vacuo and the residue was taken up in ethyl acetate.The phase was washed with water, dried with magnesium sulfate, concentrated 0.51 g of crude product obtained were purified with medium pressure liquid chromatography. (2 d) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-110 ° C): acetone 4: 1 as eluent. The fractions of the product of Rr = 0.152 were collected using the CCF system used for the monitoring of the reaction and after the removal of the solvent 0.22 g of (3-trifluorornethyl) -anilide of 5-rnetyl-ieoxazole-4 ~ were isolated. fl-^ "carboxylic acid Melting point: 115-120 ° C. ^ -H-NMR (pprn, acetone-d6) RrH + NH 7.42-7.88 4H () CH = 8.50 H (s) CH 8.50 H (e) "R12 ^ j A12 can be? Rep > plowing as described by Patterson et al., 3. Med. Chem. 35: 507-510 (1992) fll3: N-tri-fluororhenyl phenyl .3,5-dimethylisoxazole-4-carboxylated ida A mixture of 320 g of 4-carbonylchloride of 3,5-dirnethylisoxazole and 1 ml of 4-trifluoromethylaniline in 2 nrn of dichloromethane were stirred at ternp > Eratura atmosphere all night. The mixture was then treated with ethyl acetate and water. The crude product was crystallized with ethanol and water to "* provide 330 mg of N-trifluoromethylphenyl, 3,5-dirnethylisoxazole-4- ca box mi a.

Compounds of 2 B10 BIO grunt was synthesized in 2 steps, a) Synthesis of cyanoacetyl- (-nitro) anilide 1.38 g (10 rnrnol) of 4-nitroaniline were dissolved in ^^ 30 rnl of absolute pyridine, it was then cooled to -30 ° C, 0.43 rnl (5 mmol) of phosphorus trichloride was added in the form of drops with continuous stirring to avoid the temperature increase above -20 ° C. After 0.5 hours, 0.85 g of cyanoacetic acid was added, and the solution was stirred for 0.5 hours at -20 ° C and then for 12 hours at room temperature.The solvent was evaporated in vacuo.The residue was covered with HCl 1 N and extracted with ethyl acetate, which was filtered and evaporated, the residue was triturated with ether, filtered and dried in vacuo, yielding 1.70 g (83%) of product, the product having the following characteristics. : Melting point: 81-83 ° CR ^: 0.95 (hexane-EtORc; 1: 1) b) Cylaceacetyl- (-nitro) anilide Rcetylation. - ^ 0.82 g (4 nmol) of cyanoacetyl 4- *. Nitroanilide was dissolved in 2 ml of absolute pyridine, followed by the addition of 20 mg of the catalyst-4-amino-iridine and 0.50 mg of tetramethylguanidine. The reaction mixture was stirred while the nitroanilide was dissolved. Then 5 rnl of acetic anhydride were added dripping at D ° C. After 2 days the pyridine was evaporated, ethyl acetate was added and the organic layer was extracted with % NaHC03, in IN HCl and water. The organic layer was dried over ^ Naa SO ^ and ee evaporated. The residue was crystallized from ether and 210 g of the product were obtained (21% yield). The product had the following characteristics: Rf = 0.35 (EtoAc) Melting point: 260 ° C It was synthesized using two different methods: method R and method B. neo-do A A mixture of 27 grams of RIO in 150 ml of ethanol was combined with 16.2 grams of 1,8-diazabicycloCS .4. Olundequenoí). The reaction mixture was then stirred at room temperature for 30 minutes and the ethanol was removed by evaporation. The resulting solid was suspended in 500 nmol of ethyl ether and combined with 200 ml of 0.6 N hydrochloric acid solution. All solids were filtered by suction, washed with 200 ml of ethanol and dried by suction to provide 25 g of product.

Method B 2- cyano-3-hydroxy-N-C (4-t-ri fl uoro-ethyl) phenyl) -2-butenarnide (I) in two steps as follows: ai Preparation fj = cyanoacetyl- (4-tri fluoromethyl) - anuide. A mixture of 3 g (18.6 rnM) of a, a, α-trifluoro-β-toluidine and 3.37 g (29.8 M, 1.5 equivalents) of cyanoacetic acid ethyl ester was stirred in a 50-milliliter flask, equipped with magnetic stirrer, thermometer and nitrogen ventilation, on an oil bath at 180 ° C for 5 hours. The progress of the reaction was monitored by TLC (Merck Kieselgel plate 60 234, eluent: petroleum ether (90-110 ° C fraction): acetone, 1: 1. The reaction mixture was purified with medium pressure liquid chromatography (2). atm) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-110 ° C): acetone, 1: 1 as eluent The fractions of the product were collected using the CCF system used for the monitoring of the reaction and after removing the solvent, 2.11 g of cyanoacetyl- (4-trifluorornethyl) -anilide were isolated Melting point: 192-194 ° C. ^ -H-NMR (pprn, acetone-d6) RrH 7.68-7.84 4H ( dd) NH 9.84 H (s) Cl- 3.90 2H (s) Preparation: d = 2-cyano-3-hydroxy-N-C (4-t-ri-1-chloro-1-phenyl) -2-butene. In a 50-millimeter round-bottomed flask equipped with a magnetic stirrer, thermometer, a rubber stopper and a T-key (with connections for vacuum and balloon with a Rrgón blanket), 1.78 g (0.89 g, 37.1 rnM) of a Oily dispersion of 50% NaH was suspended in 4 rnl of dry acetonitrile (from P3Os) .The suspension was cooled to 10 ° C and while stirring at this temperature 2.72 g (11.9 rnM) of cyanoacetyl (4-trifluoronetil) were added. -anilide dissolved in 25 rnl of dry tetrahydrofuran (from LiRlH ^.) in 10 minutes.The reaction mixture turned yellow and was cooled after addition to -10 ° C, and then 1.05 grams (13.11 rnM, 1.1) were added. Equiv) of acetyl chloride in 20 minutes During the addition the temperature of the reaction mixture can not be higher than -5 ° C. The progress of the reaction was monitored by TLC (Merck plate Kieselgel 60 F254 eluent: petroleum ether (fraction 90-110 ° C): acetone 1: 1. When the reaction was complete, the The reaction mixture was stirred at 0 ° C for 30 minutes, at 35 ° C for 30 minutes and at 65 ° C for another 30 minutes. Then the reaction mixture was subjected to vacuum. The residue was dissolved in 30 rnl of distilled water, stirred with charcoal at 80 ° C and filtered. The resulting pale yellow filtrate was acidified with a 10% hydrochloric acid solution, the precipitated crystals were filtered, washed with water and dried. The crude crystals (2.51 grams were purified with medium pressure liquid chromatography (2 atr) using silica gel 60 as the fixed phase and petroleum ether (f action 90-110 ° C): acetone 1: 1 as eluent. of the product of R + - = 0.138 were collected using the CCF system used for monitoring the reaction and after removal of the solvent 1.99 g of 2-cyano-3-hydroxy-NC (4-tri fluoromethyl) phenyl-2 were isolated. -butenamide, CCF petroleum ether (90-110 ° C) / acetone, 1/1 = 0.138. 1 H-NMR (ppm, DMSO-d6) RrH 7.65-7.78 4H (dd9 NH 10.85 HH (s) Ha 2.25 3H (s) OH 6.39 HH (s) £ 12 The preparation of 2-cyano, 3-hydroxy, 3- (4-fluorophenyl) -N-C4-trifluoromethyl) phenyl] -2-propenamide, (C3_-7H aF4.2.O2, PM .: 350.3) was carried out as follow: ,, a) Preparation of Cyanoacetyl- (-tri luormethyl) anilide: f A mixture of 3g (18.6 rnM) of a, a, a ~ Trifluoro-p-toluidine and 3.37 g (29.8 M, 1.6 equivalents) of ester was stirred. of cyanoacetic acid in a 50-milliliter flask, equipped with a magnetic stirrer, thermometer and nitrogen vent, in an oil bath at 180 ° C for 5 hours The progress of the reaction was monitored by TLC (Merck Kieselgel plate 60 F-254, eluent: petroleum ether (fraction 90-110 ° C): acetone 1: 1. The reaction mixture was purified with # liquid chromatography of medium pressure (2 attrn) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-110 ° C): acetone 1: 1 as eluent. The fractions of the product were collected after the removal of the solvent, 2.11 g of cyanoacetyl- (4-trifluorornethyl) anilide were isolated. The product had the following characteristics: Melting point: 192-194 ° C.

? -I- NMR (pprn, acetone-dd) fHPrH 7 .68-7. 85, 4H (dd) NH 9. 84 ÍH (s) CHs. 3 . 90 2H (s) hl Preparation d = 2-cyano-3-hydroxy-3- (4- fl-orophenyl) -N- C (4- • fcrjfiMQr? Roethyl) phenyl3-2-PrQpenamides In a round bottom flask of 50 rnl equipped with magnetic stirrer, thermometer, a rubber stopper and a T-key (with connections for vacuum and ball with Rrgón blanket) were 4 ~, suspended 0.55 g (0.275 g, 11.4 rnrols) of a dispersion in * 50% NaH oil in 1 ml of dry acetonitrile (from P20a). The suspension was cooled to 10 ° C and under stirring, at this temperature, 1 g (4.4 rnM) of cyanocetyl- (4-trifluorornethyl) -anilide die- lated in 10 milliliters of dry tetrahydrofuran (of LiAlH ^. The reaction was then cooled to -10 ° C and at this temperature, 0.77 g (4.8 mM, 1.1 equiv.) of 4-fluorobenzyl chloride were added in 20 minutes.During the addition, the temperature of the # Reaction mixture rose to above -5 ° C. The progress of the reaction was monitored by TLC (Merck Kieselgel 60 F254 plate, eluent: petroleum ether (90-110 ° C fraction): acetone 1: 1. The reaction mixture was then stirred at 0 ° C for 30 minutes, at 35 ° C for 30 minutes and at 65 ° C for another 30 minutes, then the reaction mixture was subjected to vacuum.The residue was dissolved in 30 ml of distilled water, stirred with charcoal at 80 ° C and filtered. The resulting pale phase was acidified with 10% hydrochloric acid solution, the precipitated crystals were filtered washed with water and dried.The crude crystals (2.47 g) were purified with medium pressure liquid chromatography (2 atrn) using silica gel 60 as the fixed phase and petroleum ether (fraction 90-110aC): acetone 1: 1 as eluent The fractions of Rf = 0.216 were collected and after removal of the solvent, 1.05 g of 2 ^ -cyano-3-hydroxy were isolated. 3- (4-fluo ofenyl) -NC (4-thyloromethyl) phenyl) -2-prop enarnida. The product had the following characteristics: Melting point: 195 ° C (del). ? -I-NMR (pprn, acetone-dd) ArH 7.36-8.13 8H (rn) NH 9.5 OH (s) OH 16.5 2H (s) B13 The preparation of 2-cyano-3-hydroxy-3-cyclohexyl-N- (4-tri fluoromethyl) phenyl3-2-Propenamide PM .: 338.3) was carried out as described for B12 substituting 0.71 g (4.8 mM). , 1.1 equiv.) Of cyclohexylacetyl chloride by 4-fluorobenzoyl chloride. The resulting product had the following characteristics: TLC: petroleum ether (90-110 ° C): acetone, 1: 1 Melting point: 212 ° C (del, B14 The preparation of 2-cyano-3-hydroxy-3- (2, 2, 3, 3-tetrarnethylcyclopropyl) -propanol-4- (trifluoromethyl) anilide was carried out as described above for B12, using 2-chloride, 2, 3, 4-tetrarneylcyclopropylcarboxyl per ljB chloride of 4-fluorobenzoyl.

The preparation of 2-cyano-3-hydroxy-3- (pentafluorophenyl) -NC (4-t ifluo omethyl) phenyl] -2-propenarnide was carried out as described for B12, substituting 1.5 g (6.51 nm) , 1.1 equiv.) Of pentafluorobenzoyl chloride in place of 4-fluorobenzoyl chloride. The resulting product had the following characteristics: TLC: R ^ 0.360 (petroleum ether (90-110 ° C): acetone, 1: 1) melting point: 157-158 ° C.

The preparation of 2-cyano-3-hydroxy-3-C (3-phenoxy) phenyl-NC (4-tri fluoromethyl) phenyl] -2-propenarnide (CasHis sNsOs PM: 424.4) was carried out as described for B12, substituting 1.55 g (4.8 rnM, 1.1 equiv.) of 3-phenoxybenzoyl chloride in place of 4-fluorobenzoyl chloride. The resultant had the following characteristics: TLC: Rf = 0.300 (petroleum ether (90-110 ° C): acetone, 1: 1) melting point: 197-198 ° C.

MIZ The preparation of 2-cyano-3-hydroxy-4-phenyl-N-C (4-trifluoromethyl) phenyl] -2-butenarnide was carried out as described for B12, substituting 0.68 g (4.8 rnM, 1.1 equiv.) . * -_ of chlorur- or phenyl-acetyl in place of 4-fe X-fluorobenzoyl chloride. The resulting product had the following characteristics: TLC: Rf = 0.165 (petroleum ether (90-110 ° C): acetone, 1: 1) melting point: 156-158 ° C. £ 1 =. The preparation of 2-cyano-3-hydroxy-5-rnethyl-NC (4-trifluoromethyl) phenyl] -2-hexenoamide (C sHa.sFa sOs PM: 312.3) is carried out as described for El2, substituting 0.58 g fl (4.8 rnM, 1.1 equiv.) of isovaleryl chloride in place of 4-fluorobenzoyl chloride. The resulting product had the following characteristics: "CCF: R ^ = 0.323 (petroleum ether (90-110 ° C): acetone, 1: 1) melting point: 151-163 ° C.

B19 The preparation of 2-cyano-3-hydroxy-4, 4-diphenyl-N- (4-trifluoromethyl) phenyl-2-butenamide PM: 422.4) carried out as described for B12, substituting 1.12 g (4.8 rnM, 1.1. equiv.) of diphenylacetyl chloride in place of 4-fluorobenzoyl chloride. The resulting product had the following characteristics: CCF: R1 == 0.354 (petroleum ether (90-110 ° C): acetone, 1: 1) melting point: 195-202 ° C.

Group 3 compounds refluxed for 6 hours 340 ing (1.5 rnM) of l-phenyl-3-amino-4-cyano-5-cyanomethyl-2-urazole, 210 rng (1.5 rnM) of 3,4-dihydroxy-benzaldehyde and 4 Piperidine drops in 30 rnl of ethanol. Cooling and filtration gave 145 g of a yellow solid. Evaporation of the solvent and trituration with CH3-Cl.a-acetone gave another 145 g of the yellow solid. (55% yield). The product had a melting point of 147 ° C. 'RMN acetone ú d-7.87 (H, S, Vinyl), 7.68 (H, d, 3 = 2.2 Hz, Hs) 7.66-7.45 (5H, pv, Ph), 7.28 (ÍH, dd, 3 = 8.3.2.2 Hz, l-). 6.92 (ÍH, d, 3 = 8.3 Hz, H3) " £ 11 Cll was synthesized using a procedure of doe O 'steps a. Synthesis of 3-amino-4-cyano-5-cyanomethyl-2- 2.2 g of a rnalonitrile dirner and 0.9 rnl of N2H were heated in 20 rnl of water for 15 minutes at 100 ° C. Cooling and filtration gave 1.5 g (61% yield) of a white solid having a melting point of 187 ° C. (NMR acetone d ^ 6 3.88 (s).) (Cf. Carboni et al., 3. Arn. Chem. Soc. 80: 2838 (1958), reporting p.f. 197 ° C. b. Condensation with dihydroxybenzaldehyde: R 0.28 g (2 rnM) of 3,4-dihydroxybenzaldehyde and 1.33 g (2.2 rnM) of 3-amino-4-cyano-5-cyanomethyl-2 pyrazole in 20 rnl of ethanol were added 3 drops of piperidine and the reaction was refluxed for 3 hours. Cooling, filtration and washing with ethanol gave 1.3 g (55% yield) of a yellow solid having a melting point of 300 ° C. £ 12 0.7 g, 3 rnM of 3,5 'di-t-butyl-4-hydroxyaldehyde, 0.46 g, 3.1 rnM of 3-arnino-4-cyano-5-cyanomethyl pyrazole (prepared according to Carboni) were refluxed for 15 hours. and others, 3. Chern, Soc, 80: 2838, 1958) and 40 g of β-alanine. By cooling and filtration 0.5 g, 46% was obtained. of yield of a yellow solid, p.f. 255 ° C. NMR CDCls or 7.92 (1. H, S, vinyl), 7.80 (2H,?), 5.76 (1H, S, OH), 3.75 (2H, br, S, NHS), 1.48 (18H, S). MS-364 (M-1, 28), 363 (M-, 100%), 348 (M-CH 3, 58), 292 (M-56-CHa, 31), 147 (41), ? /and.

Compounds of Group 4 £ 11 Reflux for 6 hours 435 rng (3 rnM) of 3- formyl-indole, 300 mg (4.5 m) of 2-thiocarboxamido-acetonitrile and 20 mg of β-alanine in 30 ml of ethanol. By cooling and XJ ^. filtration gave 0.47 g (81% yield of a solid *, yellow that had a melting point of 238 ° C). £ 12 This was synthesized as for Dll except that 1, 1, 4-tric? Ano-2-arnino-1-propene was used in place of the acetonitrile derivative. The final product had a melting point of 293 ° C.

# D13 This was synthesized as Dll except 2-carboxamido-acetonit. i was used instead of the acetonitrile derivative. The final product had a melting point of 242 ° C.

D14 3e were refluxed for 4 hours, 0.29 g (2 rnM) of 3-forrnil-indole, 0.29 g (2 rnM), of 3-amino-4-cyano-5-cyanorhexyl-? 2 ^ - ?? and 20 rng of ß-alamine in 30 rnl of ethanol. Upon cooling and filtration it gave 0.34 g (62% yield) of a yellow solid having a melting point of 281 ° C. NMR acetone d 8.52 (1H, S, vinyl), 8.42 (lH, S, Hß), 7. 79 (lH, rn), 7.57 (lH, rn), 7.27 (2H, rn), 6.17 (lH, br.S, NH). MS- 274 (M +, 100%), 219 (14), 91 (35), rn / e. £ 15. Refluxed for 4 hours 0.3 g (1.3 mM) of 3-arnino-4-cyano-5-cyanornetyl-2-pyrazole, 0.2 g (1.36 M) of 1- (3-dirnetilarnino? Ro? Il) -3 -formyl-indole and 20 mg of (3-alanine in 20 ml of ethanol.) Evaporation, trituration with benzene and filtration gave 0.4 g of a yellow solid (94% yield) containing 10% of 3-arnino-4-cyano -5-cyanomethyl-2-pyrazole 0.4 g were chromatogenated on silica gel (70-220 mesh) eluting with rnetylene chloride: 85:15 rnetanol to give 0.12 g of a bright yellow solid having a melting point of 250 ° C. NMR acetone d? D 8.45 (lHa., S, vinyl), 8.37 (lH ?, CA, Ha), 7.78 (lH ?, m), 7.60 (lH ?, m), 7.28 (2H,., rn), 4.47 Hz), 2.24 (6H, S, N- (CH ») a). MS-360IM + 1, 8%), 359 (M +, 31), 289 (100), 261 (15), 144 (6), m / e.

D16? E were refluxed for 5 hours 0.4 g (1.7 mM) 3-ino-4-cyano-5-cyanornetyl-2-urazole, 0.3 g (1.73 mil) of l-oxo ~ (, 4-dihydroxyphenyl) -2-cyanothane and 20 g of ß-alanine in 20 rnl of ethanol. Cooling and filtering gave 0.1 g of a brown solid. Preparative chromatography gave 20 mg (3% yield) of an orange solid having a p > fusion point of 115DC. NMR acetone d * 6 8.72 (1H, S, Vinyl), 8.52 (1H3_, Sx, Ha), 7.90 (lH? Rn), 7.73 (lH, rn), 7.40 (4H, rn), 7.0 (lH, d, 3 = 8.2 Hz, Ha). 4.57 (2H, t, J = 7.2 Hz), 2.46 (2H, t, 3 = 7.2 Hz), 2.34 61-1, S, N, (CHs) a), 2.17 (2H, quintet, 3 = 7.2 Hz) . The reaction mixture was refluxed for 6 hours with 0.4 g (2 rnM) of 3-forrnil-indole, 0.36 g of 1-oxo-l- (3,4-dihydroxy phenyl) -2-cyanothane and 3 drops of p > iperí ina, in 25 rnl of ethanol. Treatment and trituration with benzene gave 0.36 g of a yellow solid having a melting point of 225 ° C. NMR acetone dß 8.77 (H H, S), 7.90 (1 H, r), 7.70 (H H, Hm, 7.40 (4 H, rn), 7.0 (2 H, t, 3 = 6.7 Hz), 4.92 (2 H, t, 3 = 6.8 Hz), 3.26 (2H, t, 3 = 6.8 Hz).

Refluxed for 4 hours 0.29 g, 2 rnM, 3 ~ forrnil-indole, 0.29, 2 mM, 3-amino-4-cyano-5-cyanomethyl-2-pyrazole, and 20 ng of β- Alanine in 30 rnl of ethanol. Cooling and filtering resulted in 0.34 g, 62% yield, of a yellow solid, melting point 281 ° C. (r NMR acetone dß 6 8.52 (1H, S, Vinyl), 8.42 (1H, S, Ha), 7.79 (1H, rn), 7.75 (HH, rn), 7.27 (2H, m), 6.17 (HH, Br.S, NH), MS-274 (M-, 100%), 219 (14) , 91 (35), m / e. £ 211 80 rng, 0.55 rnM, were refluxed for 8 hours 3-formyl-indole, 130 rng, 0.6 mM of 3-amino-4-cyano-5-cyanomethyl-1-phenylpyrazole and 2 drops of piperidine in 10 rnl of ethanol. Cooling and filtering gave 120 rng, 62% yield, a yellow-green solid, m.p. 258 ° C. NMR acetone d6 & 8.56 (ÍH, S), 8.52 (ÍH, S), 7.84 (1H, rn, 7.60-7.25 (8H, m).

Group 5 The compounds of group 5 were prepared in three steps. jf a) Preparation of N-aryl oxamic acid esters (= ethyloxyalil anuides): 0.025 mol (3.4 ml) of diethyl oxalate and 0.1 mol of the appropriate aniline were mixed and refluxed at 190 ° C for 15 minutes. minutes The resulting solution was cooled and left overnight to crystallize the product. The crystals were filtered, washed with ethanol and extracted with hot ethanol. The insoluble material was removed by filtration and the solution was put in the refrigerator. The resulting crystals were filtered and dried.

TABLE xxym l b) Preparation of N-aryl-oxamic acid hydrazides (N-aryl-oxamoyl hydrazides) mf 0.05 mol of the appropriate N-aryl-oxarnic acid ester (the ... le) was dissolved in 200 rnl of ethanol and added slowly to a well-stirred solution of 7.5 rnl (ca.0.0.15 mol) of hydrated hydrazine in 50 ml of ethanol. The mixture was left at room temperature for 48 hours. The resulting heter-oene solution was refluxed for 15 minutes and the hot solution was filtered. After cooling to room temperature the precipitated substance was filtered and washed with ethanol and dried.

TABLE XXIX bc) Preparation of N-aryl oxamoyl hydrazones: EU1 0.001 mole (0.222 g) of N- (4-irnethylamino) -phenyl-oxamoyl hydrazide (2a) was dissolved in 5 ml of acetic acid and heated to "100 ° C. with 0.001 mmol (0.138 g) of 3,4-dihydroxy-benzaldehyde for 20-25 minutes in the presence of a "*" * "Catalytic amount of sodium acetate The mixture was then cooled and left overnight at room temperature The separated crystals were filtered and washed with acetic acid and water. The yield of the pure product was 0.23 g (68%) m.p. 253 ° C. PM: 342.36). Elemental analysis [%]: found C, 59.51: H, 5.28; N, 16.25. Calculated C, 59.64; H, 5.30; N, 16.37.

• Eli 0.001 mmol (0.195 g) of N-3-hydroxy-phenyloxyamoyl hydrazide (2b) was dissolved in 5 mL of acetic acid and heated to 100 ° C with 0.001 mol (0.138 g) of 3,4-dihydroxy benzaldehyde per 20-25 minutes in the presence of a catalytic amount of sodium acetate. The mixture was then cooled and left overnight at room temperature. The separated crystals were filtered and washed with acetic acid and water. ? The yield of the pure product was 0.142 g (45%) ^^ .f. > 260DC. (Cx5Hx3N.aa.Os, PM: 315.29). Elementary analysis C%] -. Found C, 57.05: H, 4.10; N, 13.20. Calculated C, 57.14; H, 4. 16; N, 13.33.

E12 0.001 mole (0.195 g) of N-3-hydroxyphenyl-oxamoyl hydrazide (2b) was dissolved in 5 ml of acetic acid and heated to 100 ° C cor. 0.0-01 rnol (0.122 g) of 2-hydroxy Fi benzaldehyde for 20-25 minutes in the presence of a catalytic amount of sodium acetate. The mixture was then cooled and left overnight at room temperature. The separated crystals were filtered and washed with acetic acid and water. The yield of the pure product was 0.224 g (75%) m.p. 264-266 ° C. (CxsHxsNaO, PM: 299.29). Elemental analysis C%]: found C, 60.11: H, 4.40; N, 13.76. Calculated C, 60.20; H, 4.38; N, 14.04. 0.001 mole (0.21 g) of N-2-methoxyphenyloxanoyl hydrazide (2c) was dissolved in 5 mL of acetic acid and heated to 100 ° C with 0.001 mmol (0.138 g) of 3,4-dihydroxy benzaldehyde per -25 minutes in the presence of a catalytic amount of sodium acetate. The mixture was then cooled and left overnight at room temperature. The separated crystals were filtered and washed with acetic acid and water. The yield of the pure product was 0.21 g (64%) f. 232-238 ° C. (Cl? HX5N3Os, MW: 329.31). Elemental analysis C%]: found C, 60.01: H, 4.51; N, 12.59. Calculated C, 58. 36; H, 4.59; N, 12.72.

(E i 0.001 mol (0.22 g) of N-2-ethoxyphenyloxanoyl hydrazide (2d) was dissolved in 5 mL of acetic acid and heated to 100 ° C with 0.001 mmol (0.138 g) of 3,4-dihydroxy benzaldehyde. per aBf 20-25 minutes in the presence of a catalytic amount of sodium acetate.The mixture was then cooled and left overnight at room temperature.The separated crystals were filtered and washed with acetic acid and water.The yield of the pure product was 0.15 g (44%) mp 208-214 ° C. (CX7HX7N30SJ PM: 343.34) Elemental analysis [%]: found C, 59.78: H, 4.81; N, 12.10, Calculated C, 59. 47; H, 4.99; N, 12.24. 0.001 mmol (0.22 g) of N-3-nitrophenyloxanoyl hydrazide (2e) was dissolved in 5 ml of acetic acid and heated to 100 ° C with 0.001 mmol (0.138 g) of 3,4-dihydroxy benzaldehyde per 20- 25 minutes in the presence of a catalytic amount of sodium acetate. The mixture was then cooled and left overnight at room temperature. The separated crystals were filtered and washed with acetic acid and water. The yield of the pure product was 0.19 g (56%) m.p. >260 ° C. (CxsHxs ^ O ^, PM: 344286. Elementary elementary [%]: found C, 52.08: H, 3.47, N, 16.10, Calculated C, 52.32, H, 3.51, N, 16.27.The £ "0.001 rnol ( 0.22 g) of N-3-nitrophenyloxanoyl hydrazide (2e) in 5 ml of acetic acid and heated at 100 ° C with 0.001 mmol (0.122 g) of 4-hydroxy-benzaldehyde for 20-25 minutes in the "presence of a catalytic amount of sodium acetate The mixture was then cooled and left overnight at room temperature The crystals were separated and filtered and washed with acetic acid and water The yield of the pure product was 0.19 g (61%) pf> 260 ° C. (CxsHxaN ^ Oa, MW: 328.29) Elemental analysis C%]: found C, 54.80: H, 3.59; N, 16.86, Calculated C, 54.88; H, 3.68; N, 17.07.

FIO can be prepared using a two-step approach. a) Preparation of 2-methyl-3-hydroxyethyl-suinazolin-4-one z 1.37 g (0.01 mole) of anthranilic acid with 8 ml of acetic anhydride were refluxed for 3 hours. The acid formed was continuously removed by atmospheric distillation. After the acetic acid formation was complete, the mixture was evaporated in vacuo to dryness. The resulting oil was mixed with 2 rnl of ethanolamine and heated at 160 ° C for 3 hours. After the reaction was complete the substance was cooled, mixed with alcohol and left at room temperature overnight. The precipitated crystals were collected by filtration, m.p. 159-60 ° C; 1.40 g (65%). * Step h 1.08 g (0.005 mol) of 2-rnethyl-3-hydroxyethyl-quinazolin-4-one and 0.69 g of 3,4-dihydroxybenzaldehyde were melted at 160 ° C and heated for an additional 30 minutes. The resulting product was dissolved in isopropanol, decolorized with charcoal and left at room temperature overnight The precipitated crystals were filtered and dried The yield of the pure product was 0.79 g (49%) mp 221-223 ° C. CxsHxeaNaC, PM: 324.34), elemental analysis C%]: found C, 66.48: H, 4.86; N, 8.62, C, 66.66; H, 4.97; N, 8.64.

FU V F12 1.01 g (5 rnrnol) of 3,4-dihydro-l, 4-oxazin-C3, 4-b] quinazolin-d-one were melted with 6 mmol of the corresponding benzaldehyde derivative on an oil bath at a I00-200 ° C temperature. After the elimination, the resulting mixture water was dissolved in ethanol and clarified with charcoal, the solvent was evaporated and the product recrystallized.For the preparation of Fll, 3,4-dihydroxybenzaldehyde was used to obtain the product. (85% yield) having a melting point of 290-292 ° C. For the preparation of F12, 3- dihydroxybenzaldehyde was used to obtain the product (63% yield) having a melting point of 208-214 ° C.

COMPOUNDS OF GROUP 7 Benzoyl hydroxyiminoethylacetate R 10 g of benzoyl ethylacetate in 20 ml of acetic acid, cooled with ice, was added 3.7 g of NaNOs. After 10 minutes, 5 rnl of water were added. After 3 hours 100 ml of water were added and the solid was filtered to • ^ Pa 7.7 g 84% yield, p.f. 110 ° C. NMR CDC13 6 7.90 (2H, rn), 7.6-7.5 (3H, rn), 4.30 (2H, q, 3 = 7.4 Hz), 1.24 (3H, +., 3 = 7.4 Hz). 2-Ethoxycarbonyl-6,7-dimethyl-3-phenyl-Quinoxaline Reflux was allowed for 6 hours, 5 g, 22.6 rnM of benzoyl hydroxy-inethylacetate and 3.1, 22.8 rnM, of 4, 5-dirnethyl-1,2-phenylenediarin in 20 rnl of ethanol and 5 rnl of HCl. With water, NaHC03, CHaCla, chromatography and hexane gave 2 g, 29% yield, of a white solid, m.p. 100 ° C. NMR CDCls d 7.96 (1H, S), 7.93 (1H, S), 7.7 (2H, rn), 7.5 (3H, rn) 4.30 (2H, q, 3 = 7.0 Hz), 2.53 (6H, S), 1.17 (3H, t, = 7.0 Hz). 6.7-Dimethyl Quinoxalin-2-one 2 hours, 2 g, 15 m, of 4,5-dirnethyl 1,2-diaminobenzene and 1.5 g, 16 M, of glyoxalic acid hydrated in 30 ml of ethanol were refluxed for 2 hours. Cooling and filtering yielded 1.2g, 46% yield, of a white solid mp 263 ° C, soluble in acetone DMSO NMR D6 6 mixture 60:40 higher - 8.07 (1H, S), 7.55 (1H ,?) , 7.06 (1H, S), 2.30 (6H, S), minor - 0.02 (1H, S), 7.42 (1H, S), 7.28 (1H, S), 2.28 (6H, S) Observation - the reaction of 4.1 g gave 3 g, 57% 2-Chloro-6.7 Dimethyl Ouinoxaline Reflux for 2 hours l.lg, 6.2 mM of 6,7-dimethylquinoxalin-2-one, 1 rnl of P0C13 and 1 ml of dimethyl Fniline in 20 ml of toluene. Treatment (NH3, CHsCla) and chromatography gave 0.4g, 33% yield, white solid, m.p. 86 ° C. NMR CDCl 3 d 8.68 (lH, S, Hβ) 7.85 (1H, S), 7.76 (1H, S), 2"50 (6H, S). At reflux for 3 hours 0.4 g (4 rnM) of phenylendia ina and 0.6 g (4 M) of phenylglyoxal monohydrate in 20 rnl of ethanol, and 10 rnl of acetic acid. Treatment using 50 ml of water and 80 ml of CHaCla followed by trituration '^ "with hexane gave 0.38 g (46% yield) of a white solid having a melting point of 65 ° C. NMR CDCI3 or 9.44 (1H, S), 8.1 (4H, rn), 7.8 (2H, rn), 7.6 (3H, m). MS-206 (M +, 100%), 179 (M-HCN, 25), 152 (37), 103 (M -Ph-CN, 42), rn / e. £ 12 fl 3 rnl of DMF and 16 rnl of PCC13, 2.7 g (10 rnM) of N- (3,4-dirnetoxyphenyl) phenylacetamide was added. The reaction was heated at 90 ° C for 4 hours, decanted on ice, filtered and washed with water to give 2.9 g (96% yield) of a white solid having a melting point of 234 ° C. NMR CDC13): d 8.26 (1H, S *, 8.0 (H, e, Hß), 7.15 (5H, e Ph), 7.15 (1H, e, ha), 4.13, 4.05 (6H, 2β, 0CH3) - MS : 301, 299 (M +, 33%, 100%), 286, 284 (M-CH3, 2%, 6%), 258, 256, (6%, 18%), 220 (9%), 215, 213 (4%, 13%), rn / e.

Gil ^ r) The compound was synthesized with the "procedure used for G12, except that the reagent N- (3,4,5-trirnetoxifen.il) phenylacetarnide was replaced.The final product had a melting point of 103 ° C. G13 2.4 g (16 rnM) of phenylglyoxal hydrate and 2.2 g (16 M) of 3,4-dirnethyl-l, 2-phenylenediarnine in 20 rnl of ethanol were refluxed for 1.5 hours, cooling and giving 3.25 g ( 88% yield) of a solid had a melting point of 124 ° C NMR CDCls d 9.23 (1H,?, H2), 8.19 (1H, d, 3 = 1.6Ha), 8.15 (1H, d, 3 = 1.7 Ha), 7.90 (2H, d, 3 = 9.0 HH), 7.57 (3H, rn) 2.52 (6H, S, CHa), MS-234 (M +, 100%), 210 (M-CH3, 11), 207 (M-HCH, 12), 165 (M-2HCN-CH3.2), 131 (M-ph-CN-3) rn / e G14 7 g of veratol (51 mM) were added to 19 rnl of HN03 at 70% cooled with ice After 0.5 hours in the cold, 10 ml of HaSO * was slowly added in 0.5 hours The resulting dark suspension was stirred for 3 hours at room temperature and water and water were added. Ice to the suspension to precipitate the product. Filtration, washing with water and drying gave 10.2 g (96% yield) of a yellow solid having a melting point of 120 ° C (NMR CDC13 d 7.35 (2H, S), 4.06 (6H, S. OCH3)). (Cf: 3. Org Chern 12: 522 (1947), p.f. reported 130 ° C, and 3. Med. Chem. 36,331 (1993) p.f. reported 122 ° C. The compound is sold by Lancaster Co., (p.f.

Two grams of 1,2-dinitro-4,5-dirnetoxybenzene were hydrogenated over 0.3 g of PtO for 1 hour, then filtered and evaporated to give 1.5 g of a black solid (Cf. 3. Med. Chem. 36: 331 (93), red brown solid reported, mp 151 ° C). The black solid was mixed with 1.3 g of phenylglyoxal, 15 ml of absolute ethanol and 15 ml of concentrated HCl and refluxed for 5 hours. The treatment, as for G10, gave a g, dark solid, which was recrystallized from "ethanol to give (31% yield) of a white solid having a melting point of 134 ° C. RMNMR CDCI3 d 9.13 ( lH, S, Ha), 8.16 (IH,, 3 = ld.6H «), 7.60-7.40 (5H, m) 4.09 (6H, S, 0CH3) - MS-266 (M +, 100% 1, 251 (M -CHa, 12), 223 (M-CH3-C0, 13), 196 (M- £ 15 Thofen-2-glyoxal-ibis-thiosemicarbazone (3.4 rnM) and 0.6 g (4 rnM) were refluxed for 6 hours. The solvent was removed by vacuum distillation, and the residue was dissolved in CHaCla V, washed with water, the organic layer was evaporated in vacuo and the residue was triturated with benzene-hexane to give an white solid (23% yield) mp 104 ° C "NMR CDC13 d 9.25 (H, s, H3), 8.07, 7.72 (4H, m, H3_T), 7.85, 7.56, 7.21 (3H, rn, thiophene) 100% ), 185 (M-HCN, 25%), 141 (6%), 106 (8%), rn / e 2, 3-diarnino-iridin and phenyl glyoxal were reacted with G1 3 to give a white solid (77% yield) having a melting point of 135 ° C. NMR CDClg d 9.47 (1H, S, H3), 9.21 8.50, 7.71 (line 8 RBC rn, H7, Ha, H6), 8.35 (2H, rn, Ph), 7.60 (3H, rn). MS-207 (M +, 100), 180 (H-HCN, 8), 179 (11), 104 (23), 77 (14), rn / e. CDCla d 7.B7Í1H, dd), 6.89 (dd), 6.62 (dd), 4.25, 3.30 (br.S.) ^ 1Z G17 was synthesized in two steps as follows: a. If there is 2-methyloxy-5-dyritrophine, 3.3 g of 1,2-dirnetoxy-4,5-d-nitrobenzene in 20 ml of 48% HBr were refluxed for 16 hours. Water was added and the reaction was extracted with CHaCla to give 1.1 g of an orange solid. Chromatography on silica gel, eluting with 2% CH3OH in CH2Cl gave 0.42 g (13% yield) of a yellow solid which turned red with KOH. NMR (CDCl 3): or 7.44 (lH, s), 7.42 (lH, s), 6.30 (lH, s), 4.07 (3H, s). Extraction of the aqueous phase with ethyl acetate gave 2 g of a red oil. Chromatography on silica gel, eluting with 5% CH30H in CHaCla gave a yellow solid, 0.1 g (3.5% yield) having a melting point of 160 ° C with a violet color, with KOH, corresponding to 1, 2-dihydroxy-4,5-dinotrobenzene.

NMR (acetone-d ^) d 7.51 (2H, s). X Reduction and condensation with phenylalioxal. Hydrogenated strength 0.2 g of 2-rnetoxy-4,5-dinitrophenol on Pd / C in 20 ml of ethanol for 1 hour. The P was filtered, 0.3 g of phenylglyoxal were added and the reaction was refluxed for 3 hours. Evaporation and chromatography on silica gel, eluting with 1% CH30H in CHaCla gave 0.1 g of an orange oil. NMR (CDC1): d 8.10, 7.6 (7H, rn), 3.54 (3H, S) "s were refluxed for 5 hours 0.55 g (4 rnM) of 4, 5-dimethyl-1,2-diaminobenzene and 0.6 g (4 mM) of benzol-1-ferric acid in 15 ml of ethanol, cooling and filtering gave 0.8 g (80% yield) of a yellow solid. which had a melting point of 275 ° C. NMR (CDCl 3): d 8.38 (2H, rn), 7.51 (3H, m), 7.70 (1H, s), 7.06 (1H, s) 2.40 (3H, s), 2.37 (3H, s). Irradiation at 8.38 ppm gave an individual band at 7.51 pprn. 3, 4-Diarninotoluene and phenylglyoxal were reacted, as for GI3, to give a slightly brown solid (31% yield) with a melting point of 114 ° C. NMR CDCI3 d 9.29, 9.26 (2S, 2: 1, Ha), 8.2, 8.17 (2br.S), 8.07-7"90 (3H, rn), 7.60 (3H, m), 2.62 (3H, S). 2a 5e reflux p > or 23 hours 0.15 g of G14 in 5 rnl of 48% HBr. Cooling and filtration gave 95 rng! 53% yield) of a green-yellow solid corresponding to the HBR salt of the quinazoline derivative, m.p. 280 ° C. The HBr was determined by elemental analysis. NMR (DMSO-D, *): or 9.25 (HH, s, H2), 8.24 (HH, d, 3 = 1.9 Hz), 8.20 (HH, d, 3 = 1.9 Hz), 7.50 (3H, rn ), 7.35 (2H, rn). The mother liquor was neutralized with NaHCO3. Extraction with Etflc gave 20 rng (15% yield) of an orange solid, m.p. 305 ° C corresponding to the free base. NMR (acetone dß): d 9.19 (HHs, Ha), 8.29 (1H, d, 3 = 1.5 Hz), 8.25 (HH, d, 3 = 1.5 Hz), 7.6 (3H, rn), 7.40 (2H) , rn). MS: 238 (M +, 54%), 211 (M-HCN, 10%), 154 (7%) 108 (1,2-benzoquinone, 100S), rn / e. £ 21 Reacted 4-benzoyl 1,2-phenylenediarnine and phenylglyoxal, co or for G13, to give a white solid (59% yield), with a melting point of 133 ° C NMR: CDCla d 9. 0 ( ÍH, S, HS), 8.49 (ÍH, S, Ha), 8.27 (4H, r, S, H sHa.-aS-) 7.90 (2H, d, 3 = 7.6 Hs), 7.60 (6H, m ). G22 Reflux for 1.5 hours 0.47 g (3 rnM) of 2,3-diarninoaphthalene and 0.47 g of phenylgloxal hydrated in 20 rnl of ethanol were refluxed. Cooling and filtering gave 0.5 g (65% yield) of a slightly brown solid having a melting point of 1S3 ° C. NMR: CDCI3 or 9.38 (1H, S, HS), 8.71, 8.67 (2H, 2d, H3.? 0), 8.25.3.10 (4H, flfl'BB 'rn., H, *. < =,), 7.58 (5H, rn, Ph). 256 (H '+ -, 100%), 229ÍH-CN, 122%), 126 (71), rn / e. G23 0.6 g, 4 rnM, phenylglyoxal and 0 ^ 6 g, 4 rnM, of 4-nitrophenylenediarine in 15 rnl of ethanol were refluxed for 0.5 hours. Cooling and filtration gave 0.9 g, 90% yield, white solid, m.p. 203DC. NMR CDCl 3 d 9.49 (lH, S, Ha), 9.02 (1H, d, 3 = 2.5 Hz, H.),, 8.54 (1H, dd, 3 = 9. "" 2, 2.5 Hz, H- ^),? 8"27 (3H, rn, Ph + W-,), 7.60 (3H, rn, Ph). G24 1.4 g, 10.3 rnM, of 4,5-dimethyl-1,2-phenylenediamine and 1.9 g, 10.2 rnM, of a-chloro 3,4-dihirox acetophan in 25 rnl of ethanol were refluxed for 2 hours. Cooling and filtration gave 0.76 g, 18% yield, strong yellow solid, m.p. 278 ° C as the HCl salt. Q23 2.4 g of 2-ethoxycarbonyl-6,7-dirnethyl-3-phenylquinoxaline and 5 g of KOH in 20 rnl of ethanol and 20 rnl of water were stirred for 20 hours at room temperature. Acidification with HCl, filtration and washing with water gave 2.1 g, 96% yield, slightly yellow solid, m.p. 153 ° C NMR acetone dß d 7.92 (1H, S), 7.30 (1H, S), 7.85 (2H, rn), 7.50 (3H, rn), 2.56 (6H, S) "G27 = 2- (4-nitrophenyl) ) 6.7 ditnetylauinoxaline fW-4-nitrophenylglyoxal 6.60 (40 mmol) of 4-nitroacetophenone in 40 ml of dioxane and 5 g, (45 mmol) of selenium dioxide in 2.2 ml of water were dissolved and mixed. The mixture was refluxed for 16 hours with continued stirring. The reaction mixture was passed through an alumina column to remove the selenium. The solvent evaporates > He prayed to the void. The crude product was used in the next step without further purification. ± RO »80 (EtOAc) - ^ - fc" R: 7.00 (85%) IR "3): 1730 (CO), 1520, 1330 (NOa) 2, 2- (4-nitp? Phenyl) 5, 7 dinuetilq inoxalina 0.50 was dissolved. g (3 mmol) of 4-nitrophenylglyoxal in 20 nmol of ethanol, and 0.34 g (2.5 nmol) of 1,2-dimethyl-4,5-dimethylenebenzene in ethanol was dissolved. The reaction mixture was stirred and refluxed for one hour. The product crystallized after cooling and filtering by washing with ethanol and then ether. G28 G28 can be produced using the protocol described for G29 infra, except that 3-bromoaniline is used in place of rnetaiodoaniline. G29 150 rng, 0.8 mM, of 2-chloro-6,7-dimethyl quinoxaline and 0.8g, 3.5 M, of -iodoaniline were heated at 100 ° C for 3-5 hours. Chromatography gave 100 g, 35% yield, solid - Flanarillo, m.p. 185 ° C. NMR CDCla 6 8.33 (1H, S), 8.22 (lH, rn), 7.7 (2H,), 7.40 (lH, rn), 7"10 (2H, rn), 2.45 (3H, S), 2.43 (3H, S). G30 210 rng, 1.1 M, of 2-chloro-6,7 dimethyl quihoxaline and 0.8g, 3.6 M, of p-iodoaniline were heated at 100 ° C for 4 hours. Chromatography gave 245 mg, 60% yield, light green solid, m.p. 228 ° C. ^^ NMR CDCI3 d 8.32 (1H, S), 7.67 (1H, S), 7.64 (2H, S), 7.68, X ^ ~ 7.55 (4H, ABq, 3ab = 9.0 HZ).

GROUP 8 H10 0.01 mmol (1.07 g) of benzylamine and 0.01 mol (1.28 g) of 3,4-dihydroxybenzaldehyde were mixed together in 15 ml of ethanol and refluxed on a water bath for 15 minutes then 0.01 mol (3.44 g) of 2- (1 '-tosiloxyethyl) -quinazolin-4-one and one drop of pyridine were added and the mixture was refluxed for 6 hours. The resulting solution was evaporated and extracted with one. 5% sodium bicarbonate solution in water. The remaining crystals were filtered, washed with water and recrystallized from isopropanol. Yield: 3.07 g (77%) P. f. : 209-211 ° C Formula: Ca «.Haa.N3? 3 Elementary analysis C%] Calculated: C: 72.17 H-.5.30 N: 10.52 Found: C: 72.12 H: 5.26 N: 10.46 n 0.01 mol ( 1.07 g) of benzylamine and 0.01 mmol (1.22 g) of salicylaldehyde, together, in 15 rnl of ethanol and refluxed on a water bath for 15 minutes then 0.01 mol (3.44 g) of 2- (1 ') was added. -tosyloxyethyl) -quinazolin-4-one and one drop of pyridine and the mixture was refluxed for 6 hours. The resulting solution was evaporated and extruded with a 5% solution in water of sodium bicarbonate. The remaining crystals were filtered, washed with water and recrystallized from isopropanol. Yield: 2.99 g (78%) P. f. : 189-192 ° C Formula: C24.Ha N302 flnálieis elemental C%] Calculated: C: 75.18 H: 5.52 N: 10.96 Found: C: 75.09 H: 5.49 N: 10.90 Ü12 0 were rnixed. 01 rnol (1. 07 g) of benzilarnin and 0. 01 rnol (1.22 g) of 3, -hydroxybenzaldehyde, together, in 15 rnl of tetanol and put to re flow on a water bath for 15 minutes then 0.01 mol (3.44 g) of 2- (l ') was added. -tosyloxyethyl) -quinazolin-4-one, and one drop of pyridine, and the mixture was refluxed for 6 hours. The resulting solution was evaporated and extracted with a solution in 5% water of sodium bicarbonate. The remaining crystals were filtered, washed with water and recrystallized from isop > Spanish Yield: 2.72 g (71%) P. f. : 184-185 ° C Formula: C2 H21N302 Elemental analysis C%] Calculated: C: 75. 18 H: 5. 52 N: 10. 96 Found: C: 75. Q2 H: 5.45 N: 11.08 H13 0.01 mol (1.07 g) of benzylamine and 0.01 mmol (1.22 g) of 4, -hydroxybenzaldehyde were mixed together in 15 ml of ethanol and refluxed on a water bath for 15 minutes then 0.01 mol (3.44 g) of 2- (l-tosyloxyethyl) -quinazolin-4-one, and a drop of pyridine were added, and the mixture was refluxed for 6 hours . The solution The resultant was evaporated and extracted with a 5% solution in water of sodium bicarbonate.The remaining crystals were filtered, washed with water and rectanstalised with water, and yield: 3.40 g (89 g. %) P. . : 217 - 219 ° C Formula: Ca ^ HaxNaOs » Elemental analysis l "% 3 Calculated: C: 75.18 H: 5.52 N: 10.96 Found: C: 75.26 H: 5.47 N: 10.88 Üli 0.01 mmol of (1.07 g) of benzylamine was mixed and 0. 01 rnol (1.54 g) of 3, 4, 5-trihydroxybenzaldehyde, together, in 15 rnl of ethanol and refluxed on a water bath for 15 minutes, then "0.01 mol (3.44 g) of 2- ( l '~ tosyloxyethyl) -quinazolin-4-one, and a drop of p> iridine, and the mixture was refluxed for 6 hours.The resulting solution was evaporated and extracted with a 5% solution in water of sodium bicarbonate.

Ffiltraron, washed with water and recrystallized from isopropanol. Yield: 3.36 g (81%) P. F. : 223-225 ° C Formula: Ca ^ Ha NsO. ,, Elementary ratio C%] Calculated: C: 69.39 H: 5.10 N: 10.11 Found: C: 69.51 H: 5.07 N: 10.08 GROUP 9, il? 3 hours 0.3 g (2 rnM) of 5-forrnil-indole and 0.4 g (2 rnM) of 2-cyano-H- (1- (+) phenylethyl) acetarni were refluxed in 3 rnl of ethanol and 2 drops of pyridine. Water and HCl were added and the reaction was extracted with ethyl acetate to give a viscous oil. Chromatography on silica gel gave "0.42 g (66% yield) of a slightly yellow solid having a melting point of 76 ° C. MS-315 (M +, 24%), 196 (M-NCH (CH3) C ^ H-., 22), - 195 (25), 188 (21), 173 (24), 168 (13), 149 (57), 145 (100), 134 (92), 116 (53), rn / e GROUP 10 aaaHQ 230 g, 1.06 rnM, 5-bromo 3, 4, dihydroxy-benzaldehyde, 76 rnG, 0.53 rnM, of diactonitrilsulfone and 10 rng of β-alanine in 10 ml of ethanol were refluxed for 5 hours. For cooling ~ and filtration gave 220 rng, 76% yield, orange solid, m.p. > 300 ° C. NMR acetone d * d 8.18 (21-1, S, vinyl), 7.90 (2H, d, J = 1.6Hz), X 7.78 (2H, d, 3 = 1.6 H z). F 311 = 2- (3-bromo-4,5-dihydroxyphenyl) -1-cyano-l-cyanomethyl-s-l-phutylenete A mixture of 500 rng of 5-brorno-3, 4-dihydroxybenzaldehyde and 700 rng of sulfonyl-diacetonitrile in 5 rnl of ethanol was refluxed with a few drops of pipdin for "4 hours." The ethanol was removed in a rotary evaporator and the mixture was treated with ethyl acetate, dilute acid and brine.

Part of the crude product was then purified by HPLC on a C-18 column to yield approximately 50 rng of 2- (3-bromo-4,5-dihydroxyphenyl) -1-cyano-l-c-anornetylsulphyl-ethene.

GROUP 11? ^ R 6.7-Oxyethoxy-quinazolin-4-one 7 g of 4,5-dimethoxy, 2-arninobenzoic acid and 8 ml of formaldehyde were heated at 170 ° C for 2 hours. Cold water was added and the solid was filtered to give 0.9 g, 12% yield, slightly brown solid, m.p. 308 ° C. NMR-DMSO t d 8.0 (1H, S) 7.43 (1H, S), 7.12 (1H, S) 3.89 (3H, S), 3.85 (3H, S). Ai 4-Chloro-6.7. dimethoxy-auinalozine F 3.5 hours, 0.8 g of 6,7-dirnetoxy-quinazolin-4-one, 1 ml of P0C13 and 1 ml of dirnethylaniline in 20 ml of toluene were refluxed for 3.5 hours. Treatment and trituration with hexane gave 0.5 g of a slightly gray solid, 0.5 g, 57% yield, m.p. 188 ° C. NMR CDC13 6 8.88 (1H, S), 7.41 (1H, S), 7.3d (lH, S), 4.09 (3H, S), 4. 08 (3H ,?).

CHEEP Reflux was carried out for 3 hours, 0.3 g, 1.4 rnM, of 3,4-dihydroxy-5-bromo-benzaldehyde, 0.15 g, 0.7 M, of N -3-cyanornethylcarbonylamino-N-propylcyanoacetamide, and 25 mg of β-alanine in 20 ml of ethanol. Cooling and filtration gave 0.24 g 57% yield, yellow solid, mp 283 ° C.

P12; 3, 5-bis (trifluoromethyl) anilide of dichlorophenophenol-2. 0.45 nmol (2.9 nmol) of 3,5-bis- (tri fluoromethyl) aniline was dissolved in 5 ml of absolute pyridine, then cooled to 10 ° C. 0.12 rnl (1.5 m ol) of phosphorus trichloride was added with continuous agitation in the form of drops. After 0.5 hour 0.51 g (4 mmol) of thiophene-2-carboxylic acid was added and stirred for 12 hours at ternp > environmental environment. The solvent was evaporated in vacuo, IN HCl was added to the residue and it was extracted with ethyl acetate. The ethyl acetate solution was extracted with sodium bicarbonate solution, dried over sodium sulfate, filtered and evaporated, the product was triturated with ether, filtered and dried in vacuo, mp: 145-147. ° C Rf: 0.80 (Hexane-EtRc = 1: 1) R: U.62 g (80%) IR (crn-11-: 3280 (NH): 1640 (CONH); 1560 (Car); 1130 (CF) 3 g, 20.1 rnM, 3-methyl-isoquinoline were heated in 20 rnl acetic acid and 5 rnl 30% H202 at 70 ° C for 14 hours. Water was added to the cooled solution and bicarbonate was added to neutralize. Extraction with CHaCla and trituration with hexane gave 1.9 g, 57% yield, white solid, mp 128 ° C. (3. O. C .. 21: 1337 (1956), mp- 138 ° C). NMR CDCla d 8.86 (lH, s), 7.70-7.50 (5H, rn), 2.64 (3H, S) P14 a. Refluxed for 1 hour, 0.04 g, 1.8 rnM of 4-chloro-6,7, dirnetoxy-quinalozine and 0.19 g, 2 M of aniline in 15 ml of ethanol. It was cooled and filtered to yield 0.445 g, 78% yield, light yellow solid,? -268 ° C, as the HCl salt ^^ of Pl a. í ^ 'b. Free base. 0.35g of P14a was treated with HsO-NaaC03 and extracted with CHaCla to give 0.13g, 42% yield, white solid, m.p. 241 ° C. NMR CDC13 d 8.66 (1H, S, Ha), 7.67 (1H, S), 7.63 (1H, S), 7.4-7.14 (5H, m), 3.96 (3H, S), 3.93 (3H, S). £ l = J 1- (2-chloro eni 1 met i Ten) -3- (3-methoxy-n-propyl) -2. - thia = Qlidinedione) A solution of 400 g of 3- (3-rnetoxy-n-prop> il-2, 4-thiazolidinedione and 260 rng of 2-chlorobenzaldehyde in 4 rnl of ethanol with a drop of piperidine were placed in reflux for 4 hours.The mixture was then treated with ethyl acetate and water.The crude product was purified on a column of silica gel (5% methanol in dichloromethane) to yield 200 mg of 1- (2-chlorophenyl) ethylene) -3- (3-methoxy-n-propyl) -24-thiazolidinedione. (P15 can also be obtained from Aldrich Chemical). PIB were refluxed for 5 hours, 0.69 g, 2.5 nM of 5-iodovaniline, N-3-phenyl-N-propylcyanoacetamide (prepared as described by Gazit et al., 3. Med Chem 34: 1896, 1991 ) and 50 rng of ß-alanine in 30 rnl of ethanol. Evaporation gave an oil which was triturated with benzene-hexane and filtered to give a bright yellow solid, 0.82g, 71% yield, ad 8.12 (1H, S), 7.75 (IH, d, 3 = 2.0 Hz), 7.68 (IH, d, 3 = 2.0 Hz), 7.30-7.1 (5H, rn), 3.96 (3H, S, 0, CHa), 3.45 (2H, q, 3 = 6.0 Hz), 2.70 (2H, t, 3 = 6.0 Hz), 1.95 (2H, quin, 3 = 6.0 Hz). MS-462 (M-+ -; "53), 357 (M-CHS) aPh, 18), 335 (M-1, 100), 327 (M-NH (CHa) 3 ph, 31), rn / e P 17 a. It was placed in reflux for two hours 0.4g, 1.8 rnM, 4-chloro-6,7, dimethoxy-quinolone, and 0.24 g, 2 nM, of indoline in 10 ml of ethanol, was cooled and filtered to give 0.46 g, 74% yellow solid yield (P17a), m.p. 238 ° C. b. Free base - 0.3 g of AG P17a was treated with HzO-MaaC0a and extracted with CHaCla to give 0.13g, 48% yield, white solid m.p. 158 ° C. NMR CDC13 or 8.79 (1H, S, HS), 7.30 (1H, S), 7.28 (1H, S), 7.14-6.80 (4H, rn), 4.36 (2H, t, 3 = 7.6 Hz), 4.06 (3H , S, 0CH3), 3.85Í3H, S, 0CH3), 3.22 (2H, t, 3 = 7.6 Hz). P 18: l-cyano-2- (3-ethoxy-4-hydroxyphenyl) -l-methoxycarbonyl-ethene A mixture of 20 g of 3-ethoxyl-4-hydroxybenzaldehyde and 13 g of methyl cyanoacetate in 100 rnl of ethanol was refluxed with a piperidine lrnl for 4 hours. The crude mixture was allowed to cool to room temperature and, with stirring, water was added until the solid started to washed with a cold mixture of ethanol and water (1: 2) and dried by suction to yield 25 g of l-cyano-2- (3-ethoxy-4-hydroxy phenyl) -1-methoxycarbonyl-ethene.

P19: N-2-chlorophenyl (2-cyano-2-N-morpholinylcarbonyl) ioacetamide A solution of 1.5 g of N-morpholinyl-cyanoacetarnide 'sn 20 nrn of tetrahydrofuran at 0 ° C was added with 680 rng of sodium ethoxide. This mixture was stirred at 0 ° C for one hour and 1.7 g of 2-chlorophenyl isothiocyanate in 5 ml of tetrahydrofuran in the form of drops were added. After the addition, the mixture was heated to room temperature and then heated to 50 ° C for 6 hours. Upon cooling, all ethanol was removed and the resulting solid suspended in 10 mL of water. Then 3 ml of IN sodium hydroxide solution was added thereto, vigorously stirred and washed with 50 ml of co-ether. The aqueous layer was then acidified with IN hydrochloric acid until pH 1. The solid was then collected by filtration by suction. This produced 750mg of N-2-chlorophenyl (2-cyano-2-N-morpholinylcarbonyl) -thioaceta ida. (P19 can also be obtained from Ryan Scienti fic.) P20: N-2-chlorophenyl (2-cyano-β-N-3-tri-fluorophenylaminocarbonyl) thioacetamide * P20 was synthesized using conditions similar to those described "for P19 but starting with N -3-trifluoromethyl phenyl-cyanoacetamide. (P2Q can also be obtained from Ryan Scientific.) P21; N-3-methoxyphenyl- (2-cyano-2-N-pyrrolidinecarbonyl ti Q-acetamide) P21 was synthesized using conditions similar to those described for P19, but starting with N-pyrrolidinyl-cyanoacetarnide and 3-methoxyphenylisothiocyanate as the reactants. (P19 can also get from Ryan Scientific.) P22 = N-4-tri-fluoromethylbenzyl 3, 5-dimethylisoxazole-4-carboxatide P22 was made using the same conditions as those described for N-tri fluoromethyl phenyl-3,5-dirnethyldisoxazole-4-arboxarnide (R13), but starting with 4-tri-fluorophenylbenzyllanine.

P23: N-trifluoromethylbenzyl-3-methylisoxazole-4-carboxamya A solution of 3 g of 3-rnethyl-isoxazole-4-carboxylic acid and 4. 8 g of 1,3-dicyclohexylcarbodimide in 30 ml of dichloromethane were stirred at room temperature for 30 minutes. This was then added with 8 rnl of 4-trifluoromethylbenzyllanine in the form of drops, and the mixture was stirred at room temperature overnight. The mixture was then diluted with ethyl acetate (100 nmol) and treated with dilute hydrochloric solution and solutions of saturated sodium bicarbonate and sodium chloride; dried over sodium sulfate, filtered and concentrated. The crude product was then crystallized with ethanol and water to yield 1.5 g of N-tri fluoromethylbenzyl-3-methylisoxazole-4-carboxamide. ? P2; 1 -trifluoromethyl-1-benzylamino-bonyl-1-cyano-2-hydroxypropene? A solution of 500 mg of N-trifluoromethylbenzyl 3-rnethylisoxazole-4-carboxamide in 5 ml of ethanol was added to 300 mg of 1,8-diazabicyclo C5.4.03 undequene- (7). The mixture was then stirred at room temperature for 1 hour and acidified with 2 ml of 2N hydrochloric solution. The solid It was collected by filtration to yield 350 mg of 1-t-ri fl uororneti-benzylcarbonyl-1-cyano-2-hydroxypropene.

P25 They were refluxed for 5"hours, 0.42 g, 3 rnM, 3,4-dihydroxybenzaldehyde, Od g, 3.1 rnM, of N-4-fluorobenzylcyanoacetarnide and 2.0 rng of β-alanine, 0.89 g, 95% by concentration and filtration. yield, yellow solid ", pf.212 ° C, NMR acetone d« s or 8.10 (1H, S, Vinyl), 7.68 (lH, d, 3 = 2.3Hz, Ha), 7.40 (3H, m, H? , H ?, H *), 7.09 (2H, t, 3 = 8.8Hz), H.3., A,) 6.98 (H, d, 3 = 8.3Hz, HS), 4.56 (2H, br, S) . MS-312 (M-, 46%), 311 (30), 295 (H-OH, 35), 161 (17), 124 (NHCH ^ CsH ^ F, 100%)., Other modalities are within the following claims.

Claims (7)

1. NOVELTY OF THE INVENTION CLAIMS 1. - The use of a composition consisting of a compound selected from the group consisting of: a) a compound of group 1 having the chemical structure shown in the basic structure of group 1, Rx, Ra, R'a, R "'s , and R '' 'a are independently selected from the group consisting of halogen, trihalomethyl and NOa, and s is selected to consist of hydrogen, carboxy, or carbalkoxy, b) a group 2 compound having the chemical structure reentracted in the basic structure of group 2, characterized in that R ^ and Rs are independently, halogen, hydrogen, trihalogenomethyl, or NOs, VR < is either aryl, alkyl, = ilkenyl or alkynyl, c) a group compound 3 which has the chemical structure shown in the basic structure of the group 3, further characterized in that R 'R. Re ion independently selected from the group consisting of; and R * is aryl or hydrogen; d) a compound of group 4 having the chemical structure shown in the basic structure of group 4, characterized in that R or is, either = S, = 0, SH, OH, or NHa; and Ra_? is SH, OH, NHS, = C (CN) a or aryl; or R? 0 and Ra.a. Tornadoe together are aril; and Ri2 is hydrogen, aryl, alkyl, alkenyl or alkynyl, e) a compound of group 5 having the chemical structure shown in the basic structure of the group, R? S, R? < s, R? -7, and ene, alkoxy, OH, amino, alkylamino or SH; f) a compound of group 6 having the chemical structure shown in the basic structure of group 6, further characterized in that it is aryl, alkyl, alkenyl or alkynyl; Rao is an alkyl; o and Rso are together an aryl, g) a compound of group 7 having the chemical structure shown in the basic structure of group 7, further characterized in that b is an optional pi bond, Y and Z carbon or nitrogen; RE and R22 are halogen, halogen, OH, γH, NHa, NOa, alkyl, alkenyl, alkynyl, alkoxy, benzoyl, COOH, or carbalkoxy, or R = x and R s together form an aromatic ring to give an aryl; is hydrogen, halogen, = 0, OH, SH, NHS, alkoxy, COOH, aryl; Ra ^. is H or aryl and RaS is hydrogen, halogen, = S, or 0 =; provided that if b is not a bond, the adjacent nitrogen optionally has a substituent from the group consisting of hydrogen, alkyl, , alqui. lenamm oalqui lo and alqui. lenci.ano; h.) a compound of group 8 having the chemical structure shown in the chemical structure shown in the basic structure of group 8, R * ", * and Ss are independently alkyl, aryl, alkenyl or alkynyl; and R27 is alkyl, and i) a compound of group 9 having the structure shown in the basic structure of "group 9, further characterized in that Ra is either hydrogen or halogen, R30 is either alkyl, alkenyl, or alkynyl, Ra is aryl, and Raa is either = 0 or = S, j) a tax of group 10 having the structure shown in the basic structure of group 10 further characterized because R33 is alkyl or aryl, and R34, R3s , and R3a is each independently selected from the group consisting of: halogen, OH, hydrogen, alkoxy, SH, Ha or C (CHa) a; k) a cornp> which belongs to group 11 having a structure shown in the fornulae of group 11; 1) the active drug of said cornproduct, or a pharmaceutically acceptable salt of said corncom, in the preparation of a medicament for treating a patient suffering from a cell proliferation disorder or by inappropriate PDGF-R activity, by significant inhibition of one or more activities of PDGF-R in vivo or in vitro.
2. 2. The use of a composition according to claim 1, further characterized in that said constituent compounds are selected from the group consisting of: RIO, All, R12, A13, B10, Bll, B12, B13, B14, B15 , B16, B17, dlß, B19, CIO, CU, C13, Dll, D12, D13, D14, D15, D16, D17, D18, D20, E10, Eli, E12, E13, E14, E15, E16, FIO, Fll , F12, G10, Gil, G12, G13, G14, G15, GIS, G17, G18, G19, G20, G21, G22, G23, G24, G25, G26, G27, G28, G29, G30, H12, 110, 311 , PlO, P12, P13, P14, P15, P16, P17 P18, P19, P20, P21, P22, P23, P24, P25, the active drug of said compound or a pharmaceutically acceptable salt of said compound.
3. 3. The method to treat a patient suffering from a cell proliferation disorder in accordance with the - A claim 1, further characterized p > or that said compound selectively inhibits PDGF-R activity.
4. 4. The method for treating a patient suffering from a cell proliferation disorder according to claim 3, further characterized in that said compound also selectively inhibits one or more related tyrosine kinase members from the group consisting of KDR, Flt-1 and Flk-1.
5. 5. The method for treating a patient suffering from a cell proliferation disorder according to claim 1, further cited because said patient is a human and said cell proliferation disorder is a cancer selected from the group consisting of intraxial brain cancer, ovarian cancer, colon cancer, prostate cancer, lung cancer, Kaposi's sarcoma and rnelanoma.
6. 6. The method for treating a patient suffering from a cell proliferation disorder according to claim 5, further characterized in that said cancer is either glioma, ovarian cancer, prostate cancer or Kapiosi sarcoma.
7. 7. The method for treating a patient suffering from a cell proliferation disorder according to claim 1, further characterized in that said patient is a human and said disorder is either blood vessel proliferation disorder or a fibrotic disorder. 8. - The method to treat a patient suffering from a - cell proliferation disorder according to claim 7"," further characterized in that said fibrotic disorder is a "hepatic fibrotic disorder or a cell proliferation disorder - rnesangial." 9. The method for treating a patient suffering from a disorder of cell proliferation according to claim 8, further characterized in that said blood vessel proliferation disorder is atherosclerosis. tfS 10. The method for treating a patient suffering from a cell proliferation disorder according to claim 5, further characterized because it comprises the step of determining whether said cancer has an inappropriate PDGF-R activity 11.- The method for treating a patient suffering from a cell proliferation disorder- in accordance with claim I, further characterized in that said compound further comprises the physiologically acceptable vehicle that is • PBTE: D5W 12.- One method pair to treat a patient suffering from a cell proliferation disorder further characterized by inappropriate PDGF-R activity, comprising the step of administration to said patient. a therapeutically effective amount of a composition comprising a compound selected from the group consisting of AlO, A12 or Bll. 13. - The method for treating a patient suffering from fl-asthenic cell proliferation - according to claim 12, further characterized in that said patient is a human and said cell proliferation disorder is a cancer selected from the group consisting of cancer of intraxial brain, ovarian cancer, colon cancer, prostate cancer, lung cancer, Kaposi's sarcoma and rnelanone. 14. The method for treating a patient suffering from a tr-asthenic cell proliferation in accordance with the A'-claim 13, further characterized in that said cancer is either glioma, cancer- ovarian, prostate, or sarcoma of Kaposi 15"- The method for treating a patient suffering from a cell proliferation disorder according to claim 12, further characterized in that said compound is RIO or Bll. 16. The method for treating a patient suffering from a cell proliferation disorder in accordance with the JHL claim 15, further characterized in that said cancer is gliorna 17. The method for treating a patient suffering from a cell proliferation disorder according to claim 15, further characterized in that said cancer is ovarian cancer. The method for treating a patient suffering from a cell proliferation disorder in accordance with that characterized in that said cancer is 19. The method for treating a patient suffering from a cell proliferation disorder in accordance with claim 12., characterized in that said patient is a human and said disorder is either a blood vessel proliferation disorder or a fibrotic disorder. 20. The method for treating a patient suffering from a cell proliferation disorder in accordance with claim 19, further characterized in that said fibrotic disorder is a hepatic fibrotic disorder or a rnsangial cell proliferation disorder. 21. The method for treating a patient suffering from a cell proliferation disorder "in accordance with claim" 20, further characterized in that said blood vessel proliferative disorder is arteriosclerosis. 22. The method for treating "a patient suffering from a cell proliferation disorder according to claim 12, further characterized in that it comprises the step of determining whether said cancer has an inappropriate PDGF-R activity. treating a patient suffering from a cancer characterized by inappropriate PDGF-R activity, comprising the step of administering to said patient a therapeutically effective amount of a composition comprising an agent capable of significantly inhibiting the ... PDGF-R activity, and a cytotoxic agent. The method for treating a patient suffering from a cancer according to claim 23, further characterized in that said cytotoxic agent ee selects from the group that connects VP-16 and cisplatin. 25. The method for treating a patient suffering from a cancer according to claim 24, further characterized in that said cytotoxic agent is cisplatin and said cancer is lung cancer. 26.- A method to evaluate the susceptibility of a cancer- to the inhibition of its growth by a cornp > since it comprises the steps of: a) culturing said cells canceling a medium containing PDGF; b) a culture of said cancer cells in a medium containing PDGF and said compound; c) measuring and comparing the growth of said cells in (a) and < b) 27.- The method for evaluating the susceptibility of an inhibition of its growth by a compound of "- • conformity with" claim 26, further characterized in that it comprises the passage of cultured non-cancerous cells in a medium containing said compound and the measurement of cell growth. 28.- A method of p > The invention relates to a compound capable of inhibiting PDGF-R activity and the activity of a testicine kinase related to PDGF-R comprising the steps of: a) measuring the capacity of said compound to inhibit the measurement of capacity said activity F1K-1 29.- The test method for a compound capable of inhibiting PDGF-R activity and the activity of a tyrosine kinase according to claim 28, further characterized in that said compound significantly inhibits the activity PDGF-R and Flk-1 30.- A compound selected from the group consisting of BIO, B12, CIO, Cll, E10, Eli, E12, E13, E14, E.15, E16, FIO, ¥ ^ feFll, F12, G21, G22, Hll, H12, H13 and H14. 31.- A composition that includes either RIO, R12 or Bll and a PBTE vehicle: D5U. F