USE OF BENZYLIDENE-MALONONITRILE DERIVATES FOR THE TREATMENT OF LEUKEMIA

Abstract: The present invention relates to products and methods useful for treating leukemia, in particular acute lymphoblastomic leukemias. Compounds described herein are effective in inhibiting activities associated with ALL both in vitro and in vivo. Methods for modifying such compounds and screening other compounds are also provided in order to identify additional compounds with similar or better inhibition properties.

Description: DESCRIPTION USEOFBENZYLIDENE-MALONONITRILEDERIVATESFORTHETREATMENTOFLEUKEMIA Related Applications This application is a continuation-in-part application of U.S. application Serial No. 08/231,717, filed April 22, 1994, incorporated herein by reference in its entirety, including any drawings. Field of the Invention This invention relates to compositions and methods useful for diagnosing and treating leukemia. Background of the Invention None of the following is admitted to be prior art to the invention. Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal trans¬ duction involves the reversible phosphorylation of tyro- sine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs) . Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been impli- cated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentia¬ tion. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed. RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phos- phorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self- phosphorylation (autophosphorylation and/or transphos- phorylation) of the receptor on tyrosine residues. Their intrinsic tyrosine kinase is activated upon ligand binding, thereby initiating a complex signal transduc- tion pathway that begins with receptor autophosphoryla- tion and culminates in the tyrosine phosphorylation of a variety of cellular substrates and ultimately in the initiation of nuclear events necessary for the overall cell response. Individual phosphotyrosine residues of the cytoplasmic domains of receptors (e.g.. EGF, PDGF, and insulin) may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways. Some receptors, such as the lymphokine receptors IL-3, G -CSF, and IL-7 which are expressed in pre-B acute lymphoblatic leukemia (ALL) , do not possess a kinase domain and use cytoplasmic PTKs such as the src family of PTKs to transduct a signal. The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains (SRC homology domain 2) and SH3 domains (SRC homology domain 3) . The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146-1153, 1991; Schlessinger, Trends Biochem. Sci . , 13:443-447, 1988; and Ullrich and Schlessinger, Cell , 61:203-212, 1990. Oncogenic mutations in PTKs block normal cell differentiation and induce cellular transformation. The enhanced PTK activity or the overexpression of their normal counterparts was found to be essential for their transforming activity. Hyperproliferation of cells leading to non-malignant growth is also often associated with enhanced PTK activity; for example, the enhanced PTK activity of PDGF receptor which results from its exposure to sustained levels of PDGF seen in atherosclerosis and restenosis. Ross, New England J. Med.. 314:488-500, 1986. Pre-B ALL may be due to aberrations in the developmental program of cells at early stages of B lymphocyte differentiation. Such aberrations could include dysregulation of growth factor, receptors and/or signalling molecules that are associated with the receptor system. The most common form of leukemia in children is pre-B ALL. Despite intensive chemotherapy, 20% of children and up to 70% of adults with ALL will relapse and their prognosis is generally poor. The phenotype of the clones from such patients corresponds to the early stages of B lymphocyte differentiation (HLA" DR + , and CD10 + ) . Compounds that inhibit certain activities of protein tyrosine kinses include particular tyrphostins. Some tyrphostins have been proposed for treating leukemia. See U.S.Patent No. 5,217,999, issued June 8, 1993, incorporated herein by reference in its entirety, including any drawings. In addition, some tyrphostins have been proposed for treating abl associated leukemias. See International patent application WO 94/26260, published November 24, 1994, incorporated herein by reference in its entirety, including any drawings. Summary of Invention The present invention relates to products and methods useful for treating leukemia, in particular acute lymphoblastomic leukemias. Compounds described herein are effective in inhibiting activities associated with ALL both in vitro and in vivo. Methods for modifying such compounds and screening other compounds are also provided in order to identify additional compounds with similar or better inhibition properties. Tyrphostins described herein gave complete suppression of proliferation of human acute lymphoblastic leukemia cells in a clonogenic assay system. These compounds are effective in suppressing proliferation of all types of leukemia examined, including chronic leukemia, T cell leukemia and B cell leukemia and both fresh leukemia cells and leukemia cell lines. These compounds, for example ALL 7, similarly suppress leukemia cell growth in vivo in an animal model in which human ALL cells injected into irradiated SCID mice grow and disseminate in a pattern similar to the terminal stage of the human disease. Animals treated with ALL 7 were examined seven weeks after injection of human ALL cells and such cells could not be detected. In contrast, untreated controls showed massive ALL cell infiltration in all tissues studied at seven weeks after injection. In accordance with one embodiment of the invention, a method is provided for treating leukemia in a mammal comprising administering to the mammal an effective amount of a composition which comprises as active ingredient a compound of the formula wherein R x is hydrogen, hydroxy, nitro, alkoxy or halo; and R 2 is hydrogen, hydroxy, halo, nitro, alkoxy, - CN, - COOH, - (NH 2 ) =C (CN) 2 , - CONHCH 2 wherein R 3 is hydrogen, hydroxy, nitro, alkoxy or halo and n is 2 to 4, in a mixture with a pharmaceutically acceptable diluent or carrier. By "nitro" is meant a N0 2 group. The alkoxy group may be any length, preferably less than 10 carbon atoms, more preferably less than 7, most preferably less than 4. In preferred embodiments the mammal has acute lymphoblatomic leukemia and/or is not an abl associated leukemia. A group of tyrphostins shown herein to be very effective in controlling or inhibiting growth of human leukemia cells without suppressing normal hemopoietic cell lineages have the formula: wherein R x is hydrogen, hydroxy, nitro, alkoxy or halo; and R 2 is hydrogen, hydroxy, halo, nitro, alkoxy, - CN, - COOH, -_ (NH,)=C(CN),, or CONH wherein R 3 is hydrogen, hydroxy, nitro, alkoxy or halo and n is 2 to 4, in a mixture with a pharmaceutically acceptable diluent or carrier. Suitable substituents for the tyrphostin compounds include hydroxy, alkyl, alkoxy, nitro or halo. Halo may be chlorine, bromine or iodine. A preferred group of compounds for treatment of human leukemia are 3,5-dimethoxy,4-hydroxybenzylidene malononitrile (ALL 1); 3,4-dihydroxybenzylidene malononitrile (ALL 2); o>- cyano-3,4-dihydroxycinnamate (ALL 3); 3-hydroxy,4- nitrobenzene malononitrile (ALL 4) ; γ-cyano- β-amino-3- hydroxy,4-nitrocinnamal malononitrile (ALL 5); γ-cyano- S-amino-3,4-dihydroxy-5-methoxycinnamal malononitrile (ALL 6); α;-cyano-3,4-dihydroxycinnamal benzylamide (ALL 7 ) ; N-bis- [ α-cyano-3-methoxy-4-hydroxy-5-nitro-cinnam] - butylene diamine (ALL 8) . An especially preferred compound is α-cyano-3,4-dihydroxycinnamal benzylamide (ALL 7) . The compounds of the present invention may be modified and tested for their ability to inhibit proliferation of leukemia cells and inhibit other activities associated with leukemias. Other compounds may also be assayed for these activities. The compounds and compositions described above and found by the screening methods described herein may be used to inhibit the proliferation and colony formation of ALL and other immune cells (such as B cells, T-cells, etc.), preferably by at least 50%, more preferably, 80%, most preferably 90%. The development of drugs to inhibit tyrosine kinases is reviewed in Levitzki and Gazit, Science, 267:1782, 1995, incorporated herein by reference in its entirety, including any drawings. In preferred embodiments, inhibition of signal transduction is by agents with molecular weight less than 3000, preferably less than 1500 such as quinazolines, tyrphostins, guinoxalines, and extracts from natural sources. The quinazolines, tyrphostins, quinolines, and quinoxalines referred to above include well known compounds such as those described in the literature. For example, representative publications describing quinazoline include Barker et al., EPO Publication No. 0 520 722 Al; Jones et al., U.S. Patent No. 4,447,608; Kabbe et al., U.S. Patent No. 4,757,072; Kaul and Vougioukas, U.S. Patent No. 5, 316,553; Kreighbaum and Comer, U.S. Patent No. 4,343,940; Pegg and Wardleworth, EPO Publication No. 0 562 734 Al; Barker et al. , Proc. of Am. Assoc. for Cancer Research 32:327 (1991); Bertino, J.R., Cancer Research 3:293-304 (1979); Bertino, J.R., Cancer Research 9(2 part 1) :293- 304 (1979); Curtin et al., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44 (2) :173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et al., Cancer Research 51:5579-5586 (1981) ; Jones et al. J. Med. Chem. 29 (6) :1114-1118; Lee and Skibo, Biochemistry 26 (23) :7355-7362 (1987) ; Lemus et al. , J. Or . Chem. 54:3511-3518 (1989) ; Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et al. , Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330 (1985) ; Phillips and Castle, J. Heterocvclic Chem. 17 (19) .1489-1596 (1980); Reece et al., Cancer Research 47 (11) :2996-2999 (1977); Sculier et al., Cancer Immunol. and Immunother. 23:A65 (1986) ; Sikora et al., Cancer Letters 23:289-295 (1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all of which are incorporated herein by reference in their entirety, including any drawings. Quinoxaline is described in Kaul and Vougioukas, U.S. Patent No. 5,316,553, incorporated herein by reference in its entirety, including any drawings. Quinolines are described in Dolle et al. , J. Med. Chem. 37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al. , J. Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem. Letters 2:1771-1774 (1992), all of which are incorporated by reference in their entirety, including any drawings. Tyrphostins are described in Allen et al. , Clin. EXP. Immunol. 91:141-156 (1993); Anafi et al. , Blood 82:12:3524-3529 (1993); Baker et al. , J. Cell Sci. 102:543-555 (1992); Bilder et al. , Amer. Phvsiol. Soc. pp. 6363-6143 :C721-C730 (1991) ; Brunton et al. , Proceedings of Amer. Assoc. Cancer Rsch. 33:558 (1992) ; Bryckaert et al., Experimental Cell Research 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol. 151 (5) :2717-2724 (1993) ; Gazit et al. , J. Med. Chem. 32:2344-2352 (1989) ; Gazit et al., " J. Med. Chem. 36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994) ; Kaur et al. , King et al., Biochem. J. 275:413-418 (1991); Kuo et al. , Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282 (1992); Lyall et al. , J. Biol. Chem. 264:14503-14509 (1989); Peterson et al. , The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer 50:80-85 (1992); Posner et al. , Molecular Pharmacology 45:673-683 (1993); Rendu et al. , Biol. Pharmacology 44 (5) :881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J\. Pharm. and Experimental Therapeutics 267(3) :119-1125 (1993); Wolbring et al., J. Biol. Chem. 269(36) :22470- 22472 (1994); and Yoneda et al., Cancer Research 51:4430-4435 (1991); all of which are incorporated herein by reference in their entirety, including any drawings. The above-noted groups of compounds are particularly useful for screening in assays for inhibition of activities associated with disorders such as ALL. Such assays can be used to identify those compounds that are useful in treatment of the noted disorders. Those skilled in the art can use the assays described herein or equivalents of such assays for routine screening of such molecules to find those which are active in inhibition of the signal transduction pathway discussed above and thus useful for treatment of those immune disorders, such that the life expectancy of an individual affected with such a disorder will be increased or that one or more symptoms of the disease will be reduced. As noted above, some compounds have already been identified that inhibit activities associated with ALL, both in vitro and in an in vivo animal model. Other such compounds can be identified using the techniques described herein. In other preferred embodiments the agent identified in the assay is therapeutically effective and has an EC 50 or IC 50 as described below. An EC 50 or IC 50 of less than or equal to 5 μM is preferable, and even more preferably less than or equal to 1 μM, 100 nmolar, 10 nmolar, or 1 nmolar. Such lower EC 50 's or IC 50 's are advantageous since they allow lower concentrations of molecules to be used in vivo or in vitro for therapy or diagnosis. The discovery of molecules with such low EC 50 's and IC 50 's enables the design and synthesis of additional molecules having similar potency and effectiveness. In addition, the molecule may have an EC 50 or IC 50 less than or equal to 5 μM at one or more ALL cancer cells or other leukemia or cancer cells. By "therapeutically effective amount" is meant an amount of a pharmaceutical composition having a therapeutically relevant effect. A therapeutically relevant effect relieves to some extent one or more symptoms of the disease or condition in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition. Generally, a therapeutically effective amount to a molecule may vary depending on its EC S0 or IC 50 and on the age and size of the patient, and the disease associated with the patient. The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following descrip¬ tion of the preferred embodiments, and from the claims. Description of the Preferred Embodiments Tyrphostins and other small molecules are provided to suppress proliferation of immune cells, for example ALL cells. Particular tyrphostins have been shown to be effective both in vitro and in vivo. Methods for making and testing other compounds are provided, including methods of modifying the compounds shown to be effective in order to optimize the inhibition and other important properties such as half- life. Pharmaceutical Formulations and Modes of Administration Compounds of this invention may be useful in the form of the free base, in the form of salts and as a hydrate. All forms are within the scope of the invention. Acid addition salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of the base form. The acids which can be used to prepare the acid addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose anions are non- toxic to the animal organism in pharmaceutical doses of the salts, so that the beneficial properties inherent in the free base are not vitiated by side effects ascribable to the anions. Although pharmaceutically acceptable salts of said basic compound are preferred, all acid addition salts are useful as sources of the free base form even if the particular salt per se is desired only as an intermediate product as, for example, when the salt is formed only for purposes of purification and identification, or when it is used as an intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures. Pharmaceutically acceptable salts within the scope of the invention include those derived from the following acids; mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like. Tyrphostins are administered initially in a suitable dosage to provide a blood level of about 10 μM. The dosage may therefore be adjusted as required, depending on the clinical response. The particular compound that affects the disorder of interest can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) . In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such com¬ pounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 . Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage may vary within this range depend¬ ing upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For exam¬ ple, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disruption of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex component) . Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1) . It should be noted that the attending physi¬ cian would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxi¬ city) . The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary accord¬ ing to the age, body weight, and response of the indivi¬ dual patient. Depending on the specific conditions being treated, such agents may be formulated and administered syste ically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceu¬ tical Sciences, 18th ed., Mack Publishing Co., Easton, PA (-1990) . Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal admin¬ istration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physio¬ logically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formula¬ tion. Such penetrants are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the prac- tice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be admin- istered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceu¬ tically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Agents intended to be administered intracellu- larly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then adminis¬ tered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The lipo- somal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharma- ceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceu¬ tically. The preparations formulated for oral admini¬ stration may be in the form of tablets, dragees, cap- sules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolv¬ ing, granulating, dragee-making, levigating, emulsify- ing, encapsulating, entrapping or lyophilizing processes. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspen- sions of the active compounds may be prepared as appro¬ priate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injec- tion suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxy- methyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suit- able auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellu¬ lose, hydroxypropylmethyl-cellulose, sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP) . If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coat¬ ings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye- stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suit¬ able liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. EXAMPLES The examples below are non-limiting and are merely representative of various aspects and features of the present invention. The examples below demonstrate the ability of tyrphostins to inhibit the proliferation of immune cells involved in ALL both in vitro and in vivo. Example 1; Synthesis of Tyrphostins The compounds described herein for treatment of leukemia were prepared by Knoevenagel condensation reactions as described in U.S. Patent No. 3,149,148 and in Gazit et al. (1989), J.Med.Chem., volume 32, pp. 2344 to 2352 and (1991), J. Med. Chem., volume 34, pp. 1896 to 1907, which are incorporated herein by reference in their entirety, including any drawings. Tyrphostins were synthesised as described previously: ALL 1: Gazit (1989), Table I, compound 4. ALL 2: Gazit (1989), Table I, compound 23. ALL 3: Gazit (1989), Table II, compound 36. ALL 4: Gazit (1989), Table I, compound 10 ALL 5: Gazit (1989), Table III, compound 49 ALL 6: Gazit (1989), Table III, compound 52 ALL 7: Gazit (1991), Table III, compound 42 Example 2: Inhibition Of Immune Cell Proliferation By ALL 7 Five different pre-B leukemia cell lines derived from patients with clinical relapse of leukemia were studied. 2 x 10 5 cells/well were suspended in RPMI 1640 containing 10% FCS and were cultured for 24 h in the presence or absence of ALL 7 at various concentrations. Plates were pulsed with 1 μCi/well of tritiated thymidine [ 3 H] for the last 6 hr of culture and counts were read on a β scintillation counter. ALL 7 induced 50% inhibition of spontaneous leukemia cell proliferation at 5 μM, 80% inhibition at 10 μM, and total inhibition at 50 μM concentrations. Remarkably, this analogue did not inhibit normal mature B and T cell stimulation by PHA and STA respectively at concentrations up to 10 μM. Nevertheless, a decline in proliferation was observed more in T cells than in B cells at higher concentrations. At 50 μM, ALL 7 induced 27% inhibition in T cells and 17% in B cells. Example 3; Clonoσenic Assays A pre-B ALL cell line was derived from freshly obtained PBL composed primarily of blast cells from a child with pre-B ALL in relapse as described in Kamel- Reid et al., Leukemia 6; 1 (Jan.):8-17. 1991 incorporated by reference herein in its entirety including any drawings. The cells were cloned in semi solid methylcellulose (0.9% methocel, Dow Chemical Co, Midland, MI) . The cloned cell line maintained the immunologic markers of the original leukaemic blasts except CD10. The phenotype of the cell line maintained in culture was HLA " DR + , CD19 + , CD20 " and CD10 " , although the original patient sample expressed low levels of CD 10 in 40% of leukaemic cells. The karyotype of the cell line was as follows: 46xx, der(5), t(5;?), ql5;?), der(9) , t(9;?) , (pl3;?), -11, der(14) , t(14;?) , q(22;?) , +M1 with variations. The line was EBV and mycoplasma free and grew autonomously. Normal bone marrow control cells were isolated by Percoll (Pharmacia Piscataway, NY) density gradient centrifugation, prepared, and studied directly from the suspension cultures. Clonogenic assays were performed on normal bone marrow (BM) and G 2 pre-B ALL cells to determine the effect of ALL 7 on colony formation. Fauser and Messner, Blood 52:1243-1248, 1978; Messner and Fausner, Blut. 41:327, 1980; Estrov et al. , Blood 67;5 :1382-1387, 1986, all of which are incorporated herein by reference in their entirety including any drawings. After Percoll fractionation, 2xl0 5 bone marrow cells were suspended in a medium containing 10% FBS, IL- 3, GMCSF, and methylcellulose at a final concentration of 0.9% (vol/vol) in 35mm Petri dishes. The culture dishes were placed in 37°C with 5% C0 2 in air in a humidified atmosphere. Clonogenic assays were performed on ALL cells in semi solid medium. The cell lines (2xl0 5 ) were plated in 35 mm Lux suspension dishes (Nunc Inc., Naperville, IL) in α medium containing 10% FBS in methylcellulose. Duplicate culture dishes were incubated at 37°C with 5% C0 2 in air in a humidified atmosphere. Colonies (≥20 cells) were counted after 14 days using an inverted microscope. Table 1 shows three independent experiments whereby ALL 7 at various concentrations was added only once to dishes containing ALL cells or normal bone marrow. Dimethyl sulfoxide (DMSO) , the solvent in which the tyrphostin was dissolved, or medium was used as control. ALL 7 completely blocked colony formation by ALL cells at 2.5 μM to 10 μM concentrations. Further, ALL cells appeared dead morphologically. Similar concentrations of ALL 7 had no deleterious effect on normal haemopoiesis expressed as BFU-E, CFJ-C and CFU- MIS. DMSO at concentrations comparable to or higher than doses used as tyrphostin solvent did not inhibit ALL or normal colony formation. Example 4; Inhibition of Immune Cells By Tyrphostins Inhibition of proliferation of G 2 pre-B ALL cells by various tyrphostin compounds, each at 50 μM, was examined as described in Example 2 and compared with proliferation of untreated ALL cells as control. The percentage inhibition by the various tyrphostin compounds is as follows: ALL 1 50% ALL 2 70.3% ALL 3 52% ALL 4 71.25? ALL 5 61% ALL 6 47.5% ALL 7 98.2% ALL 8 50% The most effective compound was ALL 7 Example 5: In vivo Inhibition of ALL It has been shown previously that ALL cells have a fast rate of growth and dissemination when injected I.V. into SCID mice. Leukemia cells disseminated into several tissues including the brain leading to the death of the mice by 12-13 weeks. This pattern was reminiscent of the terminal stage of the disease in children and suggested that the murine SCID model provides a biological system for the study of the proliferation and the progression of leukemia and for the testing of new therapeutic modalities. The SCID mice were bred and maintained in a defined flora colony (Animal Lab at The Hospital for Sick Children, Toronto, Ontario, Canada) in sterile microisolator cages without any antibiotics. After injection with ALL cells, mice were given tyrphostin or control treatment by continuous subcutaneous infusion, using ALZET miniosmotic pumps 2001 (Alza Corp., Mountain View, C.A.). The pumps contain 200 μl and have a continuous infusion rate of 1 μl/hr, maintaining good levels for a week at least. We changed pumps weekly. The pumps were introduced through a small incision in the skin on animals' backs while they were under chloralhydrate 3.6% effect (injected I.P., 0.1 ml/lOgm mouse) . In the DMSO group, the pumps contained 46.6% DMSO and 15% Ethanol while for I.P., 35% DMSO was injected 0.5 ml/mouse. Various tissues (spleen, liver, kidney, lung, heart, and brain) were removed for histological examinations and DNA preparation. Suspensions of BM and spleen were also prepared for cytometry. We prepared touch preps on slides from PBL, BM, spleen and liver and stained them with Wright stain. Surface marker analysis was performed on 5 x 10 s cells obtained from BM and spleen. We used CD45 and anti-CDIO/CALLA (Coulter Electronics, Haileah, Florida, USA) ; isotype controls were run in every experiment and the percentage of positive cells and fluorescence intensity determined relative to those controls. Samples were run on the Epics profile analyzer (Coulter Electronics) . Tissues were fixed in 10% formalin, paraffin embedded and 4μm sections were cut and stained with haematoxylin and eosin. DNA was prepared from all tissues using standard procedures. Following the digestion of DNA with Eco RI, Southern blot analysis will be performed using probe, pl7H8 specific for a satellite DNA of human chromosome 17(35,36). The intensity of the characteristic 2.7 kb band in the samples will be compared with control mixtures of human:mouse DNA to quantitate the human DNA present in the murine tissues. This technique is very sensitive and can detect levels of human DNA as low as 0.1% in all tissues, whereas flow cytometry detects the 10-100% range in hemopoietic tissues only. 25μM/200μl pumps were used, providing 20μ moles of ALL 7, equivalent to 20μM ALL 7 used in proliferation and clonogenic assays. Additionally, a daily boost I.P. of 500 μg/0.5 ml/mouse was given. Pre-B ALL cells (5xl0 5 cells/mouse) were injected intravenously into 13 mice after irradiation with 200 cGy. Three mice died in the first week following anaesthesia. The 10 mice left were in the following groups - 5 mice received ALL 7 (continuously daily infusion S.C. by pump and 500 μg/0.5ml I.P. daily) while the other 5 served as controls; 2 mice received DMSO (continuously daily infusion by pump and 0.5 ml I.P. daily) and 3 mice received PBS only (0.5 ml I.P. daily) . In the fourth week of the study, one mouse of the PBS group and one of the ALL 7 group were sacrificed. No ALL infiltration was found, as by flow 5 cytometry, in BM and spleen of either mouse (Table 3) . The same results were seen in two mice (1 DMSO and l ALL 7) on the fifth week. Infiltration of ALL cells was finally documented in three control mice (1 PBS and 2 DMSO) at week seven of the study; massive infiltration 10 of ALL cells was detected in all tissues studied. Remarkably, ALL cells could not be detected in 3 mice sacrificed during the seventh week of treatment with ALL 7. On all mice, flow cytometry studies with CD45 15 were performed (Table 3) , as well as touch preps from BM, spleen, and liver and histological examination (data not shown) . On the histological examination, sections from liver, spleen, kidney, lungs, heart, and brain were stained with haematoxylin and eosin and immunostaining 20 with CD45. In all those studies, marked infiltration was found in the untreated mice but not in ALL 7 treated mice. No human DNA was detected in tissues of mice treated with AG-490, while human DNA was readily » detected in untreated mice. A similar experiment was 25 performed in which animals were treated with ALL 7 or control solvent by continuous intravenous infusion. Results were similar to those noted above with subcutaneous infusion. Other embodiments are within the following 30 claims. Medium 346±10 28 432±76 747±31 DMSO 1:1000 (DEQ-50μM 428±6 40±2 382±8 828±20 Note: DEQ - Diluent equivalent; ND - Not Done 5/28922 24 Table 2 DETERMINATION OF THE PERCENTAGE OF HUMAN ALL CELLS (CD45+) IN HAEMATOPOIEΗC TISSUES OF SCID MICE BY FLOW CYTOMETRY ♦ AG-490 LP. **AG-490 L Tune After PBS DMSO Early start Delayed star Injection of ALL BM Spleen BM Spleen BM Spleen BM Spleen Csfø f WteJa) ( ) (%) <%) (% ) 46.9 64.9 56.8 84.1 6 61.8 88.4 53.6 85 6 56.1 81.1 63.1 82.6 89.2 75.1 *** 56.4 65.9 ♦♦♦ 70.3 70.5 Note: *Early start - AG-490 LP. started 1 day following I. V. injection of ALL cells. ♦♦ Delayed start - SG-490 LP. started in the fourth week following I.V. ♦♦♦ As from week 6 tyiphostin dose was raised to 1 mgday (regular dose, 192 μg/day was started in the fourth week post &LL cells injection). Table 3 DETERMINATION OF THE PERCENTAGE OF HUMAN ALL CELLS IN HAEMATOPOIEΗC TISSUES OF SCID MICE BY FLOW CYTOMETRY CDS± CDlfl± PBS DMSO AG-490 PBS DMSO AG-490 Time After Injection BM Spleen BM Spleen BM Spleen BM Spleen BM Spleen BM Spleen of ALL Cells (weeks ) ( . ( %. (%) (%) (%) (%) (%) (%) ( % ) ( %. ( ) (%. 4 0 0 .. .. 0 0 -- -- -- - 5 0 0 .. .. 0 0 -- -- -- - 7 52.7 35.5 - - 0 0 ~ -- -- - 7 ~ ~ 41 34.6 0 0 - 0 0 -- 7 - - 33.4 22 0 0 - - - 0 0 (Equal to or less than 10% - outcome, in flow cytometiy is mentioned in the table as 0)

Claims: We claim: 1. A method for treating leukemia in a mammal comprising administering to the mammal an effective amount of a composition which comprises as active ingredient a compound of the formula wherein R x is hydrogen, hydroxy, nitro, alkoxy or halo; and R 2 is hydrogen, hydroxy, halo, nitro, alkoxy, - CN, - COOH, - (NH 2 )=C(CN) 2 wherein R 3 is hydrogen, hydroxy, nitro, alkoxy or halo and n is 2 to 4, in a mixture with a pharmaceutically acceptable diluent or carrier. 2. The method of claim 1 wherein the active ingredient is a compound of the formula wherein R x is hydrogen, hydroxy, nitro, Cl-4 alkoxy, or halo; and R 2 is hydrogen, hydroxy, halo, nitro, Cl-4 alkoxy, - CN, - COOH, - (NH 2 )=C(CN) 2 , wherein R 3 is hydrogen, hydroxy, nitro, Cl-4 alkoxy or halo. 3. The method of claim 2 wherein the active ingredient is a compound of the formula wherein R 1# R 2 and R 3 are hydrogen, hydroxy, nitro or methoxy; and R 4 is - CN, - COOH, - (NH 2 )=C(CN) 2 , wherein R 5 , R 6 and R 7 are hydrogen, hydroxy, nitro or methoxy. 4. The method of claim 3 wherein R x and R 3 are methoxy, R 2 is hydroxy and R 4 is - CN. 5. The method of claim 3 wherein R is hydrogen, R 2 and R 3 are hydroxy and R 4 is - CN. 6. The method of claim 3 wherein R x and R 2 are hydroxy, R 3 is hydrogen and R 4 is - CN. 7. The method of claim 3 wherein R is hydroxy, R 2 is nitro, R 3 is hydrogen and R 4 is - CN. 8. The method of claim 3 wherein R x is nitro, R 2 is hydroxy, R 3 is hydrogen and R 4 is - (NH 2 )=C(CN) 2 . 9. The method of claim 3 wherein R x and R 2 are hydroxy, R 3 is methoxy and R 4 is - (NH 2 )= C(CN) 2 . 10. The method of claim 3 wherein R x and R 2 are hydroxy, R 3 is hydrogen and R 4 is C0N3HCV\ 11. The method of claim 3 wherein R x is methoxy, R 2 is hydroxy, R 3 is nitro and R 4 is 12. The method of claim 1 wherein the leukemia is acute lymphoblastic leukemia. 13. A pharmaceutical composition for treatment of leukemia in a mammal comprising as active ingredient a compound of claim 1 in a mixture with a pharmaceutically acceptable diluent or carrier. 14. Method of inhibiting the proliferation of an immune cell comprising the step of contacting said cell with a compound of claim 1. 15. The method of claim 14 wherein said immune cell is a pre-B leukemia cell. 16. The method of claim 15 wherein said inhibition is at least 80%. 17. Method of inhibiting colony formation of ALL cells comprising the step of contacting ALL cells with a compound of claim 1.

Inventors: GAZIT AVIV, LEVITZKI ALEXANDER, ROIFMAN CHAIM, GAZIT, AVIV, LEVITZKI, ALEXANDER, ROIFMAN, CHAIM
Applicants: GAZIT AVIV, HOSPITAL FOR SICK CHILDREN RES, LEVITZKI ALEXANDER, ROIFMAN CHAIM, YISSUM RES DEV CO, GAZIT, AVIV, LEVITZKI, ALEXANDER, ROIFMAN, CHAIM, THE HOSPITAL FOR SICK CHILDREN RESEARCH AND DEVELO, YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW
Application year: 1995
Main classification: A61K31
IPC codes: A61K31