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WO1994026260A1 - PROCEDES ET COMPOSES INHIBANT LES TROUBLES DE LA PROLIFERATION CELLULAIRE CARACTERISES PAR L'ACTIVITE ANORMALE D'$i(abl) - Google Patents

PROCEDES ET COMPOSES INHIBANT LES TROUBLES DE LA PROLIFERATION CELLULAIRE CARACTERISES PAR L'ACTIVITE ANORMALE D'$i(abl) Download PDF

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WO1994026260A1
WO1994026260A1 PCT/US1994/005294 US9405294W WO9426260A1 WO 1994026260 A1 WO1994026260 A1 WO 1994026260A1 US 9405294 W US9405294 W US 9405294W WO 9426260 A1 WO9426260 A1 WO 9426260A1
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group
compound
alkyl
alkoxy
activity
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PCT/US1994/005294
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English (en)
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Alexander Levitzki
Aviv Gazit
Yinon Ben-Neriah
Chaim Gilon
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YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM 46 Jabotinsky Street
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Priority claimed from IL105707A external-priority patent/IL105707A0/xx
Application filed by YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM 46 Jabotinsky Street filed Critical YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM 46 Jabotinsky Street
Priority to AU69109/94A priority Critical patent/AU6910994A/en
Publication of WO1994026260A1 publication Critical patent/WO1994026260A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles

Definitions

  • the present invention concerns methods and compounds for inhibiting cell proliferative disorders characterized by abnormal abl activity.
  • cell proliferative disorders characterized by abnormal abl activity include forms of leukemia such as chronic myelogenous leukemia and acute lymphoblastic leukemia.
  • TKs tyrosine kinases
  • TPs tyrosine phosphatases
  • the family of tyrosine kinases can be further subdivided into receptor-type and cytoplasmic proteins.
  • the intracellular, cytoplasmic tyrosine kinases may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain.
  • the Philadelphia chromosome has been associated with cell proliferative disorders such as chronic leukemia and acute lymphoblastic leukemia (Pendergast et al . , Cell 75:175-185 (1993)).
  • the Philadelphia chromosome (PH + ) was found in over 90% of human chronic myelogenous leukemia patients, and in a much smaller percentage of acute lymphoblastic leukemia patients (Ramakrishnan and Rosenberg, Biochimica et Biophy ⁇ ica Acta 989:209-224, (1989) ) .
  • the Philadelphia chromosome results from a reciprocal translocation between chromosomes 9 and 22 (Ramakrishnan and Rosenberg supra) .
  • the c-abl gene located on chromosome 9q, is translocated into chromosome 22, within the Jbcr gene, resulting in the formation of a chimeric bcr-abl gene encoding a bcr-abl fusion protein.
  • Different types of bcr-abl fusion protein can result from a Philadelphia chromosome such as fusion proteins pl85 bcr-abl and p210 bcr-abl weight.
  • the p210 contains 937 bcr-encoded residues; the pl85 form appears to share the first 455 amino acids with p210.
  • the K562 cell line originally established from a patient with chronic myelogenous leukemia in the terminal blast crisis stage, can be induced to erythroid differentiation by reducing the level of p210 bcr-abl by specific antisense oligonucleotides, or inhibiting its tyrosine kinase activity by general tyrosine kinase blockers such as herbimycin A, genistein and erbstatin
  • the present invention concerns methods and compounds for inhibiting cell proliferative disorders characterized by abnormal abl activity.
  • the preferred compounds described herein inhibit cell proliferative disorders by targeting abnormal ajbl activity.
  • the preferred target is abnormal ajbl autokinase activity.
  • other mechanisms involving aJbl activity may be responsible for the observed cell proliferation inhibition described in the examples below.
  • the compounds may interact with abnormal abl substrates, such as Grab-2 (Pendergast, et al . , supra) , and, thus, inhibit the effect of abnormal a l activity.
  • the compounds described herein have other uses such as being used as lead structures for obtaining additional compounds having equivalent or better activity, screening for additional compounds having equivalent or better activity, and in helping to diagnose if a cell proliferative disorder is caused by abnormal abl activity.
  • Different groups of compounds whose members can inhibit growth of cells characterized by abnormal ajbl activity are described herein.
  • characterized by is meant that abnormal aJl activity is present in a cell.
  • inhibition of the abnormal aJbl activity, or the effect of the abnormal aJbl activity will to some extent inhibit growth of the cell having the abnormal ajbl activity.
  • Cell proliferative disorders refer to disorders wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm (e.g.. discomfort or decreased life expectancy) to the multicellular organism.
  • Cell proliferative disorders can occur in different types of animals and in humans.
  • Cell proliferative disorders include cancers, such as chronic myelogenous leukemia and acute lymphoblastic leukemia.
  • the preferred use of the described compounds is as a therapeutic agent in the treatment of a cell proliferative disorder.
  • Therapeutic agents should be administered in a dosage sufficient to have a therapeutic effect. A therapeutic effect is achieved by eliminating or inhibiting the growth, to some extent, of cells causing or contributing to a cell proliferative disorder.
  • a therapeutic effect relieves to some extent one or more of the symptoms of a cell proliferative disorder.
  • a therapeutic effect refers to one or more of the following: 1) reduction in tumor size; 2) inhibition (i.e.. slowing to some extent, preferably stopping) of tumor metastasis; 3) inhibition, to some extent, of tumor cell growth; and/or 4) relieving to some extent one or more of the symptoms associated with the disorder.
  • the compounds described herein are preferably administered with a pharmacologically acceptable carrier.
  • a pharmacologically acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration.
  • pharmacologically acceptable carriers include water, saline, physiologically buffered saline, and cyclodextrins.
  • Hydrophobic compounds are preferably administered using a carrier.
  • a factor in choosing an appropriate pharmacologically acceptable carrier is choosing a carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound.
  • an agent for treating a patient having a cell proliferative disorder characterized by abnormal aJbl activity is described.
  • the agent which can inhibit growth of a cell having abnormal aJbl activity is selected from the group consisting of:
  • R is selected from the group consisting of NH, O, and S
  • R 2 is substituted phenyl having 1 to 3 substituents selected from the group consisting of ester, amide, thioamide, thioether, halogen, trihalomethyl, OH, SH, N0 2 , alkoxy, cyano, and amino
  • n is 0 or an integer between l and 6
  • m is 0 or an integer between 1 and 6, provided that if n is 1 and m is 0 said substituted phenyl is not 2-CO(NH 2 ) -phenyl or 4-(COOCH 3 ) -phenyl
  • R 3 , R 4 , R 5 , and Rg is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 , and NH 2 ; and R 7 is either H or has the chemical formula:
  • R' where t is an integer between 1 and 12, and R' and R" is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alkoxy and aryl; c) a compound having the chemical formula:
  • R 8 , g, and R ⁇ 0 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy alkylaryl, OH, a ine, SH, halogen, hydrogen, N0 2 and NH 2 ; l is an alkylaryl; and R 12 is selected from the group consisting of further substituted aryl, aryl, CN, amide, and thioamide. d) a compound having the chemical formula:
  • Abnormal ajbl activity refers to a change in one or more aJbl activities compared to that of a normal abl protein, and includes the following: 1) an increase in kinase activity; 2) a different substrate specificity; 3) a different cellular location; and/or 4) a different duration of signal. (Anafi, M. , et al . , J. Biol . Chem . 267 :4518-4523, 1992). A normal ajbl protein is that occurring in the general population which is not associated with a cell proliferative disorder.
  • An abnormal ajbl protein has one of the following abnormalities (compared to a normal ajbl protein) : 1) fusion with another protein, such as, for example, jbcr; 2) truncation; 3) other mutations such as, for example, amino acid substitutions and internal deletions.
  • Methods of inhibiting abnormal ajbl activity such as p210 bcr-abl or pl85 bcr-abl activity includes targeting the abnormal protein autokinase activity; and inhibiting phosphorylation of substrates by the abnormal protein, particularly those substrates not phosphorylated by normal abl.
  • Effective compounds targeted to inhibit the tyrosine phosphorylation of a tyrosine kinase may also act by causing the production of an agent which inhibits cell proliferation (Anafi et al . , FEBS 330 : 260 , 1993).
  • the compound inhibits abnormal aJbl activity due to a bcr-abl fusion, such as p210 bcr-abl or pl85 bcr-abl .
  • the compounds targeted to cell proliferative disorders resulting from bcr-abl fusions preferably inhibit the kinase ability of an isolated bcr-abl fusion as measured by the methods described herein.
  • In vitro inhibition refers to an IC 50 (dose required for 50% inhibition) of 50 ⁇ M or less, more preferably 5 ⁇ M or less, even more preferably 1 ⁇ M or less. More preferably, the compound inhibits the kinase ability of the bcr-abl fusion in whole cells with an IC 50 of 50 ⁇ M or less, more preferably 5 ⁇ M or less, even more preferably 1 ⁇ M or less.
  • Compounds with effective in vitro are good candidates for therapeutic compounds. The activity of those compounds effective in vitro can be confirmed using animal models.
  • Gishizky, M, et al . , Proc . Natl . Acad . Sci . USA 90:3755-3759 (1993) describes such a model for transplantation of bcr-abl induced chronic myelogenous leukemia-like syndrome in mice.
  • Compounds which preferentially inhibit the tyrosine kinase activity of an abnormal bcr-abl fusion are preferred compounds for use as a therapeutic in the treatment of cell proliferative disorders characterized by a bcr-abl fusion, and use for diagnostic purposes.
  • Preferentially inhibition refers to at least a two fold, preferably 5 fold, more preferably 10 fold, greater inhibition on Jbcr-aJbl activity compared to the total tyrosine kinase activity or epidermal growth factor receptor (EGF-R) activity.
  • EGF-R epidermal growth factor receptor
  • Such compounds are preferred because their use in a patient can reduce adverse side reactions resulting from using compounds having a wide range of activities on cellular processes.
  • such compounds may be used to determine if a disorder is to some extent driven by abnormal bcr-abl activity, by assessing the effect of the compound on total tyrosine kinase activity and on EGF-R activity.
  • a compound selected from the group of compounds consisting of AI-10, AI-11, AI-12, AI- 14, AI-15, AII-20, AII-21, AII-22, AIII-35, AIII-37, AIV- 41, and AIV-42 is described. These compounds have been found to inhibit growth of cells having abnormal aJbl activity. Of these compounds, compounds AI-10, AI-11, AI- 12, AI-14, AI-15, and AII-20 are preferred compounds. These preferred compounds have a strong inhibitory effect on p210 bcr-abl kinase activity.
  • composition containing a therapeutically effective amount of a compound mentioned above, and a pharmacologically acceptable carrier is described.
  • a method of treating a patient having a cell proliferative disorder characterized by abnormal aJbl activity involves administering to the patient a therapeutically effective amount of a compound selected from the group consisting of: a) a compound having the chemical formula:
  • R x is selected from the group consisting of NH, 0, and S, R 2 is an aryl, n is 0 or an integer between 1 and 6, and m is 0 or an integer between 1 and- 6; .
  • R 3 , R 4 , R 5 , and R-_ is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 , and NH 2 ; and R 7 is either H or has the chemical formula:
  • R' and R' ' is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alkoxy and aryl; c) a compound having the chemical formula:
  • R 8 , Rg, and R 10 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 and NH 2 ;
  • R u is selected from the group consisting of an H, alkyl, and alkylaryl;
  • R 12 is selected from the group consisting of aryl, further substituted aryl, CN, amide, and thioamide, d) AIV-40; e) AIV-41; and f) AIV-42.
  • Figure 2 illustrates the chemical structure of Group Ilia compounds.
  • Figures 3A-D illustrate the chemical structures of exemplary compounds belonging to Groups I-IV. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention features compounds and methods for inhibiting cell proliferative disorders characterized by abnormal ajbl activity. Data is presented below illustrating the ability of exemplary compounds, belonging to different compound Groups, to inhibit the growth of cells characterized by abnormal aJbl activity
  • the preferred compounds are those compounds which can inhibit the kinase activity of abnormal aJbl proteins. Using the present application as a guide, one skilled in the art can obtain other compounds having equivalent or better activity.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkenyl group refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkynyl refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkoxy group refers to an "-0-alkyl” group, where “alkyl” is defined as described above.
  • aryl group refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
  • the preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, N0 2 , amine, thioether, cyano, alkoxy, alkyl, and amino groups.
  • a "further substituted aryl” refers to an aryl in which the preferred substituent(s) include those mentioned above for an aryl and an additional aryl.
  • An alkylaryl group refers to an alkyl (as described above) , covalently joined to an aryl group (as described above) .
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from
  • heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • amide refers to an -C(0)-NH-R, where R is alkyl, aryl, alkylaryl or hydrogen.
  • a “thioamide” refers to -C(S)-NH-R, where R is alkyl, aryl, alkylaryl or hydrogen.
  • ester refers to an -C(0)-0R', where R' is alkyl, aryl, or alkylaryl.
  • amine refers to a -N(R / )R // , where R" and R' ' ' , is each independently either hydrogen, alkyl, aryl, or alkylaryl, provided that R" and R'" are not both hydrogen.
  • substituted phenyl refers to a phenyl having
  • a thioether refers to -S-R, where R is either alkyl, aryl, or alkylaryl.
  • the present invention provides methods of inhibiting or decreasing proliferation of cells having enhanced proliferation due to abnormal aJbl activity and compounds useful in these methods.
  • Proliferation of cells, particularly leukemic cells having enhanced proliferation due to abnormal aJbl activity may be inhibited or decreased by exposing the cells to an amount of one of the compounds described herein (see Section III, infra) effective to inhibit or decrease activity of a Jbcr- aJbl fusion protein.
  • the present invention has application in different self-proliferative disorders characterized by abnormal aJbl activity, such as hematopoietic cell proliferative disorders including forms of leukemia.
  • Blood cells such as T and B lymphocytes, granulocytes, macrophages, mast cells, egakaryocytes, erythrocytes and eosinophils originate from a self-renewing population of multi-potential hemopoietic stem cells, located mainly in the bone marrow, which generate progenitor cells committed irreversibly to one or another of the various hemopoietic lineages.
  • Progenitor cells may each generate clones of lineage-restricted cells that mature into specialized cells.
  • cytoplasmic tyrosine kinases are expressed in, and may have important functions in, hematopoietic cells including src, lyn, fyn, blk, lck, csk and hck.
  • T-cell activation for example, is associated with activation of lck.
  • the signaling activity of lyn may be stimulated by binding of allergens to IgE on the surface of basophils. (Eisenian, supra) .
  • Abnormalities in tyrosine kinase regulated signal transduction pathways can result in hematopoietic cell proliferative disorders.
  • mutations in the cytoplasmic tyrosine kinase atk are responsible for the agammaglobulinemia, (Ventrie, D., et al, Nature 361 : 226 , 1993) .
  • This defect appears to prevent the normal differentiation of pre-B cells to mature circulating B cells and results in a complete lack of serum immunoglobulins of all isotypes.
  • the present invention is directed to methods and compounds particularly useful for treating leukemia characterized by abnormal aJbl activity.
  • Leukemia refers to a progressive proliferation of abnormal leukocytes found in hemopoietic tissues, other organs, and usually in the blood in increased numbers (Stedman's Medical Dictionary 25th edition (Hensyl ed. 1990) ) .
  • leukemia Different forms of leukemia are known in the art and include acute promyelocytic, adult T-cell, basophilic, embryonal, eosinophilic, granulocytic, hairy cell, leukopenic, lymphoblastic, lymphocytic, mature cell, megakaryocytic, meningeal, micromyeloblastic, mixed cell, monocytic, myeloblastic, myelomonocytic, neutrophilic, plasma cell, polymorphocytic, Reider cell, splenic, stem cell, and subleukemic.
  • Leukemia's are targeted by the present invention by directly inhibiting cell growth or inducing differentiation.
  • “Differentiation” refers to the maturation process of immature cells. The failure of cells to properly differentiate can lead to the build up of immature cells resulting in a cell proliferative disorder. The differentiated cells are not immortal. Inducing differentiation results in inhibiting cell growth because the terminally differentiated cells do not proliferate.
  • Group I compounds have the general structure:
  • R- is selected from the group consisting of NH, 0, and S, R 2 is aryl, n is an integer between 0 and 6, and m is an integer between 0 and 6;
  • n is 1-3, preferably 1; m is 0-3, preferably 0; R 2 is a substituted phenyl having 1 to 3 substituents independently selected from the group consisting of COOH, ester, amide, thioamide, thioether, halogen, trihalomethyl, OH, SH, N0 2 , alkoxy, cyano, and amino; preferably R 2 is a substituted phenyl having one substituent selected from the group consisting of COOCH 3 , COOH, and CO(NH 2 ); more preferably if n is 1, and m is 0 the substituted phenyl is not 2-CO(NH 2 )-phenyl or 4- (COOCH 3 )-phenyl.
  • the exemplary Group I compounds shown in Table 1 are all very effective in inhibiting p210 bcr-abl kinase activity (see the examples described below) .
  • novel Group I compounds include AI- 10, AI-11, AI-12, AI-14 and AI-15. These novel compounds define a subset of Group I compounds (see, Figure 1, Group I) where R x is selected from the group consisting of NH, O, and S, R 2 is a substituted phenyl having 1 to 3 substituents independently selected from the group consisting of ester, amide, thioamide, thioether, halogen, trihalomethyl, OH, SH, N0 2 , alkoxy, cyano, and amino, n is an integer between 0 and 6, and m is an integer between 0 and 6, provided that if n is 1, and m is 0, R 2 is not 2-CO(NH 2 )-phenyl or 4-(COOCH 3 )-phenyl.
  • n is 1-3, preferably 1; is 0-3 preferably 0; Rj is S or NH, and R 2 is substituted phenyl containing 1 to 3 substituents independently selected from the group consisting of ester, COOH, and CO(NH 2 ), preferably R 2 is substituted phenyl having one substituent selected from the group consisting of COOCH 3 , COOH, and CO(NH 2 ).
  • R 3 , R ⁇ R 5 , and ⁇ is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 , and NH 2 ; and R 7 is selected from the group selected from H or:
  • R' where t is an integer between 1 and 12, and R' and R' ' is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alkoxy and aryl;
  • Examples of Group II compounds are listed in Table 2 and shown in Figure 3b.
  • the compounds listed in Table 2 have the Group II generic structure where, R 7 is not hydrogen, and R 3 , R 4 , R 5 , and R is hydrogen.
  • An example of a Group II compound where R 7 is H is AII-20.
  • R 3 , R 4 , R 5 , and Rg is each independently selected from the group consisting of hydrogen, alkyl, and OH, preferably H; and when R 7 has the chemical formula:
  • R' and R' ' is each independently hydrogen, alkyl, or halogen, preferably methyl.
  • Group III compounds have the general structure:
  • R 8 , ,, and R 10 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 and NH 2 ;
  • R n is selected from the group consisting of an H, alkyl, and alkylaryl;
  • R 12 is selected from the group consisting of aryl, further substituted aryl, CN, amide, and thioamide.
  • R 8 is alkoxy, OH, halogen or H, preferably OCH 3 , OH or H;
  • R is alkoxy, OH, halogen or H, preferably OH;
  • Rj 0 is alkoxy, OH, halogen or H, preferably OH, H, or Br;
  • R n is H or alkylaryl, preferably H or CH 2 - phenyl; and
  • R 12 is a thioamide or amide having the formula:
  • X 3 is S or 0, and r is an integer between 1-12, preferably 1-6, and the aryl is preferably a substituted phenyl.
  • Two novel subsets of Group III compounds are: 1) those having the generic Figure of Group III where R 8 , j, and R j0 , are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 and NH 2 ; R n is an alkylaryl; and R 12 is selected from the group consisting of aryl, further substituted aryl, CN, amide, and thioamide, and 2) compounds having the cher-iical formula:
  • R 8 , Rg, and R 10 is each independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, OH, amine, SH, halogen, hydrogen, N0 2 and NH 2 ; l is H; and R 13 is a substituted phenyl independently having 1 to 3 substituents selected from the group consisting of ester, amide, thioamide, thioether, halogen, trihalomethyl, OH, SH, N0 2 , alkoxy, cyano, and amino, or phenyl.
  • the present disclosure also relates to the identification of other specific compounds belonging to the groups described herein which are useful in the present invention. Identification can be carried out by assaying the ability of a compound to inhibit abnormal abl tyrosine kinase activity, and preferably, the ability of the compound to inhibit growth of cells having a cell proliferative disorder characterized by abnormal aJbl tyrosine kinase activity. Such assays can be preformed as described in the art, or as described in the examples below.
  • Therapeutic compounds should be more potent in inhibiting cell having abnormal ajbl activity than in exerting a cytotoxic effect.
  • a measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index: LD 50 /IC 50 .
  • IC 50 the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein.
  • LD 50 the dosage which results in 50% toxicity, can also be measured by standard techniques, such as using an MTT assay as described by Mossman J. Immunol . Methods 55:55-63 (1983), by measuring the amount of LDH released (Korzeniewski and Callewaert, J. Immunol .
  • Methods 64 313 (1983); Decker and Lohmann-Matthes, J. Immunol . Methods 115:61 (1988), or by measuring the lethal dose in animal models.
  • Compounds with a large therapeutic index are preferred.
  • the therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.
  • mice Animal model systems known in the art and deemed predictive of human in vivo activity can be used to further confirm the therapeutically effective compounds belonging to the groups described herein.
  • Gishizky M. describes transplantation of bcr-abl induced myelogenous leukemia-like syndrome in mice.
  • the mice described by Gishizky et al . can be used as an animal model for bcr-abl induced myelogenous leukemia.
  • Another example of an animal model is described by Heisterkamp, N. , et al . , Nature 344 : 251-251 , 1990.
  • Heisterkamp et al describes a transgenic model in which mice expressing a bcr-abl mutant protein develop lymphoid malignancies.
  • plasma half-life and bio-distribution of the drug and metabolites in plasma, tumors, and major organs can be determined to facilitate the selection of drugs most appropriate for the inhibition of a disorder.
  • Such measurements can be carried out, for example, using HPLC analysis on extracts of tissues or blood of treated animals.
  • Compounds that show potent inhibitory activity in the screening assays but have poor pharmacokinetic characteristics can be optimized by altering the chemical structure to produce additional compounds, preferably within the described groups. The additional compounds can be test. In this regard, compounds displaying good pharmacokinetic characteristics can be used as models.
  • Toxicity studies can also be carried out by measuring the blood cell composition.
  • toxicity studies can be carried out as follows: 1) the compound is administered to mice (an untreated control mouse should also be used) ; 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition, and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.
  • Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness, or toxicity. Gross abnormalities in tissue are noted, and tissues are examined histologically. Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.
  • the compounds of this invention can be administered to a patient preferably in a pharmaceutical composition comprising the active compound and a carrier or excipient.
  • the compounds also can be prepared as pharmaceutically acceptable salts (i.e. , non-toxic salts which do not prevent the compound from exerting its effect) .
  • Pharmaceutically acceptable salts can be acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. (See, e.g. supra . PCT/US92/03736) .
  • Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.
  • salts can be prepared by standard techniques. For example, the free base form of the compound is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In another example, the salt is prepared by reacting the free base and acid in an organic solvent.
  • Carriers or excipient can be used to facilitate administration of the compound, for example, to increase the solubility of the compound.
  • carriers and excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physio- logically compatible solvents.
  • the compounds or pharma ⁇ ceutical composition can be administered by different routes including intravenously, intraperitoneally, subcu- taneously, and intramuscularly; orally, topically, or transmucosally.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologi ⁇ cally compatible buffers such as Hanks's solution, Ring- er's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice 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 administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically 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 intracellularly 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 administered 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 liposomal 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, many small organic molecules may be directly administered intracellularly.
  • 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 within the capability of those skilled in the art in light of the detailed disclosure provided herein.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g.. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate 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 injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl 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.
  • compositions for oral use can be obtained, for example by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable 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 cellulose, hydr- oxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • PVP polyvinylpyrrolidone
  • disinte- grating 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 coatings.
  • suitable coatings 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.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and 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 lubri ⁇ cants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • PBTE:D5W consists of PBTE diluted 1:1 in a solution of 5% dextrose in water.
  • the therapeutically effective dose can be estimated initially from cell culture and animal models.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture.
  • Such information can be used to more accurately determine useful doses in humans.
  • the use of hydrophobic compounds can be facilitated by different techniques such as combining the compound with a carrier to increase the solubility of the compound and using frequent small daily doses rather than a few large daily doses.
  • the composition can be administered at short time intervals, such as by the methods described above or using a pump to control the time interval or achieve continuous administration.
  • Suitable pumps are commercially available (e.g.. the ALZET® pump sold by Alza corporation, and the BARD ambula ⁇ tory PCA pump sold by Bard MedSystems) .
  • the proper dosage depends on various factors such as the type of disease being treated, the particular composition being used, and the size and physiological condition of the patient.
  • the expected daily dose is between 1 to 2000 mg/day, preferably 1 to 250 mg/day, and most preferably 10 to 150 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.
  • Drugs should be administered at doses ranging from 0.02 to 25 mg/kg/day, preferably 0.02 to 15 mg/kg/day, most preferably 0.2 to 15 mg/kg/day.
  • drugs can be administered at 0.5 to 1200 mg/m 2 /day, preferably 0.5 to 150 mg/m 2 /day, most preferably 5 to 100 mg/m 2 /day.
  • the average plasma level should be 50 to 5000 ⁇ g/ml, preferably 50 to 1000 ⁇ g/ml, and most preferably 100 to 500 ⁇ g/ml. Plasma levels may be reduced if pharmacological effective concentrations of the drug are achieved at the site of interest.
  • Example 1 Compounds which induce differentiation:
  • This example describes compounds which induce differentiation in cells having abnormal aJbl activity and techniques which can be used to obtain additional compounds able to induce differentiation and/or inhibit cell proliferation belonging to the different groups of compounds described herein.
  • the K562 cell line was originally established from a pleural effusion of a chronic myelogenous leukemia
  • CML CML patient in the terminal blast crisis stage.
  • Cells were cultured in RPMI 1640, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and
  • DMSO dimethylsulfoxide
  • K562 cells Exponentially growing K562 cells (approximately 7 X 10 s cells/ml) , were treated with non-toxic levels of compounds (up to 100 ⁇ M) . The cells were then washed twice with HANKS buffered solution, and the pellet was resuspended with protein lysis buffer containing 10 mM Tris pH 8, 50 mM NaCl, 5 mM EDTA, 30 mM Na-pyrophosphate, 50 M NaF, 1% Triton X-100, 2 mM phenylmethylsolfonyl fluoride, 100 KlU/ l aprotinin, 5 ⁇ g/ml leupeptin and 100 ⁇ M sodium vanadate.
  • protein lysis buffer containing 10 mM Tris pH 8, 50 mM NaCl, 5 mM EDTA, 30 mM Na-pyrophosphate, 50 M NaF, 1% Triton X-100, 2 mM phenylmethyl
  • Cell lysates were prepared on ice for 1 hour with short vortexing every 10 minutes. Cell lysates were cleared by centrifugation at 12,000 g for 30 minutes. The protein concentrations of the supernatants were measured using a Bio-RadTM protein assay, and the cell lysate was boiled for 6 minutes in SDS gel sample buffer. Extracts (60 ⁇ g) were loaded onto different lanes of 7.5% SDS polyacrylamide gel, electrophoresed and blotted to nitrocellulose paper (0.2 ⁇ , Schleicher and Schuell Inc.).
  • Protein blots were blocked overnight with TBST (50 mM Tris base pH 7.5, 150 mM sodium chloride and 0.05% Tween-20) , 5% bovine serum albumin and 1% chicken egg albumin, then incubated for 2 hours at room temperature with phosphotyrosine antibodies (Zymed Inc.) in blocking solution.
  • the filters were washed and incubated with protein A-peroxidase for 40 minutes at room temperature, washed again and subjected to ECL reaction (Amersham Inc.) and autoradiography. Stripping of the filters for further antibody reaction was done at 50°C in 10 mM Tris, pH 7.5, 2% SDS and 100 mM 2-3-mercaptoethanol.
  • the assays were performed using the copolymer Glu 6 Ala 3 Tyr 4 (Sigma Inc.) as previously described by Anafi et al . , J. Biol . Chem . 267:4518 (1992) and Yaish et al . , Science 242:933 (1988).
  • the effective dose of 50% of maximal percentage of differentiation was calculated from dose response curve of K562, treated with different compound concentra ⁇ tions.
  • the minimal toxic concentration is the first concentration in which the cells were arrested or killed prior to their differentiation.
  • AII-20, AIII-30, AIII-34, and AIV-40 were more effective in inducing erythroid differentiation than killing cells (i.e. , ED 50 ⁇ minimal toxic concentration) .
  • AIII-30 and AIV-40 did not significantly inhibit tyrosine kinase activity in intact K562 cells.
  • the compounds AIII-34 and AII-20 were found
  • AII-20 and AIII-34 were added to cells and the onset of erythroid differentiation was followed for 8 days in parallel with cell growth measurements. Cell mortality throughout the experiment for treated and untreated cultures was about 5%. From day 3, cell differentiation was accompanied by growth arrest, and its degree correlated to the level of cell differentiation. After one day of treatment all the AII- 20 concentrations tested, and up to 100 /xM AIII-34, had no effect on cell proliferation and just a small effect on differentiation. After five days of treatment, the differentiation level reached 60% in the treated cells (differentiation was less than 1% in untreated cells) . At low compound concentrations a good correlation was found between the degree of differentiation and the induction of growth arrested.
  • tyrosine phosphorylated proteins were identified in western blots of lysates of K562 cells. Several phosphorylated bands diminished following treatment at concentration of AII-20 and AIII-34, which were effective in inducing cell differentiation along with growth arrest. A 210 kDa band was affected by these compounds and by herbimycin A. Herbimycin is a non- selective phosphotyrosine blocker which can induce K562 differentiation (Cancer Res . 49:331 (1989)). Herbimycin A treatment resulted in a concomitant decrease of the 210 kDa protein detected with monoclonal anti-aJbl antibodies (8E9) , while no similar decrease was evident following treatment with AII-20 or AIII-34.
  • AII-20 and AIII-34 are potent blockers of p210 Jcr-aJ phosphotyrosine kinase in intact K562 cells. These two compounds are believed to be the first compounds for which a direct correlation has been shown for p210 bcr-abl phosphotyrosine kinase inhibitory activity and ability to induce erythroid differentiation.
  • AII-20 is a potent inhibitor for at least two tyrosine kinases, p210 jbcr-aJ and EGF receptor.
  • This example describes compounds which cell proliferation of cell having abnormal aJbl activity and techniques which can be used to obtain additional compounds able to induce differentiation and/or inhibit cell proliferation belonging to the different groups of compounds described herein.
  • K562 cells (ATCC 562, Rockville, MD) were cultured in medium (RPMI medium containing 10% fetal calf serum, 2 mM glutamine) containing 100 units/ml penicillin 38 and 100 ⁇ g/ml streptomycin) . Cells were transferred to 96 well plates (2 X 10 3 cells/well) and incubated with increasing concentrations of compounds to a final volume of 200 ⁇ l. Control cells were incubated with medium containing identical concentrations of the compound solvent (DMSO) .
  • DMSO compound solvent
  • K562 cells were measured after 6 days by measuring the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) to a blue formazan product (Mossman T. , J. Immunol . Methods 65:55 (1983)).
  • MTT 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl- 2H-tetrazolium bromide
  • K562 cells were plated at a density of 5,000 cells per well in 96 well plate in 100 ⁇ l of medium. Cells were exposed to compound for the indicated periods and pulsed with [ 3 H]-thymidine, L-[ 3 H]- leucine, and [ 3 H]-uridine at 5 ⁇ Ci/ l for the last 2 hours of drug exposure or 10 ⁇ Ci/ml for the last 30 minutes of the drug exposure. Cells were harvested and incorporation of label was assessed as described in Kaur et al . , J. National Cancer Institute 84:1736-40 (1992).
  • ATP Levels Ten million cells were collected by centrifugation and washed once with phosphate buffered saline (PBS) . To the cell pellet was added 500 ⁇ l of 60% methanol. The contents were mixed, heated at 95°C for 1.5 minutes, clarified by centrifugation and analyzed by ion- exchange HPLC on Partisal SAX column using gradient 39 elution with ammonium phosphate buffers (Ford et al . , Cancer Res . 51:3733-40, (1991).
  • PBS phosphate buffered saline
  • Cel l extraction and p210 bcr-abl immunoprecipitation Exponentially growing K562 cells (1 x 10 7 cells) were washed twice in phosphate buffered saline, and then the cell pellet was lysed in 1.0 ml of ice-cold kinase-lysis buffer (10 mM Na 2 HP ⁇ 4 -NaH 2 P0 4 [pH 7.0] 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS] 150 mM NaCl containing 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10 ⁇ g/ml of Aprotinin, and 10 ⁇ g/ml of pepstatin) , briefly vortexed and centrifuged at 35,000 rpm for 90 minutes.
  • ice-cold kinase-lysis buffer 10 mM Na 2 HP ⁇ 4 -NaH 2 P0 4 [pH
  • leupeptin was added to a final concentration of 50 ⁇ g/ml.
  • Each 1 ml of clarified extract was incubated with 5 ⁇ l of anti-Jbcr-aJbl sera (Ab-2, Oncogene Science) or with antiserum which had been incubated with immunizing peptide (10X) at room temperature for 2 hours prior to addition to extract. Incubation with antisera was overnight (16 hours) at 4°C with gentle shaking.
  • 15 ⁇ l packed volume of preswollen protein a-sepharose beads per .1 ml of extract were added and extracts were incubated for another 2 hours at 4°C with gentle shaking. Beads were pelleted by centrifugation.
  • In-vitro auto-pho ⁇ phorylation reaction (kinase activity) : The p210 Jbcr-aJbl protein immunoprecipitates were washed twice with extraction buffer lacking SDS. Precipitates were washed once with 50 mM Tris (pH 7.0) and resuspended in 20 ⁇ l of 20 M PIPES [piperzine-N,N'-bis(2- ethanesulfonic acid] (pH 7.0)-20 mM MnCl 2 . In some reactions, acid denatured rabbit muscle enolase (5 ⁇ g/5 ⁇ l) was added as an exogenous substrate for the p210 Jbcr- aJbl kinase.
  • reaction mixture Five microliters of compound were added at 8X final concentration of each reaction mixture. Reactions were initiated by adding lO ⁇ l of [ ⁇ - 32 p]ATP (10 ⁇ Ci per sample, 3000 Ci/mmole; Amersham Corp.), incubated for 20 minutes at 30°C, stopped by addition of 10 ⁇ l of 5X SDS gel loading buffer, heated at 95°C for 5 minutes and 40 analyzed on 7.5% SDS-polyacrylamide gel electrophoresis and by autoradiography (Laemmeli, UK., Nature 227:680-685 (1970) ) .
  • 32 P-orthophosphate labeling, immunoprecipitation and phosphotyrosine immunoblotting The kinase activity of p210 bcr-abl was measured using an anti-phosphotyrosine antibody. 1 X 10 7 cells were exposed to compounds for time periods of 1, 6 and 24 hours. Cells were labeled for 1 hour with 1 mCi of carrier free 32 P-orthophosphate in 5 ml phosphate free medium containing 10% dialyzed serum and appropriate concentrations of the drug.
  • Cells were centrifuged at 1,000 rpm for 5 minutes, washed 3 times, and lysed in 600 ⁇ l of 10 mM sodium phosphate (pH 7.5), 100 mM NaCl, 5 mM NaF, 100 ⁇ M Na 3 V0 4 , 1% Triton X-100, 0.5% Sodium deoxycholate, 2 mM phenylmethylsulfonly fluoride, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin. Cell lysates were centrifuged at 14,000 rpm for 15 minutes. Supernatant was removed, and proteins determined by the method of Bradford (Bradford, MM., Anal. Biochem .
  • phosphorylated proteins (15 ⁇ g) were separated by 7.5% SDS-polyacrylamide (Laemilli, supra) .
  • phosphorylated proteins were separated by 7.5% SDS-polyacrylamide (Laemilli, supra) .
  • Six hundred micrograms of labelled cell lysate protein was immunoprecipitated.
  • Immunoprecipitated proteins were separated by 7.5% SDS-polyacrylamide gels and transferred to Immobilin-P in 10 mM 3-[cyclohexylamino]-l- propanesulfonic acid (pH 11.0), 10% methanol at O.A for 2 hours at 4°C.
  • Phosphotyrosine was detected by western blotting with a mouse monoclonal anti-phosphotyrosine antibody (#05-321, UBI, NY) followed by alkaline phosphatase detection or with analogously prepared unlabelled cell extracts by 125 I-protein A.
  • IC S0 was measured using the MTT assay. Inhibition was measured using 50 ⁇ M compound concentration. Complete refers to 95-100% inhibition. Partial refers to 10-80% inhibition. No effect refers to ⁇ 10% inhibition.
  • Compounds inhibiting growth of K562 cells differed in their ability to inhibit the autokinase activity.
  • Compounds AI-10, AI-12, AII-20, AI- 13, AI-11, AI-14, AIII-36, AI-15, and AI-16 completely inhibit autokinase activity.
  • K562 cells to AI-16 for 24 hours inhibit DNA, protein and RNA synthesis completely at a concentration of 25 ⁇ M.
  • AIII-32 inhibits DNA and RNA synthesis by 80% at 25 ⁇ M, but protein synthesis is less affected (only "50% inhibition) even at 50 ⁇ M concentration of the drug.
  • Cells whose growth was arrested after 24 hours of exposure of drug were clearly viable as measured by trypan blue exclusion and by capacity to reduce MTT, which depends on intact mitochondrial electron transport (Mossman, supra) .
  • K562 cells maintained comparable levels of ATP with a similar ATP/ADP ratio compared to untreated or vehicle treated cells. Thus, inhibition of cell growth and macromolecular synthesis did not occur with gross alteration of cellular metabolic capacity.
  • AI-16 appears to manifest growth inhibition in conjunction with an early decrease in DNA synthesis.
  • AI- 16 inhibited [ 3 H]thymidine incorporation by 60% or 90% after 2 hours exposure to AI-16 to 20 ⁇ m or 40 ⁇ M 44 respectively;
  • [ 3 H]uridine and L-[ 3 H]leucine incorporation were maintained at >80% after 2 hour exposure to the same concentrations of AI-16, and even at 8 hour of exposure to drug, L-[ 3 H]leucine incorporation was largely unaffected while [ 3 H]uridine incorporation was 60% of control.
  • AI-16 and AIII-32 are both potential tyrosine kinase antagonists
  • Neither AI-16 (25 ⁇ M) nor AIII-32 (15 ⁇ M) after 24 hours of drug exposure inhibited [ 32 P0 4 ] incorporation into total proteins.
  • AI-16 (but not AIII-32) , there was a decrease in [ 32 P0 4 ] labelling of the p210 bcr-abl protein, and also a decrease in the mass of phosphotyrosine detected by anti-phosphotyrosine antibodies using alkaline phosphatase calorimetric or [ 125 I]-protein A detection technique.
  • AI-16 specifically decreased the phosphotyrosine content of immuno-precipitated p210 bcr-abl . Both of these changes occur as decrease in DNA synthesis is developing, but before significant decrease in RNA or protein synthesis.
  • p210 bcr-abl tyrosine kinase inhibition may affect a pathway leading to continued DNA synthesis, and by its inhibition AI-16 could then inhibit cell growth.
  • AI-16 could then inhibit cell growth.
  • AIII-32 does not ever inhibit p210 bcr-abl kinase activity even as it inhibits cell growth.
  • the compound was prepared according to Carboni et al . 2.2 g malononitril dimer and 0.9 ml N 2 H 4 in 20 ml water were heated 15 minutes at 100°C. Cooling and filtering gave 1.5 g; 61% yield, white solid, mp 187°C (Carboni et al., mp 187°C) . NMR acetone d 6 ⁇ J3.88(s).
  • AIII-34 was synthesized using a two step procedure.

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Abstract

La présente invention concerne des procédés et des composés inhibant les troubles de la prolifération cellulaire, caractérisés par l'activité anormale d'abl. Les composés préférés décrits inhibent les troubles de la prolifération cellulaire par ciblage de l'activité anormale d'abl. La cible préférée est l'activité anormale de l'autokinase abl.
PCT/US1994/005294 1993-05-14 1994-05-13 PROCEDES ET COMPOSES INHIBANT LES TROUBLES DE LA PROLIFERATION CELLULAIRE CARACTERISES PAR L'ACTIVITE ANORMALE D'$i(abl) WO1994026260A1 (fr)

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