WO2012012653A1 - Polythérapie basée sur des inhibiteurs de mdm2 et efgr - Google Patents

Polythérapie basée sur des inhibiteurs de mdm2 et efgr Download PDF

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WO2012012653A1
WO2012012653A1 PCT/US2011/044882 US2011044882W WO2012012653A1 WO 2012012653 A1 WO2012012653 A1 WO 2012012653A1 US 2011044882 W US2011044882 W US 2011044882W WO 2012012653 A1 WO2012012653 A1 WO 2012012653A1
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optionally containing
containing unsaturation
lower alkyl
branched
straight chain
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PCT/US2011/044882
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English (en)
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Joseph P. Errico
Benjamin Mugrage
Ignatius Turchi
Matthew Sills
Jane Ong
John Allocco
Pam Wines
Margarita Bastos
Original Assignee
Errico Joseph P
Benjamin Mugrage
Ignatius Turchi
Matthew Sills
Jane Ong
John Allocco
Pam Wines
Margarita Bastos
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Priority claimed from US12/986,146 external-priority patent/US8618302B2/en
Application filed by Errico Joseph P, Benjamin Mugrage, Ignatius Turchi, Matthew Sills, Jane Ong, John Allocco, Pam Wines, Margarita Bastos filed Critical Errico Joseph P
Priority to CA2805658A priority Critical patent/CA2805658C/fr
Priority to EP11810417.3A priority patent/EP2596366A4/fr
Publication of WO2012012653A1 publication Critical patent/WO2012012653A1/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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention generally relates to development of new chemical entities for use in the treatment of disease, and more particularly to methods of identifying lead molecules for use in quasi-rational drug design.
  • EGFR is involved in the same cellular signaling pathway as MDM2.
  • EGFR is a known cancer-associated molecule and EGFR inhibitors, such as Tarceva, provide targeted cancer treatment.
  • Significant numbers of cancer patients become resistant to treatment with approved EGFR inhibitors, such as Tarceva. There is currently no approach to overcome such resistance.
  • MDM2 inhibitor compounds useful for cancer treatment alone or in synergistic combination with an inhibitor of Epidermal Growth Factor Receptor (EGFR).
  • An MDM2 inhibitor used in combination with an EGFR inhibitor, such as Tarceva can provide treatment for patients with developed resistance.
  • Combinatorial treatment with an MDM2 inhibitor and an EGFR inhibitor, such as Tarceva can have synergistic anti-cancer effects and can overcome developed resistance.
  • One aspect provides a method of treating a proliferative disease, disorder, or condition.
  • the method of combinatorial treatment can include administering to an MDM2 inhibitor and an EGFR inhibitor to a subject.
  • the subject can be in need of such treatment.
  • the amount of the MDM2 inhibitor and the EGFR inhibitor can be an amount sufficient to produce a therapeutic effect.
  • the proliferative disease, disorder, or condition includes cancer.
  • administering the MDM2 inhibitor and the EGFR inhibitor results in a synergistic reduction in cell proliferation in a tumor of the subject or a synergistic increase in apoptosis in a tumor of the subject as compared to administration of either the MDM2 inhibitor or the EGFR inhibitor alone.
  • a pharmaceutical composition comprising an MDM2 inhibitor, an EGFR inhibitor, and a pharmaceutically acceptable carrier or excipient is administered to the subject.
  • a first pharmaceutical composition comprising an MDM2 inhibitor and a pharmaceutically acceptable ' carrier or excipient and a second pharmaceutical composition comprising an EGFR inhibitor and a
  • pharmaceutically acceptable carrier or excipient is administered to the subject.
  • the subject has one or more of (i) an inactivating P53 mutation or deletion in the subject; (ii) a defect in an upstream component of a p53 pathway; (iii) a defect in a downstream component of the p53 pathway; (iv) increased expression an MDM2 gene as compared to a control; (v) increased levels of MDM2 protein as compared to a control; or (vi) resistance to treatment with an EGFR inhibitor alone.
  • the method includes selecting or modifying a treatment on the basis of detecting in a subject one or more of (i) an inactivating P53 mutation or deletion in the subject; (ii) a defect in an upstream component of a p53 pathway; (iii) a defect in a downstream component of the p53 pathway; (iv) increased expression an MDM2 gene as compared to a control; (v) increased levels of MDM2 protein as compared to a control; or (vi) resistance to treatment with an EGFR inhibitor alone.
  • the EGFR inhibitor is selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zaiutumumab, matuzumab, potato carboxypeptidase inhibitor, gefitinib, lapatinib, and erlotinib, or a combination thereof.
  • the EGFR inhibitor is erlotinib (tradename Tarceva).
  • the MDM2 inhibitor (i) inhibits MDM2 activity; (ii) increases phosphorylated p53; (iii) re-activates p53; (iv) inhibits binding of p53 and MDM2; or a combination thereof. In some embodiments, the MDM2 inhibitor inhibits binding of p53 and MDM2.
  • the MDM2 inhibitor comprises a compound of Formula (2) as defined herein. In some embodiments, the MDM2 inhibitor comprises a compound of Formula (10) as defined herein. In some embodiments, the MDM2 inhibitor comprises a compound of Formula (11) as defined herein.
  • Another aspect provides a pharmaceutical composition including an MDM2 inhibitor; an EGFR inhibitor, and a pharmaceutically acceptable carrier or excipient.
  • FIG. 1 is a line and scatter plot showing inhibition of p53/MDM2 binding.
  • RLU is shown as a function of nm, with EC 50 determined for each of AD4-1505, AD4- 10963, AD4-11511 , AD4-10482, AD4-10942, AD4-10944, and AD4-10628.
  • FIG. 2 is a series of bar graphs showing percent inhibition of proliferation in A431 cells resulting from a combination of Tarceva and each of compounds AD4-10483, AD4-1505, AD4-10963, and AD4-10628-2.
  • CI an indication of synergy when less than 0.8, was determined for each combination.
  • the present disclosure is based, at least in part, on the discovery that combinatorial administration of a compound that inhibits binding of p53 and MDM2 along with an EGFR inhibitor (e.g., Tarceva) can provide synergistic anti-cancer effects and can overcomes developed resistance.
  • a compounds ability to inhibit binding of p53 and MDM2 can be an indicator of synergistic anti-cancer effect when in combination with an EGFR inhibitor, especially in a subject with or at risk for resistance to such EGFR inhibitor.
  • combinatorial administration of a compound that inhibits p53 binding to MDM2 and an EGFR inhibitor can provide synergistic anti-cancer effects and can overcome developed resistance.
  • administration of MDM2 inhibitors in combination with an EGFR inhibitor, such as Tarceva can result in synergistic tumor reduction and can overcome resistance to the EGFR inhibitor.
  • the EGFR and p53/MDM2 signaling are interconnected through the interaction of ERK and MDM2.
  • a compound with the ability to inhibit p53/MDM2 binding can: (a) inhibit cell proliferation in the A431 cell line that over-expresses the EGF receptor; (b) produce a synergistic effect with Tarceva, an EGFR kinase inhibitor, in a cell proliferation assay in the A431 cell line; (c) induce apoptosis in the A431 cell line as measured by an increase in caspase activity, to a similar extent as Tarceva; (d) produce a synergistic effect with Tarceva in an apoptosis assay in the A431 cell line; (e) increases apoptosis in the A549 cell line, as measured by increased DNA fragmentation; (f) produce a synergistic effect with Tarceva in an apoptosis assay in the A431 cell line; or (g) increase phosphorylated p53; or a combination thereof.
  • the present disclosure provides several novel classes of compounds that inhibit MDM2, re-activate p53, or inhibit the binding of p53 and MDM2.
  • a pharmacophore- based class of compounds that inhibits MDM2 and thereby re-activates p53 has been identified.
  • Such compounds can be used alone as anti-cancer therapeutic agents, or in synergistic combination with therapeutic EGFR inhibitors, as described further herein.
  • Also provided herein are in silico pharmacophore-based design, in vitro assays, and in vivo animal models to identify and optimize compounds that inhibit MDM2.
  • An MDM2 inhibitor can be a compound as disclosed in U.S. Application Serial No. 11/626,324, published as US Application Publication No. 2008/0015194; U.S. Nonprovisional Application Serial No. 12/986,146; International Application No.
  • An MDM2 inhibitor can be a compound as disclosed in Vassilev 2006 Trends in Molecular Medicine 13(1), 23-31.
  • an MDM2 inhibitor can be a nutlin (e.g., a cis-imidazole compound, such as nutlin-3a); a benzodiazepine as disclosed in
  • An MDM2 inhibitor can be a compound according to Formula 2 (a Type A AD4-1505-like compound) as follows:
  • R 1 of Formula (2) can represent:
  • a 2-Pyridyl ring of Formula (3) wherein R 23 is selected from the group consisting of hydrogen; fluoro; chloro; trifluoromethyl; methyl; ethyl; and methoxy; R 3 is selected from the group consisting of hydrogen; fluoro; chloro; methyl; ethyl; methoxy; a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; aryl comprising a phenyl or heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms; and alkoxy -OR 10 where R 10 is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R 24 is selected from the group consisting of hydrogen
  • an unsubstituted heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms or a heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms which has one or more optional substitution with the substituent defined as one or more of the following groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation, cycloalkyi defined as C-1 to C-6 optionally containing unsaturation, Aryl including phenyl or heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms, Alkoxy (-OR 10 where R 0 is defined as a lower alkyl group or cycloalkyi group in the above definition).
  • R 1 is a 2-pyridyl ring of Formula (3) having combinations of substituted halogens and alkyl groups
  • the resulting compound can exhibit increased antiproliferative activity.
  • R is a 2-pyridyl ring of Formula (3)
  • the following substitutions can provide increased antiproliferative activity: R 4 is hydrogen, R 24 is fluoro, R 3 is hydrogen, and R 23 is fluoro; R 4 is methyl, R 24 is chloro, R 3 is hydrogen, and R 23 is fluoro; R 4 is hydrogen, R 24 is chloro, R 3 is ethyl, and R 23 is fluoro; R 4 is hydrogen, R 24 is fluoro, R 3 is methyl, and R 23 is fluoro; R 4 is hydrogen, R 24 is chloro, R 3 is hydrogen, and R 23 is ethyl; R 4 is methyl, R 24 is chloro, R 3 is hydrogen, and R 23 is chloro; R 4 is hydrogen, R 24 is chloro, R 3 is hydrogen, and R 23 is chlor
  • R 1 is a 2-pyridyl ring of Formula (3) and R 24 is chloro and there is additionally a chloro or methyl at one or both of R 3 or R 23
  • the resulting compound can exhibit increased apoptosis.
  • R 1 is a 2-pyridyl ring of Formula (3)
  • substitutions can provide increased apoptosis: R 24 is chloro, R 3 is hydrogen, and R 23 is methyl; R 24 is chloro, R 3 is methyl, and R 23 is fluoro; R 24 is chloro, R 3 is chloro, and R 23 is hydrogen; and R 24 is chloro, R 3 is hydrogen, and R 23 is chloro.
  • R 1 of Formula (2) is a 2-Pyridyl ring of Formula (3)
  • the group at R 24 of the aminopyridine can block metabolism in cultured hepatocytes.
  • R 1 of Formula (2) can represent: an unsubstituted 2- (1 ,3-thiazoyl) ring (see Formula (6)) or a 2-(1 ,3-thiazoyl) ring with groups at the 4- or imposition of the thiazole ring, for example a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring (see Formula (6)) or a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring (see Formula (6)) or a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring (see Formula (6)) or a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring with groups at the 4- or imposition of the thiazole ring, for example a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring (see Formula (6)) or a 2-(4,5-Dimethyl-1 ,3-thiazoyl ring (see Formula (6)) or a 2-(4,5-Dimethyl-1
  • R 2 of Formula (2) can represent:
  • Trifluoromethoxy Difluoromethoxy, 3, 4-methylenedioxy, 2, 3-methylenedioxy, Nitro or Halogen (F, CI, Br, I);
  • R 2 is a phenyl ring substituted at the 2- and 4- positions
  • the resulting compound can exhibit increased stability.
  • R 2 is 4-trifluoromethylphenyl; 2-fluoro,4-trifluoromethylphenyl; or 2,4-dichlorophenyl
  • the resulting compound can exhibit increased stability.
  • R 2 is a phenyl ring substituted with a combination of halogens and trifluoromethyl groups
  • the resulting compound can exhibit increased antiproliferative activity.
  • R 2 is 4-chlorophenyl; 2-fluoro,4- trifluoromethylphenyl; 3-fluoro,4-chlorophenyl; 2-fluoro,4-chlorophenyl; 2,3-dichlorophenyl; 2,3,5-trichlorophenyl; 2,4-dichlorophenyl; 3,4-dichlorophenyl; or 3,5-dichlorophenyl
  • the resulting compound can exhibit increased antiproliferative activity.
  • the compound(s) of Formula (2) are according to R1 and R2 as provided in the following TABLE 1 , TABLE 2, TABLE 3, and TABLE 4.
  • the compound(s) of Formula (2) excludes compound AD4-1505, Formula (1).
  • An MDM2 inhibitor can be a compound according to Formula 10 (a Type B AD4-1505-like compound) as follows:
  • Trifluoromethoxy Difluoromethoxy, 3, 4-methylenedioxy, 2, 3-methylenedioxy, Nitro or Halogen (F, CI, Br, I);
  • the compound(s) are the enantiomeric isomers of
  • the compound of Formula (10) is AD4-10960.
  • X 1 and R 2 of Formula (11) are defined as above for structural sub-class Type A, Formula (2).
  • R 22 of Formula (11) can represent a lower alkyl group with one to 6 carbons (C-1 to C-6), straight chain, branched, optionally containing unsaturation, or substitution at the C-1 or C-2 carbons with one or more of the following substituents: an unsubstituted Phenyl ring or a Phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation, cycloalkyl defined as C-1 to C-6 optionally containing unsaturation or one oxygen or nitrogen atom, Heteroaryl containing from 1 to 4 N, O, or S atoms, hydroxyl (-OH), Alkoxy (-OR 10 where R 10 is defined as a lower alkyl group or cycloalkyl group as in the above definition), Dialkylamino (-NR13R14, where R 13 and R14 are independently selected from
  • a cycloalkyl is defined as five or six aliphatic ring (C-1 to C-6) optionally containing unsaturation or one oxygen or nitrogen atom.
  • the compound(s) are the enantiomeric isomers of
  • the compound of Formula (11) is AD4-10535.
  • the compound(s) of Formula (11) excludes compound AD4-1505, Formula (1).
  • An MDM2 inhibitor compound described herein can have structural features associated with one or more desired functions, such as stability, antiproliferative activity, or apoptotic activity.
  • groups at the 5-position of the aminopyridine of compounds described herein provide analogs having increased stability (e.g., more stable toward liver microsome incubation).
  • a compound substituted at the 5-position of the aminopyridine can exhibit increased stability.
  • AD4- 13053 and AD4-13041 both having a chlorine atom at the 5-position of the aminopyridine show increased stability over AD4-10628.
  • a compound substituted with a chlorine atom at the 5-position of the aminopyridine can exhibit increased stability increases stability.
  • aminopyridine ring substitutions provide increased antiproliferative activity: 3,5-diF; 3-F.5- CL,6-Me; 3-F,5-CI,6-Me; 3-F,5-CI,4-Et; and 3,5-diF,4-Me.
  • a compound with the following aminopyridine ring substitutions provide further increased antiproliferative activity: 3-Et,5-CI; 3,5-diCI,6-Me; 3-F,5-CI,4-Me; and 5-CF3.
  • a compound with the following aminopyridine ring substitutions provide even further increased antiproliferative activity: 3-Me,5-CI; 3,5-diCI; 4-Me,5-CI; and 4,5- diCI.
  • a chloro group at the 5-position of the aminopyridine ring and additional chloro or methyl groups at the 3- or 4-positions on the aminopyridine ring of compounds described herein provide compounds with increased apoptotic activity.
  • a compound with the following aminopyridine ring substitutions provide increased apoptotic activity: 3-Me,5-CI; 3-F,5-CI,4-Me; 4,5-diCI; and 3,5-diCI.
  • groups at the 2- and 4-position of the benzene ring of compounds described herein provide analogs having increased stability (e.g., more stable toward liver microsome incubation).
  • a compound substituted at the 2- and 4-position of the benzene ring of compounds can exhibit increased stability.
  • AD4-13041 , AD4-13042, AD4-13165, and AD4-13206 show increased stability.
  • a compound substituted with a halogen atom at the 2- or 4-position of the benzene ring of the aminopyridine can exhibit increased stability increases stability.
  • a compound substituted with a chlorine atom at the 2- and 4-position of the benzene ring of the aminopyridine can exhibit increased stability increases stability.
  • a compound substituted with a fluorine atom at the 2- and 4-position of the benzene ring of the aminopyridine can exhibit increased stability increases stability.
  • a compound substituted with a trifluoromethyl at the 4-position or a fluorine atom at the 2-position and a trifluoromethyl at the 4-position of the benzene ring of the aminopyridine can exhibit increased stability increases stability.
  • a compound with the following benzene ring substitutions provide increased antiproliferative activity: 4-CI; 2-F,4-CF 3 ; and 3-F.4- Cl.
  • a compound with the following benzene ring substitutions provide further increased antiproliferative activity: 2-F.4-CI; 2,3-diCI; and 2,3,5-triCI.
  • a compound with the following benzene ring substitutions provide even further increased antiproliferative activity: 2,4-diCI; 3,4-diCI; and 3,5-diCI.
  • a chloro group at the 4-position of the benzene ring and additional chloro or fluoro groups at the 2- or 3-positions on the benzene ring of compounds described herein provide compounds with increased apoptotic activity.
  • a compound with the following benzene ring substitutions provide increased apoptotic activity: 2,4-diCI (see e.g., AD4-13130, AD4-13178); and 2-CI.4-F (see e.g., AD4-13185).
  • an AD4-1505-like compound can be synthesized by reacting an amino pyridine intermediate compound, an aldehyde intermediate compound and a hydroxyquinoline, as further described in U.S. Application Serial No. 12/986,146 and WO 2011/085126.
  • the reaction can include combining the amino pyridine intermediate compound, the aldehyde intermediate compound and the hydroxyquinoline in ethanol (e.g., absolute ethanol).
  • the MDM2 inhibitor is selected from one or more of the following compounds:
  • alkyl denotes a Ci-i 2 alkyl group, suitably a C1-6 alkyl group, e.g. d-4 alkyl group.
  • Alkyl groups may be straight chain or branched. Suitable alkyl groups include, for example, methyl, ethyl, propyl (e.g. n-propyl and isopropyl), butyl (e.g. n-butyl, iso-butyl, sec-butyl and tert-butyl), pentyl (e.g. n-pentyl), hexyl (e.g. n-hexyl), heptyl (e.g.
  • alk for example in the expressions "alkoxy”, “haloalkyl” and “thioalkyl” should be interpreted in accordance with the definition of "alkyl”.
  • alkoxy groups include methoxy, ethoxy, propoxy (e.g. n-propoxy), butoxy (e.g. n-butoxy), pentoxy (e.g. n-pentoxy), hexoxy (e.g. n-hexoxy), heptoxy (e.g. n-heptoxy) and octoxy (e.g. n-octoxy).
  • cycloalkyl denotes a C 3- io cycloalkyl group (i.e., 3 to 10 ring carbon atoms), more suitably a C3-8 cycloalkyl group, for example, a cycloalkyl group.
  • exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • a preferred number of ring carbon atoms is three to six.
  • aryl denotes a Ce-12 aryl group, suitably a Ce-io aryl group, more suitably a Ce-e aryl group.
  • Aryl groups will contain at least one aromatic ring (e.g. one, two or three rings).
  • An example of a typical aryl group with one aromatic ring is phenyl.
  • An example of a typical aryl group with two aromatic rings is naphthyl.
  • heteroaryl denotes an aryl residue, wherein one or more (e.g., 1 , 2, 3, or 4, suitably 1 , 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O, or else a 5-membered aromatic ring containing one or more (e.g., 1 , 2, 3, or 4, suitably 1 , 2 or 3) ring atoms selected from N, S and O.
  • Exemplary monocyclic heteroaryl groups having one heteroatom include: five membered rings (e.g., pyrrole, furan, thiophene); and six membered rings (e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl).
  • five membered rings e.g., pyrrole, furan, thiophene
  • six membered rings e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl.
  • Exemplary monocyclic heteroaryl groups having two heteroatoms include: five membered rings (e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-1-yl, imidazol-2-yl imidazol-4-yl); six membered rings (e.g., pyridazine, pyrimidine, pyrazine).
  • Exemplary monocyclic heteroaryl groups having three heteroatoms include: 1 ,2,3-triazole and 1 ,2,4-triazole.
  • Exemplary monocyclic heteroaryl groups having four heteroatoms include tetrazole.
  • bicyclic heteroaryl groups include: indole (e.g., indol-6-yl), benzofuran, benzthiophene, quinoline, isoquinoline, indazole, benzimidazole, benzthiazole, quinazoline and purine.
  • a saturated group is generally understood as having no double or triple bonds.
  • each carbon atom is attached to two hydrogen atoms, except those at the ends of the chain, which bear three hydrogen atoms.
  • an unsaturated hydrocarbon is generally understood as a carbon structure containing one or more double or triple bonds.
  • halogen or halo includes fluorine (F), chlorine (CI) bromine (Br) or iodine (I).
  • amino refers to the group -NH 2 .
  • the pharmaceutically acceptable salt can take a form in which a basic side chain is protonated with an inorganic or organic acid.
  • Representative organic or inorganic acids include hydrochloric, hydrobromic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic, 2- naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, saccharide or trifluoroacetic acid.
  • an acidic side chain forms a salt with a metal ion (e.g., sodium, potassium ions and the like) or other positive ion such as ammonium.
  • a metal ion e.g., sodium, potassium ions and the like
  • other positive ion such as ammonium.
  • Some of the crystalline forms of the compounds may exist in more than one polymorphic form and as such all forms are intended to be included in the present disclosure.
  • some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this disclosure.
  • the compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
  • the present disclosure further includes within its scope prodrugs of the compounds described herein.
  • prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound.
  • the term "administering" shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed compounds, but which converts to the above specified compound in vivo after administration to the subject.
  • composition is intended to encompass a product comprising a claimed compound(s) in a therapeutically effective amount, as well as any product which results, directly or indirectly, from combinations of the claimed compounds.
  • an EGFR inhibitor in combination with an MDM2 inhibitors can result in synergistic inhibition proliferation or increased death of cancer cells.
  • An EGFR inhibitor can be an anti-proliferative or pro-apoptotic compound.
  • An EGFR inhibitor can be selected so as to show a synergistic anti-proliferative or pro- apoptotic effect when co-administered with an MDM2 inhibitor (e.g., an inhibitor of p53 and MDM2 binding).
  • MDM2 inhibitor e.g., an inhibitor of p53 and MDM2 binding.
  • Quantitative methods e.g., protein phosphorylation detection
  • An EGFR inhibitor can be a monoclonal antibody, such cetuximab
  • Erbitux is a humanized monoclonal antibody that binds to an extracellular epitope on EGFR. Erbitux blocks activation of the receptor by preventing both ligand binding and receptor dimerization.
  • An EGFR inhibitor can be a protein, such as potato carboxypeptidase inhibitor (PCI).
  • An EGFR inhibitor can be a small molecule inhibitor, such as gefitinib (tradename Iressa, N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4- ylpropoxy)quinazolin-4-amine); lapatinib (tradename Tykerb, N-[3-chloro-4-[(3- fluorophenyl)methoxy]phenyl]-6-[5-[(2-ethylsulfonylethylamino)methyl]-2-furyl]quinazolin-4- amine); erlotinib (tradename Tarceva, N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine). Tykerb, Iressa, and Tarceva are kinase inhibitors that block EGFR tyrosine kinase activity.
  • gefitinib trade
  • an EGFR inhibitor can be an selected from lapatinib (tradename Tykerb), gefitinib (tradename Iressa), erlotinib (tradename Tarceva), or cetuximab (tradename Erbitux), or a combination thereof.
  • MDM2 inhibitors in combination with EGFR inhibitor Tarceva can result in synergistic inhibition proliferation or increased death of cancer cells.
  • Such a combinatorial therapeutic approach can overcome resistance to conventional EGFR inhibitors, which have been reported to occur in a significant number of patients.
  • An MDM2 inhibitor optionally used in combination with an EGFR inhibitor, such as Tarceva, can provide treatment for patients with developed resistance. Such therapy can treat a variety of cancers, including NSCLC, colon, pancreatic cancers and head and neck tumors, in patients where an EGFR inhibitor alone would not be effective.
  • Combinatorial treatment with an MDM2 inhibitor and an EGFR inhibitor can result in a synergistic anti-cancer effect or can overcome developed resistance.
  • Such combinatorial therapy is especially useful in a subject having an inactivating P53 mutation, as described further herein. Synergistic effects or overcoming developed resistance can allow lower doses, significantly reducing therapy cost in a substantial patient population.
  • compositions of the invention include pharmaceutical formulations of the various compounds described herein.
  • compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety.
  • Such formulations will contain a therapeutically effective amount of a biologically active agent(s) described herein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal.
  • the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
  • Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
  • Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • inducers e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below.
  • therapies described herein one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
  • Another aspect provided herein is a process of treating a proliferative disease, disorder, or condition with an MDM2 inhibitor and an EGFR inhibitor.
  • a proliferative disease, disorder, or condition is associated with EGFR, MDM2, or p53.
  • the therapeutic method can include administration of a therapeutically effective amount of an MDM2 inhibitor and an EGFR inhibitor, either as individual compositions or a jointly formulated composition.
  • An MDM2 inhibitor can be used with, or formulated with, known therapeutic compounds.
  • Combination therapy is understood as a therapeutic regimen comprising, e.g., an MDM2 inhibitor described herein and a second agent, an MDM2 inhibitor and a second agent can be formulated for separate administration or may be formulated for administration together.
  • An MDM2 inhibitor can be combined with another anti-proliferative compound, such as the EGFR kinase inhibitors, Tykerb, Iressa, and Tarceva, or Erbitux, a humanized monoclonal antibody to the EGF receptor, to produce a greater therapeutic effect than either agent alone.
  • another anti-proliferative compound such as the EGFR kinase inhibitors, Tykerb, Iressa, and Tarceva, or Erbitux, a humanized monoclonal antibody to the EGF receptor
  • An MDM2 inhibitor can be used or formulated with an EGFR inhibitor approved for treatment of an EGFR-related condition or disorder.
  • an MDM2 inhibitor can be used with or formulated with one or more of Tykerb, Iressa, Tarceva, or Erbitux.
  • Tykerb, Iressa, and Tarceva are kinase inhibitors that block EGFR tyrosine kinase activity.
  • Erbitux is a humanized monoclonal antibody that binds to an extracellular epitope on EGFR. Erbitux blocks activation of the receptor by preventing both ligand binding and receptor dimerization.
  • an MDM2 inhibitor and known EGFR inhibitors, such as those described above can act in a complementary or synergistic fashion.
  • Methods described herein are generally performed on a subject in need thereof.
  • a subject in need of the therapeutic methods described herein can be diagnosed with a proliferative disease, disorder, or condition, or at risk thereof.
  • a subject in need of the therapeutic methods described herein can be diagnosed with a disease, disorder, or condition associated with EGFR, MDM2, or p53, or at risk thereof.
  • a subject in need of the therapeutic methods described herein can have, be diagnosed with, thought to have, or suspected of having an inactivating mutation or deletion in p53 (e.g., deletion or mutation of TP53 gene) (see Vassilev 2006 Trends in Molecular Medicine 13(1), 23-31). Determination of p53 status in tumor cells can be according to, for example, a DNA microarray-based p53 GeneChip (see Ahrendt et al. 1999 Proc Natl Acad Sci USA 96, 7382-7387).
  • a subject in need of the therapeutic methods described herein can have, be diagnosed with, thought to have, or suspected of having a defect in an upstream or a downstream component of the p53 pathway (see Vassilev 2006 Trends in Molecular Medicine 13(1), 23-31).
  • a subject in need of the therapeutic methods described herein can have, be diagnosed with, thought to have, or suspected of having aberrant MDM2 expression (see Vassilev 2006 Trends in Molecular Medicine 13(1), 23-31).
  • a subject in need of the therapeutic methods described herein can have, be diagnosed with, thought to have, or suspected of having overexpression of the MDM2 gene or overexpression of MDM2 protein without gene amplification, which may suppress p53 function (see Vassilev 2006 Trends in Molecular Medicine 13(1), 23-31).
  • a subject in need of the therapeutic methods described herein can have, be diagnosed with, thought to have, suspected of having, or be at risk for a resistance to a conventional therapeutic treatment, such as treatment with an EGFR inhibitor.
  • a determination of the need for treatment can be assessed by a history and physical exam consistent with the disease, disorder, or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art.
  • the subject can be an animal subject, preferably a mammal, more preferably horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, and most preferably a human.
  • proliferative diseases or conditions treatable with compositions described herein include, but are not limited to, cancer; blood vessel proliferative disorders; fibrotic disorders; mesangial cell proliferative disorders; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by virus infections, such as papilloma virus infection.
  • Various compounds described herein can be effective for inhibiting EGFR, and thus, effective against diseases or conditions associated with EGFR, including, but not limited to, proliferative diseases.
  • the proliferative disease treated by a compound described herein is a condition caused by excessive growth of cancer or non-cancer cells that express a member of the EGFR family of receptors.
  • the excess cells generated by a proliferative disease can express EGFR at normal levels or can overexpress EGFR.
  • Particularly suitable diseases or conditions associated with EGFR can be those stimulated by a ligand of EGFR or mutations of such ligands.
  • Examples of such ligands that stimulate EGFR include, but are not limited to, EGF, TGF- alpha, heparin-binding growth factor (HBGF), ⁇ -cellulin, and Cripto-1.
  • Examples of proliferative disease associated with EGFR include, but are not limited to, cancer; blood vessel proliferative disorders; fibrotic disorders; mesangial cell proliferative disorders; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by virus infections, such as papilloma virus infection.
  • Cancer refers generally to any malignant neoplasm or spontaneous growth or proliferation of cells.
  • a subject having "cancer" may have a leukemia, lymphoma, or other malignancy of blood cells.
  • the subject methods are used to treat a solid tumor.
  • Exemplary solid tumors include but are not limited to non small cell lung cancer (NSCLC), testicular cancer, lung cancer, ovarian cancer, uterine cancer, cervical cancer, pancreatic cancer, colorectal cancer (CRC), breast cancer, as well as prostate, gastric, skin, stomach, esophageal, and bladder cancer.
  • NSCLC non small cell lung cancer
  • testicular cancer lung cancer
  • lung cancer ovarian cancer
  • uterine cancer cervical cancer
  • pancreatic cancer colorectal cancer
  • breast cancer as well as prostate, gastric, skin, stomach, esophageal, and bladder cancer.
  • Treatment of cancer or treating a subject having cancer can include inhibition of replication of cancer cells, inhibition of spread of cancer, reduction in tumor size, lessening or reducing the number of cancerous cells in the body of a subject, or amelioration or alleviation of symptoms of cancer.
  • a treatment can be considered therapeutic if there is a decrease in mortality or morbidity, and can be performed prophylactically, or therapeutically.
  • Methods described herein can be used to treat (e.g., reduce tumor size, decrease the vascularization, and/or increase the permeability of) an established tumor.
  • An established tumor is generally understood as a solid tumor of sufficient size such that nutrients, e.g., oxygen, can no longer permeate to the center of the tumor from the subject's vasculature by osmosis and therefore the tumor requires its own vascular supply to receive nutrients.
  • Methods described herein can be used to treat a solid tumor that is not quiescent and is actively undergoing exponential growth.
  • a therapeutic protocol can be modified according to permeability of a solid tumor.
  • Permeability of a solid tumor generally refers to the permeability of a solid tumor to a therapeutic.
  • a solid tumor may be said to be permeable to a therapeutic if the therapeutic is able to reach cells at the center of the tumor.
  • An agent that increases the permeability of a tumor may for example, normalize, e.g., maintain, the vasculature of a solid tumor.
  • Tumor vascularization or tumor permeability can be determined by a variety of methods known in the art, such as, e.g. by immunohistochemical analysis of biopsy specimens, or by imaging techniques, such as sonography of the tumor, computed tomography (CT) or magnetic resonance imaging (MRI) scans.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • EGFR Tuzi et al., 1991 , Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719
  • HER2/neu Slamon et al., 1989, Science 244:707-712
  • PDGF-R Kiumabe et al., 1992, Oncogene 7:627-633
  • Overexpression of the receptor and autocrine loops have been demonstrated in most common and severe cancers (see e.g., Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci.
  • Overexpression of EGFR is known to be associated with cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney, (see e.g., Atalay et al., 2003, Ann. Oncology 14:1346-1363; Herbst and Shin, 2002, Cancer 94:1593-161 1 ; Modjtahedi et al., 1996, Br. J.
  • Cancer 73:228-235 Overexpression of EGFR can be correlated or associated with poor prognosis of the patients (see e.g., Herbst and Shin, 2002, Cancer 94.1593-1611; Modjtahedi et al., 1996, Br. J. Cancer 73:228-235).
  • HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.
  • An inhibitor compound described herein can be used therapeutically either as exogenous materials or as endogenous materials.
  • Exogenous agents are those produced or manufactured outside of the body and administered to the body.
  • Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery to within or to other organs in the body.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal,
  • a therapeutically effective amount of and MDM2 inhibitor and an EGFR inhibitor can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient.
  • the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to provide a sufficient therapeutic outcome, as described further herein.
  • an effective amount of a compound described herein is generally that which can exhibit an anti-proliferative effect to an extent such as to ameliorate the treated condition.
  • an effective amount of a compound described herein may inhibit MDM2 or EGFR to an extent such as to ameliorate the treated condition.
  • an effective amount is that amount of therapy (or combination therapy) that is sufficient to affect a desired result on a cancerous cell or tumor, including, but not limited to, for example, reducing tumor size, reducing tumor volume, decreasing vascularization of a solid tumor, or increasing the permeability of a solid tumor to an agent, either in vitro or in vivo.
  • an effective amount of therapy is the amount that results in a percent tumor inhibition of more than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%.
  • an effective amount of therapy (or combination therapy) is sufficient to achieve a desired clinical result, including but not limited to, for example, ameliorating disease, stabilizing a subject, preventing or delaying the development of, or progression of cancer in a subject.
  • An effective amount of therapy (or combination therapy) can be determined based on one administration or repeated administration. Methods of detection and measurement of the indicators above are known to those of skill in the art. Such methods include, but are not limited to measuring reduction in tumor burden, reduction of tumor size, reduction of tumor volume, reduction in proliferation of secondary tumors, decreased solid tumor
  • tumor burden can be determined.
  • Tumor burden also referred to as tumor load, generally refers to a total amount of tumor material distributed throughout the body of a subject.
  • Tumor burden can refer to a total number of cancer cells or a total size of tumor(s), throughout the body, including lymph nodes and bone barrow.
  • Tumor burden can be determined by a variety of methods known in the art, such as, for example, by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) scans.
  • Tumor size can be determined, for example, by determining tumor weight or tumor volume.
  • compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
  • Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5 o, (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio
  • LD50/ED50 where large therapeutic indices are preferred.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
  • the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
  • an MDM2 inhibitor or an EGFR inhibitor as described herein can occur as a single event, a periodic event, or over a time course of treatment.
  • agents can be administered daily, weekly, bi-weekly, or monthly.
  • agents can be administered in multiple treatment sessions, such as 2 weeks on, 2 weeks off, and then repeated twice; or every 3rd day for 3 weeks.
  • An MDM2 inhibitor and an EGFR inhibitor can have the same or different administration protocols.
  • One of ordinary skill will understand these regimes to be exemplary and could design other suitable periodic regimes.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease, disorder, or condition associated with a target biomolecule for which the compound is specific.
  • Simultaneous administration can occur through administration of one composition containing three or more of an MDM2 inhibitor, an EGFR inhibitor, an antibiotic, an antiinflammatory, or another agent.
  • a combination of an MDM2 inhibitor and an EGFR inhibitor can be administered sequentially with an antibiotic, an antiinflammatory, or another agent.
  • a combination of an MDM2 inhibitor and an EGFR inhibitor can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent.
  • Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art.
  • the agents and composition can be used therapeutically either as exogenous materials or as endogenous materials.
  • Exogenous agents are those produced or manufactured outside of the body and administered to the body.
  • Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • Agents can be encapsulated and administered in a variety of carrier delivery systems.
  • carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and
  • Carrier- based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.
  • Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
  • Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxy I or carboxyl group, and usually at least two of the functional chemical groups.
  • the candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • a candidate molecule can be a compound in a library database of compounds.
  • One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182).
  • Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds.
  • a lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943- 3948).
  • a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249).
  • initial screening is performed with lead-like compounds.
  • a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.
  • kits can include the compositions of the present invention and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein, for example, treatment methodologies or screening methodologies.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to one or more compounds described herein, vectors, diagnostic reagents, assay reagents, and/or combinations thereof.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, or sterile saline, each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10:
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • the following example describes an assay that measured the ability of compounds to inhibit the binding of p53 to MDM2 using the AlphaScreen assay technology (PerkinElmer).
  • the following protocol is an adaptation of the method described by H.R. Lawrence et al. (Bioorg. Med. Chem. Lett. 19 (2009) 3756-3759).
  • Recombinant, truncated, human, N-terminal GST-MDM2 (aa 1-150) was obtained from GeneScript. Wild-type, full length human N-terminal 6-his p53 was purchased from SignalChem.
  • Tissue Culture On day 1 , seed A549 cells (10,000 cells/well at 200 ⁇ ) in tissue culture media (RPMI-1640 with 1% sodium pyruvate, 1% Pen-Strep, 1% L- Glutamine and 10% FBS) were placed in 96-well, tissue culture-treated plates. The plates were allowed to incubate overnight @ 37°C, 5% C0 2 . On day 2, the media was removed from the plates and 160 ⁇ media containing 5% FBS was added. 40 ⁇ of media- containing test compound in 100% DMSO (prepared at 5x the dosing concentration) was added to the existing media resulting in a final DMSO concentration of 0.5%. Cells were incubated in the presence of a compound for 24 hrs @ 37°C, 5% C0 2 .
  • tissue culture media RPMI-1640 with 1% sodium pyruvate, 1% Pen-Strep, 1% L- Glutamine and 10% FBS
  • DNA Fragmentation Assay After 24 hrs, the abovementioned plates were centrifuged at 200 x g for 10 min. The media was removed by gently inverting the plate and disposing of the media onto a paper towel. The plates were tapped gently to remove excess media. 200 ⁇ lysis buffer was added to each well, and mixed by shaking at 300 rpm. The plates were incubated at room temperature for 30 min. The plates were centrifuged at 200 x g for 10 min and 20 ⁇ of lysis supernatant was removed for use in ELISA analysis to detect cell death.
  • ELISA Analysis 20 ⁇ of the cell lysis supernatant was placed into streptavid in-coated plates with 20 ⁇ positive control and 20 ⁇ incubation buffer negative control. Add 80 ⁇ Immunoreagent (for DNA fragment detection) to each well. The wells were covered with foil adhesive and shaken at 300 rpm for 2 h at room temperature. The supernatant solution was removed and the wells were washed 3 times with 300 ⁇ incubation buffer. 100 ⁇ of ABTS detection substrate was added to each well. The wells were incubated on a plate shaker at 250 rpm for approximately 10-20 min. 100 ⁇ ABTS of stop buffer was then added. The absorbance was read at 400 and 492 nm on PolarStar plate reader. The absorbance measures the colored product spectrophotometrically, correlating with apoptosis. EXAMPLE 3: PROTEOME PROFILE, MEASUREMENT OF APOPTOSIS
  • This example describes an assay that measured the relative expression levels of 35 apoptosis-related proteins in a single sample of whole cell extract. Methods are according to Examples 1-2 unless otherwise specified.
  • the protocol and reagents were purchased from R&D systems (Human apoptosis array kit; cat. # ARY009).
  • the kit consisted of an antibody array spotted on nitrocellulose membranes with each specific antibody printed in duplicate.
  • the detection antibodies were biotinylated so they could be used with a streptavidin-HRP conjugate designed for chemiluminescent imaging.
  • the protocol was modified to utilize the LiCor infrared imaging technology by substituting an infrared 680 nm-tagged streptavidin.
  • A549 NSCLC cells were seeded in 6 well tissue culture plates in RPMI-1640 supplemented with 5% FBS at a density of 1x10 6 cells / well, 2 ml / well and allowed to incubate overnight at 37 °C, 5 % C0 2 , and 85% relative humidity.
  • This example describes an assay that measures the relative expression of human p53 and phospho-p53 (S15) in intact, formaldehyde-fixed, A549 NSCLC cells in a 96 well format. Methods are according to Examples 1-3 unless otherwise specified.
  • A549 NSCLC cells were seeded in black 96 well clear bottom tissue culture plates at a density of 25,000 cells/well, 200 ⁇ well, in RPMI-1640 supplemented with 5 % FBS and allowed to incubate overnight at 37 °C, 5 % C0 2 , and 85 % relative humidity.
  • the blocking buffer was removed, then incubated with 50 ⁇ /well of a 1/250 dilution of either: (1) polyclonal Goat anti-human p53 or; (2) a 1/40 dilution of polyclonal Rbt anti human phospho (S15) p53 for 16 hrs at room temperature with shaking.
  • the wells were washed 5x for 5 min each with 0.1 % Tween-20 in PBS with shaking.
  • the cells were then incubated with 50 ⁇ /well of either: (1) a 1/5000 dilution of Donkey anti Goat IgG IR 800 nm or; (2) Goat anti-Rbt IR 800 nm containing a 1/600 dilution of DRAQ7 and 1/1000 dilution of Sapphire 700 for 2-3 hrs at room temperature with shaking in the dark.
  • This example describes an assay that measures the ability of compounds to inhibit the proliferation of cultured cells. This assay can also be used to assess whether combining two or more compounds produces additive, synergistic, or antagonistic effects on cell growth. Methods are according to Examples 1-4 unless otherwise specified.
  • This example describes an assay that measures the ability of compounds to stimulate p53-induced reporter gene activity. In order for p53 activity to be observed, the interaction of p53 with MDM2 must be inhibited. Methods are according to Examples 1-5 unless otherwise specified.
  • HEK293 cells were transiently transfected with the inducible p53-responsive firefly luciferase reporter and constitutively-expressing Renilla construct. After 16 hrs of transfection at 37 °C, 5% CO2, the transfection media was removed and 200 ⁇ well of assay media was added. The cells were then incubated for 8 hrs at 37°C, 5% C0 2 .
  • the lysis buffer supernatant was transferred to a white/opaque CoStar 96- well plate and 100 ⁇ of firefly luceriferase assay substrate was added. The luminescence in each well was determined using a Polarstar plate reader.
  • This example describes an assay that measures the ability of compounds to increase the activity of caspase 3/7 activity. Methods are according to Examples 1-6 unless otherwise specified.
  • the cells were treated with the compound(s) diluted in 25 ⁇ N ⁇ of DMEM, with 1mg/ml BSA. (no FBS). The plates were incubated for 5.5 hr at 37°C. The plates were removed from the incubator and allowed to equilibrate to room temperature for 30 min. 25 ⁇ of Capsase Glo reagent was added to each well. The plates were covered and allowed to incubate at room temperature for 60 min. The luminescence in each well was determined using a plate reader.
  • EXAMPLE 8 AD4 COMPOUNDS INHIBIT CELL PROLIFERATION AND DEMONSTRATE SYNERGISTIC EFFECT WITH TARCEVA
  • the following example provides preliminary assessment of the ability of selected AD4 compounds to inhibit EGF-mediated cell proliferation using the A431 cell proliferation assay and to demonstrated synergistic effects with Tarceva. Methods are according to Examples 1-7 unless otherwise specified. Compound structures are as disclosed in U.S. Application Serial No. 12/986,146 and WO 2011/085126. [0227]
  • the A431 cell line over-expresses the EGF receptor, and was utilized to assess the ability of AD4 compounds to inhibit EGF-mediated cell proliferation. A number of compounds inhibit cell proliferation in the A431 cell line, with IC50 values ranging from 1.0-3.7 ⁇ (see e.g., TABLE 6A). Tarceva inhibits cell proliferation with an IC50 value of 0.8 ⁇ (see e.g., TABLE 6A).
  • AD4 compounds inhibit cell proliferation by interfering with a pathway that can potentiate the effects of the EGF inhibitor.
  • AD4 compounds demonstrated synergistic effects with Tarceva (see e.g., TABLE 6B).
  • CI values ⁇ 0.8 indicate a synergistic effect.
  • all of the tested AD4 compounds produced synergy when combined with Tarceva.
  • AD4-1505 and AD4-10963 produced the greatest effects.
  • AD4 compounds in combination with Tarceva were shown to produce a synergistic effect.
  • a number of compounds in the AD4 series were evaluated for their ability to produce synergistic effects (CI value ⁇ 0.8) with Tarceva in the A431 cell proliferation assay (see Example 5).
  • Results show that most AD4 compounds tested in combination with Tarceva produce a CI value ⁇ 0.8, which indicates synergy (see e.g., TABLE 7B).
  • AD4 compounds inhibit cell proliferation by interfering with a pathway that can potentiate the effects of EGF receptor inhibitors.
  • EXAMPLE 11 COMBINATION AD4 TARCEVA INHIBIT BINDING OF P53 AND MDM2
  • EXAMPLE 12 AD4 COMPOUNDS INHIBIT BINDING OF P53 WITH MDM2
  • the following example demonstrates a series of identified compounds that inhibit binding of p53 with MDM2. Methods are according to Examples 1-7 unless otherwise specified.
  • the assay was performed as described in Example 1 , except that recombinant full length human N-terminal GST-MDM2 was used and obtained from ABNOVA, and the wild type, full length human N-terminal 6-his p53 was purchased from Fisher Scientific Co.
  • Compound structures are as disclosed in U.S. Application Serial No. 12/986,146 and WO 2011/085126.
  • the compounds studied in this example were AD4 1505, AD4 10963, AD4 11511 , AD4 10482, AD4 10942, AD4 10944, and AD4 10628.
  • EXAMPLE 13 AD4 COMPOUNDS INHIBIT INTERACTION OFP53AND NIDM2
  • AD4-1505, AD4-10953 and AD4-10944 stimulated p53 reporter gene activity by 42.5-, 9.7- and 7.5-fold, respectively.
  • a correlation plot shows the relationship between the ability of compounds to inhibit p53/MDM2 binding (see Example 13) and the ability to produce synergy with Tarceva (see Example 11) in the A431 cell proliferation assay.
  • Compound structures are as disclosed in U.S. Application Serial No. 12/986,146 and WO 2011/085126.
  • EXAMPLE 15 AD4 COMPOUNDS INDUCE APOPTOSIS AS MEASURED BY DNA
  • EXAMPLE 16 AD4 COMPOUNDS INDUCE APOPTOSIS AS MEASURED BY INCREASES DNA FRAGMENTATION
  • AD4-13243 which increased DNA fragmentation and increased phosphorylated p53 in the proteome profile assay, significantly increased both total p53 expression and S15p-p53 at concentrations of about 1-3 ⁇ . These results demonstrate that AD4-13243 can induce apoptosis by increasing the expression of p53.
  • EXAMPLE 17 COMBINATION AD4/TARCEVA SYNERGISTICALLY INDUCE APOPTOSIS
  • EXAMPLE 18 COMBINATION AD4/TARCEVA COMPOUNDS INDUCE APOPTOSIS WITH NO MEASURABLE CYTOTOXICITY
  • AD4-13178 The results from this study indicate that 6 AD4 compounds (AD4-13178, AD4-13225, AD4-13243, AD4-13130, AD4-13229 and AD4-13165) at a concentration of 5 ⁇ , increased all three phosphorylated forms of p53, similar to nutlin, which inhibits the binding of p53 and MDM2.
  • AD4 compounds produced their effect on p53 by a different mechanism than nutlin. More importantly, all six of the compounds that increased the phosphorylated forms of p53 in the proteome profiler assay increased apoptosis, as measured by DNA fragmentation.
  • AD4 compounds in this example increased all three phosphorylated forms of p53 which inhibit the binding of p53 and MDM2.

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Abstract

La méthode ci-décrite permet de traiter une maladie, une affection, ou un trouble prolifératif chez un sujet par administration d'une combinaison d'un inhibiteur de la liaison p53-MDM2 et d'un inhibiteur d'EGFR. Divers modes de réalisation des méthodes décrites exercent un effet anti-prolifératif et anti-apoptique synergique, comparé à l'administration d'un de ces agents seul.
PCT/US2011/044882 2010-07-21 2011-07-21 Polythérapie basée sur des inhibiteurs de mdm2 et efgr WO2012012653A1 (fr)

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PCT/US2011/020418 WO2011085129A2 (fr) 2010-01-06 2011-01-06 Méthodes et compositions pour le développement de médicaments ciblés
US12/986,146 2011-01-06
PCT/US2011/020414 WO2011085126A2 (fr) 2010-01-06 2011-01-06 Méthodes et compositions pour le développement de médicaments ciblés
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EP2788004A4 (fr) * 2011-12-07 2015-12-30 Univ Duke Procédés d'identification et d'utilisation d'inhibiteurs de mdm2
EP3388419A1 (fr) * 2017-04-12 2018-10-17 Leadiant Biosciences SA Inhibiteurs de gli1 et utilisations associées
US10413549B2 (en) 2012-11-21 2019-09-17 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with isoquinoline and quinoline derivatives
US10640803B2 (en) 2013-10-30 2020-05-05 Eutropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
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EP2788004A4 (fr) * 2011-12-07 2015-12-30 Univ Duke Procédés d'identification et d'utilisation d'inhibiteurs de mdm2
US9937178B2 (en) 2011-12-07 2018-04-10 Duke University Methods of identifying and using MDM2 inhibitors
WO2013192423A2 (fr) 2012-06-20 2013-12-27 Eutropics Pharmaceuticals, Inc. Méthodes et compositions utiles pour traiter les maladies impliquant des protéines de la famille bcl-2 avec des dérivés de quinoline
WO2013192423A3 (fr) * 2012-06-20 2015-06-25 Eutropics Pharmaceuticals, Inc. Méthodes et compositions utiles pour traiter les maladies impliquant des protéines de la famille bcl-2 avec des dérivés de quinoline
US10765673B2 (en) 2012-06-20 2020-09-08 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with quinoline derivatives
US10413549B2 (en) 2012-11-21 2019-09-17 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with isoquinoline and quinoline derivatives
US10732182B2 (en) 2013-08-01 2020-08-04 Eutropics Pharmaceuticals, Inc. Method for predicting cancer sensitivity
US11656230B2 (en) 2013-08-01 2023-05-23 Eutropics Pharmaceuticals, Inc. Method for predicting cancer sensitivity
US10640803B2 (en) 2013-10-30 2020-05-05 Eutropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
US11519015B2 (en) 2013-10-30 2022-12-06 Entropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
EP3388419A1 (fr) * 2017-04-12 2018-10-17 Leadiant Biosciences SA Inhibiteurs de gli1 et utilisations associées

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