WO2013138341A1 - Composés inhibiteurs de topk - Google Patents

Composés inhibiteurs de topk Download PDF

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Publication number
WO2013138341A1
WO2013138341A1 PCT/US2013/030531 US2013030531W WO2013138341A1 WO 2013138341 A1 WO2013138341 A1 WO 2013138341A1 US 2013030531 W US2013030531 W US 2013030531W WO 2013138341 A1 WO2013138341 A1 WO 2013138341A1
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alkyl
compound
cycloalkyl
aryl
heteroaryl
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PCT/US2013/030531
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English (en)
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Yong-Yeon CHO
Zigang Dong
Ann M. Bode
Dong Joon KIM
Myoung Ok KIM
Kanamata Srinivasa REDDY
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Regents Of The University Of Minnesota
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Publication of WO2013138341A1 publication Critical patent/WO2013138341A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • T-LAK-cell-originated protein kinase is a serine-threonine kinase that is a member of MAPKK family and is involved in many cellular functions, including tumor development, cell growth, apoptosis and
  • TOPK is highly expressed in many cancers such as lymphoma, leukemia, melanoma, colorectal, breast cancer, lung and cholangiocarcinoma (Zhu F, et al., Gastroenterology, 2007, 133, 219-31; Abe Y, et al., J Biol Chem, 2000, 275, 21525-31; He F, et al., Hum Pathol, 2010, 41, 415-24; Park JH, et al., Cancer Sci, 2010, 01, 403-11 ; and Simons-Evelyn M, et al, Blood Cells Mol Dis, 2001, 27, 825-9).
  • TOPK interacts with hDlg through TOPK's C-terminal PDZ-binding motif (Ayllon V, O'Connor R.,
  • TOPK expression corresponds with H-Ras-induced cell transformation, UVB-induced JNKs activation and DNA damage-induced p53 expression (Hu F, et al., Oncogene, 2010, 29, 5464- 74; and Oh SM, et al., Cancer Res, 2007, 67, 5186-94).
  • TOPK was identified as a downstream target of EWS-FLI1 chimeric fusion protein (Herrero-Martin D, et al., Br J Cancer, 2009, 101, 80-90).
  • TOPK interacts with p53 and promotes tumorigenesis by inhibiting p53 functions (Hu F, et al., Oncogene, 2010, 29, 5464-74).
  • the mitogen-activated protein kinase kinase 1 and 2 (MEK 1/2) signaling pathway is a major component of the RAS/RAF/MEK/ERKs signaling axis that regulates tumorigenesis and cancer cell growth.
  • MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in MAPK signaling.
  • the invention provides a compound of formula I:
  • R 1 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C C ⁇ alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(Ci-C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl,
  • R is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C6)alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C r C 6 )alkanoyl,
  • R 4 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (Q-C ⁇ alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C6)alkyl, (C r C 6 )alkoxy, (C r C 6 )alkanoyl,
  • X is N or CR m ;
  • each R a is independently H, (d-C ⁇ alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 - C 6 )alkyl, aryl, or ary ⁇ d-C ⁇ alkyl;
  • each R c is independently H, (Ci-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 - C 6 )alkyl, aryl, or ary ⁇ C C ⁇ alkyl;
  • each R d is independently H, (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C )cycloalkyl(Cr C 6 )alkyl, aryl, or aryliQ-C ⁇ alkyl;
  • each R f is independently H, (Q-C ⁇ alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(Ci- C 6 )alkyl, aryl, or aryl(C ! -C 6 )alkyl;
  • R m is H, cyano, (C 1 -C 6 )alkoxycarbonyl, heterocycle, or heteroaryl;
  • each R ba is independently (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl, heteroaryl, aryl(d- C 6 )alkyl, or heteroary ⁇ d-C ⁇ alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl,
  • each R bb and R bc is independently selected from H, (Ci-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C C ⁇ cycloalkyliQ-C ⁇ alkyl, aryl, heteroaryl, aryl(C]-C 6 ) alkyl and heteroaryliC Ce) alkyl; or R bb and R bc together with the nitrogen to which they are attached form a aziridino, azetidino, morpholino, piperazino, pyrrolidino or piperidino;
  • each R ea is independently (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl, heteroaryl, aryl(Ci- C 6 )alkyl, or heteroaryl(CrC 6 )alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl,
  • each R e and R ec is independently selected from H, (d-C ⁇ alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycIoalkyl(C 1 -C 6 )alkyl, aryl, heteroaryl, aryliCi-Ce) alkyl and alkyl; or R eb and R ec together with the nitrogen to which they are attached form a aziridino, azetidino, morpholin
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
  • the invention also provides a method to treat cancer in an animal comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal.
  • the invention also provides a method to treat hair loss in a mammal comprising administering a TOPK inhibitor to the mammal.
  • the invention also provides a method to treat hair loss in a mammal comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the mammal.
  • the invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of cancer.
  • the invention also provides a TOPK inhibitor for the prophylactic or therapeutic treatment of hair loss.
  • the invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of hair loss.
  • the invention also provides the use of a compound of formula (I) or a
  • the invention also provides the use of a TOPK inhibitor to prepare a medicament useful for treating hair loss in a mammal.
  • the invention also provides the use of a compound of formula (I) or a
  • Representative compounds of the invention were also found to inhibit TOPK. Representative compounds of the invention were also found to be highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and - independent cell growth. The inhibitory activity was associated with markedly reduced ERKs and RSK phosphorylation. Additionally, a representative compound of the invention inhibited colon cancer cell growth in an in vivo xenograft mouse model.
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of the invention or salts thereof.
  • Compound 1 is also referred to as HI-TOPK-032.
  • FIG. 1 Knock-down of TOPK expression suppresses anchorage-dependent and -independent colon cancer cell growth.
  • A Colon cancer cells stably expressing knockdown of TOPK were established. The expression of TOPK was determined by Western blotting.
  • B Knocking down TOPK suppresses anchorage-dependent proliferation of colon cancer cells.
  • C Murine embryonic fibroblasts (MEFs) deficient in TOPK protein expression exhibit decreased proliferation. Anchorage-dependent cell growth was determined at 1, 2 and 3 days using the MTS assay.
  • D Knocking down TOPK suppresses anchorage-independent growth of colon cancer cells. HCT116 colon cancer cells stably expressing shMock or shTOPKwere incubated in 0.3% agar for 3 weeks.
  • FIG. 1 Compound 1 suppresses TOPK kinase activity.
  • A Chemical structure of Compound 1.
  • B The effect of Compound 1 on TOPK and MEK1 kinase activities.
  • FIG. 3 Computer modeling results indicate that Compound 1 binds to the TOPK active site.
  • A Docking model of Compound 1 and the TOPK protein structure. Compound 1 is shown in sphere representation. TOPK is shown as a cartoon model.
  • B Binding site of TOPK with Compound 1. The ATP-binding site of TOPK is shown in surface representation. Compound 1 is shown in stick representation.
  • C Surface representation of TOPK with Compound 1. Compound 1 is shown in stick representation. TOPK is shown in surface representation ite.
  • D Interaction between TOPK and Compound 1. Compound 1 is shown in stick representation.
  • FIG. 4 Compound 1 exerts anti-cancer activity against colon cancer cells.
  • B Compound 1 inhibits anchorage-independent cancer cell growth. Colon cancer cells were incubated in 0.3% agar for 3 weeks with Compound 1. Colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. Data are represented as means ⁇ S.D.
  • the effect of Compound 1 was examined in shMock and knockdown cell lines with medium expression of TOPK (#3 shTOPK) or low expression of TOPK (#2 shTOPK). Cells were incubated for 72 h and growth was determined by MTS assay.
  • (B) Effect of Compound 1 on growth of wildtype TOPK MEFs and (C) TOPK knockout MEFs. Cell growth at 1, 2 or 3 days was measured by MTS assay. Data are shown as means ⁇ S.D. (N 5) and similar results were obtained from two independent experiments. The asterisk (*) indicates a significant (p ⁇ 0.05) difference between Compound 1 treated cells and untreated control ceils.
  • Colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. Data are represented as means ⁇ S.D. of values from triplicate values and similar results were obtained from 2 independent experiments.
  • the asterisk (*) indicates a significant (p ⁇ 0.05) decrease in colony formation induced by Compound 1 compared to untreated control cells.
  • FIG. 6 Compound 1 prevents xenograft tumor growth.
  • the asterisk (*) indicates a significant difference between tumors from untreated and treated mice as determined by t test (p ⁇ 0.05).
  • B Compound 1 has no effect on mouse body weight. Body weights from treated or untreated groups of mice were obtained once a week.
  • C Compound 1 inhibits TOPK-target protein expression in HCT116 colon tumor tissues. The tumor tissues from groups treated with vehicle, 1 mg or 10 mg/kg/BW Compound 1 were immunoblotted with antibodies to detect total TOPK, p-TOPK, p53, total ERK, p-ERK, total RSK, p-RSK and ⁇ -actin. ⁇ -Actin was used to verify equivalent loading of protein.
  • D Representative signaling pathway of TOPK mediated multifunction by Compound 1.
  • FIG. 7 TOPK directly phosphorylates SRD5A2.
  • A TOPK phosphorylates SRD5A2 in vitro. The phosphorylation of SRD5 A2 by TOPK was assessed by an in vitro kinase assay using TOPK (active, 500 ng) and GST tagged full-length SRD5A2 with [ ⁇ -32 ⁇ ] ⁇ . The TOPK inhibitor was used a negative control.
  • FIG. 1 TOPK promotes the development of hair pores.
  • A Representative photographs of mice treated or not treated with a TOPK inhibitor. The TOPK inhibitor (400 nmol) in acetone was topically applied 3 times weekly until the termination of the experiment at 16 weeks.
  • B Effect of a TOPK inhibitor on inner hair pores or
  • C outer hair pores. The number of inner or outer hair pores was counted after H&E staining.
  • FIG. 9 CInQ inhibitors suppress MEK kinase activity.
  • A Respective chemical structures of CInQ-01, -03 and -06.
  • CInQ inhibitors (CInQ-01, -03 and -06) substantially suppress
  • B MEKl and
  • C MEK2 kinase activities in a dose-dependent manner.
  • the effect of CInQ inhibitors or U0126, a well-known MEK inhibitor, on MEK activity was assessed by an in vitro kinase assay using MEKl (active, 250 ng) or MEK2 (active, 500 ng) and inactive ERK2 (MEKl or 2 substrate, 500 ng) proteins with [ ⁇ - P]ATP. All data are represented as means ⁇ S.D.
  • FIG. 10 Computer models of CInQ inhibitors docked with MEK.
  • A Docking models of MEKl with CInQ-01, -03 or -06..
  • B Docking models of MEK2 with CInQ-01, - 03 or -06..
  • CInQ -01, -03 or -06 exerts anti-cancer activity.
  • CInQ inhibitors dose-dependently inhibit colon cancer cell growth. Cells were treated with the individual CInQ inhibitor or U0126 for 1, 2 or 3 days.
  • the asterisk (*) indicates a significant (p ⁇ 0.05) difference in growth of cells treated inhibitor compared to untreated or only EGF-treated control.
  • C CInQ inhibitors dose- dependently suppress anchorage independent colon cancer cell growth. Cells were treated with individual CInQ inhibitors or U0126 in 0.3% agar and incubated for 3 weeks.
  • D Effect of individual CInQ inhibitors or U0126 on EGF-induced transformation of HaCaT cells. Cells were co-treated with EGF and CInQ inhibitors or U0126 in 0.3% agar and incubated for 2 weeks. Colonies were counted using a microscope and the Image-Pro PLUS (v6) computer software program. Data are shown as means ⁇ S.D.
  • FIG. 12 Effect of CInQ -01, -03 or -06 on AP-1 promoter activity.
  • CInQ inhibitors suppress AP-1 reporter activity in colon cancer cells. Cells were transfected with the APl-lucif erase reporter and CMV-renilla plasmids. At 1 day after transfection, cells were treated with individual CInQ inhibitors or U0126 and incubated for 2 days. The AP-1 reporter activity was assessed.
  • CInQ inhibitors suppress AP-1 signaling in colon cancer cells. Colon cancer cells were treated with individual CInQ inhibitors or U0126 for 2 days.
  • D Effect of CInQ inhibitors on EGF-induced AP-1 signaling in HaCaT cells.
  • FIG. 13 The anti-cancer activity exerted by CInQ inhibitors is dependent on MEK expression.
  • A Colon cancer cells stably expressing knockdown of MEKl 12 were established using lenti viral infection. The expression of MEKl and MEK2 was determined by Western blotting. ⁇ -Actin was used to verify equal protein loading. Band density was measured using the Image J (NIH) software program.
  • B The effect of CInQ inhibitors or U0126 on cell growth was not as efficient in knockdown MEK 1/2 cells compared to control cells. Cells were treated with CInQ inhibitors or U0126 for 3 days and cell growth was analyzed by MTS assay. Data are shown as means ⁇ S.D.
  • C CInQ inhibitors or U0126 were not as effective to inhibit anchorage-independent cell growth in knockdown MEK1/2 cells compared to control cells. Cells were treated with CInQ inhibitors or U0126 in 0.3% agar and incubated for 3 weeks at 37°C/5% C0 2 . Data are represented as means ⁇ S.D. of values from triplicate values and similar results were obtained from two independent experiments. The asterisk (*) indicates a significant (p ⁇ 0.05) decrease in colony formation induced by CInQ inhibitors or U0126 compared to untreated control cells.
  • FIG. 14 CInQ-03 prevents xenograft tumor growth.
  • A Representative photographs of tumor-bearing athymic nude mouse treated or not treated with CInQ-03.
  • B left panel
  • CInQ-03 suppresses colon tumor growth.
  • HCT116 colon cancer cells were injected subcutaneously into the dorsal right flank of mice. Mice were injected with CInQ-03 or vehicle 3 times a week for 11 days. Mice were monitored until tumors reached 1 cm total volume, at which time mice were euthanized and tumors were extracted.
  • C Hematocylin & eosin (H&E) staining and immunohistochemistry analysis of tumor and skin tissues. Treated or untreated groups of mice were euthanized and tumors extracted. Colon tumor tissue slides were prepared from paraffin sections after fixation with formalin and then stained with H&E or anti-Ki67. Expression of Ki67 was visualized by light microscope (X200).
  • CInQ-03 inhibits MEK-target protein expression in HCT116 colon tumor tissues.
  • Tumor tissues from groups treated with vehicle, 1 or 5 mg CInQ-03 per kg B.W. were immunoblotted with antibodies to detect total MEK, phosphorylated MEK, total ERKs, phosphorylated ERKs, total RSK, phosphorylated RSK and ⁇ -actin.
  • ⁇ -Actin was used to verify equal protein loading. Band density was measured using the Image J (NIH) software program.
  • the 4 th panel in C is from mice not injected with cells but with compound only-no tumors developed.
  • FIG. 17 Expression of total and phosphorylated TOPK in colon cancer cell lines. Cells were incubated for 48 h in medium containing 10% FBS and analyzed by Western blot. Similar results were observed from 2 independent experiments. Long and short indicates exposure time.
  • FIG. 18 Effect of Compound 1 on transformation of JB6 cells.
  • A Compound 1 inhibits cell transformation. JB6-Mock or JB6-TOPK overexpressing cells were incubated in 0.3% agar for 2 weeks with compound 1. Similar results were observed from 2 independent experiments.
  • Figure 19. Effect of Compound 1 on API, NF- ⁇ or COX2 reporter activity in colon cancer cells.
  • API activity is strongly inhibited by Compound 1 in HCT116 colon cancer cells.
  • B NF- ⁇ activity is markedly inhibited by Compound 1 in HCT116 colon cancer cells.
  • C COX2 activity is strongly inhibited by Compound 1 in HCT15 colon cancer cells. These reporter activities in colon cancer cells were analyzed using the substrates included in the reporter assay system. Data are represented as means ⁇ S.D. of triplicate values from 2 independent experiments and the asterisk (*) indicates a significant (p ⁇ 0.05) effect of Compound 1 compared to untreated controls.
  • FIG. 20 Representative photographs of tumor-bearing athymic nude mouse treated or not treated with Compound 1.
  • FIG. 21 Screening of CInQ inhibitors against MEK1 kinase activity.
  • A Chemical structure of CInQ-01 to -06.
  • B Effect of CInQ inhibitors (CInQ-01 to -06) on MEK1 kinase activity as determined by an in vitro kinase assay. All data are represented as means ⁇ S.D. of values from two independent experiments. Band density was measured using the Image J (NIH) software program. The asterisk (*) indicates a significant (p ⁇ 0.05) decrease induced by CInQ-01 to -06 compared to untreated control.
  • FIG. 22 Effect of CInQ inhibitors on TOPK, a MAPKK family member.
  • the effect of CInQ inhibitors on TOPK activity was assessed by an in vitro kinase assay using TOPK (active, 500 ng) and histone H2AX (TOPK substrate, 500 ng) proteins with [ ⁇ - 32 P]ATP. All data are represented as means ⁇ S.D. of values from two independent experiments. Band density was measured using the Image J (NIH) software program.
  • the asterisk (*) indicates a significant (p ⁇ 0.05) decrease induced by CInQ-01, -03 or -06 compared to untreated control.
  • FIG. 23 Establishing knockdown MEK1 and MEK2 stable cell lines.
  • HCT1 16 colon cancer cells were stably infected with shMock, shMEKl or
  • shMEK2 The expression of MEK1 and MEK2 was analyzed by Western blot.
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.
  • Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C ! -C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms comprising one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X).
  • heterocyclyl or “heterocycle” as used herein refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below.
  • the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring.
  • the ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms.
  • exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl.
  • heterocycle also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from heterocycles (to form for example a 1,8-decahydronapthyridinyl ), carbocycles (to form for example a decahydroquinolyl) and aryls to form the multiple condensed ring system.
  • a heterocycle a single saturated or single partially unsaturated ring or multiple condensed ring system
  • Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the multiple condensed ring.
  • the rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another. It is also to be understood that the point of attachment of a multiple condensed ring system (as defined above for a heterocycle) can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring.
  • the point of attachment for a heterocycle or heterocycle multiple condensed ring system can be at any suitable atom of the heterocycle or heterocycle multiple condensed ring system including a carbon atom and a heteroatom (e.g., a nitrogen).
  • a heteroatom e.g., a nitrogen
  • the atom range is for the total ring atoms of the heterocycle and includes carbon atoms and heteroatoms.
  • a 3-membered heterocycle would include an aziridinyl and a 10-membered heterocycle would include a 1,2,3,4- tetrahydroquinolyl.
  • heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4- tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1 ,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro [cyclopropane- ⁇ , ⁇ - isoindolinyl]-3'-one, isoindolinyl-l-one, 2-oxa-6-azaspiro[3.3]hept
  • treating hair loss may include one or more of the following:
  • (Q-C ⁇ alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl, or hexyl;
  • (C 3 -C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • (C3-C 6 )cycloalkyl(C 1 -C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2- cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl;
  • (d-C 6 )alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy
  • R b is not 2-thienyl when R 1 is H, R 2 is H, R 3 is H, R 4 is H, and R a is H.
  • R b is not 2-thienyl.
  • R b is not thienyl.
  • R 1 and R 4 are each H.
  • R 2 and R 3 are each H.
  • R a is H.
  • R 1 is H
  • R 2 is H
  • R 3 is H
  • R 4 is H
  • R 5 is H
  • R b is aryl or heteroaryl wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(d- C 6 )alkyl, (d-C 6 )alkoxy, (d-C 6 )alkanoyl, (C 1 -C6)alkoxycarbonyl, and (C 2 -C 6 )alkanoyloxy;.
  • Processes for preparing compounds of formula I are provided as further embodiments of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as given above unless otherwise qualified.
  • R b is -NR bb R bc .
  • R b is -NR bb R bc ;
  • R bb is H; and
  • R bc is aryl.
  • the invention provides a compound of formula (la):
  • R 1 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (d-C 6 )alkoxy, (C 1 -C 6 )alkanoyl,
  • R is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(Ci-C 6 )alkyl, (d-C 6 )alkoxy, (d-C 6 )alkanoyl,
  • R 4 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (d-C 6 )alkanoyl,
  • R c is H, (C C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, aryl, or aryl(d-C 6 )alkyl;
  • R d is H, (d-C ⁇ alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, aryl, or arylCd-C f alkyl;
  • R ba is (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl, heteroaryl, aryl(d-C 6 )alkyl, or heteroaryl(d-C )alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl, trifluoromethoxy, (d- C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (Cj-Cejalkoxycarbonyl, and (C2-C 6 )alkanoyloxy;
  • each R bb and R bc is independently selected from H, (C 1 -C 6 )alkyl, (C3-C6)cycloalkyl, (C 3 -C )cycloalkyl(Ci-C 6 )alkyl, aryl, heteroaryl, aryl(d-C 6 ) alkyl and heteroaryl(d-C6) alkyl; or R bb and R bc together with the nitrogen to which they are attached form a aziridino, azetidino, morpholino, piperazino, pyrrolidino or piperidino;
  • R ea is (d-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl, heteroaryl, aryl(Ci-C 6 )alkyl, or heteroaryl(C 1 -C 6 )alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl, trifluoromethoxy, (d- C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (d-C 6 )alkoxy, (d-C 6 )alkanoyl, (d-C 6 )alkoxycarbonyl, and (C 2 -C 6 )alkanoyloxy; and
  • each R cb and R ec is independently selected from H, (d-C 6 )alkyl, (C 3 -C )cycloalkyl, (C 3 -C6)cycloalkyl(d-C 6 )alkyl, aryl, heteroaryl, aryl(d-C 6 ) alkyl and heteroaryl(d-C ) alkyl; or R eb and R ec together with the nitrogen to which they are attached form a aziridino, azetidino, morpholino, piperazino, pyrrolidino or piperidino;
  • R 1 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (d-C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl,
  • R is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(d-C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl,
  • R is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (d-C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (d-C ⁇ alkoxy, (C 1 -C 6 )alkanoyl,
  • R 4 is H, halo, nitro, cyano, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, (d-C 6 )alkoxy, (d-C 6 )alkanoyl,
  • R a is H, (C C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, aryl, or arylCd- ⁇ alkyl;
  • R ba is (d-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl, heteroaryl, aryl(C 1 -C6)alkyl, or
  • heteroaryl(d-C6)alkyl wherein any aryl or heteroaryl is optionally substituted with one or more groups independently selected from halo, nitro, trifluoromethyl, trifluoromethoxy, (d- C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(d-C 6 )alkyl, (d-C 6 )alkoxy, (d-C 6 )alkanoyl, (C 1 -C 6 )alkoxycarbonyl, and (C 2 -C 6 )alkanoyloxy; and
  • each R bb and R bc is independently selected from H, (d-C 6 )alkyl, (C 3 -C 6 )cycloalkyl, (C 3 -C6)cycloalkyl(d-C 6 )alkyl, aryl, heteroaryl, aryl(d-C6) alkyl and heteroaryl(d-C 6 ) alkyl; or R b and R° together with the nitrogen to which they are attached form a aziridino, azetidino, morpholino, piperazino, pyrrolidine or piperidino;
  • the invention provides compounds of formula I that exclude compounds of formula lb.
  • R is -OR .
  • the compound is not: or a salt thereof.
  • a compound of formula (I) or (la) can be prepared as illustrated in the following Schem
  • a compound of formula I can also be prepared as illustrated in the following
  • the DBA mouse is the oldest of all inbred strains. It was developed in 1909 by Little, who, around 1929, divided it into two lines: DBA/1 and DBA/2. It was introduced into The Jackson Laboratory in 1948 at the F-26th generation, and into Charles River France in February 1982 at the F- 140th generation. It has a coat color of non-agouti dilute brown.
  • mice While establishing a breeding colony of these mice homozygote TOPK male and female mice were obtained by mating; they were confirmed through genotyping by PCR and Western blotting. The mice were filtered into the SKH-1 hairless background to facilitate skin cancer UV studies. During this process, it was found that TOPK heterozygote mice grew hair even though their genetic background had been filtered to 93.75% of the SKH-1 hairless background. In contrast, the wildtype mice did not grow hair. This result strongly indicates that inhibition of TOPK might be an effective methodology to treat baldness in humans.
  • a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I.
  • administration of a compound of formula I as a pharmaceutically acceptable acid or base salt may be appropriate.
  • pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following:
  • binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form.
  • tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • the ability of a compound of the invention to treat cancer may be determined using pharmacological models which are well known to the art, or using the tests described in Example 1 below.
  • the ability of a compound of the invention to treat hair loss may be determined using pharmacological models which are well known to the art.
  • MEK1 substrate Active MEK1, inactive ERK2 (MEK1 substrate), active ERK1, active JNK1, active p38, ATF2(p38 substrate), histone H2AX (TOPK substrate) human recombinant protein and MBP (Myelin Basic Protein; TOPK substrate) for kinase assays were purchased from MBP (Myelin Basic Protein; TOPK substrate) for kinase assays.
  • the inactive N-terminal RSK2 (ERK1 substrate) and C-terminal c-Jun (JNK1 substrate) human recombinant protein for kinase assays were purified from E. Coli.
  • the active TOPK human recombinant protein for the kinase assay was purchased from SignalChem (Richmond, BC).
  • Antibodies to detect total TOPK, phosphorylated TOPK (T9), total CDC2, phosphorylated CDC2 (T15), total ERK, phosphorylated ERK (T202/Y204), total RSK, phosphorylated RSK (T356/S360) and caspase 7 were purchased from Cell Signaling Technology (Beverly, MA).
  • Antibodies to detect p53, PARP and ⁇ -actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • DNA stat-60 for genomic DNA isolation was obtained from Tel Test (Friendswood, TX).
  • All cell lines were purchased from American Type Culture Collection (ATCC) and were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 8 weeks. Enough frozen vials were available for each cell line to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less.
  • ATCC American Type Culture Collection
  • HCEC human colonic epithelial cells
  • basal media HyClone, Logan, UT
  • EGF 25 ng/mL
  • insulin 10 ⁇ g/mL
  • gentamicin sulfate 50 ⁇ g/mL
  • transferrin 2 ⁇ g/mL
  • hydrocortisone 1 ⁇ g/mL
  • sodium selenite 5 nM
  • cosmic calf serum HyClone, Logan, UT
  • HCT116 and HT29 human colon cancer cells were cultured in McCoy's 5 A medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic- antimycotic.
  • HCT15 and DLD1 human colon cancer cells were cultured in RPMI1640 medium supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.
  • JB6 mouse skin epidermal cells were cultured in MEM supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.
  • TOPK-WT or KO MEFs mouse embryonic fibroblasts were cultured in DMEM
  • the lentiviral expression vectors including Gipz-shTOPK and packaging vectors, including pMD2.0G and psPAX, were purchased from Addgene Inc. (Cambridge, MA).
  • each viral vector and packaging vectors (pMD2.0G and psPAX) were transfected into HEK293T cells using JetPEI following the manufacturer's suggested protocols.
  • the transfection medium was changed at 4 h after transfection and then cells were cultured for 36 h.
  • the viral particles were harvested by filtration using a 0.45 mm syringe filter, then combined with 8 ⁇ g/ml of polybrane (Millipore, Billerica, MA) and infected into 60% confluent HCT-116 cells overnight.
  • the cell culture medium was replaced with fresh complete growth medium for 24 h and then cells were selected with puromycine (1.5 ⁇ g/ml) for 36 h. The selected cells were used for experiments.
  • a TOPK structure was modeled using comparative modeling.
  • the sequence of TOPK was downloaded from NCBI (GI: 83305809) and BLAST was used to search for homologous proteins in the RCSB Protein Data Bank. Results indicated that the sequence identity between the sequences of TOPK and proteins with known structures is below 30% and the sequence similarity is about 45%.
  • the protein structure from 2F4J (PDB entry) was selected as the template structure to model the TOPK structure.
  • the alignment of sequences of TOPK and 2F4J was generated by BLAST and edited in Prime v3.0.
  • the secondary structure of TOPK was predicted by SSpro.
  • the TOPK structure was built with Prime v3.0 followed by refining and minimizing loops in the binding site.
  • Glide v5.7 was used for docking of TOPK and Compound 1.
  • Compound 1 was prepared using LigPrep v2.5 and then assigned AMSOL partial atom charge. Flexible docking was performed with extra precision (XP) mode as described (17). The number of poses per ligand was set to 10 in post-docking minimization and at most 5 poses would be output. The other parameters were kept as default.
  • Transient transfection was conducted using jetPEI (Qbiogene, Carlsbad CA), and assays for the activity of firefly luciferase and Renilla activity were performed according to the manufacturer's manual (Promega, Madison, WI).
  • Cells (1 x 10 4 per well) were seeded the day before transfection into 12-well culture plates.
  • Cells were co-transfected with reporter plasmid (250 ng) and internal control ⁇ CMV-Renilla, 50 ng) in 12-well plates and incubated for 24 h. Colon cancer cells were treated with Compound 1 for 48 h. Cells were harvested in Promega Lysis Buffer.
  • the Luciferase and Renilla activities were measured using substrates in the reporter assay system (Promega). The luciferase activity was normalized to Renilla activity.
  • Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 x Protease inhibitor tablet). Equal amounts of protein were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were separated by SDS/PAGE and transferred to
  • polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. After washing with PBS containing 0.1% Tween 20, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody at a 1 :5,000 dilution and the signal was detected with a chemiluminescence reagent (Amersham Biosciences Corp).
  • Cells were seeded (1 x 10 3 cells per well) in 96- well plates and incubated for 24 hours and then treated with different doses of each compound. After incubation for 1, 2 or 3 days, 20 ⁇ of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% C0 2 incubator. Absorbance was measured at 492 nm.
  • the kinase assay was performed in accordance with instructions provided by Upstate Biotechnology (Billenca, MA). Briefly, the reaction was carried out in the presence of 10 of [ ⁇ - P] ATP with each compound in 40 ⁇ of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 10 mM MnCl 2 , and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 ⁇ protein loading buffer and the mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each experiment was repeated twice. The relative amounts of incorporated radioactivity were assessed by autoradiography.
  • Compound 1 directly suppresses TOPK kinase activity.
  • Thirty-six compounds were screened (30 ⁇ concentration) by in vitro TOPK kinase assay (Fig. 16 A) and by cell proliferation assay (4 or 20 ⁇ concentration) (Fig. 16B).
  • Six compounds were selected to test for inhibition of TOPK (10 ⁇ concentration) (Fig. 16C), Based on screening results, Compound 1 (Fig. 2A) was identified as a potent TOPK inhibitor.
  • an in vitro TOPK or MEK1 kinase assay was performed with increasing concentrations of Compound 1.
  • Compound 1 effectively inhibited TOPK kinase activity.
  • Compound 1 occupies the ATP-binding site of TOPK (Fig. 3 A, B) and fits the binding site very well (Fig. 3C). The compound forms hydrogen bonds with GLY83 and ASP151 and has a hydrophobic interaction with LYS30 (Fig. 3D). These results suggest that Compound 1 binds to the TOPK active site.
  • Compound 1 inhibits colon cancer cell growth and induces apoptosis.
  • Western blot analysis was performed. Results showed that HCT116 colon cancer cells highly expressed phosphoiylated TOPK compared with other colon cancer cells (Fig. 17A) and these cells were used in further studies.
  • growth was measured using the MTS assay at 1, 2 or 3 days after treatment with Compound 1. Results indicated that colon cancer cell growth was significantly decreased by Compound 1 in a dose-dependent manner (Fig. 4A). Additionally, the effect of Compound 1 on anchorage-independent cell growth was determined.
  • Colon cancer cells were seeded with Compound 1 in 0.3% agar and incubated for 3 weeks. Data showed that anchorage-independent cancer cell growth was strongly suppressed by Compound 1 in a dose- dependent manner (Fig. 4B).
  • HCT116 colon cancer cells were treated with Compound 1 and then incubated for 3 days. Results showed that DNA fragmentation induced by Compound 1 was substantially increased in HCT116 colon cancer cells compared with untreated control cells (Fig. 4C). Previous studies suggested that overexpressing TOPK can induce neoplastic cell transformation.
  • TOPK was shown to inhibit the p53 signaling pathway, involved the G2/M transition, and activated the ERK signaling pathway. Therefore, inhibition of TOPK kinase activity by a TOPK inhibitor should be able to induce abundance of the p53 protein and its downstream target proteins, G2/M phase marker proteins (i.e., CDC2 phosphorylation) and reduce ERKs phosphorylation.
  • the inhibition of TOPK by Compound 1 is dependent on the abundance of TOPK.
  • the effect of Compound 1 on growth of shMock, #3 shTOPK (medium expression of TOPK) or #2 shTOPK (low expression of TOPK) colon cancer cells was assessed by MTS assay at 72 h. Results indicated that cells expressing shTOPK were resistant to Compound l's inhibitory effect on growth compared to cells expressing shMock (Fig. 5 A). Similar resistance to Compound 1 was observed in TOPK knockout MEFs (Fig. 5B, C). Additionally, the effect of Compound 1 on anchorage-independent colon cancer cell growth was examined. Results showed that the inhibitory effect of Compound 1 on anchorage-independent cell growth in shTOPK cells was much less than its effect on shMock growth (Fig. 5D). These findings showed that the anticancer activity induced by Compound 1 is dependent on TOPK protein expression.
  • Compound 1 inhibits colon cancer tumor growth in a xenograft mouse model.
  • HCT116 colon cancer cells were injected into the flank of athymic nude mice. Mice were injected with vehicle or Compound 1 at 1 or 10 mg/kg 3 times a week over a period of 25 days. Treatment of mice with 1 or 10 mg/kg of Compound 1 significantly inhibited HCT116 tumor growth by over 60% relative to the vehicle-treated group (Figs. 20A, Fig. 20B; p ⁇ 0.05). Additionally, mice seemed to tolerate treatment with Compound 1 without overt signs of toxicity or significant loss of body weight similar to the vehicle-treated group (Fig. 6B).
  • TOPK serine/threonine kinase
  • a TOPK inhibitor has not yet been found, possibly because the TOPK crystal structure has not yet been reported.
  • a sequence-based homology search was performed. It was found that the sequences of TOPK and MEKs are highly conserved. Based on this sequence homology, the effect of U0126, a well-known inhibitor of MEK, on the in vitro TOPK kinase activity was tested.
  • U0126 inhibited TOPK activity by 25% at the highest concentration (20 ⁇ ; data not shown). Based on these preliminary data, 36 compounds with a similar structure to a MEK inhibitor were selected and Compound 1 was identified as a potent TOPK inhibitor. In addition, a homology model based on the known structure of MEK was built and a docking simulation between Compound 1 and the modeled TOPK protein was performed. Compound 1 was docked to the active site of TOPK (Fig. 3). The inhibitory effect of various Compound 1 analogues on TOPK kinase activity in vitro were then compared. However, none of the analogues had any effect (data not shown).
  • TOPK directly interacts with the DBD domain of tumor suppressor p53.
  • TOPK's downstream target, tumor suppressor activated pathway-6 (TSAP6,) reportedly binds to myelin transcription factor 1 (MYT1), which induces phosphorylation of CDC2 (Tyrl5).
  • MYT1 myelin transcription factor 1
  • p53 expression or phosphorylation of CDC2 (Tyrl5) is affected by Compound 1 was investigated.
  • Compound 1 is a novel and specific TOPK inhibitor both in vitro and in vivo. These findings should be useful for further development of drugs targeted against TOPK. Additionally Compound 1 may possess therapeutic potential against colorectal cancer and other human cancers.
  • DHT 5-alpha reductase
  • SRD5A2 is the gene that codes for the protein product that is responsible for the pathogenesis of male pattern baldness.
  • DHT androgen dihydrotestosterone
  • SRD5A2 is the gene that codes for the protein product that is responsible for the pathogenesis of male pattern baldness.
  • the hair follicles become miniaturized; resulting in fine, short hair that is prone to fall out (Kaufman KD., Mol Cell Endocrinol, 2002, 89-95).
  • Much classical evidence pinpoints the direct involvement of DHT in male pattern baldness. Studies of
  • pseudohermaphrodites (lacking 5 AR) showed protection from male pattern baldness throughout life.
  • inhibitors of the enzyme SRD5A2 have slowed the rate of hair loss, indicating its direct involvement (Nyholt DR, et al., J Invest Dermatol. 2003, 121, 1561).
  • a TOPK inhibitor (Compound 1, 400 nmol) in acetone was topically applied 3 times weekly until the termination of the experiment at 16 weeks.
  • the number of inner or outer hair pores was counted after H&E staining.
  • Data are shown in Figure 8 as means ⁇ S.E. of values (n > 5) and the asterisk (*) indicates a significant difference (p ⁇ 0.05) between TOP inhibitor treated groups compared to the vehicle treated group.
  • CInQ-01 N-( 12-cyanindolizino [2,3 -b] quinoxalin-3 -yl)-4-fluorobenzamide, purity: 95%)
  • CInQ-02 N-(12-cyanindolizino[2,3-b]quinoxalin-3-yl)-2-thiophenecaboxamide, purity: 95%)
  • CInQ-03 (2-chloro-N-(12-cyanindolizino[2,3-b]quinoxalin-2-yl)benzamide, purity: 95%)
  • CInQ-04 N-(12-cyanindolizino[2,3-b]quinoxalin-2-yl)-4-methylbenzene- sulfonamide, purity: 95%)
  • CInQ-05 (2-(l,l-dimethylethyl)-indolizino[2,3-b]quinoxaline-12- carbonitrile, purity: 95%)
  • CInQ-06 N-(12-cyanindolizino[2,
  • phosphorylated MEK total ERKs, phosphorylated ERKs, total RSK and phosphorylated RSK were purchased from Cell Signaling Technology (Beverly, MA).
  • HCT116 human colon cancer cells were cultured in McCoy's 5 A medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.
  • HCT15 human colon cancer cells were cultured in RPMI1640 medium supplemented with 10% FBS (Atlanta Biologicals) and 1% antibiotic-antimycotic.
  • HaCaT (human keratinocyte) cells were cultured in DMEM medium supplemented with 10% FBS (Atlanta Biologicals) and 1% antibiotic- antimycotic.
  • the crystal structures of MEK1 and MEK2 were obtained from the RCSB Protein Data Bank [PDB entry: lS9J and 1S9I (Ohren JF, et al., Nat Struct Mol Biol. 2004, 11, 1192- 7)].
  • the crystal structures were prepared using the Protein Preparation Wizard in Maestro v9.2. Hydrogens were added consistent with a pH of 7. All water molecules were removed and then the structure was minimized with an RMSD cutoff value of 0.3 A.
  • Three compounds were prepared using LigPrep v2.5 and then assigned AMSOL partial atom charge.
  • the program Glide v5.7 (Friesner RA, et al., J Med Chem. 2006, 49, 6177-96) was used for ligand docking.
  • the receptor grid was created with the centroid of the crystal ligand as the center of the grid. Flexible Docking was performed with extra precision (XP) mode. The number of poses per ligand was set to 10 in post-docking minimization and at most 5 poses would be output. The other parameters were kept as default.
  • the lentiviral expression vectors including Gipz-shMEKl or shMEK2 and packaging vectors, including pMD2.0G and psPAX, were purchased from Addgene Inc. (Cambridge, MA).
  • each viral vector and packaging vectors pMD2.0G and psPAX
  • the transfection medium was changed at 4 h after transfection and then cells were cultured for 36 h.
  • the viral particles were harvested by filtration using a 0.45 mm sodium acetate syringe filter, then combined with 8 ⁇ g/ml of polybrane (Millipore, Billerica, MA) and infected into 60% confluent HCT116 cells overnight.
  • the cell culture medium was replaced with fresh complete growth medium for 24 h and then cells were selected with puromycine for 36 h (1.5 ⁇ g/ml of puromycine). The selected cells were used for
  • Transient transfection was conducted using jetPEI (Qbiogene, Carlsbad CA), and assays to determine firefly luciferase and Renilla activities were performed according to the manufacturer's manual (Promega, Madison, WI).
  • Cells (1 x 10 4 per well) were seeded the day before transfection into 12-well culture plates.
  • Cells were co-transfected with the AP-1 reporter plasmid (250 ng) and an internal control (CMV-Renilla, 50 ng) in 12-well plates and incubated for 24 h.
  • Colon cancer cells were treated with individual CInQ inhibitors or U0126 for 2 days. Keratinocytes were treated with CInQ inhibitors or U0126 for 2 h before EGF treatment for 12 or 24 h.
  • Cells (8 x 10 3 per well) suspended in complete growth medium (McCoy's 5 A, RPMI1640 or DMEM supplemented with 10% FBS and 1% antibiotics) were added to 0.3% agar with EGF alone or with different doses of each compound in a top layer over a base layer of 0.6% agar with EGF alone or with different doses of each compound.
  • the cultures were maintained at 37°C in a 5% C0 2 incubator for 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v.4) program (Media Cybernetics).
  • Cell lysates were prepared with RIP A buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 x protease inhibitor tablet). Equal amounts of protein were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were separated by SDS/PAGE and transferred to
  • polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. After washing with PBS containing 0.1% Tween 20, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody at a 1 :5,000 dilution and the signal was detected with a chemiluminescence reagent (Amersham Biosciences Corp). Cell proliferation assay
  • Cells were seeded (1 x 10 cells per well) in 96-well plates and incubated for 24 h and then treated with different doses of each compound. After incubation for 1, 2 or 3 days, 20 ⁇ of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% C0 2 incubator. Absorbance was measured at 492 nm.
  • the kinase assay was performed in accordance with instructions provided by Upstate Biotechnology (Billerica, MA). Briefly, the reaction was carried out in the presence of 10 ⁇ ' of [ ⁇ - P] ATP with each compound in 40 ⁇ of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 10 mM MnCl 2 , and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 ⁇ protein loading buffer and the mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each experiment was repeated twice and the relative amounts of incorporated radioactivity were assessed by autoradiography.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Tumor and skin tissues from mice were embedded in paraffin blocks and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry.
  • Tumor tissues were de- paraffinized and hydrated then permeabilized with 0.5% Triton X-100/1 x PBS for 10 min. They were then hybridized with Ki-67 (1 :500) as the primary antibody and horse-radish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG antibody was used as the secondary antibody. After developing with 3, 3'-diaminobenzidine, the sections were counterstained with H&E. All sections were observed by microscope and the Image-Pro Plus software (v. 4) program (Media Cybernetics).
  • HCT116 cells 1.5xl0 6 cells/100 ⁇ ) were suspended in serum free McCoy's 5 A medium and inoculated subcutaneously into the right flank of each mouse.
  • ClnQ-inhibitors suppress MEKl and MEK2 kinase activities.
  • CInQ-01 to -06 compounds (30 ⁇ concentration) were screened using an in vitro MEKl kinase assay and results showed that MEKl kinase activity was strongly inhibited by CInQ-01, -03 or -06.
  • CInQ-01, -03 and -06 are potent MEK inhibitors (Fig. 9 A and 9B). Therefore these three compounds (5 and 10 ⁇ ) were selected and were evaluated further by in vitro MEKl and MEK2 kinase assays.
  • Results showed that CInQ-01, -03 and -06 significantly suppressed MEKl and MEK2 kinase activities in a dose dependent manner (Fig. 9B and 9C).
  • CInQ-01, -03 or -06 affected the kinase activity of TOPK, a MAPKK family member
  • an in vitro TOPK kinase assay was performed with the ClnQ- inhibitors.
  • Results indicated that CInQ-01 and -03 had little effect of TOPK kinase activity (Fig. 10) and CInQ-06 suppressed TOPK activity only by about 20%.
  • the docking score of MEKl with PD318088 is -9.45, and the docking scores of MEKl with CInQ-01, -03 and - 06 are -7.84, -7.29 and -7.83, respectively. Additionally, in the docked structure of MEK2 with the ClnQ-inhibitors, all compounds formed a hydrogen bond with Ser216 (Fig. 10B). CInQ-03 forms an additional hydrogen bond with LyslOl, which also forms a hydrogen bond with PD334581 in the crystal structure of MEK2. The docking score of MEK2 with -9.45, and the docking scores of MEKl with CInQ-01, -03 and - 06 are -7.84, -7.29 and -7.83, respectively. Additionally, in the docked structure of MEK2 with the ClnQ-inhibitors, all compounds formed a hydrogen bond with Ser216 (Fig. 10B). CInQ-03 forms an additional hydrogen bond with LyslOl, which also forms a hydrogen bond with
  • PD334581 is -8.92, and the docking scores of MEK2 with CInQ-01, -03 and -06 are -8.54, -6.49, and -8.58, respectively.
  • CInQ-inhibitors suppress anchorage-dependent and -independent cell growth.
  • the effect of the CInQ-inhibitors on colon cancer cell growth and on the growth of EGF-induced HaCaT keratinocytes was examined. Cell growth was measured using the MTS assay at 1, 2 or 3 days after treatment with EGF alone or EGF and individual CInQ-inhibitors. Results indicated that colon cancer cell growth was significantly decreased by the respective CInQ- inhibitors (Fig. 11 A). EGF-induced cell growth was also strongly suppressed by CInQ- inhibitors (Fig. 11B).
  • CInQ-inhibitors suppress activator protein-1 (AP-1) activity. It was then determined whether CInQ-inhibitors had an effect on activator protein-1 (AP-1) reporter activity in HCT116 cells or HaCaT cells stimulated with EGF. HCT116 cells were treated with individual CInQ-inhibitors for 48 h and HaCaT cells were pre-treated with CInQ-inhibitor for 2 h before stimulation with EGF for 12 or 24 h. AP-1 reporter activity was strongly suppressed by CInQ-inhibitors in colon cancer cells (Fig. 12 A) and in EGF-treated HaCaT cells (Fig. 12B). The effect of these inhibitors on downstream signaling of MEK in colon cancer cells and EGF-induced HaCaT cells was also examined.
  • HCT116 cells stably expressing mock (shGipz) or knockdown of MEK 1/2 (shMEKl/2) were established and analyzed MEK expression by Western blot (Fig. 23 A, 23, and 13 A).
  • the effect of inhibitors on growth of shGipz or shMEKl/2 colon cancer cells was assessed by MTS assay at 72 h. Results indicated that cells expressing shMEKl/2 were resistant to the anti-growth effect of the CInQ-inhibitors compared to s/iG/pz-expressing cells (Fig. 13B).
  • CInQ-03 inhibits colon cancer tumor growth in a xenograft mouse model. Based on the results described above, CInQ-03 was selected for further study in vivo. HCT116 colon cancer cells were injected into the flank of athymic nude mice and mice were treated with CInQ-03 at 1 or 5 mg/kg or vehicle 3 times a week over a period of 11 days after the average tumor volume grew to about 70 mm . Treatment of mice with 1 or 5 mg/kg of CInQ-03 strongly suppressed HCT116 tumor growth by over 70% relative to the vehicle-treated group (Fig. 14A, 14B, left panel; p ⁇ 0.05).
  • mice seemed to tolerate treatment with CInQ-03 without overt signs of toxicity or significant loss of body weight similar to the vehicle-treated group (Fig. 14B, right panel). Furthermore, the effects of CInQ-03 on a tumor proliferation marker were evaluated by immunohistochemistry and H&E staining of HCT116 tumor and skin tissues after 11 days of treatment. The expression of Ki67 was markedly decreased by treatment with CInQ-03 (Fig. 14C, upper panel). However, Ki67 expression in skin tissues in CInQ-03 treated tissues was similar to the vehicle-treated group (Fig. 14C, lower panel).
  • the CInQ inhibitors may provide clinical advantage such as reduced toxicities compared with current MEK inhibitors.
  • CInQ-03 is a novel and specific MEK inhibitor both in vitro and in vivo. These results should be useful for development of novel MEK inhibitors. Future studies will investigate the efficacy of CInQ-03 and pharmacological characterization and examination of dermatologic toxicities.
  • Example 4 Preparation of Compound 102:
  • the intermediate compound 101 was prepared as follows,
  • This compound was prepared according to the method described in Example 6, employing pyridine-2-acetonitrile (118 mg, 1 mmol) and 2, 3-dichloroquinoxaline (199 mg, 1 mmol).
  • Example 10 The following illustrate representative pharmaceutical dosage forms, containing a compound of formula I ('Compound X'), for therapeutic or prophylactic use in humans.

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Abstract

L'invention concerne l'utilisation d'inhibiteurs de TOPK pour traiter la perte de cheveux, ainsi que l'utilisation d'un composé de la formule (I), où R1- R8 et X ont l'une quelconque des valeurs définies par les présentes, ou un sel de qualité pharmaceutique de celui-ci, pour traiter la perte de cheveux ou le cancer.
PCT/US2013/030531 2012-03-15 2013-03-12 Composés inhibiteurs de topk WO2013138341A1 (fr)

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