AU2009215073A1 - Inhibitors of checkpoint kinases - Google Patents
Inhibitors of checkpoint kinases Download PDFInfo
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- AU2009215073A1 AU2009215073A1 AU2009215073A AU2009215073A AU2009215073A1 AU 2009215073 A1 AU2009215073 A1 AU 2009215073A1 AU 2009215073 A AU2009215073 A AU 2009215073A AU 2009215073 A AU2009215073 A AU 2009215073A AU 2009215073 A1 AU2009215073 A1 AU 2009215073A1
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- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/10—Spiro-condensed systems
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- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
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- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- 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
- C07D487/08—Bridged systems
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- 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
- C07D487/10—Spiro-condensed systems
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- C—CHEMISTRY; METALLURGY
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
Description
WO 2009/102537 PCT/US2009/031633 TITLE OF THE INVENTION INHIBITORS OF CHECKPOINT KINASES BACKGROUND OF THE INVENTION 5 Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions. They ensure that critical events such as DNA replication and chromosome segregation are completed in high fidelity. The regulation of these cell cycle checkpoints is a critical determinant of the manner in which tumor cells respond to many chemotherapies and radiation. Many effective cancer therapies work by causing DNA damage; 10 however, resistance to these agents remains a significant limitation in the treatment of cancer. Of the several mechanisms of drug resistance, an important one is attributed to the prevention of cell cycle progression through the control of critical activation of a checkpoint pathway. This arrests the cell cycle to provide time for repair, and induces the transcription of genes to facilitate repair, thereby avoiding immediate cell death. By abrogating checkpoint arrests at, for example, 15 the G2 checkpoint, it may be possible to synergistically augment tumor cell death induced by DNA damage and circumvent resistance. Human CHKl plays a role in regulating cell cycle arrest by phosphorylating the phosphatase cdc25 on Serine 216, which may be involved in preventing activation of cdc2/cyclin B and initiating mitosis. Therefore, inhibition of CHKI should enhance DNA damaging agents 20 by initiating mitosis before DNA repair is complete and thereby causing tumor cell death. It is an object of the instant invention to provide novel compounds that are inhibitors of CHK1 (also refered to as Chek1). It is also an object of the present invention to provide pharmaceutical compositions that comprise the novel compounds that are inhibitors of CHK1. 25 It is also an object of the present invention to provide a method for treating cancer that comprises administering such inhibitors of CHKI activity. SUMMARY OF THE INVENTION The instant invention provides for compounds which comprise substituted 30 thioquinazolinones that inhibit CHK1 activity. The invention also provides for compositions comprising such inhibitory compounds and methods of inhibiting CHKI activity by administering the compound to a patient in need of treatment of cancer. DETAILED DESCRIPTION OF THE INVENTION 35 The compounds of the instant invention are useful in the inhibition of the activity of CHK1. In a first embodiment of this invention, the inhibitors of CHK1 activity are illustrated by the Formula A: -I1- WO 2009/102537 PCT/US2009/031633 0 N R2 A N R3 S N(R4% 0 wherein: a is 0 or 1; b is 0 or 1; m is 0, 1, or 2; n is 0, 1,2,3,4,5 or 6; andp is 0, 1,2,3,4, 5 or 6; 5 Ring Z is selected from: aryl, heteroaryl, heterocyclyl, (C4-C8)cycloalkenyl and (C4-C8)cycloalkyl; No is selected from heteroaryl and heterocyclyl, including spirocyclic moieties;
R
1 is independently selected from: CF3, oxo, (C=O)aObC1-CIO alkyl, (C=O)aOb 10 aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=0)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m-(Cl C1O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
2 is selected from: Ci-C1o alkyl, C2-Clo alkenyl, C2-Cl0 alkynyl, CO2H, C3 15 C8 cycloalkyl, aryl and heterocyclyl, said alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
3 is selected from: H, CF3, OxO, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-ClO alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=O)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 RS, S(O)m-(Ci 20 C10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
4 is independently selected from: H, CF3, oxo, (C=O)aObC-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCI C6 perfluoroalkyl, (C=O)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m 25 (CI-CO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
6 is: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2 C1o alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl, Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=O)R 7
R
8 , S(O)mNR7R 8 , S(O)m-(C1-C1O)alkyl, SH or 30 (C=O)aObC3-CS cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6a; -2- WO 2009/102537 PCT/US2009/031633 R6a is selected from: CF3, (C=O)aOb(Cl-C1o)alkyl, Oa(Cl-C3)perfluoroalkyl, (Co-C6)alkylene-S(O)mRa, oxo, OH, halo, CN, (C2-Clo)alkenyl, (C2-C10)alkynyl, (C3 C6)cycloalkyl, (CO-C6)alkylene-aryl, (CO-C6)alkylene-heterocyclyl, (C0-C6)alkylene-N(Rb)2, C(O)Ra, (C0-C6)alkylene-CO2Ra, C(O)H, and (C0-C6)alkylene-CO2H, said alkyl, alkenyl, 5 alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (C1 -C6)alkoxy, halogen, CO2H, CN, O(C=O)CI -C6 alkyl, oxo, and N(Rb)2;
R
7 and R 8 are independently selected from: H, (C=O)ObC1-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, Ct-C1o alkyl, aryl, C2-C1o 10 alkenyl, C2-Clo alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb 2 , said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R6a, or R 7 and R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected 15 from N, 0 and S, said monocylcic or bicyclic heterocycle optionally substituted with one or more substituents selected from R6a; Ra is H, (C1-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocycly; and Rb is independently H, (CI-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCI-C6 alkyl, (C=O)CI-C6 alkyl or S(O)mRa; 20 or a pharmaceutically acceptable salt or a stereoisomer thereof. In a second embodiment of this invention, the inhibitors of CHK1 activity are illustrated by the Formula B: O N (R 5)g (Rikn B N S (R4)p R N 0 wherein: 25 N is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; q is 0, 1, 2, 3, 4 or 5;
R
5 is independently selected from: CF3, oxo, (C=O)aObCI-Cl0 alkyl, (C=O)aObaryl, C2-Co alkenyl, C2-Co alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, 30 ObC1-C6 perfluoroalkyl, Oa(C=O)bNR 7 R8, oxo, CHO, (N=O)R7R 8 , S(O)mNR 7
R
8 , S(O)m -3 - WO 2009/102537 PCT/US2009/031633 (Cl-C10)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6a; and all substituents and variables are as defined in the first embodiment; 5 or a pharmaceutically acceptable salt or a stereoisomer thereof. In a third embodiment of this invention, the inhibitors of CHKI activity are illustrated by the Formula B, wherein: No is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; 10 ais0orl;bis0orl;mis0, l,or2;nis0,1,2,3,4or5; pis0,l,2,3,4,5or 6; and q is 0, 1, 2, 3, 4 or 5; R1 is independently selected from: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C1O alkenyl, (C=O)aObC2-C10 alkynyl, C02H, halo, OH, ObCl-C6 perfluoroalkyl, (C=0)aNR 7 R8, CN, (C=O)aObC3-CS cycloalkyl, S(O)mNR 7 R8, S(O)m-(Cl 15 C1 o)alkyl, and (C=)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R6;
R
5 is independently selected from: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7 R8, oxo, CHO, (N=O)R 7 R8, S(O)mNR7R 8 , S(O)m 20 (Cl-C1O)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6;
R
3 is selected from: H, CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C1O alkenyl, (C=O)aObC2-Clo alkynyl, CO2H, halo, OH, ObCl-C6 25 perfluoroalkyl, (C=O)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m-(Cl C 10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
4 is independently selected from: H, CF3, oxo, (C=O)aObC1-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-Co alkynyl, CO2H, halo, OH, ObCl 30 C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O) 1 mNR 7
R
8 , S(O)m (CI-CO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
6 is: CF3, oxo, (C=0)aObCl-C10 alkyl, (C=0)aObaryl, C2-Cl0 alkenyl, C2 C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, 35 Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=O)R 7 R8, S(O)mNR 7 R8, S(O)m-(Cl-C10)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; -4- WO 2009/102537 PCT/US2009/031633
R
7 and R 8 are independently selected from: H, (C=O)ObC1-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, CI-C0 alkyl, aryl, C2-Clo alkenyl, C2-Clo alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb 2 , or R 7 and R8 can be taken together with the nitrogen to which they are attached to form a monocyclic or 5 bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (C1 -C6)alkyl; and Rb is independently H or (C I-C6)alkyl; or a pharmaceutically acceptable salt or a stereoisomer thereof. 10 In a fourth embodiment of this invention, the inhibitors of CHK1 activity are illustrated by the Formula B, wherein: Ni is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; aisOor l;bis Oorl;mis 0, 1,or2;nis0, 1,2,3,4or5; pisO, 1,2,3,4, 5or 15 6; and q is 0, 1, 2, 3, 4 or 5; R1 is independently selected from: CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-Co alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCI-C6 perfluoroalkyl, (C=O)aNR 7 R8, CN, (C=O)aObC3-CS cycloalkyl, S(O)mNR 7 R8, S(O)m-(Ci CiO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and 20 heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
5 is independently selected from: CF3, oxo, (C=O)aObC1-C10 alkyl, (C=O)aObaryl, C2-Cl0 alkenyl, C2-Cl0 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=O)R 7
R
8 , S(O)mNR 7 R8, S(O)m (CI-CO)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, 25 heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6;
R
3 is: Cl-C6 alkyl, said alkyl is optionally substituted with one or more substituents selected from R 6 ;
R
4 is independently selected from: H, CF3, oxo, (C=O)aObCl-C10 alkyl, 30 (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCI C6 perfluoroalkyl, (C=0)aNR 7
R
8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7
R
8 , S(O)m (C I-C IO)alkyl, and (C=0)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
6 is: CF3, oxo, (C=0)aObCl-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2 35 C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, -5- WO 2009/102537 PCT/US2009/031633 Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=0)R 7
R
8 , S(O)mNR 7
R
8 , S(O)m-(C-C10)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; R7 and R 8 are independently selected from: H, (C=O)ObCl-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, CI-C1o alkyl, aryl, C2-ClO 5 alkenyl, C2-Clo alkynyl, heterocyclyl, C3-Cg cycloalkyl, S(O)mRa, and (C=O)NRb 2 , or R 7 and R8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (Cl-C6)alkyl; and 10 Rb is independently H or (Cj -C6)alkyl; or a pharmaceutically acceptable salt or a stereoisomer thereof In a fifth embodiment of this invention, the inhibitors of CHKI activity are illustrated by the Formula B, wherein: N is selected from heteroaryl and heterocyclyl, including spirocyclic 15 moieties; ais0orl;bis0orl;mis0,l,or2;nislor2; pis0,1,2,3,4,5or6;andqis I or2;
R
1 is independently selected from: H, S(O)2-(Cl-C6)alkyl, (C1-C6)alkyl, and (C=O)O(C1-C6)alkyl; 20 R 5 is independently selected from: H, halo, C3-Cg cycloalkyl, C3-C8 cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl are optionally substituted with one or more substituents selected from R 6 ;
R
3 is: Cl-C6 alkyl, said alkyl is optionally substituted with one or more substituents selected from R 6 ; 25 R 4 is independently selected from: H, CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-ClO alkenyl, (C=O)aObC2-C1O alkynyl, CO2H, halo, OH, ObCl C6 perfluoroalkyl, (C=O)aNR 7
R
8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m (CI-CO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; 30 R 6 is: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aObaryl, C2-Co alkenyl, C2 C10 alkynyl, (C=0)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl, Oa(C=O)bNR 7 R8, oxo, CHO, (N=O)R 7
R
8 , S(O)mNR 7
R
8 , S(O)m-(Cl-C10)alkyl, SH or (C=O)aObC3-C8 cycloalkyl;
R
7 and R 8 are independently selected from: H, (C=O)ObCl-Clo alkyl, 35 (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-Clo alkyl, aryl, C2-Clo alkenyl, C2-C1O alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb 2 , or R 7 and -6- WO 2009/102537 PCT/US2009/031633 R8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (CI-C6)alkyl; and 5 Rb is independently H or (C1 -C6)alkyl; or a pharmaceutically acceptable salt or a stereoisomer thereof. Specific compounds of the instant invention include: 3-(3-chlorophenyl)-2- {[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-8 methylquinazolin-4(3H)-one (1-8); 10 1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl}hexanoyl)piperidine-3 carboxamide (1-9); 2-{2-[(4-acetylpiperazin-1-yl)carbonyl]hexyl}-3-(3-chlorophenyl)-quinazolin-4(3H)-one (1-10); 3-(3-chlorophenyl)-2-{2-[(4-phenylpiperazin-1-yl)carbonyl]hexyl}quinazolin-4(3H)-one (1-11); 1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yljmethyl } hexanoyl)-N,N 15 diethylpiperidine-3-carboxamide (1-12); 3-(3-chlorophenyl)-2- (2-[(4-pyridin-2-ylpiperazin-1-yl)carbonyl]hexyl}-quinazolin-4(3I)-one (1-13); 2-(2-{[4-(aminomethyl)-piperidin-1-yl]carbonyl}-hexyl)-3-(3-chloro-phenyl)quinazolin-4(3H) one (1-14); 20 3-(3-chlorophenyl)-2- {2-[(7-methyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]hexyl}quinazolin 4(3H)-one (1-15); 2-(2-{[4-(1H-benzimidazol-2-yl)piperidin-1 -yl]carbonyl}hexyl)-3-(3-chlorophenyl)quinazolin 4(3H)-one (1-16); 3-(3-chlorophenyl)-2- {2-[(1,l -dioxidothio-morpholin-4-yl)-carbony]hexyl} quinazolin-4(3H) 25 one (1-17); 3-(3-chlorophenyl)-2-(2-{[4-(methylsulfonyl)-piperazin-1-yl]carbonyl}-hexyl)quinazolin-4(3H) one (1-18); 3-(3-chlorophenyl)-2-(2-{[4-(IH-imidazol-1-ylmethyl)piperidin-1-yljcarbonyl}hexyl)quinazolin 4(3H)-one (1-19); 30 3 -(3 -chlorophenyl)-2-(2- { [4-(methylsulfonyl)-piperi din-i -yl] carbonyl} -hexyl)quinazolin-4(3H) one (1-20); 3-(3-chlorophenyl)-2-[2-(2,8-diazaspiro[5.5}-undec-2-ylcarbonyl)-hexyllquinazolin-4(3H)-one (1-21); 3-(3-chlorophenyl)-2-(2-{[4-(2-fluorophenyl)-piperazin-1 -yl]carbonyl}-hexy)quinazolin-4(3H) 35 one (1-22); 3-(3-chlorophenyl)-2-(2-{[4-(4-fluorophenyl)-piperazin-1-yl]carbonyl}-hexyl)quinazolin-4(3H) one (1-23); -7- WO 2009/102537 PCT/US2009/031633 3-(3-chlorophenyl)-2-{2-[(4,4-diphenylpiperidin-1-yl)carbonyllhexyl}-quinazolin-4(3H)-one (1 24); 3-(3-chlorophenyl)-2- {2-[(4-pyridin-4-ylpiperidin-1 -yl)carbonyl]hexyl}-quinazolin-4(3H)-one (1-25); 5 3-(3-chlorophenyl)-2-{2-[(3,3-diphenylpiperidin-1-yl)carbonyl]hexyl}-quinazolin-4(3H)-one (1 26); 3-(3-chlorophenyl)-2- { [1 -(2,8-diazaspiro[5.5]-undec-2-ylcarbonyl)-pentyl]thio}-7-(methyl sulfony1)quinazolin-4(3H)-one (1-27); methyl 3-(3-chloro-phenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-4-oxo 10 3,4-dihydro-quinazoline-7-carboxylate (1-28); 3-(3-chlorophenyl)-2-({3-methyl-1-[(4-pyridin-2-ylpiperazin-1 -yl) carbonyllbutyl}thio)quinazolin-4(3H)-one (1-29); 3-(3-chlorophenyl)-2-[(1-{[4-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazin-1-yl]carbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-30); 15 3-(3-chloropheny)-2-[(1-{[4-(4-fluorophenyl)-piperazin-1-yl]carbonyl}-3-methylbutyl)thio] quinazolin-4(3H)-one (1-31); 3-(3-chlorophenyl)-2-[(1-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-32); 3-(3-chlorophenyl)-2-[(1- {[7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-ylJcarbonyl}-3 20 methylbutyl)thioquinazolin-4(3H)-one (1-33); 2-({ 1 -[(7-benzyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]-3-methylbutyl}thio)-3-(3 chlorophenyl)quinazolin-4(3H)-one (1-34); 3-(3-chloropheny)-2-[(1- {[7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-yljcarbonyl}-3 methy1butyl)thio]quinazolin-4(3H)-one (1-35); 25 3-(3-chlorophenyl)-2-[(1- {[7-(4-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-36); 1-(2-{[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (1-37); 1-( {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}(phenyl)acetyl]piperidine-4 30 carboxamide (1-38); 1-(2-{[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}-4-methylpentanoyl) piperidine-4-carboxamide (1-39); 3-(3-chlorophenyl)-2-[( 1- {[7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yllcarbonyl} pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-40); 35 3-(3-chlorophenyl)-2-[(1-{[7-(4-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}pentyl) thio}-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-41); 3-(3-chlorophenyl)-2-[(1-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-y1]carbony1}pentyl) thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-42); -8- WO 2009/102537 PCT/US2009/031633 3-(3-chlorophenyl)-7-(methylsulfonyl)-2-({1-[(7-phenyl-2,7-diazaspiro{4.4]non-2-y)carbony] pentyl}thio)quinazolin-4(3H)-one (1-43); 3-(3-chlorophenyl)-2-[(1- { [7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-ylcarbonyl} pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-44); 5 methyl 3-(3-chlorophenyl)-2-({1-[(3-cyano-3-phenylazetidin-1-yl)carbonyl]pentyl}-thio)-4-oxo 3,4-dihydroquinazoline-7-carboxylate (1-45); methyl 3-(3-chlorophenyl)-2-{[1-(2,6-diazaspiro[3.3]hept-2-ylcarbonyl)pentyl]thio}-4-oxo-3,4 dihydro-quinazoline-7-carboxylate (1-46); 3-(3-chlorophenyl)-2-[(1-{[(2R)-2-(trifluoro-methyl) pyrrolidin-1-yl]-carbonyl} pentyl)thio] 10 quinazolin-4(3H)-one (1-47); 3-(3-chlorophenyl)-2-[(1- {[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl} pentyl)thio]quinazolin-4(3H)-one (1-48); 3-(3-chlorophenyl)-2-[(1-{ [5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1]hept-2-y1]carbonyl} pentyl)-thio]quinazolin-4(3H)-one (1-49); 15 3-(3-chlorophenyl)-2-[(1- {[5-(3-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-yl]carbonyl} pentyl)-thio]quinazolin-4(3H)-one (1-50); 3-(3-chlorophenyl)-2-[(1-([4-(4-methyl-1,2,5-oxadiazol-3-yl)piperazin-1-yl]carbonyl}pentyl) thio]quinazolin-4(3H)-one (1-51); 3-(3-chlorophenyl)-2-[(1-{ [3-(4-fluorophenyl)-1-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl}pentyl) 20 thio]quinazolin-4(3H)-one (1-52); 3-(3-chlorophenyl)-2-[(1-{[(3S,4S)-3,4-difluoropyrrolidin-1-yl]carbonyl)pentyl)thio]quinazolin 4(3H)-one (1-53); 3-(3-chlorophenyl)-2- {[2-(2,8-diazaspiro(5.5]undec-2-yl)-1-(1H-imidazol-4-ylmethyl)-2 oxoethyl]thio}quinazolin-4(3H)-one (1-54); 25 2-(3-(1H-imidazol-4-yl)-1-oxo-1-(2,8-diazaspiro[5.5]undecan-2-yl)propan-2-ylthio)-3-(3 chlorophenyl)-8-methylquinazolin-4(3H)-one (1-55); 3-(3-chlorophenyl)-8-methyl-2-(1-oxo-I-(4-phenylpiperazin-1-yl)hexan-2-ylthio)quinazolin 4(3H)-one (1-56); 3-(3-chlorophenyl)-2-(1-(7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxohexan-2 30 ylthio)-8-methylquinazolin-4(3H)-one (1-57); 3-(3-chlorophenyl)-2-(1-(7-(4-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)- 1 -oxohexan-2 ylthio)-8-methylquinazolin-4(3H)-one (1-58); 3-(3-chlorophenyl)-2-(1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxohexan-2 ylthio)-8-methylquinazolin-4(3H)-one (1-59); 35 3'-[2-[(1-{[4-(Amino-carbonyl)piperidin-1-yl]carbonyl}pentyl)thio]-4-oxoquinazolin-3(4H) yl]biphenyl-4-carboxylic acid (2-3); 1-(2- {[3-(2'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-4); -9- WO 2009/102537 PCT/US2009/031633 1-(2- { [3-(3'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-5); 1-(2- {[3-(4'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine- 4 carboxamide (2-6); 5 1-(2- {[3-(2'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio)hexanoyl)piperidine-4 carboxamide (2-7); 1-(2-{[3-(3'-aminobiphenyl-3-y)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoy1)piperidine- 4 carboxamide (2-8); 1-(2- ([3-(4'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 10 carboxamide (2-9); 1-(2- {[4-oxo-3-(3-pyridin-3-ylphenyl)-3,4-dihydroquinazolin-2-yl]thio)hexanoyl)piperidine-4 carboxamide (2-10); 1-(2- ([4-oxo-3-(3-pyridin-4-ylphenyl)-3,4-dihydroquinazolin-2-yl]thio)hexanoyl)piperidine-4 carboxamide (2-11); 15 3'-[2-[(1- {[4-(aminocarbonyl)piperidin-1 -yl]carbonyl}pentyl)thio]-4-oxoquinazolin-3(4H) yl]biphenyl-3-carboxylic acid (2-12); 1-[2-({3-[3-(3-fury1)phenyl]-4-oxo-3,4-dihydroquinazolin-2-yl}thio)hexanoyl]piperidine-4 carboxamnide (2-13); 1-[2-({3-[3-(1-methyl-1H-pyrazol-4-y1)phenyl]-4-oxo-3,4-dihydroquinazolin-2-yl)thio) 20 hexanoyl]piperidine-4-carboxamide (2-14); 1-(2-{[3-(3-cyclohex-1-en-1-ylphenyl)-4-oxo-3,4-dihydroquinazolin-2-y1]thio}hexanoyl) piperidine-4-carboxamide (2-15); 1-[2-({4-oxo-3-[3-(2-thienyl)phenyl]-3,4-dihydroquinazolin-2-yl)thio)hexanoyl]piperidine-4 carboxamide (2-16); 25 2-[(1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}-3-methylbutyl)thio]-3-[3-(3 furyl)pheny1Jquinazolin-4(3H)-one (2-17); 2-[(1-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yllcarbonyl}-3-methylbutyl)thio]-3-(3 cyclohex- 1-en-i -ylphenyl)quinazolin-4(3H)-one (2-18); 1-(2-{ [3-(4-fluoro-2'-methybiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-yljthio~hexanoyl) 30 piperidine-4-carboxamide (2-19); 2-[( 1-{ [5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-yJ]carbonyl}pentyl)thio]-3-[3-(3 furyl)phenyl]quinazolin-4(3H)-one (2-20); 2-[(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl}pentyl)thio]-3-[3-(3 furyl)phenyl]quinazolin-4(3H)-one (2-21); 35 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[5-(4-fluorophenyl)-2,5-diazabicyclo{2.2.1]hept-2 yl]carbonyllpentyl)thio]quinazolin-4(3H)-one (2-22); 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8 yl]carbonyl)pentyl)thio]quinazolin-4(3H)-one (2-23); - 10- WO 2009/102537 PCT/US2009/031633 3-(3-cyclohex- 1-en-i -ylphenyl)-2-[(1- {[4-(2,4-difluorophenyl)piperazin-1 yl]carbonyl}pentyl)thio]quinazolin-4(3H)-one (2-24); 2-[(1-{[4-(2,4-difluorophenyl)piperazin-1-yl]carbonyl}pentyl)thio)-3-[3-(3-furyl) phenyl] quinazolin-4(3H)-one (2-25); 5 3-(3-cyclohex-1-en-I -ylphenyl)-2-{ [I -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl] thio}quinazolin-4(3H)-one (2-26); 2-([3-amino-1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)propyl]thio)-3-(3-chlorophenyl) quinazolin-4(3H)-one (3-7); 2-{[4-amino-i -(2,8-diazaspiro [5.5]undec-2-ylcarbony1)butyl]thio} -3-(3-chlorophenyl)-8 10 methylquinazolin-4(3H)-one (3-8); 2-{[5-amino-i -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-3-(3-chlorophenyl) quinazolin-4(311)-one (3-9); 2-[(4-amino-i - { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}butyl)thio]-3-(3 chlorophenyl)quinazolin-4(3H)-one (3-10); 15 2-(5-amino-1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxopentan-2-ylthio)-3-(3 chlorophenyi)-8-methylquinazolin-4(3H)-one (3-11); 2-[(5-amino- 1- ([7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-ylcarbonyl)pentyl)thio]-3-(3 chlorophenyl)quinazolin-4(3H)-one (4-6); and 2-{[5-amino-1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-3-(3-chlorophenyl)-8 20 methylquinazolin-4(3H)-one (4-7); or a pharmaceutically acceptable salt or a stereoisomer thereof. TFA salts of the compounds of the instant invention include: 3-(3-chlorophenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-8 methylquinazolin-4(3H)-one (1-8); 25 1-(2- { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl}hexanoyl)piperidine-3 carboxamide (1-9); 2- (2-[(4-acetylpiperazin-1 -yl)carbonyl]hexyl}-3-(3-chlorophenyl)-quinazolin-4(3H)-one (1-10); 3-(3-chlorophenyl)-2-{2-[(4-phenylpiperazin-1-yl)carbonyljhexyli}quinazolin-4(3H)-one (1-11); 1-(2-{[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yllmethyl}hexanoyl)-NN 30 diethylpiperidine-3-carboxamide (1-12); 3-(3-chloropheny1)-2-(2-{(4-pyridin-2-ylpiperazin-1-yl)carbonyljhexyl)-quinazolin-4(3H)-one (1-13); 2-(2-{[4-(aminomethyl)-piperidin-1-y1]carbonyl}-hexyl)-3-(3-chloro-phenyl)quinazolin-4(3H) one (1-14); 35 3-(3-chlorophenyl)-2-{2-[(7-methyl-2,7-diazaspiro[4.4]non-2-y1)carbonyl]hexyl}quinazolin 4(3H)-one (1-15); 2-(2- {[4-(1H-benzimidazol-2-y1)piperidin-1-yl]carbonyl}hexyl)-3-(3-chlorophenyl)quinazolin 4(3H)-one (1-16); - 11 - WO 2009/102537 PCT/US2009/031633 3-(3-chlorophenyl)-2- {2-[(1,1 -dioxidothio-morpholin-4-yl)-carbonyljhexyl}quinazolin-4(3H) one (1-17); 3-(3-chlorophenyl)-2-(2-{[4-(methylsulfonyl)-piperazin-1 -yl]carbonyl}-hexyl)quinazolin-4(3H) one (1-18); 5 3-(3-chlorophenyl)-2-(2-{ [4-(IH-imidazol-1-ylmethyl)piperidin-1-yl]carbonyl}hexyl)quinazolin 4(3H)-one (1-19); 3-(3-chlorophenyl)-2-(2-{[4-(methylsulfonyl)-piperidin-1-yl]carbonyl}-hexyl)quinazolin-4(3H) one (1-20); 3-(3-cblorophenyl)-2-[2-(2,8-diazaspiro[5.5]-undec-2-ylcarbonyl)-hexyl]quinazolin-4(3H)-one 10 (1-21); 3-(3-chlorophenyl)-2-(2-{[4-(2-fluorophenyl)-piperazin-1-yl carbonyl}-hexyl)quinazolin-4(3H) one (1-22); 3-(3-chlorophenyl)-2-(2-{[4-(4-fluorophenyl)-piperazin-1-yl]carbonyl}-hexyl)quinazolin-4(3H) one (1-23); 15 3-(3-chlorophenyl)-2-{2-[(4,4-diphenylpiperidin-1-yl)carbonyl]hexyl}-quinazolin-4(3H)-one (1 24); 3-(3-chlorophenyl)-2-{2-[(4-pyridin-4-ylpiperidin-1-yl)carbonyl]hexyl}-quinazolin-4(3H)-one (1-25); 3-(3-chlorophenyl)-2-{2-[(3,3-diphenylpiperidin-1-yl)carbonyljhexyl)-quinazolin-4(3H)-one (1 20 26); 3-(3-chlorophenyl)-2-([1-(2,8-diazaspiro[5.5]-undec-2-ylcarbonyl)-pentyl]thio)-7-(methyl sulfonyl)quinazolin-4(3H)-one (1-27); methyl 3-(3-chloro-phenyl)-2- { [1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyljthio}-4-oxo 3,4-dihydro-quinazoline-7-carboxylate (1-28); 25 3-(3-chlorophenyl)-2-({3-methyl-1-[(4-pyridin-2-ylpiperazin-1-yl) carbonyl]butyl)thio)quinazolin-4(3H)-one (1-29); 3-(3-chlorophenyl)-2-[(1-{[4-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazin-1-yl]carbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-30); 3-(3-chlorophenyl)-2-[(1-{[4-(4-fluorophenyl)-piperazin-1-yljcarbonyl}-3-nethylbutyl)thio] 30 quinazolin-4(31)-one (1-31); 3-(3-chlorophenyl)-2-[(1-{ [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-32); 3-(3-chlorophenyl)-2-[(1-{[7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-ylIcarbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-33); 35 2-({1-[(7-benzyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]-3-methylbutyl}thio)-3-(3 clorophenyl)quinazolin-4(3H)-one (1-34); 3-(3-chloropheny)-2-[(1-{[7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-ylcarbonyl}-3 methylbutyl)thio]quinazolin-4(3H)-one (1-35); - 12 - WO 2009/102537 PCT/US2009/031633 3-(3-chlorophenyl)-2-[(1- {[7-(4-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl} -3 methylbutyl)thio]quinazolin-4(3H)-one (1-36); 1-(2- {{3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (1-37); 5 1-[ { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}(phenyl)acetyl]piperidine-4 carboxamide (1-38); 1-(2- { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio} -4-methylpentanoyl) piperidine-4-carboxamide (1-39); 3-(3-chlorophenyl)-2-[(1-{[7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl} 10 pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-40); 3-(3-chlorophenyl)-2-[( 1- {[7-(4-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}pentyl) tbio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-41); 3-(3-chlorophenyl)-2-[(1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-y]carbonyl)pentyl) thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-42); 15 3-(3-chlorophenyl)-7-(methylsulfonyl)-2-({ 1-[(7-phenyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl] pentyl}thio)quinazolin-4(3H)-one (1-43); 3-(3-chlorophenyl)-2-[( 1- {[7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl} pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-44); methyl 3-(3-chlorophenyl)-2-({1-[(3-cyano-3-phenylazetidin-1-yl)carbonyl]pentyl}-thio)-4-oxo 20 3,4-dihydroquinazoline-7-carboxylate (1-45); methyl 3-(3-chlorophenyl)-2-{[1-(2,6-diazaspiro[3.3]hept-2-ylcarbonyl)pentyl]thio}-4-oxo-3,4 dihydro-quinazoline-7-carboxylate (1-46); 3-(3-chlorophenyl)-2-[(1-{[(2R)-2-(trifluoro-methyl) pyrrolidin-l-yl]-carbonyl} pentyl)thio] quinazolin-4(3H)-one (1-47); 25 3-(3-chlorophenyl)-2-[(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl} pentyl)thio]quinazolin-4(3H)-one (1-48); 3-(3-chlorophenyl)-2-[(1-{[5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1]hept-2-yl]carbonyl} pentyl)-thio]quinazolin-4(3H)-one (1-49); 3-(3-chlorophenyl)-2-[(1-{[5-(3-fluorophenyl)-2,5-diazabicyclo[2.2.1]hept-2-yl]carbonyl} 30 pentyl)-thio]quinazolin-4(3H)-one (1-50); 3-(3-chlorophenyl)-2-[( 1-{[4-(4-methyl-1,2,5-oxadiazol-3-yl)piperazin-1-yl]carbonyl}pentyl) thio]quinazolin-4(3H)-one (1-51); 3-(3-chlorophenyl)-2-[(1-{[3-(4-fluorophenyl)-1-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl}pentyl) thio]quinazolin-4(3H)-one (1-52); 35 3-(3-chlorophenyl)-2-[(1-{ [(3S,4S)-3,4-difluoropyrrolidin-1-yl]carbonyl}pentyl)thio]quinazolin 4(3H)-one (1-53); 3-(3-chlorophenyl)-2-{[2-(2,8-diazaspiro[5.5]undec-2-yl)-1-(1 H-imidazol-4-ylmethyl)-2 oxoethyl]thio)quinazolin-4(3H)-one (1-54); - 13 - WO 2009/102537 PCT/US2009/031633 2-(3-(1 H-imidazol-4-yl)- 1 -oxo- 1 -(2,8-diazaspiro[5.5)undecan-2-yl)propan-2-ylthio)-3-(3 chlorophenyl)-8-methylquinazolin-4(3H)-one (1-55); 3-(3-chlorophenyl)-8-methyl-2-(1-oxo-1-(4-phenylpiperazin-1-y1)hexan-2-ylthio)quinazolin 4(3H)-one (1-56); 5 3-(3-chlorophenyl)-2-(I-(7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]nonan-2-yl)- 1 -oxohexan-2 yltbio)-8-methylquinazolin-4(3H)-one (1-57); 3-(3-chlorophenyl)-2-(1-(7-(4-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1 -oxohexan-2 ylthio)-8-methylquinazolin-4(3H)-one (1-58); 3-(3-chlorophenyl)-2-(1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxohexan-2 10 ylthio)-8-methylquinazolin-4(3H)-one (1-59); 3'-[2-[(1-{[4-(Anino-carbonyl)piperidin-I -yl]carbonyl}pentyl)thio]-4-oxoquinazolin-3(4H) yl]biphenyl-4-carboxylic acid (2-3); 1-(2-{[3-(2'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-y1]thio}hexanoyl)piperidine-4 carboxamide (2-4); 15 1-(2-{[3-(3'-chlorobiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-5); 1-(2- {[3-(4'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-6); 1-(2-{[3-(2'-aminobiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-y1]thio}hexanoy1)piperidine-4 20 carboxamide (2-7); 1-(2-{ [3-(3'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-8); 1-(2- {[3-(4'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio)hexanoyl)piperidine-4 carboxamide (2-9); 25 1-(2-{[4-oxo-3-(3-pyridin-3-ylphenyl)-3,4-dihydroquinazolin-2-yljthio}hexanoyl)piperidine-4 carboxamide (2-10); 1-(2- {[4-oxo-3-(3-pyridin-4-ylphenyl)-3,4-dihydroquinazolin-2-yl]thio}hexanoyl)piperidine-4 carboxamide (2-11); 3'-{2-[(1- {[4-(aminocarbonyl)piperidin-1-yl]carbonyl}pentyl)thio]-4-oxoquinazolin-3(4H) 30 yljbiphenyl-3-carboxylic acid (2-12); 1-[2-({3-[3-(3-fiiryl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-yl}thio)hexanoyl]piperidine-4 carboxamide (2-13); 1-[2-({3-{3-(1-methyl-1H-pyrazol-4-yl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-yl)thio) hexanoyl]piperidine-4-carboxamide (2-14); 35 1-(2-{[3-(3-cyclohex-1-en-1-ylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl) piperidine-4-carboxamide (2-15); 1-[2-({4-oxo-3-[3-(2-thienyl)phenyl]-3,4-dihydroquinazolin-2-yl}thio)hexanoyl]piperidine-4 carboxamide (2-16); -14- WO 2009/102537 PCT/US2009/031633 2-[(1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl} -3-methylbutyl)thio]-3-[3-(3 furyl)pheny1]quinazolin-4(3H)-one (2-17); 2-[(1-{[7-(3-chlorophenyl)-2,7-diazaspiro{4.4]non-2-y1]carbonyl}-3-methylbutyl)thiol-3-(3 cyclohex-1-en-l -ylphenyl)quinazolin-4(3H)-one (2-18); 5 1-(2-{[3-(4'-fluoro-2'-methylbiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoyl) piperidine-4-carboxamide (2-19); 2-[(1- {[5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-yljcarbonyl)pentyl)thio]-3-{3-(3 furyl)phenyl]quinazolin-4(3H)-one (2-20); 24(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro{4.5]dec-8-yljcarbonyl)pentyl)thiol-3-[3-(3 10 furyl)phenyl]quinazolin-4(3H)-one (2-21); 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[5-(4-fluorophenyl)-2,5-diazabicyclo{2.2.1]hept-2 yl]carbonyl}pentyl)thio]quinazolin-4(3H)-one (2-22); 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8 yl]carbonyl}pentyl)thiolquinazolin-4(3H)-one (2-23); 15 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(I- { [4-(2,4-difluorophenyl)piperazin-1 yljcarbonyl}pentyl)thio]quinazolin-4(3H)-one (2-24); 2-[(1-{[4-(2,4-difluorophenyl)piperazin-1-yl]carbonyl}pentyl)thio]-3-[3-(3-furyl) phenyl] quinazolin-4(3H)-one (2-25); 3-(3-cyclohex-1-en-1 -ylphenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl] 20 thio}quinazolin-4(3H)-one (2-26); 2-{[3-amino-i -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)propyl]thio}-3-(3-chlorophenyl) quinazolin-4(3H)-one (3-7); 2- ([4-amino-i -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)butyl]thio) -3-(3-chlorophenyl)-8 methylquinazolin-4(3H)-one (3-8); 25 2- (5-amino-I -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-3-(3-chlorophenyl) quinazolin-4(3H)-one (3-9); 2-{(4-amino- 1- { [7-(3-chlorophenyl)-2,7-diazaspiro{4.4]non-2-yl]carbonyl}butyl)thio]-3-(3 chlorophenyl)quinazolin-4(3H)-one (3-10); 2-(5-amino- 1 -(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)- I -oxopentan-2-ylthio)-3-(3 30 chlorophenyl)-8-methylquinazolin-4(3H)-one (3-11); 2-[(5-amino-I-{ [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-ylcarbonyl}pentyl)thio]-3-(3 chlorophenyl)quinazolin-4(3H)-one (4-6); and 2- ([5-amino-I -(2,8-diazaspiro [5.5]undec-2-ylcarbonyl)pentyljthio } -3-(3 -chlorophenyl)-8 methy1quinazolin-4(3H)-one (4-7); 35 or a stereoisomer thereof The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: EL. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, - 15 - WO 2009/102537 PCT/US2009/031633 racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention. In addition, the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is 5 depicted. When any variable (e.g. R1, R 6 , R 6 a, etc.) occurs more than one time in any constituent, its definition on each occurrence is independent at every other occurrence. Also, combinations of substituents and variables are pennissible only if such combinations result in stable compounds. Lines drawn into the ring systems from substituents indicate that the 10 indicated bond may be attached to any of the substitutable ring atoms. If the ring system is bicyclic, it is intended that the bond be attached to any of the suitable atoms on either ring of the bicyclic moiety. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that 15 are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. The phrase "optionally substituted with one or more substituents" should be taken to be equivalent to the 20 phrase "optionally substituted with at least one substituent" and in such cases the preferred embodiment will have from zero to three substituents. It is understood that one or more silicon (Si) atoms can be incorporated into the compounds of the instant invention in place of one or more carbon atoms by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by 25 techniques known in the art from readily available starting materials. Carbon and silicon differ in their covalent radius leading to differences in bond distance and the steric arrangement when comparing analogous C-element and Si-element bonds. These differences lead to subtle changes in the size and shape of silicon-containing compounds when compared to carbon. One of ordinary skill in the art would understand that size and shape differences can lead to subtle or 30 dramatic changes in potency, solubility, lack of off target activity, packaging properties, and so on. (Diass, J. 0. et al Organometallics (2006) 5:1188-1198; Showell, GA. et al. Bioorganic & Medicinal Chemistry Letters (2006) 16:2555-2558). As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For 35 example, Cl-C10, as in "CI-C10 alkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, "CI-Clo alkyl" specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on. -16- WO 2009/102537 PCT/US2009/031633 The term "spirocyclic" means two rings which are conjoined at a single carbon atom, for example: N -N NNN N N N d N-~ andN ,NCF The term "cycloalkyl" means a monocyclic or bicyclic saturated aliphatic 5 hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" inlcudes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. The term "cycloalkenyl" means a monocyclic or bicyclic partially unsaturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, 10 "cycloalkenyl" inicudes cyclopropenyl, methyl-cyclopropenyl, 2,2-dimethyl-cyclobutenyl, 2 ethyl-cyclopentenyl, cyclohexenyl, and so on. "Alkoxy" represents either a cyclic or non-cyclic alkyl group of indicated number of carbon atoms attached through an oxygen bridge. "Alkoxy" therefore encompasses the definitions of alkyl and cycloalkyl above. 15 If no number of carbon atoms is specified, the term "alkenyl" refers to a non aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl groups include 20 ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three 25 carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl, 3 methylbutynyl and so on. The straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. In certain instances, substituents may be defined with a range of carbons that 30 includes zero, such as (CO-C6)alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as -CH2Ph, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph, and so on. As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, 35 anthryl or acenaphthyl. - 17- WO 2009/102537 PCT/US2009/031633 The term heteroaryl, as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from I to 4 heteroatoms selected from the group consisting of 0, N and S. Heteroaryl groups within the scope of this definition include: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, 5 indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofaranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. Attachment of a heteroaryl substituent can occur via a carbon atom or via a heteroatom. 10 As appreciated by those of skill in the art, "halo" or "halogen" as used herein is intended to include chloro (Cl), fluoro (F), bromo (Br) and iodo (I). The term "heterocycle" or "heterocyclyl" as used herein is intended to mean a 3 to I 0-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of 0, N and S, and includes bicyclic groups. "Heterocyclyl" 15 therefore includes the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof. Further examples of "heterocyclyl" include: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, chromanyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, 20 oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, 25 dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisochromenyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, 30 tetrahydroquinolinyl and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl substituents may be unsubstituted or unsubstituted, unless specifically defined otherwise. For example, a (CI-C6)alkyl may be substituted with one, two or three substituents selected from 35 OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl, piperidinyl, and so on. In this case, if one substituent is oxo and the other is OH, the following are included in the definition: -(C=0)CH2CH(OH)CH3, -(C=0)OH, -CH2(OH)CH2CH(O), and so on. - 18.- WO 2009/102537 PCT/US2009/031633 In certain instances, R 7 and R 8 are defined such that they can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3 7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S, said heterocycle optionally substituted with one 5 or more substituents selected from R6a. Examples of the heterocycles that can thus be formed include the following, keeping in mind that the heterocycle is optionally substituted with one or more substituents chosen from R 6 a: RRa Ra 6 a R 6 a R 6 a Rea Rea N W-)N N\N N'\N NV\ 7 ]N 6 N N -N HN'< N NNN, Rea Rea Rea ~ z- Rea -N FN O -N N-Rea -N -N , Rea Rsa 'a /N -N j -N a N Rea Rea O0 C 0 j+ t /\ -N Rea FN \ S -N \jSO2 N -N R6a Rea R6@ N Rea N'N Nan r an In an embodiment of Formula A or B, is selected from: - 19 - WO 2009/102537 PCT/US2009/031633 N -N N NN N N N N N N S \N , N N and} g In an embodiment of Formula B: No is selected from: r:CN N N N N N \ N --- N rN r \N N N NGN andN 5 a is 0 or 1; b is 0 or l; m is 0, 1, or 2; n is 0, 1, 2,3,4 or 5; p is 0, 1, 2,3,4,5 or 6; and q is 0, 1, 2,3,4 or 5; RI is independently selected from: CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCI-C6 perfluoroalkyl, (C=0)aNR7R 8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7
R
8 , S(O)m-(C1 10 C1 o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
5 is independently selected from: CF3, oxo, (C=O)aObC1-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=O)R 7
R
8 , S(O)mNR 7
R
8 , S(O)m 15 (C1-C1o)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6;
R
3 is selected from: H, CF3, oxo, (C=O)aObCl-ClO alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6 20 perfluoroalkyl, (C=O)aNR 7
R
8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7RS, S(O)m-(Cl C1 O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
4 is independently selected from: H, CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObC1 25 C6 perfluoroalkyl, (C=O)aNR 7
R
8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7
R
8 , S(O)m - 20 - WO 2009/102537 PCT/US2009/031633 (CI-C1o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
6 is: CF3, oxo, (C=O)aObCl-Cl0 alkyl, (C=O)aObaryl, C2-Clo alkenyl, C2 C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl -C6 perfluoroalkyl, 5 Oa(C=0)bNR 7
R
8 , oxo, CHO, (N=O)R 7
R
8 , S(O)mNR7R 8 , S(O)m-(Ci-Clo)alkyl, SH or (C=O)aObC3-C8 cycloalkyl;
R
7 and R 8 are independently selected from: H, (C=0)ObCl-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, CI-Clo alkyl, aryl, C2-CbO alkenyl, C2-C1 0 alkynyl, heterocyclyl, C3-Cg cycloalkyl, S(O)mRa, and (C=O)NRb2, or R 7 and 10 R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (Cl-C6)alkyl; and Rb is independently H or (Ci I-C6)alkyl; 15 or a pharmaceutically acceptable salt or a stereoisomer thereof. In another embodiment of Formula B: is selected from: N N NN\N N N N 0 'D N S C :N NN N aisoorl; bis0or l;mis0, 1,or2;nis 1 or2; pis 0,1,2,3,4,5 or6; andqis 20 1 or 2;
R
1 is independently selected from: H, S(O)2-(Cl-C6)alkyl, (Ci-C6)alkyl, (C=0)O(Cl-C6)alkyl;
R
5 is independently selected from: H, halo, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl are optionally 25 substituted with one or more substituents selected from R 6 ;
R
3 is: CI-C6 alkyl, said alkyl is optionally substituted with one or more substituents selected from R 6 ;
R
4 is independently selected from: H, CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-ClO alkenyl, (C=O)aObC2-ClO alkynyl, CO2H, halo, OH, ObCl 30 C6 perfluoroalkyl, (C=O)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7
R
8 , S(O)m -21- WO 2009/102537 PCT/US2009/031633 (Cl-CIO)alkyl, and (C=0)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ;
R
6 is: CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2 C10 alkynyl, (C=O)aOb heterocyclyl, C02H, halo, CN, OH, ObC1-C6 perfluoroalkyl, 5 Oa(C=0)bNR 7
R
8 , oxo, CHO, (N=0)R7R 8 , S(O)mNR 7
R
8 , S(O)m-(C-CiO)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; R7 and R 8 are independently selected from: H, (C=0)ObCI-C10 alkyl, (C=0)ObC3-C8 cycloalkyl, (C=Q)Obaryl, (C=O)Obheterocyclyl, Cj-C1o alkyl, aryl, C2-C1O alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb2, or R 7 and 10 R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (C1-C6)alkyl; and Rb is independently H or (CI-C6)alkyl; 15 or a pharmaceutically acceptable salt or a stereoisomer thereof. In another embodiment of Formula B: is selected from: N N N N N N N N N, nd %,, N N ~ N \NG\N an Ng ais Oor 1;bis0or 1;mis0, l,or2; nis 1 or2; pis 0, 1,2,3,4,5 or 6; andqis 20 1 or 2;
R
1 is independently selected from: H, S(O)2-(C1-C6)alkyl, (Cl-C6)alkyl, (C=0)O(C1-C6)alkyl;
R
5 is independently selected from: H, halo, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl are optionally 25 substituted with one or more substituents selected from R 6 ;
R
3 is: C I-C6 alkyl, said alkyl is optionally substituted with NH2;
R
4 is independently selected from: H, CF3, Oxo, (C=O)aObCl-Clo alkyl,
(C=
0 )aOb aryl, (C=0)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, C02H1, halo, OH, ObC1 C6 perfluoroalkyl, (C=0)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m 30 (CI-C1o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; - 22 - WO 2009/102537 PCT/US2009/031633
R
6 is: CF3, oxo, (C=O)aObCi-C6 alkyl, (C=0)aObaryl, C2-C6 alkenyl, C2-C6 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7
R
8 , oxo, CHO, (N=O)R 7
R
8 , S(O)mNR 7
R
8 , S(O)m-(Cl-C6)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; 5 R 7 and R 8 are independently selected from: H, (C=0)ObC-C6 alkyl, (C=O)ObC3-CS cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-C6 alkyl, aryl, C2-C6 alkenyl, C2-C6 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb2, or R 7 and R8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the 10 nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (Cl -C6)alkyl; and Rb is independently H or (C1 -C6)alkyl; or a pharmaceutically acceptable salt or a stereoisomer thereof Included in the instant invention is the free form of compounds of Formula A, as 15 well as the pharmaceutically acceptable salts and stereoisomers thereof Some of the isolated specific compounds exemplified herein are the protonated salts of amine compounds. The term "free form" refers to the amine compounds in non-salt form. The encompassed pharmaceutically acceptable salts not only include the isolated salts exemplified for the specific compounds described herein, but also all the typical pharmaceutically acceptable salts of the free form of 20 compounds of Formula A. The free form of the specific salt compounds described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free forms may differ from their respective salt fonns somewhat in certain physical properties, such as solubility in polar solvents, 25 but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention. The pharmaceutically acceptable salts of the instant compounds can be synthesized from the compounds of this invention which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic compounds are prepared either 30 by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base. Thus, pharmaceutically acceptable salts of the compounds of this invention 35 include the conventional non-toxic salts of the compounds of this invention as formed by reacting a basic instant compound with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids -23- WO 2009/102537 PCT/US2009/031633 such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic (TFA) and the like. 5 When the compound of the present invention is acidic, suitable "pharmaceutically acceptable salts" refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, 10 calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, N,N'-dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, 15 N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, bistidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like. The preparation of the pharmaceutically acceptable salts described above and 20 other typical pharmaceutically acceptable salts is more fully described by Berg et al, "Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19. It will also be noted that the compounds of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then 25 be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom. UTILITY The compounds, compositions and methods provided herein are particularly deemed useful for the treatment of cancer. Cancers that may be treated by the compounds, 30 compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma, non-small 35 cell; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, -24 - WO 2009/102537 PCT/US2009/031633 lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colorectal, rectal ; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate 5 (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, 10 Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, 15 ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma}, granulosa-thecal cell tumors, Sertoli-Leydig cell 20 tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood myeloidd leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, 25 myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lyrmphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions. 30 Cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: breast, prostate, colon, colorectal, lung, non-small cell lung, brain, testicular, stomach, ovarian, pancrease, skin, small intestine, large intestine, throat, head and neck, oral, bone, liver, bladder, kidney, thyroid and blood. Cancers that may be treated by the compounds, compositions and methods of the 35 invention include: breast, prostate, colon, ovarian, colorectal, lung and non-small cell lung. Cancers that may be treated by the compounds, compositions and methods of the invention include: breast, colon, (colorectal) and lung (non-small cell lung). - 25 - WO 2009/102537 PCT/US2009/031633 Cancers that may be treated by the compounds, compositions and methods of the invention include: lymphoma and leukemia. The compounds of the invention are also useful in preparing a medicament that is useful in treating cancer. 5 The compounds of this invention may be administered to mammals, including humans, either alone or, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration. 10 The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or 15 more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium 20 phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption 25 in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed. Formulations for oral use may also be presented as hard gelatin capsules wherein 30 the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions contain the active material in admixture with excipients 35 suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products - 26 - WO 2009/102537 PCT/US2009/031633 of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of 5 ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame. 10 Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be 15 preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional 20 excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying 25 agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants. 30 Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant. The pharmaceutical compositions may be in the fon of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's 35 solution and isotonic sodium chloride solution. The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the - 27 - WO 2009/102537 PCT/US2009/031633 active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulsion. The injectable solutions or microemulsions may be introduced into a patient's blood-stream by local bolus injection. Alternatively, it may be advantageous to administer the 5 solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD PLUSTM model 5400 intravenous pump. The pharmaceutical compositions may be in the form of a sterile injectable 10 aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, 15 fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Compounds of the instant invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by 20 mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. For topical use, creams, ointments, jellies, solutions or suspensions, etc., 25 containing the compound of the instant invention are employed. (For purposes of this application, topical application shall include mouth washes and gargles.) The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. 30 To be administered in the forn of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. 35 When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms. - 28 - WO 2009/102537 PCT/US2009/031633 The dosage regimen utilizing the compounds of the instant invention can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt 5 thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease. For example, compounds of the instant invention can be administered in a total daily dose of up to 1000 mg. Compounds of the instant invention can be administered once daily (QD), or divided into multiple daily doses such as twice 10 daily (BID), and three times daily (TID). Compounds of the instant invention can be administered at a total daily dosage of up to 1000 mg, e.g., 200 mg, 300 mg, 400 mg, 600 mg, 800 mg or 1000 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above. In addition, the administration can be continuous, i.e., every day, or intermittently. 15 The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration of a compound of the instant invention may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days. 20 In addition, the compounds of the instant invention may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period. For example, the compounds of the instant invention may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, or twice daily at a dose of 100 - 500 mg for three to five days a week. In another 25 particular embodiment, the compounds of the instant invention may be administered three times daily for two consecutive weeks, followed by one week of rest. Any one or more of the specific dosages and dosage schedules of the compounds of the instant invention, may also be applicable to any one or more of the therapeutic agents to be used in the combination treatment (hereinafter refered to as the "second therapeutic agent"). 30 Moreover, the specific dosage and dosage schedule of this second therapeutic agent can further vary, and the optimal dose, dosing schedule and route of administration will be determined based upon the specific second therapeutic agent that is being used. Of course, the route of administration of the compounds of the instant invention is independent of the route of administration of the second therapeutic agent. In an embodiment, 35 the administration for a compound of the instant invention is oral administration. In another embodiment, the administration for a compound of the instant invention is intravenous administration. Thus, in accordance with these embodiments, a compound of the instant invention is administered orally or intravenously, and the second therapeutic agent can be -29- WO 2009/102537 PCT/US2009/031633 administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form. 5 In addition, a compound of the instant invention and second therapeutic agent may be administered by the same mode of administration, i.e. both agents administered e.g. orally, by IV. However, it is also within the scope of the present invention to administer a compound of the instant invention by one mode of administration, e.g. oral, and to administer the second therapeutic agent by another mode of administration, e.g. IV or any other ones of the 10 administration modes described hereinabove. The first treatment procedure, administration of a compound of the instant invention, can take place prior to the second treatment procedure, i.e., the second therapeutic agent, after the treatment with the second therapeutic agent, at the same time as the treatment with the second therapeutic agent, or a combination thereof. For example, a total treatment 15 period can be decided for a compound of the instant invention. The second therapeutic agent can be administered prior to onset of treatment with a compound of the instant invention or following treatment with a compound of the instant invention. In addition, anti-cancer treatment can be administered during the period of administration of a compound of the instant invention but does not need to occur over the entire treatment period of a compound of the instant invention. 20 The instant compounds are also useful in combination with therapeutic, chemotherapeutic and anti-cancer agents. Combinations of the presently disclosed compounds with therapeutic, chemotherapeutic and anti-cancer agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6 th edition (February 15, 2001), Lippincott Williams & Wilkins 25 Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis 30 inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, y-secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints. The instant compounds are particularly useful when co-administered with radiation therapy. 35 "Estrogen receptor modulators" refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY 117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1 - 30 - WO 2009/102537 PCT/US2009/031633 piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4' dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646. "Androgen receptor modulators" refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen 5 receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate. "Retinoid receptor modulators" refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a 10 difluoromethylomithine, ILX23-7553, trans-N-(4'-hydroxyphenyl) retinamide, and N-4 carboxyphenyl retinamide. "Cytotoxic/cytostatic agents" refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia 15 activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, 20 topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors. Examples of cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, 25 estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2 methyl-pyridine)platinum, benzylguanine, glufosfamide, GPXI 00, (trans, trans, trans)-bis-mu (hexane-1,6-diamine)-mu-[diamine-platinum(lI)]bis[diamine(chloro)platinum (fI)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1 -(11-dodecylamino- 1 0-hydroxyundecyl)-3,7 30 dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-deoxo-10 hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino 3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and mTOR inhibitors (such as Wyeth's CCI-779). 35 An example of a hypoxia activatable compound is tirapazamine. Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade). - 31 - WO 2009/102537 PCT/US2009/031633 Examples of microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPRI 09881, BMS 184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene 5 sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L proline-t-butylarnide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS188797. In an embodiment the epothilones are not included in the microtubule inhibitors/microtubule-stabilising agents. Some examples of topoisomerase inhibitors are topotecan, hycaptamine, 10 irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzylidene-chartreusin, 9-methoxy-N,N dimethyl-5-nitropyrazolo(3,4,5-kl]acridine-2-(6H) propanamine, 1 -amino-9-ethyl-5-fluoro-2,3 dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':b,7]-indolizino(1,2b]quinoline 10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1 100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2' 15 dimethylamino-2'-deoxy-etoposide, GL331, N- [2-(dimethylamino)ethyl]-9-hydroxy-5,6 dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2 (dimethylamino)ethyll-N-methylamino] ethylj-5-[4-hydro0xy-3,5-dimethoxyphenyl] 5,5a,6,8,8a,9-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5 methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2 20 aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2 (2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1 [2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2 (dimethylamino)ethyl)acridine-4-carboxamide, 6-[ [2-(dimethyl amino)ethyl] amino]-3-hydroxy 7H-indeno[2,1-c] quinolin-7-one, and dimesna. 25 Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications W003/039460, WO03/050064, W003/050122, W003/049527, W003/049679, W003/049678, W004/039774, W003/079973, W003/09921 1, WO03/105855, WO03/106417, W004/037171, W004/058148, W004/058700, W004/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190, 30 US2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLPI, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL. Examples of "histone deacetylase inhibitors" include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone 35 deacetylase inhibitors may be found in the following manuscript; Miller, T.A. et al. J. Med. Chem. 46(24):5097-5116 (2003). "Inhibitors of kinases involved in mitotic progression" include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of -32- WO 2009/102537 PCT/US2009/031633 PLK-1), inhibitors of bub- 1 and inhibitors of bub-RI. An example of an "aurora kinase inhibitor" is VX-680. "Antiproliferative agents" includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and 1NX3001, and antimetabolites such as 5 enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2' fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4 dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyllglycylamino]-L-glycero-B 10 L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-{2-amino-4-oxo 4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, 11 -acetyl-8-(carbamoyloxymethyl)-4-formyl- 6 methoxy-14-oxa- 1,11 -diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl- 1-B-D 15 arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab. Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar. 20 "HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3 methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR@; see U.S. Patent Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR@; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL@; see U.S. Patent Nos. 4,346,227, 4,537,859, 25 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL@; see U.S. Patent Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR@; see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. 5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are 30 described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996) and US Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such 35 salts, esters, open-acid and lactone forms is included within the scope of this invention. "Prenyl-protein transferase inhibitor" refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein -33- WO 2009/102537 PCT/US2009/031633 transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl protein transferase type-II (GGPTase-I, also called Rab GGPTase). Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 5 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No. 10 5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, 15 WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Patent No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.
1394
-
14 0 1 (1999). 20 "Angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt- 1 (VEGFRI) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, 25 interferon-c, interleukin- 12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. 30 Endocrinol., Vol. 16, p.
10 7 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-0-chloroacetyl-carbonyl)-fumagillol, 35 thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp.
96 3
-
9 68 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186). -34- WO 2009/102537 PCT/US2009/031633 Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis 5 pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIaJ) (see Thrombosis Res. 101:329-354 (2001)), TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed August 8, 2001) and 60/349,925 (filed January 18, 2002). 10 "Agents that interfere with cell cycle checkpoints" refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHKI and CHK2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7 hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032. 15 "Agents that interfere with receptor tyrosine kinases (RTKs)" refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001. "Inhibitors of cell proliferation and survival signalling pathway" refer to 20 compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 25 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf kinase (for example BAY-43-9006 ), inhibitors of MEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of P13K (for example LY294002). As described above, the combinations with NSAID's are directed to the use of 30 NSAID's which are potent COX-2 inhibiting agents. For purposes of this specification an NSAID is potent if it possesses an IC5 for the inhibition of COX-2 of 1p 1 M or less as measured by cell or microsomal assays. The invention also encompasses combinations with NSAID's which are selective COX-2 inhibitors. For purposes of this specification NSAID's which are selective inhibitors of 35 COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays. Such compounds include, but are not limited to those disclosed in U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S. Patent 6,020,343, U.S. - 35 - WO 2009/102537 PCT/US2009/031633 Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S. Patent 5,550,142, U.S. Patent 5,604,260, U.S. 5,698,584, U.S. Patent 5,710,140, WO 94/15932, U.S. Patent 5,344,991, U.S. Patent 5,134,142, U.S. Patent 5,380,738, U.S. Patent 5,393,790, U.S. Patent 5,466,823, U.S. Patent 5,633,272 and U.S. Patent 5,932,598, all of which are hereby incorporated by reference. 5 Inhibitors of COX-2 that are particularly useful in the instant method of treatment are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; or a pharmaceutically acceptable salt thereof. Compounds that have been described as specific inhibitors of COX-2 and are 10 therefore useful in the present invention include, but are not limited to, the following: parecoxib, BEXTRA@ and CELEBREX® or a pharmaceutically acceptable salt thereof. Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpimase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl] 1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-I -[[3,5-dichloro-4-(4 15 chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3 naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416). As used above, "integrin blockers" refers to compounds which selectively 20 antagonize, inhibit or counteract binding of a physiological ligand to the av@3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the avP5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the ccvP3 integrin and the avP5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary 25 endothelial cells. The term also refers to antagonists of the avP6, av18, 1 11, a2@l, c511, 961 and a604 integrins. The term also refers to antagonists of any combination of avp3, uv@5, avI6, avp8, al1PI, a211, a51l, a601 and a6@4 integrins. Some specific examples of tyrosine kinase inhibitors include N (trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5 30 yl)methylidenyl)indolin-2-one, 17-(allylamino)- 1 7-demethoxygeldanamycin, 4-(3-chloro-4 fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl) 6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10 (hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-IH-diindolo[1,2,3-fg:3',2',I'-kljpyrrolo[3,4 i][1,6]benzodiazocin-1-one, SH268, genistein, ST1571, CEP2563, 4-(3-chlorophenylamino)-5,6 .35 dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino 6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI57 IA, N-4-chlorophenyl-4-(4-pyridylmethyl)- 1 -phthalazinamine, and EMD 121974. -36- WO 2009/102537 PCT/US2009/031633 Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the instantly claimed compounds with PPAR-y (i.e., PPAR-gamma) agonists and PPAR-6 (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies. PPAR-y and PPAR-5 are the nuclear 5 peroxisome proliferator- activated receptors y and 6. The expression of PPAR-y on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J Cardiovasc. PharmacoL 1998; 31:909-913; J. Biol. Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-y agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the 10 development of retinal neovascularization in mice. (Arch. Ophthamol 2001; 119:709-717). Examples of PPAR-y agonists and PPAR- y/ct agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP01 10, DRF4158, NN622, G1262570, PNU182716, 15 DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in USSN 60/235,708 and 60/244,697). Another embodiment of the instant invention is the use of the presently disclosed 20 compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer see Hall et al (Am. J. Hum. Genet. 61:785-789, 1997) and Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. 25 Patent No. 6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J. Immunol. 2000;164:217-222). The compounds of the instant invention may also be administered in combination 30 with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar). A compound of the present invention may be employed in conjunction with anti 35 emetic agents to treat nausea or nemesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin- 1 -37- WO 2009/102537 PCT/US2009/031633 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 5 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In another embodiment, conjunctive therapy with an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosed for the treatment or prevention of emesis that may result upon administration of the instant compounds. 10 Neurokinin-1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 15 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 20 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93118023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 25 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 30 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incorporated herein by reference. 35 I an embodiment, the neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(1-(R)-(3,5 bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)- 4
-(
3 -(5-oxo-1H,4H-1,2,4 - 38 - WO 2009/102537 PCT/US2009/031633 triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719,147. A compound of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous 5 erythropoiesis receptor activator (such as epoetin alfa). A compound of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include 10 filgrastim. A compound of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin. A compound of the instant invention may also be useful for treating or preventing cancer in combination with P450 inhibitors including: xenobiotics, quinidine, tyramine, 15 ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin, troleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine, verapamil, cortisol, itraconazole, mibefradil, nefazodone and nelfinavir. A compound of the instant invention may also be useful for treating or preventing 20 cancer in combination with Pgp and/or BCRP inhibitors including: cyclosporin A, PSC833, GF120918, cremophorEL, fumitremorgin C, Kol32, Ko134, Iressa, Imatnib mesylate, EKI-785, C11033, novobiocin, diethylstilbestrol, tamoxifen, resperpine, VX-710, tryprostatin A, flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine, verapamil, terfenadine, ketoconazole, nifidepine, FK506, amiodarone, XR9576, indinavir, amprenavir, cortisol, 25 testosterone, LY335979, OC144-093, erythromycin, vincristine, digoxin and talinolol. A compound of the instant invention may also be useful for treating or preventing cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids). Examples of bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), 30 alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate and tiludronate including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof. A compound of the instant invention may also be useful for treating or preventing 35 breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane. A compound of the instant invention may also be useful for treating or preventing cancer in combination with siRNA therapeutics. -39- WO 2009/102537 PCT/US2009/031633 The compounds of the instant invention may also be administered in combination with -secretase inhibitors and/or inhibitors of NOTCH signaling. Such inhibitors include compounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, 5 WO 2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139). A compound of the instant invention may also be useful for treating or preventing cancer in combination with PARP inhibitors. 10 A compound of the instant invention may also be useful for treating cancer in combination with the following therapeutic agents: abarelix (Plenaxis depot®; aldesleukin (Prokine@); Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen@); amifostine (Ethyol®); anastrozole (Arimidex@); arsenic trioxide (Trisenox®); asparaginase (Elspar@); azacitidine (Vidaza@); 15 bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin@); bleomycin (Blenoxane®); bortezomib (Velcade@); busulfan intravenous (Busulfex@); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU@); carmustine (Gliadel@); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil 20 (Leukeran@); cisplatin (Platinol@); cladribine (Leustatin®, 2-CdA@); clofarabine (Clolar@); cyclophosphamide (Cytoxan@, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet@); cytarabine (Cytosar-U@); cytarabine liposomal (DepoCyt@); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp@); daunorubicin liposomal (DanuoXome®); daunorubicin, 25 daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard@); docetaxel (Taxotere@); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection@); doxorubicin liposomal (Doxil®); DROMOSTANOLONE PROPIONATE (DROMOSTANOLONE®); DROMOSTANOLONE PROPIONATE (MASTERONE INJECTIONS); Elliott's B Solution 30 (Elliott's B Solution®); epirubicin (Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide, VP- 16 (Vepesid®); exemestane (Aromasin®); Filgrastim (Neupogen@); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil@); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine (Gemzar@); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate 35 (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implants); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX@); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); irinotecan (Camptosar®); lenalidomide (Revlimid®); letrozole (Femara®); - 40- WO 2009/102537 PCT/US2009/031633 leucovorin (Wellcovorin@, Leucovorin@); Leuprolide Acetate (Eligard@); levamisole (Ergamisol®); lomustine, CCNU (CeeBU@); meclorethamine, nitrogen mustard (Mustargen@); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs@); methotrexate (Methotrexate@); 5 methoxsalen (Uvadex@); mitomycin C (Mutamycin®); mitotane (Lysodren@); mitoxantrone (Novantrone@); nandrolone phenpropionate (Durabolin-50@); nelarabine (Arranon@); Nofetumomab (Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia@); pegademase (Adagen (Pegademase Bovine)®); 10 pegaspargase (Oncaspar@); Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta@); pentostatin (Nipent®); pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®); Rasburicase (Elitek@); Rituximab (Rituxan®); sargramostim (Leukine@); Sargramostim (Prokine@); sorafenib (Nexavar®); streptozocin (Zanosar@); sunitinib maleate (Sutent®); talc (Sclerosol@); 15 tamoxifen (Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone (Teslac@); thioguanine, 6-TG (Thioguanine@); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar@); Tositumomab/I-131 tositumomab (Bexxar@); Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine 20 (Oncovin@); vinorelbine (Navelbine®); vorinostat (Zolinza®) and zoledronate (Zometa®). Thus, the scope of the instant invention encompasses the use of the instantly claimed compounds in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase 25 inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-7 agonists, PPAR-b agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, y-secretase and/or NOTCH 30 inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above. The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the 35 invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents. -41- WO 2009/102537 PCT/US2009/031633 As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. 5 The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term "treating cancer" or "treatment of cancer" refers to administration to a 10 mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, but also to an effect that results in the inhibition of growth and/or metastasis of the cancer. In an embodiment, the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an 15 inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon-a, interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF. In an embodiment, the estrogen receptor modulator is tamoxifen or 20 raloxifene. Also included in the scope of the claims is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of the instant invention in combination with radiation therapy and/or in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid 25 receptor modulator, a cytotoxiccytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR-y agonists, PPAR-b agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic-enhancing drug, an 30 inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, -y-secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above. And yet another embodiment of the invention is a method of treating cancer that 35 comprises administering a therapeutically effective amount of a compound of the instant invention in combination with paclitaxel or trastuzumab. - 42 - WO 2009/102537 PCT/US2009/031633 The invention further encompasses a method of treating or preventing cancer that comprises administering a therapeutically effective amount of a compound of the instant invention in combination with a COX-2 inhibitor. The instant invention also includes a phannaceutical composition useful for 5 treating or preventing cancer that comprises a therapeutically effective amount of a compound of the instant invention and a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMNG-CoA reductase inhibitor, an HIfV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, a PPAR-y 10 agonist, a PPAR-6 agonist, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, y-secretase and/or NOTCHinhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell cycle checkpoint, and any of the therapeutic agents listed above. All patents, publications and pending patent applications identified are hereby 15 incorporated by reference. Abbreviations that may be used in the description of the chemistry and in the Schemes that follow are: Ac2O (acetic anhydride); AcOH (acetic acid); AEBSF (p aminoethylbenzenesulfonyl fluoride); BSA (bovine serum albumin); BuLi (n-Butyl lithium); CDC13 (chloroform-d); CuJ (copper iodide); CuSO4 (copper sulfate); DBU (1,8 20 diazabicyclo[5.4.0]undec-7-ene); DCE (dichloroethane); DCM (dichloromethane); DEAD (diethyl azodicarboxylate); DIPEA (diisopropylethylamine); DMF (N,N-dimethylformamide); DMP (Dess-Martin periodinane); DMSO (dimethyl sulfoxide); DPPA (diphenylphosphoryl azide); DTT (dithiothreitol); EDTA (ethylene-diamine-tetra-acetic acid); EGTA (ethylene glycol-tetra-acetic acid); Et2O (diethylether); EtOAc (ethyl acetate); EtOH (ethanol); HOAc 25 (acetic acid); HPLC (high-performance liquid chromatography); IRMS (high resolution mass spectrum); LAH (lithium aluminum hydride); LCMS (liquid chromatograph-mass spectrometer); LHMDS (lithium bis(trimethylsilyl)amide); LRMS (low resolution mass spectrum); mCPBA (3 chloroperoxybenzoic acid); MeOH (methanol); MP-B(CN)H3 (Macroporous cyanoborohydride); NaHC03 (sodium bicarbonate); Na2S04 (sodium sulfate); Na(OAc)3BH (sodium 30 triacetoxyborohydride); NH40Ac (ammonium acetate); NBS (N-bromosuccinamide); NMP (1 methyl-2-pyrrolidinone); NMR (nuclear magnetic resonance); PBS (phosphate buffered saline); PCR (polymerase chain reaction); Pd(dppf) ([1,1'-bis(diphenylphosphino)ferrocene] palladium); Pd(Ph3)4 (palladium(0) tetrakis-triphenylphosphine); POCl3 (phosphorous oxychloride); PS DIEA (polystyrene diisopropylethylamine); PS-PPh3 (polystyrene-triphenyl phosphine); PTSA 35 (para-toluene sulfonic acid); ); PyBOP (benzotriazol- 1 -yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate); Pyr (pyridine); Selectfluor (1 -chloromethyl-4-fluoro- 1,4 diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate); RP HPLC (reverse phase high-performance liquid chromatography); TBAF (tetrabutylammonium fluoride); t-BuOH (tert-butanol); THF - 43 - WO 2009/102537 PCT/US2009/031633 (tetrahydrofuran); Tf (trifluoromethanesulfonyl); TFA (trifluoroacteic acid); and TMSCH2N2 (trimethylsilyldiazomethane). The compounds of this invention may be prepared by employing reactions as shown in the following Reaction Schemes, in addition to other standard manipulations that are 5 known in the literature or exemplified in the experimental procedures. The illustrative Reaction Schemes below, therefore, are not limited by the compounds listed or by any particular substituents employed for illustrative purposes. Substituent numbering as shown in the Reaction Schemes do not necessarily correlate to that used in the claims and often, for clarity, a single substituent is shown attached to the compound where multiple substituents are optionally 10 allowed under the definitions of Formula A hereinabove. Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in Reaction Schemes A-C. SYNOPSIS OF REACTION SCHEMES As shown in Reaction Scheme I, the substituted antrhranilic acid (A-1) can 15 undergo a condensation with substituted isothiocyanates to provide mercaptopyrimidinone (A-2). Compounds A-2 can be further derivatized by coupling to a-halo carboxylic acids to furnish compounds denoted by A-3. Reaction Scheme A O N-S X 0
(R
1
)
5 A OH
(R
1 )R H R OH (R1)5
NH
2 N SH N is A-1 A-2 A-3 R3 OH 0 20 Reaction Scheme II illustrates the preparation of thioquinazolinones (B-1) by amide coupling of substituted mercaptopyrimidinone (A-3) with various amines (B-2). Reaction Scheme B 0 0 NR2 HNR2N
(R
1
)
5 A
R
6
(R)
5 A R' NISB- N S R 5 A-3 R 3 OH B-1 R 3 KR 25 As shown in Reaction Scheme III, Suzuki Coupling of iodide C-1 with boronic ester (C-2) can afford compounds denoted by C-3. -44 - WO 2009/102537 PCT/US2009/031633 Reaction Scheme C Suzuki Coupling R7
(R
1
)
5
A(R?
1
)
5 Al ( ) S R |0) N S R 5 C-1 R 3 R6 R C-3 R3 NR 4 C-2 EXAMPLES Examples provided are intended to assist in a further understanding of the 5 invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof. The reagents utilized in synthesizing the compounds depicted in the following Tables are either commercially available or are readily prepared by one of ordinary skill in the art. SCHEME I 0 N===S OOH 0 - OH -EtOM N C 1-4o - N 01 I75C ' N ji
NH
2 C NSH Et 3 N, DMF N 1-1 1-2 1-3 1-5 OH 0 NBOC o o 1-6 N C NH N S T N PyBOP, EtN, 1-7, N DCM 1-8 O N DMF, rt 0 N 0 N BOO H 10 3-(3-chlorophenvl)-2-mercapto-8-methylquinazolin-4(3h)-one (1-3) 2-amino-3-methylbenzoic acid (1-1) (3 g, 21.88 mmol, 1.0 equiv) was dissolved in EtOH (44 mL), and heated to 75 C. 3-Chlorophenyl isothiocyanate (1-2) was added in 0.2 eq. portions over the course of 78 hours until disappearance of benzoic acid. The mixture was 15 cooled, concentrated, and the slurry was filtered and washed with cold ethanol to afford pure 3 (3-chlorophenyl)-2-mercapto-8-methylquinazolin-4(3H)-one (1-3). LRMS m/z (M+H) 303.0 found, 303.1 required. 2- {[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-yl]thio} hexanoic acid (1-5) 20 3-(3-Chlorophenyl)-2,3-dihydro-2-thioxo-4(IH)-8-methylquinazolinone (1-3, 145 mg, 0.50 mmol, 1.0 equiv), 2-bromohexanoic acid (1-4, 93 PL, 0.65 mmol, 1.3 equiv.), and triethylamine (420 pL, 3.0 mmol, 6 equiv.) were dissolved in anhydrous DMF (1 mL) and stirred at 75 *C. When LCMS indicated the reaction was complete, the crude reaction mixture was partitioned between ethyl acetate and 0.1 M HCI, the aqueous phase drained and the organic - 45 - WO 2009/102537 PCT/US2009/031633 layer washed twice more with 0.1 M HCI followed by brine to yield the product (1-5). LRMS m/z (M+H) 417.0 found, 417.1 required. tert-Butyl 8-(2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2 yllthio}hexanovl)-2.8-diazaspiro[5.5]undecane-2-carboxylate (1-7) 5 2-{{3-(3-Chlorophenyl)- 8-methyl-4-oxo-3,4-dihydroquinazolin-2 yljthio}hexanoic acid (1-5, 174 mg, 0.43 mmol, 1.0 equiv.), tert-butyl 2,8 diazaspiro[5.5]undecane-2-carboxylate (1-6, 220 mg, 0.864 mmol, 2 equiv.), PyBOP (337 mg, 0.648 mmol, 1.5 equiv.) and triethylamine (0.6 mL, 4.32 mmol, 10 equiv.) were dissolved in DMF (1 mL) and stirred at rt. Once LCMS showed reaction was complete, the reaction mixture 10 was filtered and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product (1-7). LRMS m/z (M+H) 653.1 found, 653.3 required 3-(3-chlorophenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyljthio}-8 methylquinazolin-4(3h)-one (1-8) tert-Butyl 8-(2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2 15 yl]thio}hexanoyl)-2,8-diazaspiro[5.5]undecane-2-carboxylate (1-7, 200 mg, 0.31 mmol, 1.0 equiv.) was dissolved in DCM (2 mL). Trifluoroacetic acid (1 mL) was added and the mixture stirred at room temperature. Once LCMS showed reaction was complete, toluene was added, the solvent and TFA were removed at reduced pressure and the resulting crude was filtered and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to 20 yield the product. 1H NMR (500 MHz, CDC1 3 ) 6 8.09 (d, 111, J= 8.0 Hz), 7.61 (d, 1H, J= 7.0 Hz), 7.46 (m, 2H), 7.32 (in, 2H), 7.21 (m, 1H), 3.73 (m, 2H), 3.42 (br m, 1H), 3.23 (in, 1H), 2.92 (m, 111), 2.77 (in, 11), 2.73 (d, 1H, J= 13 Hz), 2.67 (s, 2H), 2.60 (in, 3H), 2.50 (br dd, 1H, J= 4.5, 12Hz), 2.08 (m, 111), 1.85 (m, 1H), 1.59 (br m, 4H), 1.46 (m, 2H), 1.36 (br t, 4H, J= 4.0 Hz), 1.25 (s, 2H), 0.88 (t, 3H, J= 7.0 Hz). LRMS m/z (M+H) 553.1 found, 553.3 required. 25 The compounds shown in Table 1 were synthesized as TFA salts according to Scheme 1. Table I Cmp Structure Name LRMS rn/z (M+H) 1-9 1-(2-{[3-(3- LRMS m/z (M+H) chlorophenyl)-4-oxo-3,4- 513.0 found, 513.1 N Cdihydroquinazolin-2-yl] required. N S methyl}hexanoyl)piperidi
NH
2 ne-3-carboxamide o 0 -46 - WO 2009/102537 PCT/US2009/031633 1-10 2- {2-ft4-acetylpiperazin- LRMS m/z (I±H) o 1 1-yl)carbonyljhexyl} -3- 513.0 found) 513.1 N CI (3-chiorophenyl> required. N S N quinazolin-4(3H-one 1-11 03-(3-chlorophenyl)-2-{2- LRMS m/z (M+1J) N Cl [(4-phenylpiperazin- 1- 547.0 found> 547.1 N S N yl)carbonyljexylIquinaz required. N,_) oli-n-4(31t-one N 1-12 1-(2-[3-(3- LRMS m/z (M+H) ychlorophenyl4-oxo-3,4- 569.1 found, 569.2 N S dihydroquinazolin-2- required. NqN yl]methylhexanoyl) of 0 NN-diethylpiperidine-3 carboxamide 1-13 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+I1) [(4-pyridin-2-ylpiperazin- 548.0 found, 548.1 NI'a I -y1)carbonyI]hexy}- required. S Nl qu inazolin-4(3H) -one N,_ 0 1-14 2-(2- {[4-(amino ethyl)- LRMS m/z (M+-H) o piperidin- -yl]carbonyl - 499.1 found, 499.1 N Cd NH 2 hexyl)-3u3-chloro- required. N N s N&phenyl)quinazolin-4(3TH N one r 0 1-15 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+J) o0 [(7-methyl-2,7-diazaspiro 525.0 found, 525.1 N A' Na CI [4.4]non-2-yl)carboniylj required. NIN N S hexylquinazolin-4(3H N,\_ N\ one 0 C -47- WO 2009/102537 PCT/US2009/031633 1-16 2-(2-{[4-(IH- LRMS m/z (M+H) 0 benzimidazol-2- 586.0 found, 586.2 N Cl N yl)piperidin-1- required. N S N yl]carbonyl}hexyl)-3-(3 NH chlorophenyl)quinazolin 0 4(3Hl)-one 1-17 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H) o [(1,1-dioxidothio- 520.0 found, 520.1 A NN Cl morpholin-4-yl)- required. N -N S carbonyljhexyl}quinazoli N n-4(3H)-one O 1-18 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H) o ([4-(methylsulfonyl)- 549.0 found, 549.1 N Cl 0 piperazin-1-ylcarbonyl}- required. N S N hexyl)quinazolin-4(3H) N one 0 1-19 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+IH) o {[4-(1H-imnidazol-1- 549.0 found, 549.1 Na CI N ylmethyl)piperidin-1- required. N S yl]carbonyl}hexyl)quinaz N olin-4(3H)-one 0 1-20 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+-H) o ([4-(methylsulfonyl)- 548.0 found, 548.1 N Cl O piperidin-1-yllcarbonyl}- required. N NS o hexyl)quinazolin-4(3H) N one -]0 -48- WO 2009/102537 PCT/US2009/031633 1-21 3-(3-chlorophenyl)-2-[2- LRMS m/z (M+H) o0 (2,8-diazaspiro[5,5]- 539.0 found, 539.1 N cI undec-2-ylcarbonyl)- required. N s hexyljquinazolin-4(3H) N one N N 1-22 3-(3-ehlorophenyl)-2-(2- LRMS znz (M+l) o0 {[4-(2-fluorophenyl)- 565.0 found, 565.1 NC -xpprzin-1Kyl] carbonyll}- required. N s 'N exyl)quainazolin-4(3T N F one 0 1-23 3-(3-chiorophenyl)-2-(2- LRMS inz (M+f) 0{[4-(4-fluorophenyl)- 565.0 found, 565.1 NcI F piperazin-1-yl]carbonyl}- required. 'N N sN N.,hexyl)quinazolin-4(3N) N SNF None 0 1-24 3-(3-chlorophenyl)-2-{2- LRMS m/z (M±H) o [(4,4-diphenylpiperidin- 622.1 found, 622.2 N N CI __I -yl)carbonyl]hexyl} required. O N C N s qiaoi-(T-n N 0 1-25 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H) X [(4-pyridin-4-ylpiperdiin- 547.0 found, 547.1 N ' CueN Ic-yl)carbonylhexyl- required. hxylquinazolin-4(3H)-one N S Na 0 -h49- WO 2009/102537 PCT/US2009/031633 1-26 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H) o [(3,3-diphenylpiperidin- 622.1 found, 622.2 N C1 1-yl)carbonyl]hexyl}- required. N S| quinazolin-4(3H)-one N 1-27 3-(3-chlorophenyl)-2- LRMS m/z (M+H) o {[1-(2,8-diazaspiro[5.5]- 617.1 found, 617.2 Na C1 undec-2-ylcarbonyl)- required. e N S pentyl]thio} -7-(methyl o N sulfonyl)quinazolin O N 4(3H)-one H 1-28 methyl 3-(3-chloro- LRMS m/z (M+H) o pheny1)-2-{[1-(2,8- 597.2 found, 597.2 N CI diazaspiro[5.5]undec-2- required. o 'N N S ylcarbonyl)pentyl]thio} O N 4-oxo-3,4-dihydro O N quinazoline-7 H carboxylate 1-29 3-(3-chlorophenyl)-2- LRMS m/z (M+H) o ({3-methyl-1 -[(4-pyridin- 548.1 found, 548.2 N C N 2-ylpiperazin- l-yl)- required. a'N N S N carbonyl]butyl}thio)quin N azolin-4(3H)-one 0 1-30 3-(3-chlorophenyl)-2-[(1- LRMS m/z (Mi-H) O {[4-(2,3-dihydro-1,4- 548.1 found, 548.2 N CI > benzodioxin-5- required. N S N 0 yl)piperazin-1 N 0 yl]carbonyl}-3 O methylbutyl)thiojquinazo ._ _ .................... lin-4(3H )-one - 50 - WO 2009/102537 PCT/US2009/031633 1-31 03-(3-chlorophenyl)-2-f(1 - LRMS m/z (M+fl) 1 -31 Nc ~ F 'N N{l[4-(4-fluorophenyb)- 565.2 found, 565.2 JaN piperazin-1 -yl]carbonyl}- required. N N S C1 N N-ehlutltil 1-33 0 -3hlopey)2[ - RM z MH 7 N cI {[7-(3-chlorophenyl)-2,7- 62 1. 0 found, 621.2 N S diazaspiro[4.4non-2- required. N N yljcarhonyl}-3 o / K methylbulyl)thio]quinazo lin-4(3L1)-one 1-33 0 ~3-(3-chlorophenyl)-2-(I- LRMS m/z (M+H) N 'N CI {[7-(2-chlorophenyl)-2,7- 621.0 found, 621.2 N" S diazaspiro[4.4]non-2- required. NpNi1 yl-carbonyle-3 o / K 3methylbutyl)thioquinazo lin-4(3H)-one 1-34 3 2-({-o7-benzyl-2,7- LRMS m/z (M+H) N 'N CI diazaspiro[4.4]non-2- 601.2 found, 601.2 N S yl)carbonyl-3- required. NO N methylbutylthio)-3-(3 0 chlorophenyl)quinazolin ln4(31H)-one 1-35 03-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H) ci {1[7-(3-methoxyphenyl)- 61,0 found, 61.2 N~ 2,7-diazaspiro[4.4]non-2- required. NN N N yflcarbonyl}-3 o ,methylbutyl)thio]quinazo lin-4(3H)-one 1-36 0 3-(3-chlorophenyl)-2-[( - LRMS m/z (M+H) 7 N Cl {[f7-(4-r'nethoxyphenyl)- 617.2 found, 617.2 2,7-diazaspiro[4.4]non-2- required. N N yl]carbonyl-3 methylbutyl)thio-quinazo Iin-4(3H)-one - 51 - WO 2009/102537 PCT/US2009/031633 1-37 0 1-(2-{[3-(3- LRMS m/z (M+H) N Cl NH 2 chlorophenyl)-4-oxo-3,4- 513.0 found, 513.2 N o dihydroquinazolin-2- required. 1-39 r -1N yl~thiolhexanoyl)piperidi 00 ne-4-carboxamide 1-38 0 1-[ 3-(3-chloropheny1>- LRMS m/z (M+H) N Cl NH 2 4-oxo-3,4- 513.0found, 513.2 N S 0 dihydroquinazolin-2- required. N S N yl]thio (phenyl)acetyl]pi 1 peridine-4-carboxamide 1-39 0 1-(2-{[3-(3- LRMS m/z (M+H) NN Cl NH 2 chlorophenyl)-4-oxo-3,4- 513.0 found, 513.2 N S 0 dihydroquinazolin-2- required. f N yl]tio-4 o methylpentanoyl)piperidi ne-4-carboxamide 1-40 0 A13-(3-chlorophenyl)-24(1 - LRMS m/z (M+H) N Cl 4[7-(2-chlorophnyl)-2,7- 699.0 found, 699.1 0 - &diazaspiro4.4non-2- required. o N N yl]carbonyl}pentyl)thiol] o / o 7-(methylsulfonyl) uinazolin-4(3H-one 1-41 0 13-(3-chlorophenyl)-2-[(- LRMS m/z (M+H) SCl [74chlorophenyl)-oo -2,7- 699.0 found, 699.1 N a N 1S diazaspiro[4.4]non-2- required. o NO N ylcarbonyl}pentyl)thio] o / 7-(methylsulfonyl) CI quinazoxin-4(3d-one 1-42 0 A3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H) N CI {{[7-(3-chlorophenyl)-2,7- 699.0 found, 699.1 0 N diazaspiro[4.4]non-2- required. 0 N O yl]carbonyl}pentyl)thio] '7-(methylsulfonyl) quinazolin-4(3H)-one - 52 - WO 2009/102537 PCT/US2009/031633 1-43 0 >3-(3-chlorophenyl)-7- LRMS m/z (M+H) N (methylsulfonyl)-2-({1- 665.0 found, 665.2 O NN- Al " [(7-phenyl-2,7- required. N S O N N diazaspiro[4.4]non-2 0 / ' yl)carbonyl]pentyl}thio)q uinazolin-4(3H)-one 1-44 0 3-(3-chlorophenyl)-2-[(1 - LRMS m/z (M+H) N c {[7-(3-methoxyphenyl)- 695.1 found, 695.2 N S 2,7-diazaspiro[4.4]non-2- required. yl]carbonyl}pentyl)thio] 0 7-(methylsulfonyl) quinazolin-4(3H)-one 1-45 0 ~methyl 3-(3- LRMS m/z (M+H) N a Cl chlorophenyl)-2-({1-[(3- 601.1 found, 601.1 N S cyano-3-phenylazetidin- required. N~ / 1-yl)carbonyl]pentyl} thio)-4-oxo-3,4 dihydroquinazoline-7 carboxylate 1-46 methyl 3-(3- LRMS m/z (M+H) CI chlorophenyl)-2-{[1-(2,6- 541.0 found, 541.1 O ' N S-NH diazaspiro[3.3]hept-2- required. N ylcarbonyl)pentyl]thio} 4-oxo-3,4-dihydro quinazoline-7 carboxylate 1-47 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H) N CI {[(2R)-2-(trifluoro- 524.0 found, 523.1 N S methyl) pyrrolidin-1-ylj- required. N carbonyl) pentyl)thio] F7F quinazolin-4(3H)-one -53 - WO 2009/102537 PCT/US2009/031633 1-48 0 N 3-(3-chlorophenyl)-2-[(- LRMSm/z(M±H) N {l0 [3-(4-fluorobenzyl)-2- 648.1 found, 648.1 N Soxo-1 -oxa-8- required. N azaspiro[4.5]dec-8 0 C'0 1ylcarbonyl }pentyl)thio]q -11 46 uinazolin-4(311)-one 1-493-3-horopheny1)-2-[(1 - LRMS m/z (M±H) N cl{ [5-(4-fluoropheny)2 ,5- 577.1 found, 576.2 diazabicyclo[2.2. 1 Jhept- required. S N F thioquinazolin-4(3H) 01 one 1-503-(3-chlorophenyl)-2-[(- LRMS mz (M+H) N l{ [5-(3-fluorophenyl)-2,5- 577.1 found, 576.2 SF diazabicyelo[2.2.I ]hept- required. N- 2-yllcarbonyllpentyl) N thioquinazolin-4(3H) one ________ 1-51 3-(3-clorophenyl)-2-[(- LRMS m/z (M+) N Cl N- [4-(4-methyl-1,2,5- 553.0 found, 552.2 N S N N oxadiao-3-y)piperazin- required. N 1 -yl]carbonyl~pentyl) o0 thio]quinazolin-4(3H) one _______ _ 1-52 3-(3-ehlorophenyl)-2-V1- LRMS m/z (M+1) o /{[3-(4-fluorophenyl)-- 620.1 found, 619.2 oxa-8-azaspiro4.5]dec- required. N 8-yl]carbonyl}pentyl) N S tfiojquinazolin-4(3H ) one f 0 - 54 - WO 2009/102537 PCT/US2009/031633 1-53 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H) N CI {[(3S,4S)-3,4- 492.0 found, 492.1 N S difluoropyrrolidin-l- required. N F yllcarbonyl}pentyl)thio]q o0 uinazolin-4(3H)-one 1-54 Cl 3-(3-chlorophenyl)-2- LRMS m/z (M+H) o / {[2-(2,8- 563.0 found, 563.2 - N diazaspiro[5.5]undec-2- required. e N :S :=:-\yl)-1I-(lH-imidazol-4 o NH ylmethyl)-2 oxoethyl]thio} quinazolin N -4(3H)-one N H 1-55 c 2-(3-(1H-imidazol-4-yl)- LRMS m/z (M+H) o -1-oxo-1-(2,8- 577.0 found, 577.2 Nb diazaspiro[5.5]undecan- required. N Na, 2-yl)propan-2-ylthio)-3 o NH (3-chlorophenyl)-8 N methylquinazolin-4(3H) one N 1-56 0 3-(3-chlorophenyl)-8- LRMS m/z (M+H) N c x/ methyl-2-(1-oxo-1-(4- 561.1 found, 561.2 N N phenylpiperazin- 1- required. N yl)hexan-2 ylthio)quinazolin-4(3H) one 1-57 0 3-(3-chlorophenyl)-2-(1- LRMS m/z (M+H) N cI (7-(3-methoxyphenyl)- 631.1 found, 631.2 N A 2,7-diazaspiro[4.4] required. N N nonan-2-yl)- I -oxohexan 0 \ 2-ylthio)-8-methyl quinazolin-4(3H)-one - 0 .......... -55- WO 2009/102537 PCT/US2009/031633 1-58 0 3-(3-chlorophenyl)-2-(1- LRMS m/z (M+H) N cI (7-(4-chlorophenyl)-2,7- 635.0 found, 635.2 N diazaspiro[4.4]nonan-2- required. NO N y1)-1-oxohexan-2-ythio) 0 / 8-methylquinazolin Ci 4(3H)-one 1-59 0 W3-(3-chlorophenyl)-2-(1- LRMS m/z (M+H) N -CI (7-(3-chlorophenyl)-2,7- 635.0 found, 635.2 N diazaspiro[4.4]nonan-2- required. NO N yl)-1-oxohexan-2-ylthio) K Ci 8-methyl quinazolin 4(3H)-one SCHEME 2 CO 2 H O HO 2 C B 0 N I o 2-2 O N 0 N S NH 2 PdCJ 2 (dppf), CsCO 3 N S NH 2 N THF, MW, 120 C,1h N O 2-1 O 2-3 3'-[2-[(1 -{ [4-(Amino-carbonyl)piperidin- 1-yl]carbonyl}pentyl)thio]-4 5 oxoguinazolin-3(4H11)-yllbiphenvl-4-carboxylic acid (2-3) 1-(2- f[3-(3-iodophenyl)-4-oxo-3,4-dihydroquinazolin-2 yl]thio}hexanoyl)piperidine-4-carboxamide (2-1, 30 mg, 0.05 mmol, 1.0 equiv.), 4-(4,4,5,5 tetramethyl-1,3,2-dioxaborolan-2-yl)- benzoic acid (2-2, 24 mg, 0.1 mimol, 2 equiv.), PdC12(dppf) (4 mg, 0.005 mmol, 0.1 equiv.) and 2M aqueous Cs 2
CO
3 (0.15 mL, 0.3 mmol, 6 equiv.) were 10 suspended in THF (0.4 mL) and the reaction mixture was irradiated in microwave at 120 *C for 60 minutes. The crude mixture was treated with Quadrapure overnight, filtered, concentrated and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield 3' [2-[( 1- { [4-(amino-carbonyl)piperidin-1-yl]carbonyl}pentyl)thioJ-4-oxoquinazolin-3(4H) yl]biphenyl-4-carboxylic acid (2-3). LRMS m/z (M+H) 599.1 found, 599.2 required. -56- WO 2009/102537 PCT/US2009/031633 The compounds shown in Table 2 were synthesized as TFA salts according to Scheme 2. Table 2 Cmp Structure Name LRMS mnk (M+H) 2-4 1-(2-f[3-(2'- LRMS /z (MAH) -~c1 chlorobiphenyl-3-yl)-4- 589.1 found, 589.2 o0 oxo-3,4- required. N NH 2 dihydroquinazolin-2 N NH2 yl]thiolhexanoyl)piperidi N S ne-4-carboxamide N 0 2-5 C1 1-(2-{[3-(3'- LRMS rn/z (M+J) xchlorobiphenyl-3-yD)-4- 589.1 found, 589.2 oxo-3,4- required. dihydroquinazolin-2 N NH 2 yI]thiohexanoyl)piperidi N S 0 ne-4-carboxamide N 0 2-6 C) 1-42-{I3-(4- LRMS inz (M±H) chlorobiphenyl-3-yl)-4- 589.1 found, 589.2 oxo-3,4- required. o0 dihydroquinazolin-2 yljthiojhexanoyl)piperidi N NH2 A NJ NH 2 ne-4-carboxamide 2-7A N S 0 0 2-7 N.1-(2-{[3-(2'- LRMS m/z (M+H)
NH
2 aminobiphenyl-3-yl)-4- 570.2 found, 570.2 o -oxo-3,4- required. dihydroquinazolin-2 N
NH
2 yl]thio)hexanoyl)piperidi N S 0 ne-4-carboxamide 0 -57- WO 2009/102537 PCT/US2009/031633 2-81-(2-{3-(3'- LRMS m/z (M+H) aminobiphenyl-3-yl)-4- 570.2 found, 570.2 oxo-3,4- required. 0 dihydroquinazolin-2 N NH 2 yl]thio}hexanoyl)piperidi N S N( "0 ne-4-carboxamide N 2-9 NH 2 1-(2-{[3-(4'- LRMS m/z (M+H) aminobiphenyl-3-yl)-4- 570.2 found, 570.2 oxo-3,4- required. o dihydroquinazolin-2 yl]thio}hexanoyl)piperidi N
NH
2 ne-4-carboxamide N S 0 N 0 2-10 N 1-(2-{[4-oxo-3-(3- LRMS m/z (M+H) pyridin-3-ylphenyl)-3,4- 556.2 found, 556.3 o dihydroquinazolin-2- required. yl]thio}hexanoyl)piperidi N
NH
2 ne-4-carboxamide N S 0 N 0 2-11 N 1-(2-{[4-oxo-3-(3- LRMS m/z (M+H) pyridin-4-ylphenyl)-3,4- 556.2 found, 556.3 dihydroquinazolin-2- required. 0 yl]thio}hexanoyl)piperidi N NH 2 ne-4-carboxamide N S 0 -58N 0 - 58 - WO 2009/102537 PCT/US2009/031633 2-12 3'-[2-[(1-{[4- LRMS m/z (M+H) OH (aminocarbonyl)piperidin 599.2 found, 599.2 -1- required. O yl~carbonyl}pentyl)thio] 4-oxoquinazolin-3(4H) N
NH
2 yl]biphenyl-3-carboxylic N S N acid 2-13 0 1-[2-({3-[3-(3- LRMS m/z (M+H) furyl)phenyl]-4-oxo-3,4- 545.2 found, 545.2 O dihydroquinazolin-2- required. N
NH
2 yl}thio)hexanoyl]piperidi ne-4-carboxamide N S O N O-,Y 2-14 N-Ni 1-[2-({3-{3-(1-methyl- LRMS m/z (M+H) 1H-pyrazol-4-yl)phenyl]- 559.2 found, 559.3 4-oxo-3,4- required. O Xdihydroquinazolin-2 N NH 2 yl}thio)hexanoyljpiperidi N _S 0 ne-4-carboxamide N S N 0 2-15 1-(2- {[3-(3-cyclohex-1 - LRMS m/z (M+H) en-1-ylphenyl)-4-oxo- 559.2 found, 559.3 O 3,4-dihydroquinazolin-2- required. yl]thio}hexanoyl)piperidi
NNH
2 ne-4-carboxamide N S 0 N 0 - 59 - WO 2009/102537 PCT/US2009/031633 2-16 LRMS m/z (M+H) 1-[2-({4-oxo-3-{3-(2- 561.1 found, 561.2 o ~thienyl)phenyl]-3,4- required. N
NH
2 dihydroquinazolin-2 N S yl}thio)hexanoyl]piperidi N ne-4-carboxamide 0 2-17 / 02(1-{[7-3- LRMS m/z (M+H) chlorophenyl)-2,7- 653.3 found, 653.3 o diazaspiro[4.4]non-2- required. N yl]carbonyl}-3 methylbutyl)thio]-3-[3 N S N N (3 furyl)phenyl]quinazolin 4(3H)-one C1 2-18 2-[(1-{[7-(3- LRMS m/z (M+H) chlorophenyl)-2,7- 667.3 found, 667.3 o ~diazaspiro[4.4]non-2- required. y1]carbonyl}-3 methylbutyl)thio]-3-(3 N S Ncyclohex-1-en-1 N ylphenyl)quinazolin 0 4(3H)-one Cl 2-19 F 1-(2-{[3-(4'-fluoro-2'- LRMS m/z (M+H) methylbiphenyl-3-yl)-4- 587.3 found, 587.3 oxo-3,4- required. o dihydroquinazolin-2 ylJthio}hexanoyl)piperidi N
NH
2 ne-4-carboxamide N S 0 N 0 - 60 - WO 2009/102537 PCT/US2009/031633 2-20 0 -~2-[(1-{[5-(4- LRMS m/z (M+H) N 0fluorophenyl)-2,5- 609.3 found, 609.3 diazabicyclo[2.2.1]hept- required. 2 N N / F yl]carbonyl}pentyl)thio] 3-[33 furyl)phenyljquinazolin 4(3H)-one 2-21 0 2-{(1-{[3-(4- LRMS m/z (M+H) fluorobenzyl)-2-oxo-1- 680.3 found, 680.3 0 oxa-8-azaspiro[4.5]dec- required. N 0 8 0 yl]carbonyl}pentyl)thio] N / 3-[3-(3 - furyl)phenyl]quinazolin F 4(3H)-one 2-22 0 3-(3-cyclohex-1-en-1- LRMS m/z (M+H) SN ylphenyl)-2-[(1-{[5-(4- 623.3 found, 623.3 N S fluorophenyl)-2,5- required. N diazabicyclo[2.2.1]hept O N F 2 yl]carbonyl)pentyl)thio]q uinazolin-4(3H)-one 2-23 3-(3-cyclohex-1-en-1- LRMS m/z (M+H) ylphenyl)-2-[(1-{[3-(4- 694.3 found, 694.3 fluorobenzyl)-2-oxo-1- required. 0 oxa-8-azaspiro[4.5]dec N 0 8 N S yl]carbonyl}pentyl)thiojq N / uinazolin-4(3H)-one F - 61 - WO 2009/102537 PCT/US2009/031633 2-24 3-(3-cyclohex-1-en-1- LRMS m/z (M+H) ylphenyl)-2-[(1-{[4-(2,4- 629.3 found, 629.3 o difluorophenyl)piperazin- required. N F N F ylcarbonyl}pentyl)thio]q N S N uinazolin-4(3H)-one N F 0 2-25 0 2-[(1-{[4-(2,4- LRMS m/z (M+H) difluorophenyl)piperazin- 615.2 found, 614.3 0 1- required. N 1F yl]carbonyl}pentyl)thiol 3-[3-(3 N S N furyl)phenyl]quinazolin 4(3H)-one 2-26 3-(3-cyclohex-1-en-1- LRMS m/z (M+H) ylpheny1)-2-{{l-(2,8- 585.4 found, 585.3 0 diazaspiro[5.5]undec-2- required. ylcarbonyl)pentyljthio}q A uinazolin-4(3H)-one N S 0 N H - 62 - WO 2009/102537 PCT/US2009/031633 SCHEME 3 0 NToluene \/PCI 3 , Br 2
H
2 N OH C reflux, 16h OH 3-1 3-2 Q O 3-3 0 OH0 C Et 3 N, DMF Ci O 0Nac 750 NA OH N; N9'H H 0 3-4 Br 1-3 3-5 O N 0 0 1-6 0 N CI OO N A ~NN CI HN NBOC N N 1. N 2
H
4 , EtOH A S PyBOP,Et 3 N, 0 2. TFA, DCM N DMFN BOC O N 3-6
H
2 N H - 0 3-7 5 4- (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoic acid (3-3) 4-Aminobutanoic acid (3-1, 2 g, 19 mmol, 1.0 equiv.) and phthalic anhydride (3 2, 2.87 g, 19 mmol, 1.0 eq.) were placed in a round bottomed flask equipped with a condenser. Toluene was added (39 mL) and the reaction mixture was heated to reflux until disappearance of the starting material. The solvent was removed under reduced pressure to yield 4-(1,3-dioxo-1,3 10 dihydro-2H-isoindol-2-yl)butanoic acid (3-3). 2-bromo-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoic acid (3-4) 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-y)butanoic acid (3-3, 4g, 17 mmol), bromine (3g, 18.9 mmol, 1.1 eq) and PCl 3 (236 mg, 1.7 mmol, 0.1 eq.) were placed in a round bottomed flask equipped with a reflux condenser, and the mixture was heated at 70 "C until 15 complete disappearance of starting material. The volatiles were evaporated and the product was carried forward without purification. LRMS m/z (M+H) 313.9 found, 313.0 required. 2- { [3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-ylthio}- 4-(1,3 dioxo-1,3-dihydro-2- H-isoindol-2-yllbutanoic acid (3-5) 3-(3-chloropheny1)-2-mercapto-8-methy1quinazolin- 4 (3H)-one (1-3) was prepared 20 as described before. 3-(3-chloropheny1)-2-mercapto-8-methylquinazolin-4(3H)-one (1-3, 211 mg, 0.70 mmol, 1.0 equiv), 2-bromo-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoic acid (3-4, 284 mg, 0.91 mmol, 1.3 equiv.), and triethylamine (588 ptL, 4.2 mmol, 6 equiv.) were dissolved in anhydrous DMF (1 mL) and stirred at 75 *C. When LCMS indicated the reaction was complete, the crude reaction mixture was partitioned between ethyl acetate and 0.1 M HCI, -63- WO 2009/102537 PCT/US2009/031633 the aqueous phase drained and the organic layer washed twice more with 0.1 M HCI followed by brine to yield the product (3-5). LRMS mn/z (M+H) 533.8 found, 533.1 required. tert-butyl 8-[2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2 yl]thio}-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butanoyl]-2,8 5 diazaspiro[5.5lundecane-2-carboxylate (3-6) 2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-yl]thio}- 4-(1,3 dioxo-1,3-dihydro-2- H-isoindol-2-yl)butanoic acid (3-5, 331 mg, 0.43 mmol, 1.0 equiv.), tert butyl 2,8-diazaspiro[5.5]undecane-2-carboxylate (1-6, 220 mg, 0.864 mmol, 2 equiv.), PyBOP (337 mg, 0.648 mmol, 1.5 equiv.) and triethylamine (0.6 mL, 4.32 mmol, 10 equiv.) were 10 dissolved in DMF (I mL) and stirred at rt. Once LCMS showed reaction was complete, the reaction mixture was filtered and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product. LRMS m/z (M+H) 770.0 found, 770.3 required. tert-Butyl 8-(4-amino-2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4 dihydroquinazolin-2-yl]thio}butanoyl)-2,8-diazaspiro[5.5]undecane-2 15 carboxylate tert-Butyl 8-[2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2 yl]thio}-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-y)butanoyl]-2,8-diazaspiro[5.5)undecane-2 carboxylate (3-6, 90 mg, 0.117 mmol, 1.0 equiv.) was dissolved in ethanol (0.585 mL). Excess IM hydrazine in THF (20 eg) was added and the mixture was stirred at rt. Once LCMS showed 20 reaction was complete, the reaction mixture was concentrated, dissolved in DMSO and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product. LRMS m/z (M+H) 640.0 found, 640.2 required 2-{[3-amino-i -(2,8-diazaspiro[5.5 ]undec-2-ylcarbonyl)propyl]thio} -3-(3 chlorophenvl)quinazolin-4(3H)-one (3-7) 25 tert-Butyl 8-(4-amino-2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4 dihydroquinazolin-2-yl]thio)butanoyl)-2,8-diazaspiro[5.5]undecane-2-carboxylate (128 mg, 0.2 mmol, 1.0 equiv.) was dissolved in DCM (lmL). Trifluoroacetic acid (0.5 mL) was added and the mixture stirred at room temperature . Once LCMS showed reaction was complete, toluene was added, the solvent and TFA were removed at reduced pressure and the resulting crude was 30 filtered and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product (3-7). 1H NMR (500 MHz, CDC 3 ) 5 8.13 (br s, 2H), 8.09 (d, 1H,J= 8.0 Hz), 7.63 (t, 1H, J= 7.0 Hz), 7.48 (m, 2H), 7.32 (m, 3H), 3.85 (m, 1H), 3.27 (m, 2H), 3.07 (m, 4H), 2.88 (br m, 6H), 2.55 (s, 3H), 2.36 (m, 2H), 1.88 (m, 1H), 1.64 (in, 5H), 1.37 (d, IH, J= 10.5 Hz). LRMS n/z (M+H) 539.9 found, 540.2 required. 35 The compounds shown in Table 3 were synthesized as TFA salts according to Scheme 3. - 64 - WO 2009/102537 PCT/US2009/031633 Table 3 3-8 2-{[4-amino-l-(2,8- LRMS m/z (M+H) ~ N a Cl diazaspiro[5.5]undec-2- 554.1 found, 554.2 N S ylcarbonyl)butyllthio} -3- required. N NH (3-cblorophenyl)-8 o rnethylquinazolin-4(311) rf one
NH
2 3-9 0 2-{[5-amino-1-(2,8- LRMS m/z (M+H) N Cl diazaspiro[5.Sjundec-2- 554.1 found, 554.3 N sykcarbonyl)pentyl~jthio) - required. -N NH o clorophe-nyl)quinazolin H2N4(3)-one 3-10 cl 2-{[4-amino-1-{[7-(3- LRMS m/z (M+H) oaa chlorophenyl)-2,7- 622.0 found, 622.2 N '6 diazaspiro[4.4jnon-2- required. N yl]carbonyl}butyl)thio]
H
2 N- o (chlorophenyl)quinazolin 4(3o)-one 3-11 Cl 2-(5-amino-1-f7-(3- LRMS m/z (M+H) o chlorophenyl)-2,7- 636.0 found, 636.2 N ' diazaspiro[4.4]nonan-2- required. N Syl)-l -oxopentan-2 N,/ \-N ylho)3-(3 H chlorophenyl)-q H2N- methylquinazolin-4(3H) one - 65 - WO 2009/102537 PCT/US2009/031633 SCHEME 4 OH NC 2 N3HN N DMF, 600C A C A OH N. Sa3 MF
N
3 4-1 B 4-3Ho PyBOP, Et 3 N, 4-1 Br 2. NaN 3 , DMF 60C 4-3 HO DMF, r 0 O 0 N CI PPh 3 , Tol, rt N Ct N S N S N CI NN N
N
3
N,\JNH
2 0 -5 0 4-6 6-azido-2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoic 5 acid (4-3) 2,6-Dibromoxanoic acid (4-1, 300 mg, 1.0 mmol, 1.0 equiv.) and 2-(3 chlorophenyl)-2,7-diazaspiro[4.4]nonane (4-4, mg, 0.9 mmol, 0.9 eq.) were placed in a round bottomed flask. DMF was added (20 mL) and the reaction mixture was heated to 40 C until disappearance of the starting material, at which point excess of NaN3 was added. Stirring was 10 continued until completion of the reaction as judged by LC-MS. The reaction mixture was carefully partitioned between Et20 and a 0. IN aqueous solution of HC1, the organic solvent separated, dried and removed under reduced pressure. The crude was purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product. LRMS m/z (M+H) 770.0 found, 770.3 required. to yield 6-azido-2-{[3-(3-chlorophenyl)-4-oxo-3,4 15 dihydroquinazolin-2-yljthio)hexanoic acid (4-3). 2-[(5-azido-1-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2 ylcarbonyl}pentvil)thiol-3-(3-chlorophenyl)quinazolin-4(3H)-one (4-5) 6-Azido-2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yljthio}hexanoic acid (4-3, 15 mg, 0.034 mmol, 1.0 equiv.), tert-butyl 2,8-diazaspiro[5.5]undecane-2-carboxylate 20 (4-4, 9 mg, 0.864 mmol, 1.2 equiv.), PyBOP (21 mg, 0.041 mmol, 1.2 equiv.) and triethylamine (0.05 mL, 0.34 mmol, 10 equiv.) were dissolved in DMF (0.2 mL) and stirred at rt. Once LCMS showed reaction was complete, the reaction mixture was filtered and purified with reverse phase HPLC (H20/ CH 3 CN gradient w/ 0.1% TFA present) to yield the product. LRMS n/z (M+H) 662.1 found, 662.2 required. 25 2-[(5-amino-I - {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2 ylcarbonyllpentyl)thiol-3-(3-chlorophenyl)quinazolin-4(3H)-one (4-6) To 2-[(5-azido- 1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2 yl] carbonyl }pentyl)thio] -3-(3-chlorophenyl)quinazolin-4(3H)-one_(10 mg, 0.016 mmol, 1.0 equiv.) was added a IM solution of PMe 3 in toluene and the mixture stirred at room -66- WO 2009/102537 PCT/US2009/031633 temperature. Once LCMS showed reaction was complete, the volatiles were removed at reduced pressure and the resulting crude was purified with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1% TFA present) to yield the product. LRMS m/z (M+H) 636.0 found, 636.2 required. The compound shown in Table 4 was synthesized as a TFA salt according to 5 Scheme 4. Table 4 4-7 0 2-{[5-amino-l-(2,8- LRMS m/z (M+H) N CI diazaspiro{5.5]undec-2- 568.1 found, 568.2 N S ylcarbonyl)pentyl]thio} - required. N NH 3-(3-chlorophenyl)-8 o methylquinazolin-4(3H) one EXAMPLES 1-8 Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for 10 practicing the invention. These examples do not limit the claimed invention. EXAMPLE 1: Identification of CHKisv1 Using Real-time PCR To facilitate the determination of compound inhibitory properties, it is desirable to identify variants of the "normal" splicing of exon regions encoding CHKI. In particular, naturally occurring splicing variations resulting in the loss of the C-terminal regulatory domain 15 of CHKI were sought. Deletion of the C-terminus confers greater kinase activity to CHKI (Chen et al., 2000, Cell 100:681-692; Katsuragi and Sagata, 2004, Mol. Biol. Cell, 15:1680 1689). Exons 2-8 encode the catalytic kinase domain and exon 9 encodes the linker region. The SQ and C-terminal regulatory domains lie within exons 10-13 (Sanchez et al., 1997, 277:1497 1501; Katsuragi and Sagata, 2004, Mol. Biol. Cell. 15:1680-1689). Real-time PCR experiments 20 and RT-PCR have been used to identify and confinn the presence of novel splice variants of human CHKJ mRNA. A naturally occurring splice variant which encodes a C-terminal truncation of the CHK1 inhibitory domain was identified, cloned, expressed and purified for use in a CHK1 kinase assay of utility for the determination of compound inhibitory properties. RT-PCR 25 The structure of CHKJ mRNA in the region corresponding to exons 8 to 11 was determined for RNA extracted from human testis using an RT-PCR based assay. Total RNA isolated from human testis was obtained from BD Biosciences Clontech (Palo Alto, CA). RT PCR primers were selected that were complementary to sequences in exon 8 and exon 11 of the reference exon coding sequences in CHK] (NM_001274). Based upon the nucleotide sequence 30 of CHKJ mRNA, the CHK1 exon 8 and exon 11 primer set (hereafter CHKI 8 1 primer set) was - 67 - WO 2009/102537 PCT/US2009/031633 expected to amplify a 478 base pair amplicon representing the "reference" CHK1 mRNA region. The CHKI 8-11 primer set was expected to amplify a 300 base pair amplicon in a transcript that possessed alternative splicing of exon 9 to exon 11. The CHK1 exon 8 forward primer has the sequence: 5' ATCAGCAAGAATTACCATTCCAGACATC 3' (SEQ ID NO 1); and the CHK1 5 exon II reverse primer has the sequence: 5' CATACAACTTTTCTTCCATTGATAGCCC 3' (SEQ ID NO 2). Total RNA from human testis was subjected to a one-step reverse transcription PCR amplification protocol using the Qiagen, Inc. (Valencia, CA), One-Step RT-PCR kit, using the following cycling conditions: 10 1) 50*C for 30 minutes; 2) 95*C for 15 minutes; 3) 35 cycles of 94*C for 30 seconds; 63.5*C for 40 seconds; 15 72*C for 50 seconds; then 724C for 10 minutes. RT-PCR amplification products (amplicons) were size fractionated on a 2% agarose gel. Selected fragments representing 250 to 350 base pair amplicons were manually extracted from the gel and purified with a Qiagen Gel Extraction Kit. The purified amplicon 20 fragments were reamplified with the CHK 8-11 primer set, and these amplicons were size fractionated on an agarose gel. Fragments representing 250 to 350 base pair amplicons were manually extracted from the gel and purified with a Qiagen Gel Extraction Kit. The purified amplicon fragments were reamplified with the CHK18.
11 primer set once more. Following size fractionation on an agarose gel and manual extraction of the 250 to 350 base pair amplicons, the 25 purified amplicon fragments (Qiagen Gel Extraction Kit) were cloned into an Invitrogen pCR2.1 vector using the reagents and instructions provided with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Clones were then plated in pools of 440 colonies per plate, onto 15 plates, for a total of 6600 clones. DNA was extracted from the pooled 440 colonies from each plate and used as template for real-time PCR. 30 Real-time PCR/TAQman To determine the presence of an alternatively spliced isoform to the CHKI reference protein (NP_001265), a real-time PCR assay was used. TAQman primers and probes used to detect the CHKsv1 isoform were designed and synthesized as pre-set mixtures (Applied Biosystems, Foster City, CA). The sequences of 35 the TAQman primers and probes used to detect the CHK1 reference form (SEQ ID NOs 3, 4, and 5) and CHK~sv] isoform (SEQ ID NOs 6, 7, and 8) are shown in Table 1. Splice junction specific probes were labeled with the 6-FAM fluorophore at the 5' end (FAM) and a non fluorescent quencher at the 3' end (NFQ). Real-time PCR was performed on human testis cDNA - 68 - WO 2009/102537 PCT/US2009/031633 using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). The TAQman reaction contained: 96-well format 384-well format 12.5 tl 5 p4l TAQman Universal MasterMix 5 1.25 k 1 0.5 i Primer-probe mix 6.25 tl 2.5 pl H20 5 pl 2 pl DNA Table 1. Primers and probes used to detect CHK1 isoforms. Name SEQ ID Sequence Specificity NO CHK1 reference SEQ ID NO GTTACTTGGCACCCCAGGA CHK1 forward primer 3 reference CHKI reference SEQ ID NO CHKI reverse primer 4 CATCCAATTTGGTAAAGAATCGTGTCA reference CHKI reference probe SEQ ID NO FAM-TCCTCACAGAACCCC-NFQ CHK1 5 reference CHKIsvl forward SEQ ID NO GCACATTCAATCCAATTTGGACTTCT CHKI svl primer 6 CHKlsv1 reverse SEQ ID NO CATCCAATTTGGTAAAGAATCGTGTCAT CHKlsv1 primer 7 CHKlsvl probe SEQ ID NO FAM-CAGTGCTTCTAGAACCC-NFQ CHKlsvl 8 The TAQman reactions were performed on an ABI Prism 7900HT Sequence 10 Detection System (Applied Biosystems, Foster City, CA). The thermocycling conditions were 50'C for 2 minutes, 95 0 C for 10 minutes, and 40 cycles of 954C for 15 seconds and 60*C for 1 minute. Data analysis of the fluorescence emission was performed by the Sequence Detector Software (SDS) (Applied Biosystems, Foster City, CA). Results of the TAQman assay indicated that pooled DNA from 13 out of 15 plates 15 appeared to possess clones that represented an alternative exon 9 to exon 11 splice junction. DNA from one of these positive pools, representing 440 colonies, was used to transform bacterial host cells. Clones were plated in pools of 55 colonies per plate onto 12 plates total. The colonies on each of the 12 plates were again pooled and used for a TAQman assay. Pooled DNA from I out of 12 plates appeared to possess a clone that represented an alternative exon 9 to 20 exon 11 splice junction. The 55 colonies on this positive plate were individually screened using a TAQman assay, and one clone was identified as possessing an alternative exon 9 to exon 11 splice junction. This positive clone was then sequenced from each end using the CHKJ exon 8 forward primer (SEQ ID NO 1) and a different exon 11 reverse primer with the sequence 5' TGCATCCAATTTGGTAAAGAATCG 3' (SEQ ID NO 9). - 69 - WO 2009/102537 PCT/US2009/031633 Sequence analysis of the clone revealed that it matched the expected sequence for alternative splicing of exon 9 of the CHK1 heteronuclear RNA to exon 11; that is the coding sequence of exon 10 is completely absent. EXAMPLE 2: Cloning of CHKIsv1 5 Real-time PCR, RT-PCR, and sequencing data indicate that in addition to the normal CHK] reference mRNA sequence, NM_001274, encoding CHK1 protein, NP_001265, a novel splice variant form of CHKI mRNA also exist in testis tissue and MOLT-4, and Daudi cell lines. Clones having a nucleotide sequence comprising the CHKl svl splice variant 10 identified in Example 1 were isolated using recombination-mediated plasmid construction in yeast. A set of two primer pairs was used to amplify and clone the entire mRNA coding sequences of CHK]svl. In the case of CHK1svl, real-time quantitative PCR analysis indicated that transcripts of this splice variant form were present at very low levels. In order to clone CHK~svJ, clones containing coding sequences of the reference CHK] (NM_00 1274) were 15 altered by an additional recombination step in yeast with 80 base pair linkers that were designed to create the desired exon 9 to exon 11 splice junction. A 5' "forward" primer and a 3' "reverse" primer were designed for isolation of full length clones corresponding to CHK~svI. The 5' "forward" CHK~sv1 primer was designed to have the nucleotide sequence of 5' TTACTGGCTTATCGAAA TTAATACGACTCACTATAG 20 GGAGGAGTCATGGCAGTGCCCTTTGT 3' (SEQ ID NO 10) and to have sequences complementary to exon 2 of the CHKJ mRNA (NM_001274). The 3' "reverse" CHK~sv] primer was designed to have the nucleotide sequence of 5' T GAAGGCACAGTCGAGGCTGA TCAGCGGGTTTAAACTCATGCATCCAATTTGGTAAAGAATCG 3' (SEQ ID NO 11) and to have sequences complementary to exon II of the CHK] mRNA (NM_001274). The 25 40 nucleotides at the 5' ends of the primer sequences indicated in italics are "tails" that were incorporated into the PCR amplicons and facilitated subsequent plasmid recombination events in yeast. These CHK~svJ "forward" and "reverse" primers were expected to amplify coding sequences of the reference CHKI mRNA (NM 001274), which was then used in a subsequent recombinational cloning step to create CHK~sv1 -specific sequence. 30 RT-PCR The CI-IK1sv1 cDNA sequence was cloned using a combination of reverse transcription (RT) and polymerase chain reaction (PCR). More specifically, about 25 ng of MOLT-4 cell line mRNA (BD Biosciences Clontech, Palo Alto, CA) was reverse transcribed using Superscript II (Gibco/Invitrogen, Carlsbad, CA) and oligo d(T) primer 35 (RESGEN/Invitrogen, Huntsville, AL) according to the Superscript II manufacturer's instructions. For PCR, I p 1 of the completed RT reaction was added to 40 pl of water, 5 pd of lOX buffer, 1 pl of dNTPs and 1 pl of enzyme from a Clontech (Palo Alto, CA) Advantage 2 PCR kit. PCR was done in a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, - 70 - WO 2009/102537 PCT/US2009/031633 CA) using the CHK~svI "forward" and "reverse" primers for CHKIsv] (SEQ ID NOs 10,11). After an initial 94*C denaturation of I minute, 35 cycles of amplification were performed using a 30 second denaturation at 94 0 C followed by a 40 second annealing at 63.5 0 C and a 50 second synthesis at 72 0 C. The 35 cycles of PCR were followed by a 10 minute extension at 724C. The 5 50 pl reaction was then chilled to 4 0 C. 10 pl of the resulting reaction product was run on a 1% agarose (Invitrogen, Ultra pure)gel stained with 0.3 pg/ml ethidium bromide (Fisher Biotech, Fair Lawn, NJ). Nucleic acid bands in the gel were visualized and photographed on a UV light box to determine if the PCR had yielded products of the expected size, in the case of the CHK1 mRNA, a product of about 1243 base pairs. The remainder of the 50 pl PCR reactions 10 from MOLT-4 cells was purified using the QIAquik Gel extraction Kit (Qiagen, Valencia, CA) following the QlAquik PCR Purification Protocol provided with the kit. About 50 p1 of product obtained from the purification protocol was concentrated to about 6 pI by drying in a Speed Vac Plus (SC1 IA, from Savant, Holbrook, NY) attached to a Universal Vacuum System 400 (also from Savant) for about 30 minutes on medium heat. 15 Cloning and assembly of CHK~svI full-length clones and yeast transformation Assembly of the full length CI-IKsv1 clone by homologous recombination cloning in yeast was performed using a cycloheximide-based counterselection scheme similar to that described previously by Raymond et al. (2002, Genome Res. 12:190-197). Assembly of the full-length CHK~svI full length clone by homologous 20 recombination between the 1243 base pair CHK] amplicon, produced using the CHK1 svl forward and reverse "tailed" primers described earlier, and the expression vector was performed by simultaneous transformation of these pieces into yeast cells. A subsequent recombination step with 80 base pair oligonucleotide linkers created the CHK~sv] exon 9 to exon 11 splice junction. All yeast transformation steps described in subsequent paragraphs were performed by 25 electroporation (Raymond et al., 2002 Genome Res. 12:190-197). 1 pg of the 1243 base pair CHK] purified amplicon was cloned directly into 100 ng of Srfl-digested pCMR1 I by cotransformation of 100 p1 of yeast strain CMY1-5 (Mata, URA3A, CYH2R). Ura*, cycloheximide resistant colonies were selected on Ura-deficient media plates containing 1 gg/ml cycloheximide (Sigma, St. Louis, MO). Standard yeast media were 30 used (Sherman, 1991, Methods Enzymol. 194:3-21). Total DNA from yeast cell culture containing the CHK] clone was used to transform E coli to chloramphenicol (Sigma, St. Louis, MO) resistance to prepare a large quantity of the recombinant plasmid as described in Hoffman and Winston (1987 Gene 57:267-72). The colonies were picked from the plates into 2 ml of 2X LB media. These liquid cultures were incubated overnight at 37 0 C. Plasmid DNA was extracted 35 from these cultures using the Qiagen (Valencia, CA) Qiaquik Spin Miniprep kit. -71- WO 2009/102537 PCT/US2009/031633 Table 2. Composition of pCMR 1 plasmid Nucleotide Functional description of sequence coordinates 1 - 6013 Copy-controlTM E coli origin of replication from pCCIFOS (Epicentre Technologies, Madison, WI). 6014 - 7884 Yeast URA3 gene, ARS4 autonomously replicating sequence and CEN6 centromere from pRS316 (Sikorski and Hieter, 1989). 7885 - 8825 Mammalian CMV promoter from InVitrogen (Carlsbad, CA) vector pcDNA3,1/mye-HIS A. 8826 - 10,774 Yeast CYH2 gene amplified from strain BY4709 (Brachmann et al. 1998) 10,775 - 10,782 Engineered Srfl restriction site. 10,783 - 13,556 Mammalian poly-adenylation sites, selectable markers, SV40 origin, etc. from pcDNA3.l /myc-HIS A. 13,557 - 13,596 DNA sequence from InVitrogen vector pENTRl l. 13,597 - 14,561 pCMR1 I - specific; chloramphenicol resistance gene from pCCIFOS. To construct the CHK~svI clone, 1 pg of 80 base pair linkers shown in Table 3 (SEQ ID NOs 12, 13) that spans the region of the alternative splicing of exon 9 to exon 11, and 100 ng of Banil-digested CHKI/pCMR 11 clone were used to cotransform 100 pil of a 5 cycloheximide sensitive yeast strain. The overlapping DNA between the linkers and CHKJ/pCMR1 I clone dictates that most yeast transformants will possess the correctly assembled construct. Ura*, cycloheximide resistant colonies were selected for subsequent preparation and transformation of E. coli. Plasmid DNA extracted from E. coli was analyzed by restriction digest to confirm the presence of the alternative splicing of exon 9 to exon 11 in the CHKIsv1 10 clone. Eight CHK~svI clones were sequenced to confirm identity, and the clones possessing the appropriate sequences are used for protein expression in multiple systems. Table 3. Linkers used to create exon 9 to exon 11 splice junction for CHKIsvl clone SEQ ID NO Linker Sequence SEQ ID NO 12 AATCCAATTTGGACTTCTCTCCAGTAAACAGTGCTTCTAGAACC CCTGGCAGCGGTTGGTCAAAAGAATGACACGATTCT SEQ ID NO 13 AGAATCGTGTCATTC'TTTTGACCAACCGCTGCCAGGGGTTCTAG AAGCACTGTTTACTGGAGAGAAGTCCAAATTGGATT - 72 - WO 2009/102537 PCT/US2009/031633 Summary of CHK~sv] polynucleotide The polynucleotide coding sequence of CHK~sv1 mRNA (Seq ID NO 14) contains an open reading frame that encodes a CHK1 svl protein (SEQ ID NO 15) similar to the reference CHK1 protein (NP 001265), but lacking amino acids encoded by a 178 base pair 5 region corresponding to exons 10 of the full length coding sequence of reference CHK1 mRNA (NM_001274). The deletion of the 178 base pair region results in a shift of the protein translation reading frame in comparison to the reference CHK1 protein reading frame, creating a carboxy terminal peptide region that is unique to CHKI svl (italicized in Seq ID NO 15). The frameshift also creates a premature termination codon 29 nucleotides downstream of the 10 exon 9/exon 11 splice junction. Therefore, the CHKl svl protein is missing an internal 59 amino acid region corresponding to the amino acid region encoded by exon 10 and is also lacking the amino acids encoded by the nucleotides downstream of the premature stop codon as compared to the reference CHK1 (NP 001265). Exon 10 encodes the SQ/TQ domains of CHK1, and exons 11-13 encode the autoinhibitory region (Sanchez et al., 1997, Science 277:1497-1501; 15 Katsuragi and Sagata, 2004, Mol. Biol. Cell. 15:1680-1689). While deletion of the autoinhibitory region confers constitutive activity to the CHKI kinase domain, when the SQ/TQ domains are also removed, CHKI enzymatic activity decreases (Ng et al., 2004, J. Biol. Chem. 279:8808 8819). Table 4. Nucleotide coding sequence and coded polypeptide for CHKl sv1 Seq ID ATGGCAGTGCCCTTTGTGGAAGACTGGGACTTGGTGCAAACCCTGGG NO 14 AGAAGGTGCCTATGGAGAAGTTCAACTTGCTGTGAATAGAGTAACTG AAGAAGCAGTCGCAGTGAAGATTGTAGATATGAAGCGTGCCGTAGAC TGTCCAGAAAATATTAAGAAAGAGATCTGTATCAATAAAATGCTAAA TCATGAAAATGTAGTAAAATTCTATGGTCACAGGAGAGAAGGCAATA TCCAATATTTATTTCTGGAGTACTGTAGTGGAGGAGAGCTTTTTGACA GAATAGAGCCAGACATAGGCATGCCTGAACCAGATGCTCAGAGATTC TTCCATCAACTCATGGCAGGGGTGGTTTATCTGCATGGTATTGGAATA ACTCACAGGGATATTAAACCAGAAAATCTTCTGTTGGATGAAAGGGA TAACCTCAAAATCTCAGACTTTGGCTTGGCAACAGTATTTCGGTATAA TAATCGTGAGCGTTTGTTGAACAAGATGTGTGGTACTTTACCATATGT TGCTCCAGAACTTCTGAAGAGAAGAGAATTTCATGCAGAACCAGTTG ATGTTTGGTCCTGTGGAATAGTACTTACTGCAATGCTCGCTGGAGAAT TGCCATGGGACCAACCCAGTGACAGCTGTCAGGAGTATTCTGACTGG AAAGAAAAAAAAACATACCTCAACCCTTGGAAAAAAATCGATTCTGC TCCTCTAGCTCTGCTGCATAAAATCTTAGTTGAGAATCCATCAGCAAG AATTACCATTCCAGACATCAAAAAAGATAGATGGTACAACAAACCCC TCAAGAAAGGGGCAAAAAGGCCCCGAGTCACTTCAGGTGGTGTGTCA GAGTCTCCCAGTGGATTTTCTAAGCACATTCAATCCAATTTGGACTTCT - 73 - WO 2009/102537 PCT/US2009/031633 CTCCAGTAAACAGTGCTTCTAGAACCCCTGGCAGCGGTTGGTCAAAAG AATGA Seq ID MAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKIVDMKRAVD NO 15 CPENIKKEICINKMLNHENVVKFYGHRREGNIQYLFLEYCSGGELFDRIEP DIGMPEPDAQRFFHQLMAGVVYLHGIGITHRDIKPENLLLDERDNLKISDF GLATVFRYNNRERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVL TAMLAGELPWDQPSDSCQEYSDWKEKKTYLNPWKKIDSAPLALLHKILV ENPSARITIPDIKKDRWYNKPLKKGAKRPRVTSGGVSESPSGFSKHIQSNL DFSPVNSASRTPGSGWSKE EXAMPLE 3: Expression of CHKlsv1 Protein The baculovirus gene expression vector system permits protein expression insect cells, which are inexpensive and easy to maintain. The proteins produced are of similar quality to that in mammalian cells (Miller, 1988, Biotechnology 10:457-465; Miller, 1989, Bioessays 5 11:91-95). Methods of protein expression using the baculovirus expression vectors in insect cells are known in the art and techniques are discussed in O'Reilly et al., Baculovirus Expression Vectors - A Laboratory Manual, W. H. Freeman and Co., New York, 1992 and Baculovirus Expression Vector System Instruction Manual, 6 th edition, Pharmingen, San Diego, 1999. Cloning CHKlsv1 for Insect Cell Expression 10 To create a CHK~svl/baculovirus transfer vector construct, the CHK1sv1/pCMR 11 clone (see Example 2) was used as template for PCR to amplify the coding sequence of CHKlsv1 (SEQ ID NO 14) using the primers listed in Table 5 (SEQ ID NOs 16, 17). The primer represented by SEQ ID NO 16 contains an optimal translation initiation sequence immediately upstream of the ATG start codon and an upstream EcoRI restriction site 15 that become incorporated into the amplicon. The primer represented by SEQ ID NO 17 contains sequence encoding six histidine residues C-terminal to the CHK1 svl coding sequence as well as an EagI restriction site that become incorporated into the CHKlsvlamplicon. The CHKlsvl amplicon was run on a 1% agarose gel. A selected amplicon fragment of the expected size, in the case of CHK]sv], a product of about 994 base pairs, was manually extracted from the gel and 20 purified with a Qiagen Gel Extraction Kit. The purified amplicon fragment was digested with EcoRIl and EagI. The EcoRI/EagI-digested amplicon was ligated into the baculovirus transfer vector pVL1 393 (Pharmingen, San Diego, CA) which had been digested with EcoRT and EagI and dephosphorylated with alkaline phosphatase. The CHK~svI/pVLI 393 construct was then transformed into E. coli strain DH5a. Plasmid DNA extracted from selected from ampicillin 25 resistant colonies was sequenced to confirm identity, and the clones possessing the appropriate sequences were used for protein expression in insect cells. - 74 - WO 2009/102537 PCT/US2009/031633 Table 5. Primers used to clone CHK~svl into baculovirus transfer vector VLI 393 SEQ ID NO Primer Sequence SEQ ID NO 16 CCCGGAATTCACCATGGCAGTGCCCTTTGTGGAAGACTGG SEQ TD NO 17 TGTGTCCGGCCGTCAGTGATGGTGATGGTGATGTTCTTTTGACC AACCGCTGCC Insect Cell Expression of CHKlsvl The CHK~svJ/pVLI 393 construct was co-transfected with linearized AcNPV BaculoGold DNA (Pharmingen, San Diego, CA) into SF9 insect cells (Invitrogen, Carlsbad, 5 CA). Individual recombinant viruses were selected by end point dilution. Virus clones were amplified to obtain high titer stocks. These virus stocks were used for protein expression tests in small scale SF9 cultures to verify production of the CHKI svl recombinant protein. Transfected SF9 cell lysates were analyzed by polyacrylamide gel electrophoresis for CHKsvl protein expression. The CHKI svl protein was visualized by Commassie staining or by Western blotting 10 using an anti-CHK1 antibody (G4 antibody; Santa Cruz Biotechnology, Inc). Based on expression, an individual virus was selected for larger scale CHK1sv1 expression. For recombinant protein expression on the liter scale, SF9 suspension cultures were grown at 27 0 C in Ex-cell 401 serum-free media (JRH Scientific, Lenexa, KS) and were infected with a recombinant virus stock using a multiplicity of infection of 0.3 virus per cell. The infected SF9 15 culture was harvested 72 hour following virus transfection, and pelleted by centrifugation. Pellets were stored at -70 0 C. Purification of CHKI svl Recombinant Protein Insect cell pellets were lysed with B-PER protein extraction reagent (Pierce, Rockford, IL) containing I pM microcystin (Sigma, St. Louis, MO), 10 pM cypermethrin (EMD 20 Biosciences, San Diego, CA), and EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) (1 tablet/50 ml lysis buffer). All manipulations during protein purification were performed at 4*C. Cells were resuspended in the lysis buffer were stirred for 45 minutes. DNAseI (Roche) was then added to a final concentration of 200 U/ml and the cell suspension was stirred for an additional 30 minutes. The lysed cell suspension was centrifuged for 25 30 minutes at 30,000 g. The lysis supernatant was decanted and centrifuged for 30 minutes at 30,000 g. For each 10 ml of cleared supernatant, 1 ml bed volume of Talon metal affinity resin (Clontech, Palo Alto, CA) was added, and the suspension was stirred for 45 minutes. The affinity resin/lysate suspension was centrifuged at 5000 g for 3 minutes and then the supernatant was discarded. The affinity resin was washed 4X with Buffer A (50 pM Tris, pH 8.0; 250 mM 30 NaCl) using 5X volumes of the resin. The washed resin was resuspended as a 2X slurry in Buffer A and packed into a chromatography column. The resin-packed column was washed with 6X bed volumes of Buffer A. CHK1svl-His-tagged protein is eluted from the column using a step-wise gradient of imidazole in Buffer A. Imidazole concentrations in the 2X bed volumen fractions were 5, 10, 20, 30, 40, 50, and 60 mM. Elution fractions were concentrated using the - 75 - WO 2009/102537 PCT/US2009/031633 Amicon Ultra 15 Centrifugal Filter Device, 30,000 Nominal Molecular Weight Limit (Millipore, Billerica, MA). The concentrated enzyme fractions were diluted 50% in glycerol and stored at 20*C. Fractions were analyzed for the presence of CHKIsvl-His-tagged protein using polyacrylamide gel electrophoresis followed by Coommassie staining and Western blotting using 5 an anti-CHKI antibody (G4 antibody; Santa Cruz Biotechnology, Inc). The CHK1 svl kinase activity of the column fractions was determined using the kinase assay described in the following section. EXAMPLE 4: CHKIsvl Kinase Assay CHK1svl activity was assayed in vitro using a synthetic peptide substrate. The 10 phosphopeptide product was quantitated using a Homogenous Time-Resolved Fluorescence (HTRF) assay system (Park et al., 1999, Anal. Biochem. 269:94-104). The reaction mixture contained 40 mM HEPES, pH 7.3; 100 mM NaCl; 10 mM MgC12; 2 mM dithiothreitol; 0.1% BSA; 0.1 mM ATP; 0.5 pM peptide substrate; and 0.1 nM CHKlsv1 enzyme in a final volume of 40 pl. The peptide substrate has the amino acid sequence amino 15 terminus-GGRARTSSFAEPG-carboxy terminus (SynPep, Dublin CA) (SEQ ID NO 18) and is biotinylated at the N-terminus. The kinase reaction was incubated for 30 minutes at 22 0 C, and then terminated with 60 pl Stop/Detection Buffer (40 mM HEPES, pH 7.3; 10 mM EDTA; 0.125% Triton X-100; 1.25% BSA; 250 nM PhycoLink Streptavidin-Allophycocyanin (APC) Conjugate (Prozyme, San Leandro, CA); and 0.75 nM GSK3cc anti-phosphoserine antibody (Cell 20 Signaling Technologies, Beverly, MA; Cat# 9338) labeled with europium-chelate (Perkin Elmer, Boston, MA). The reaction was allowed to equilibrate for 2 hours at 22 0 C, and relative fluorescent units were read on a Discovery plate reader (Packard Biosciences). Inhibitor compounds are assayed in the reaction described above, to determine compound IC50s. 1 paL of compound dissolved in DMSO was added to each 40 RL reaction in a half-log dilution series 25 covering a range of 1 nM to 100 piM. Relative phospho substrate formation, read as HTRF fluorescence units, is measured over the range of compound concentrations and a titration curve generated using a four parameter sigmoidal fit. Specific compounds of the instant invention were tested in the assay described above and were found to have IC 50 of < 50 ptM against substrate. 30 EXAMPLE 5: Inhibition of CHK1 Autophosphorylation in Cells Inhibitor compounds are assayed for their ability to inhibit CHK1 in cells by monitoring CHKI autophosphorylation in response to DNA damage. H1299 cells (ATCC, Manassas, VA) are grown in culture medium: RPMI 1640 supplemented with 10% fetal bovine serum; 10 mM HEPES; 2 mM L-glutamine; Ix non-essential amino acids; and penicillin 35 streptomycin. Cells from T-75 flasks are pooled, counted, seeded into 6 well dishes at 200,000 cells per well in 2 ml media, and incubated. Serial dilution series of compounds in DMSO or DMSO control are added to each well from a 1000x working stock in DMSO and incubated for 2 hr at 37 C. Following the 2-hr incubation period, 1OOnM camptothecin (EMD Biosciences, San - 76 - WO 2009/102537 PCT/US2009/031633 Diego, CA) is added from a 200x working stock in PBS to all drug-treated cells (except one of the high dose wells) and one DMSO control well. After a 4 hour incubation with camptothecin, each well is washed once with ice-cold PBS and 300 pL of lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 50 mM NaF, 1% NP-40, 0.5% Deoxycholic acid, 0.1% SDS, 0.5 pM Na 3
VO
4 5 and IX Protease Inhibitor Cocktail Complete - without EDTA (Roche Diagnostics, Mannheim, Germany)) is added to each well. Plates are shaken at 40 C for 10-15 min and lysates are then transferred to 1.5 ml microcentrifuge tubes and frozen at -804 C. Lysates are thawed on ice and cleared by centrifugation at 15,000 x g for 20 min and the supernatants are transferred to clean tubes. 10 Samples (20pL) are prepared for gel electrophoresis by addition of 5 pL of 5x sample loading buffer and heat-denaturation for 5 min at 100* C. Samples are electorphoresed in Tris/Glycine SDS-polyacrylamide gels (10%) and proteins are transferred onto PVDF. Blots are then blocked for 1 hr in 3% BSA in TBS and probed using an antibody against phospho-Ser-296 CHKI (Cell Signaling Technologies - Cat #2346). Bound antibody is visualized using a 15 horseradish peroxidase conjugated secondary antibody (goat anti-rabbit Jackson Labs - Cat# Ill 035-046) and enhanced chemiluminescence (ECL-plus, Amersham, Piscataway, NJ). After stripping of the first antibody set by incubation in 62.5 mM Tris HCl pH 6.7, 2% SDS and 2 mercaptoethanol to 100 pM for 30 min at 550 C, blots are re-probed for total CHK1, using a CHK1 monoclonal antibody (Santa Cruz Biotechnology Inc., Cat# SC-8408). The CHK1 20 monoclonal is detected using a a sheep anti-mouse IgG coupled to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ, Cat#NA93 1) and enhanced chemiluminescence (ECL plus, Amersham). ECL exposed films are scanned and the intensity of specific bands is quantitated with ImageQuant software. Titrations are evaluated for level of phospho-CHK1 (Ser296) signal normalized to total CHKI and IC50 values are calculated. 25 EXAMPLE 6: Functional Activity of Inhibitors in Checkpoint Escape Assay DNA damage arrest To measure functional activity of CHK1 inhibitors in cells, compounds are assayed for their ability to abrogate DNA damage induced cell cycle arrest. The assay determines cell phospho-nucleolin levels as a measure of the quantity of cells entering M-phase after cell 30 cycle arrest brought on by the DNA damaging agent camptothecin. H1299 cells (ATCC, Manassas VA) are seeded at a density of 5000 cells/well in RPMI640 media supplemented with 10% fetal bovine serum. After incubation for 24 hours at 37 0 C at 5% C0 2 , camptothecin is added to a final concentration of 200 nM and incubated for 16 hours. An equal volume of a test compound serial dilution series in growth media plus 200nM 35 camptothecin and 332nM nocodozole (final concentration: 50ng/ml) is added and incubation at 37 0 C is continued for 8 hours. Media is removed from the wells and 50 PL lysis buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 50 mM NaF, 1% Triton X-100, 10% Glycerol, I x Proteinase Inhibitor Cocktail (Roche Diagnostics, Mannheim Germany), I pl/ml DNase I (Roche - 77 - WO 2009/102537 PCT/US2009/031633 Diagnostics), 300 pM Sodium Orthovanadate, I pM Microcystin (Sigma, St. Louis, MO) added. The plate with lysis buffer is shaken for 30 min at 4C and frozen (-70"C) for 20 min. Levels of phosphonucleolin in the cell lysates is measured using the IGEN Origen technology (BioVeris Corp., Gaithersburg, MD). 5 Detection of phosphonucleolin in cell lysates 4E2 anti-nucleolin antibody (Research Diagnostics Inc., Flanders, NJ) was biotinylated using Origen Biotin-LC-NHS-Ester (BioVeris Corp.) using the protocol described by the manufacturer. Goat anti-mouse antibody (Jackson Immuno Research, West Grove, PA) was ruthenylated employing a ruthenylation kit (BioVeris Corp.; cat# 110034) according to the 10 protocol described by the manufacturer. To each well of a 96-well plate is added 25 ptL of antibody buffer (phospho buffered saline pH7.2, 1% bovine serum albumin, 0.5% Tween-20) containing 2 jig/ml biotynylated 4E2 anti-nucleolin antibody and 0.4mg/ml streptavidin coated paramagnetic Dynabeads (BioVeris Corp.) along with 25pL of cell lysate (above). The antibodies and lysate are incubated with shaking for 1 hr at room temperature. Next, 50 ng of anti 15 phosphonucleolin TG3 antibody (Applied NeuroSolutions Inc., Vernon Hills, IL) in a volume of 50 pL of antibody buffer (above) are added to each well of the lysate mix and incubation is continued for 30 min at room temperature. Lastly, 25pL of a 240ng/ml solution of the ruthenylated goat anti-mouse antibody in antibody buffer is added to each well and incubation continued for 3 hours at room temperature. The lysate antibody mixtures are read in a BioVeris 20 M-series MS analyser and EC50s for compound dependent increases in phosphor-nucleolin are determined. EXAMPLE 7: Other Biological Assays CHK1 Expression and Purification: Recombinant human CH1 can be expressed as a fusion protein with glutathione S-transferase at the amino-terminus (GST-CHK1) using 25 standard baculovirus vectors and a (Bac-to-Bac@) insect cell expression system purchased from GIBCOTM Invitrogen. Recombinant protein expressed in insect cells can be purified using glutathione sepharose (Amersham Biotech) using standard procedures described by the manufacturer. CHKI Fluorescense Polarization Assays: CHK1 kinase inhibitors can be 30 identified using fluorescence polarization to monitor kinase activity. This assay utilizes 10 nM GST-CHKI and contains 5 mM 2-(N-Morpholino)ethanesulfonic acid (MES, pH 6.5), 5 mM magnesium chloride (MgCl2), 0.05% Tween@-20, 1 pM adenosine 5' triphosphate (ATP), 2 mM 1,4-Dithio-DL-threitol (DTT), I iM peptide substrate (Biotin-ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 10 nM peptide substrate tracer (Fluorescine-GSRRP-pS-YRKI-free acid) (pS 35 = phosphorylated-Serine) (SEQ ID NO: 20), 60 ng anti-phospho-CREB(S 133) mouse monoclonal IgG purified on Protein G sepharose from crude mouse ascites purchased from Cell Signalling Technologies (Beverly, MA), 4% dimethyl sulfoxide (DMSO) and 30 pM inhibitor compound. Reactions are incubated at room temperature for 140 minutes and terminated by - 78 - WO 2009/102537 PCT/US2009/031633 addition of 25 mM EDTA (pH 8.0). Stopped reactions are incubated for 120 minutes at room temperature and fluorescence polarization values determined using a Molecular Devices/LJL Biosystems AnalystTM AD (Sunnyvale, CA) with standard fluorescine settings. CHK1 SPA Filtration Assay: Assays (25 pl) contain 10 nM GST-CHKI, 10 mM 5 MES, 2 mM DTT, 10 mM MgC12, 0.025% Tween@-20, 1 uM peptide substrate (Biotin ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 1 p.M ATP, 0.1 pCi 3 3 P-,y-ATP (New England Nuclear, NEN) and are reacted for 90 minutes at room temperature. Reactions are terminated by adding 55 pl of phosphate buffered saline containing 50 mM EDTA, 6.9 mM ATP, 0.5 mg Scintilation proximity assay (SPA) beads (Amersham Biosciences). Peptide 10 substrate is allowed to bind beads for 10 minutes at room temperature followed by filtration on a Packard GF/B Unifilter plate and washed with phosphate buffered saline. Dried plates may are sealed with Topseal T M (NEN) and 33p incorporated to peptide substrate using a Packard Topcount® scintillation counter with standard settings for 33p. CHK1 FlashPlate® Kinase Assay: Assays (25 pl) contain 8.7 GST-CHK1, 10 15 mM MES, 0.1 mM ethylene glycol-bis(p-aminoethylether)-N,N,N',N'-tetracetic acid (EGTA, pH 8.0), 2 mM DTT, 0.05% Tween 20, 3 p.M peptide substrate (Biotin-ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 1 pM ATP, 0.4 pCi 33P-y-ATP (NEN) and 4% DMSO. Reactions are incubated for 30 minutes at room temperature, terminated with 50 pl of 50 mM EDTA. 90 pl of reaction is transferred to streptavidin-coated FlashPlates@ (NEN) and incubated for 1 hour at 20 room temperature. Plates are washed with phosphate buffered saline containing 0.01% Tween 20 and 10 mM sodium pyrophosphate. Plates are dried, sealed with TopsealTM (NEN) and an amount of 33p incorporated into the peptide substrate measured using a Packard Topcount@ NXTTM scintillation counter with standard settings. CHKI DELFIA@ Kinase Assay: Assays (25 p) utilize 6.4 mM GST-CHK1 25 containing 25 mM Tris, pH 8.5, 20% glycerol, 50 mM sodium chloride (NaCI), 0.1 Surfact Amps® 20, 1 pM peptide substrate (Biotin-GLYRSPSMPEN-amide) (SEQ ID NO: 21), 2 mM DTT, 4% DMSO, 12.5 p.M ATP, 5 mM MgCl2 and are reacted for 30 minutes at room temperature. Reactions are tenninated with 100 p1 Stop buffer containing 1% BSA, 10 mM Tris, pH 8.0, 150 mM NaCl and 100mM EDTA. Stopped reactions (100 p1) are transferred to 96 well 30 neutravidin plates (Pierce) to capture the biotin-peptide substrate during a 30 minute room temperature incubation. Wells are washed and reacted with 100 pl PerkinElmer Wallac Assay Buffer containing 21.5 ng/ml anti-phospho-Ser216-Cdc25c rabbit polyclonal antibody from Cell Signalling Technology (Beverly, MA) and 292 ng/ml europium labeled anti-rabbit-IgG for 1 hour at room temperature. Wells are washed and europium released from the bound antibody by 35 addition of Enhancement Solution (100 p.) (PerkinElmer Wallac) and detected using a Wallac Victor2TM using standard manufacturer settings. Compounds of the present invention may be tested in the CHK1 FlashPlate® Kinase Assay described above. - 79 - WO 2009/102537 PCT/US2009/031633 WST Assay: HT29, HCTI 16 (5000 cells/well) or other cells are seeded (75 gl) to 96 well clear bottom plates at densities which provide linear growth curves for 72 hours. Cells are cultured under sterile conditions in appropriate media and for HT29 and HCTI 16 this media is McCoy's 5A containing 10% Fetal Bovine Serum (FBS). Following the initial seeding of 5 cells, cells are incubated at 37' C, 5% CO2 from 17 to 24 hours at which time the appropriate DNA damaging agents (camptothicins, 5-fluorouracil and etoposide) are added at increasing concentrations to a point which is capable of causing at least 80% cell killing within 48 hours, Final volume of all DNA damaging agent and compound additions are 25 pl. Assays contain <1% DMSO final. At the same time as DNA damaging agent addition, CHKl inhibitor 10 compound is added at fixed concentrations to each DNA damaging agent titration to observe enhancement of cell killing. Cell viability/cell killing under the conditions described above are determined by addition of WST reagent (Roche) according to the manufacturer at 47 hours following DNA damage and CHK1 inhibitor compound addition and following a 3.5 hour or 2.5 hour incubation at 370 C, 5% C02 wherein OD450 is measured. 15 Compounds of the present invention may be tested in the assays described above. EXAMPLE 8: Other Biological Assays Other assays that may be utilized to determine biological activity of the instant compounds include assays found in the following publications: WO 04/080973, WO 02/070494, and WO 03/101444. -80-
Claims (9)
1. A compound of the Formula A: 0 ,'R2 ZN A N S (R4) R3 N 0 5 wherein: aisOor1;bisOor 1;mis0, 1,or2;nisO, 1,2,3,4,5 or6;andpisO, 1,2,3,4,5 or6; Ring Z is selected from: aryl, heteroaryl, heterocyclyl, (C4-C8)cycloalkenyl and (C4 10 C8)cycloalkyl; No is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; R 1 is independently selected from: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, 15 (C=O)aObC2-CIO alkenyl, (C=O)aObC2-ClO alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=0)aNR 7 R 8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-(C1 C1 0)alkyl, and (C=0)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; 20 R 2 is selected from: Cl-C10 alkyl, C2-C10 alkenyl, C2-CIo alkynyl, CO2H, C3-C8 cycloalkyl, aryl and heterocyclyl, said alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; R 3 is selected from: H, CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 25 alkenyl, (C=0)aObC2-C1O alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=O)aNR 7 R 8 , CN, (C=0)aObC3-C8 cycloalkyl, S(O)mNR 7 R 8 , S(O)m-(C1-C1O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; -81- WO 2009/102537 PCT/US2009/031633 R 4 is independently selected from: H, CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-Clo alkynyl, C02H, halo, OH, ObC1-C6 perfluoroalkyl, (C=O)aNR 7 R 8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R 8 , S(O)m-(C1 C1o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and 5 heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; R 6 is: CF3, oxo, (C=O)aObCl-C10 alkyl, (C=-O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, C02H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7 R8, oxo, CHO, (N=O)R 7 R 8 , S(O)mNR 7 R 8 , S(O)m-(CI-C1o)alkyl, SH or (C=O)aObC3-C8 10 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6a; R6a is selected from: CF3, (C=0)aOb(C-ClO)alkyl, Oa(C1-C3)perfluoroalkyl, (Co C6)alkylene-S(O)mRa, oxo, OH, halo, CN, (C2-C I0)alkenyl, (C2-C10)alkynyl, (C3 15 C6)cycloalkyl, (CO-C6)alkylene-aryl, (Co-C6)alkylene-heterocyclyl, (C0-C6)alkylene-N(Rb)2, C(O)Ra, (C 0 -C6)alkylene-CO2Ra, C(O)H, and (CO-C6)alkylene-CO2H, said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Ci-C6)alkoxy, halogen, C02H, CN, O(C=O)C1-C6 alkyl, oxo, and N(Rb)2; 20 R 7 and R 8 are independently selected from: H, (C=O)ObC1-C1o alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=0)Obheterocyclyl, Ci-C1o alkyl, aryl, C2-C10 alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb 2 , said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected 25 from R 6 a, or R 7 and R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S, said monocylcic or bicyclic heterocycle optionally substituted with one or more substituents selected from R6a; 30 Ra is H, (Cl -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and Rb is independently H, (C1-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=0)OC1-C6 alkyl, (C=0)C1-C6 alkyl or S(O)mRa; 35 or a pharmaceutically acceptable salt or a stercoisomer thereof.
2. The compound according to Claim 1 of the Formula B: - 82 - WO 2009/102537 PCT/US2009/031633 0 i~ 0 r(R 5 )q -> N (R%)n B N S ND (R4)P R3 1 N 0 wherein: N is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; 5 q is 0, 1, 2, 3, 4 or 5; R 5 is independently selected from: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 10 perfluoroalkyl, Oa(C=0)bNR 7 R8, oxo, CHO, (N=O)R 7 R 8 , S(O)mNR 7 R 8 , S(O)m-(Cl CiO)alkyl, SH and (C=Q)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R6a; and all substituents and variables are as defined in Claim 1; 15 or a pharmaceutically acceptable salt or a stereoisomer thereof.
3. The compound according to Cliam 2 of the Formula B, wherein: 20 N is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; aisO or 1; b isO or 1;mis 0, 1, or2; nis 0, 1, 2,3,4 or 5; p is 0, 1, 2,3,4,5 or 6; andqis 0, 1, 2, 3, 4 or 5; 25 R 1 is independently selected from: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=0)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, C02H, halo, OH, ObC1-C6 perfluoroalkyl, (C=O)aNR 7 R 8 , CN, (C=O)aObC3-Cg cycloalkyl, S(O)mNR 7 R 8 , S(O)m-(Cl - 83 - WO 2009/102537 PCT/US2009/031633 C1 O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; R 5 is independently selected from: CF3, oxo, (C=O)aObCl-C10 alkyl, (C=O)aObaryl, C2-C10 5 alkenyl, C2-Clo alkynyl, (C=0)aOb heterocyclyl, CO2H, halo, CN, OH, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7 R 8 , oxo, CHO, (N=O)R7R8, S(O)mNR7R 8 , S(O)m-(C1 C10)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R 6 ; 10 R 3 is selected from: H, CF3, oxo, (C=O)aObC-Cl0 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCI-C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R 8 , S(O)m-(C-ClO)alkyl, and (C=0)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; 15 R4 is independently selected from: H, CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, C02H, halo, OH, ObC1-C6 perfluoroalkyl, (C=)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m-(Cl C1 o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and 20 heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; R 6 is: CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl, Oa(C=O)bNR 7 R8, oxo, CHO, (N=O)R 7 R8, S(O)mNR7R 8 , S(O)m-(CI-C10)alkyl, SH or (C=O)aObC3-C8 25 cycloalkyl; R7 and R 8 are independently selected from: I-, (C=O)ObCI-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, Cl-Clo alkyl, aryl, C2-Clo alkenyl, C2-C1O alkynyl, heterocyclyl, C3-Cg cycloalkyl, S(O)mRa, and (C=O)NRb2, or R 7 and R 8 can be taken 30 together with the nitrogen to which they are attached to fonn a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; Ra is H or (Cl -C6)alkyl; and 35 Rb is independently H or (C1 -C6)alkyl; or a pharmaceutically acceptable salt or a stereoisomer thereof. - 84 - WO 2009/102537 PCT/US2009/031633
4. The compound according to Claim 2 of the Formula B, wherein: No is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; 5 aisOor l;bis0or l;mis0, l,or2;nisO, 1,2, 3,4or5; pis 0,1,2,3,4,5 or6; andqis0, 1, 2, 3, 4 or 5; R 1 is independently selected from: CF3, oxo, (C=O)aObCl-C1O alkyl, (C=O)aOb aryl, 10 (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=O)aNR7R 8 , CN, (C=O)aObC3-Cg cycloalkyl, S(O)mNR 7 R 8 , S(O)m-(C1 C1 o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; 15 R 5 is independently selected from: CF3, oxo, (C=)aObCl-C10 alkyl, (C=O)aObaryl, C2-Cl0 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl, Oa(C=O)bNR 7 R 8 , oxo, CHO, (N=O)R 7 R 8 , S(O)mNR 7 R 8 , S(O)m-(C CIO)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R 6 ; 20 R 3 is: C1-C6 alkyl, said alkyl is optionally substituted with one or more substituents selected from R6; R 4 is independently selected from: H, CF3, oxo, (C=O)aObCI-C10 alkyl, (C=O)aOb aryl, 25 (C=O)aObC2-ClO alkenyl, (C=O)aObC2-Cl alkynyl, CO2H, halo, OH, ObCl-C6 perfluoroalkyl, (C=O)aNR 7 R 8 , CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R8, S(O)m-(C1 C1 O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; 30 R 6 is: CF3, oxo, (C=0)aObCl-Clo alkyl, (C=O)aObaryl, C2-C1O alkenyl, C2-Clo alkynyl, (C=0)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl, Oa(C=O)bNR 7 R 8 , oxo, CHO, (N=0)R 7 R 8 , S(O)mNR 7 R 8 , S(O)m-(C1-ClO)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; 35 R 7 and R 8 are independently selected from: H, (C=O)ObC1-C10 alkyl, (C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, Cl-Cl0 alkyl, aryl, C2-C10 alkenyl, C2-C10 - 85 - WO 2009/102537 PCT/US2009/031633 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=0)NRb 2 , or R 7 and R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; 5 Ra is H or (Cj-C6)alkyl; and Rb is independently H or (C I-C6)alkyl; 10 or a pharmaceutically acceptable salt or a stereoisomer thereof
5. The compound according to Claim 2 of the Formula B, wherein: N is selected from heteroaryl and heterocyclyl, including spirocyclic moieties; 15 aisOorl;bis0orl;mis0,1,or2;nislor2; pis0,1,2,3,4,5or6;andqisl or2; R 1 is independently selected from: H, S(O)2-(CI-C6)alkyl, (C1-C6)alkyl, and (C=0)O(Cl C6)alkyl; 20 R 5 is independently selected from: H, halo, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl are optionally substituted with one or more substituents selected from R 6 ; 25 R3 is: CI-C6 alkyl, said alkyl is optionally substituted with one or more substituents selected from R 6 ; R 4 is independently selected from: H, CF3, oxo, (C=)aObCl-C10 alkyl, (C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6 30 perfluoroalkyl, (C=0)aNR 7 R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR 7 R 8 , S(O)m-(Cl Cl0)alkyl, and (C=0)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R 6 ; R 6 is: CF3, oxo, (C=O)aObC1-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-CI0 alkynyl, 35 (C=O)aOb heterocyclyl, CO2H, halo, CN, 01-1, ObCl-C6 perfluoroalkyl, Oa(C=O)bNR 7 R 8 , -86- WO 2009/102537 PCT/US2009/031633 oxo, CHO, (N=O)R 7 R 8 , S(O)mNR7R 8 , S(O)m-(CIi-CO)alkyl, SH or (C=O)aObC3-C8 cycloalkyl; R 7 and R 8 are independently selected from: H, (C=O)ObCl-Cl0 alkyl, (C=O)ObC3-C8 5 cycloalkyl, (C=Q)Obaryl, (C=O)Obheterocyclyl, CI-C10 alkyl, aryl, C2-C10 alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and (C=O)NRb2, or R 7 and R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, 0 and S; 10 Ra is H or (C I-C6)alkyl; and Rb is independently H or (C1-C6)alkyl; 15 or a pharmaceutically acceptable salt or a stereoisomer thereof.
6. A compound according to Claim I which is selected from: 3-(3-chlorophenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyljthio}-8 20 methylquinazolin-4(3H)-one; 1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yllmethyl}hexanoyl)piperidine-3 carboxamide; 2- {2-{(4-acetylpiperazin-1-yl)carbonyl]hexyl}-3-(3-chloropheny)-quinazolin-4(3H)-one; 3-(3-chlorophenyl)-2- {2-[(4-phenylpiperazin-1-yl)carbonyllhexyl}quinazolin-4(3H)-one; 25 1-(2-{[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl}hexanoyl)-N,N diethylpiperidine-3-carboxamide; 3-(3-chlorophenyl)-2- {2-[(4-pyridin-2-ylpiperazin-1-yl)carbonyl]hexyl)-quinazolin-4(3H)-one; 2-(2-{[4-(aminomethyl)-piperidin-1-ylJcarbonyl}-hexyl)-3-(3-chloro-phenyl)quinazolin-4(3H) one; 30 3-(3-chlorophenyl)-2- {2-[(7-methyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]hexyl}quinazolin 4(3H)-one; 2-(2- ([4-( lH-benzimidazol-2-yl)piperidin- 1 -yl]carbonyl}hexyl)-3-(3-chlorophenyl)quinazolin 4(31)-one; 3-(3-chlorophenyl)-2- {2-[(1,1 -dioxidothio-morpholin-4-y)-carbony]hexyl} quinazolin-4(3H) 35 one; 3-(3-chlorophenyl)-2-(2- {[4-(methylsulfonyl)-piperazin-1 -yl]carbonyl}-hexyl)quinazolin-4(3H) one; - 87 - WO 2009/102537 PCT/US2009/031633 3 -(3-chlorophenyl)-2-(2- { [4-( 1 H-imidazol- I -ylmethyl)piperi din- I -yl]carbonyl 4 hexyl)quinazolin 4(311)-one; 3-(3 -chlorophenyl)-2-(2- { [4-(methylsulfonyl)-piperidin- l-yl] carbonyl)4-hexyl)quinazolin-4(3Th one; 5 3 -(3-ehlorophenyl)-2-[2-(2 ,8-diazaspiro[5 .5]-undec-2-ylcarbonyl)-hexyl] quinazolin-4(3TH-one; 3-(3 -chlorophenyl)-2-(2- {[4-(2-fluorophenyl)-piperazin- 1 -ylljcarbonyl 4-hexyl)quinazolin-4(3Th one; 3-(3-chlorophenyl)-2 -(2- {[4-(4-fl-uorophenyl)-piperazin- l-yl] carhoni) -hexyl)qninazolin-4(3H) one; 10 3-(3 -chlorophenyl)-2- {2-[(4,4-diphenylpiperidin- 1 -yl)carbonylljhexyl 4-quinazolin-4(3R)-onie; 3 -(3-chlorophenyl)-2- {2-[(4-pyridin-4-ylpiperidifl- 1 -yl)carbonyllhexyl} -quinazolin-4(311)-one; 3-(3-chlorophenyl)-2- {2- [(3 ,3 -diphenylpiperi din- I -yl)carbonyljhexyl 4 -quinazolin-4(311)-one; 3-(3 -chlorophenyl)-2- { [1-(2,8-diazaspirolS 5] -undec-2-ylcarboniyl)-pentyl]thiO 4 -'7-methyl sulfonyl)quinazolin-4(311)-one; 15 methyl 3-(3-chloro-phenyl)-2- {[1 -(2,8-diazaspirolS 5]undec-2-ylcarbonyl)pentyllthio 4-4-oxo 3 ,4-dihydro-quinazoline-7-carboxylate; 3-(3-chlorophe-nyD)-2-( {3-methyl-l1-[(4-pyridin-2-ylpiperazin- l-yl) caxbonyl]hutyl 4thio)quinazolin-4(3H)-one; 3-(3 -chlorophenyl)-2-I- {[4-(2,3 -dihydro-1I,4-benzodioxin-5-yI)piperazin- l-yfl carbonyl 4-3 20 mnethylbutyl)thio]quinazolin-4(31)-one; 3 -(3-chlorophenyl)-2- [(1- {[j4-(4-fluorophenyl)-piperazin-1I-yljcarbonyl 4-3-methylbutyl)thio] quinazolin-4(31P-o-ne; 3 -(3-chlorophenyl)-2-[( 1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4. 4 nOl- 2 Y] carbonyl 4-3 methylhutyl)thio] quinazolin-4(3TH-one; 25 3-(3 -chlorophenyl)-2-I( 1- { j7-(2-chloropheny)-2,7-diazaspiro[4.4]non- 2 -YI] carbonyl 4-3 xnethylbntyl)thio] quin-azolin-4(3TH-one; 2-({ (1 [(7-benzy1-2,7-diazaspiro[4.4]non-2-y)cabonYll- 3 -methylbutyl 4thio)-3-(3 chlorophenylquinazolin-4(311)-one; 3-(3-chlorophenyl)-2-[I1-f { 7-(3-methoxyphenyl)-2 ,7-diazaspirol4.4]non-2-ylcarbolI 4-3 30 methylbutylthiolquinazolin-4(31Th-ont 3-(3-chlorophenyl)-2II 1- { [7-(4-methoxyphenyl)-2,7-diazaSpiro[4.4]non-- 2 -Yl] carbonyl 4-3 methylbutyl)thio] quinazolin-4(3H-)-one; 1 -(2- { [3 -(3-chlorophenyl)-4-oxo-3 ,4-dihydroquinazolin-2-yl] thio) hexanoyl)piperidine-4 carboxamide; 35 1 -[ { [3-(3-chloropheniyl)-4-oxo-3 ,4-dihydroquinazolin-2-yl]thio4 (phenyl)acetyllpiperidine-4 carboxamide;, 1 -(2- { [3-(3-cblorophenyl)-4-oxo-3 ,4-dihydroquinazolin-2-yllthio 4-4-methylpentanoyl) piperidine-4-carboxamide;, - 88 - WO 2009/102537 PCT/US2009/031633 3 -(3-chlorophenyl)-2-I- { [7-(2-chloropheny)-2,7-diazaspirO[4.4flO-2-Yl]cabOl pentyl)thioj -7-(methylsulfonyl)-quinazolin-4(311)-one;, 3(3 -chlorophenyl)-2[(l 1-f [7-(4-chlor-opheny1)-2,7-diazaspio[4.4flOl-2-YlJCarbOllpentyl) thiol-7-(methylsulfonyl)-quinazolin-4(3Hb-one; 5 3-(3 -chlorophenyl)-2-I( - { [7-(3 -chloropheny)-2,7-diazaspiroI4.4]non-2-y1]CabOlPetYl) thioj-7-(methylsulfon-yl)-quinazolin-4(3H0-ofle; 3-(3-chlorophenyl)-7l(methylsulfonyl)-2-( (1-[(7-phenyl-2,7-diazaspiro[4.4]non-2-y)carbolyl] pentyl }thio)quinazolin-4(31D-one; 3-(3-chlorophenyl)-2-I(l 1- { [7-(3-methoxyphenyl)-2,7-dizaSpiO[4.4flOl-2-Y] carbonyl} 10 pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3 1 )-ofe; methyl 3(3 -chlorophenyl)-2-( (1-[(3-cyano-3-pheniylazetidin-1I-yl)carbonyl]pentyl} -thio)-4-oxo 3 ,4-dihydroquinazoline-7-carboxylate; methyl 3-(3-chlorophenyl)-2- ([1 42,6-diazaspiro[3 .3]hept-2-ylcarbonyl)pentyl]thio)}-4-oxo-3 ,4 dihydro-quinazoline-7-carboxylate; 15 3 -(3-chlorophenyl)-2-[( I- { lR2R)-2-(trifluoro-lnethyl) pyrrolidin- I -ylJ-carbonyl } pentyl)thioj quinazolin-4(3H)-one; 3 -(3-chlorophenyl)-2-[I- {[3-(4-fluorobenzyl)-2-oxo-l1-oxa-8-azaspiro[4. Sjdec-8-yllcarbonyl } pentyl)thio] quinazolin-4(3H)-one; 3 -(3-chlorophenyl)-2-[( 1- {[5-(4-fluorophenyl)-2,5-diazabieycloll2.2.l ]hept-2-yJ] carbonyl 4 20 pentyl)-thio] quinazolin-4(3H)-one; 3-(3-chlorophenyl)-2-[(1I- {[5-(3-fluorophenyl)-2,5-diazabicyclo[2.2.1 Ijhept-2-yljcarbonyl 4 pentyl)-thio] quinazolin-4(3H)-one; 3 -(3-chlorophenyl)-2-[1 - ([44(4-methyl- 1,2 ,5-oxadiazol-3-yJ)piperazin- I-yI] carbonyl 4 pentyl) thio] quinazolin-4(3 1-)-one; 25 3 -(3-chlorophenyl)-2-[(1I- {[3 -(4-fluorophenyl)- 1 -oxa-8-azaspiro[4. 5]dec-8-yl] carbonyl 4 pentyl) thio] quinazolin-4(3H)-one; 3 -(3-chloroph-enyl)-2-[I1- ([(3 S,4S)-3 ,4-difluoropyrrolidin- l -ylj carbonyl 4pentyl)thio]q-uinazolin 4(3H)-one; 3-(3-chlorophenyl)-2- { [2-(2,8-diazaspiro [5. 57undec-2-yI)- 1 -(1 H-imidazol-4-ylmethyl)-2 30 oxoethyl~thiolq-uinazolin-4(3H)-one; 24(3-(1 H-imidazol-4-yl)- 1 -oxo- I -(2,8-diazaspiro[5.5]unidecan-2-yl)propan-2-ylthio)-3-( 3 chlorophenyl)-8-rnethylquinazolin-4(3H)-one; 3-(3-chlorophenyl)-8-methyl-2-( I -oxo- I -(4-phenylpiperazin- I -yl)hexan-2-ylthio)quainazolin 4(3H)-one; 35 3-(3-chlorophenyl)-2-( 1-(7-(3-methoxyphenyl)-2 ,7-diazaspiro[4.4]nonan-2-yl)- 1 -oxohexan-2 ylthio)-8-methylquinazolin-4(3H)-one; 3-(3-chlorophenyl)-2-( 1 47-(4-chlorophenyl)-2 ,7-diazaspiro[4.4jnonan-2-yl)-l1-oxohexan-2 ylthio)-8-methylquinazolin-4(3H)-ofle; - 89 - WO 2009/102537 PCT/US2009/031633 3-(3 -chlorophenyl)-2-( 1-(7(3 -chlorophenyl)-2,7-diazaspiro[4.4]nonan- 2 -yl)-lI-oxohexan-2 ylthio)-8-rnethylquinazolin-4(3H)-one; 3t-[2-[( I-{[4-(Amnino-carbonyl)piperidin- I-yl] carhonyll}pentyl)thioj-4-oxoquinazolin-3 (4TH yl~bipheny-4-carboxylic acid; 5 14(2- { [3 -(2'-chlorobiphenyl-3-yl)-4-oxo-3 ,4-dihydroquinazolin-2-yflthio~hexalOyl)pipefldifle-4 carboxamide; 1 -(2- {[-3-booihiy--l--x-,-iyrqanzln2y~hohxny~ieiie4 carboxamide; 1 -(2- [-4clrbpey--l--x-,-iyrounzln2ylhohxny~ie'ie4 10 carboxamide;, 1 -(2- {[3-(2'-aminobiphenyl-3-yI)-4-oxo-3 ,4-dihydroquinazolin-2-yl]thiolhexanoYl)piPefldifle- 4 carboxamide;, 1 -(2- { [3-(3'-aminobipheny1-3-yl)-4-oxo-3 ,4-dihydroquinazolin-2-yl~thio} hexanoyl)piperidine-4 carboxamide;, 15 1 -(2- { [3-(4 t -anlnobiphenyl-3-yl)-4-oxo-3 ,4-dihydroquinazolin-2-yl]thio} hexanoyl)piperidine-4 carboxamide;, 1-2 {[-x--3prdn3ypey)34dhdoqunzln2y~hohxny~ieiie4 carboxamide;, 1-(2- f 4oo3( prdn4ypey)34dhdoqiaoi--lti)hxny~ieiie4 20 earboxamide; 3'-[2-[(l 1- { [4-(aminocarbonyl)piperidin- 1-yl] carbonyl }pentyl)thioll-4-oxoquinazolin-3 (4TH yl]biphenyl-3-carboxylic acid; I -[2-( {3- [3 -(3-furyl)phenylj -4-oxo-3 ,4-dihydroquainazolin-2-yl }thio)hexanoyllpiperidine-4 carboxamide; 25 1 -[2-({3- [3 -(1-methyl-i H-pyrazol-4-yI)phenylj-4-oxo-3 ,4-dihydroquinazolin-2-yl }thio) hexanoyl]piperidine-4-carboxamide; 1 -(2- {[3 -(3-cyclobex- 1-en-I -ylphen-yl)-4-oxo-3 ,4-dihydroq-uinazolin-2-yl]thio} hexanoyl) piperidine-4-carboxamide;, I -[2-( {4-oxo-3- [3 -(2-thi enyl)phenyl] -3 ,4-dihydroquinazolin-2-yl }thio)hexanoylllpiperidine-4 30 carboxamide; 2-[(1- { [7-(3 -chlorophenyl)-2,7-diazaspiro[4.4nol-2-ylcarboll-3-methylbutyl)thioj-3 -[3-(3 fttryl)phenyl]quinazolin-4(3TH-one; 2-[( 1- {[7-(3 -chlorophenyl)-2,7-diazaspirol4.4nofl-2-yl] carbonyl }-3-methylbutyl)thio] -34(3 cyclohex- I -en-I -yiphenyl)q-uinazolin-4(3TH-one; 35 1 -(2- { [3-( fuoo2meblipey -yl-4-oxo-3 ,4-dihydroquinazolin-2-yljthio }hexanoyl) piperidine-4-carboxamide; 2- [(l- { [5-(4-fhiorophenyl)-2,5-diazabicyclol2 .2.1 ]hept-2-yljcarbonyl~pentyl)thio]-3-[3-(3 fury1)phenyJlquinazolin-4(3TH-ofle; - 90 - WO 2009/102537 PCT/US2009/031633 2-[(1- {[3-(4-fluorobenzyl)-2-oxo-I-oxa-8-azaspiro[4.5]dec-8-yl]carbonyl}pentyl)thio]-3-[3-(3 furyl)phenyl]quinazolin-4(3H)-one; 3-(3-cyclohex-1-en-1-ylpheny)-2-[(1-{[5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.i]hept-2 yl]carbonyl}pentyl)thio]quinazolin-4(31)-one; 5 3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[3-(4-fluorobenzyl)-2-oxo-1-oxa-8-azaspiro[4.5]dec-8 yl]carbonyil} pentyl)thio]quinazolin-4(3H)-one; 3-(3-cyclohex-1-en-I -ylphenyl)-2-[(1-([4-(2,4-difluorophenyl)piperazin-1 yl]carbonyl}pentyl)thio]quinazolin-4(3H)-one; 2-[(1-{[4-(2,4-difluorophenyl)piperazin-1-yl]carbonyl}pentyl)thioj-3-[3-(3-furyi) phenyl] 10 quinazolin-4(3H)-one; 3-(3-cyclohex-1-en-1-ylphenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl] thio}quinazolin-4(3H)-one; 2-{[3-amino-1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)propyl]thio}-3-(3-chlorophenyl) quinazolin-4(3H)-one; 15 2-{[4-amino-1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)butyl]thio}-3-(3-chlorophenyl)-8 methylquinazolin-4(3H)-one; 2-{[5-amino-1-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-3-(3-chlorophenyl) quinazolin-4(3H)-one; 2-[(4-amino-1-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}butyl)thio]-3-(3 20 chlorophenyl)quinazolin-4(3H)-one; 2-(5-amino-1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxopentan-2-ylthio)-3-(3 chlorophenyl)-8-methylquinazolin- 4 (3H)-one; 2-[(5-amino- 1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non- 2 -yl] carbonyl) pentyl)thio]-3-(3 chlorophenyl)quinazolin-4(3H)-one; and 25 2- ({5-amino-1 -(2,8-diazaspiro [5.5]undec-2-ylcarbonyl)pentyl]thio) -3 -(3-chlorophenyl)-8 methylquinazolin-4(3H)-one; or a pharmaceutically acceptable salt or a stereoisomer thereof. 30
7. The TFA salt of a compound according to Claim 1 which is selected from: 3-(3-chlorophenyl)-2-{[1-(2,8-diazaspiro[5.5]undec-2-ylcarbony1)pentyl]thio}-8 methylquinazolin-4(3H)-one; 1-(2- ([3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yljmethyl4hexanoyl)piperidine-3 35 carboxamide; 2-{2-[(4-acetylpiperazin-l -yl)carbonylhexyl}-3-(3-chloropheny)-quinazolin- 4 ( 3 H)-one; 3-(3-chlorophenyl)-2- {2-[(4-phenylpiperazin-i -yl)carbonyl]hexyl~quinazolin-4(3H)-one; -91- WO 2009/102537 PCT/US2009/031633 1 -(2- {[3-(3 -ch-lorophienyl)-4-oxo-3 ,4-dihydroquinazolin-2-yljmethyl }hexanoyl)-NN diethylpiperidine-3-carboxamide; 3 -(3-chlorophenyl)-2- {2-[(4-pyridin-2-ylpiperazin- 1 -yl)carbonyllhexyl } -quinazolin-4(3z)-one; 2-(2- {[4-(aminomethyl)-piperidin-l -yJlcarbonyl} -hexyl)-3-(3-chloro-phenyl)quinazolin-4( 3 ID 5 one; 3-(3-chlorophenyl)-2- {2-[(7-methyl-2,7-diazaspiro[4nol-2-yl)CabonYl]heXYi }quinazolin 4(311)-one; 2-(2- { [4-( IH-benzimiclazol-2-yl)pipefldin- 1 -yljcarbonyl }hexyl)-3-(3 -chlorophenyl)quinazolin 4(3TH-one; 10 3 -(3-chlorophenyl)-2- {2-[( 1,1 -dioxidothio-morpholin-4-yl)-caholyl]hexyl } quinazolin-4(3Lb one; 3-{3 -chlorophenyl)-2-(2- ([4- (methylsul fonyl)-piperazin- I -yljcarbon-yl }-hexyl)q-uinazolin-4(31) one; 3 -(3 -chlorophenyl)-2-(2- ([4-K 1H-imidazol- 1 -ylmethyl)piperidin- I1-yl] carbonyl }hexyl)quinazolin 15 4(31-1)-one; 3-(3-chlorophenyl)-2-(2- {[4-(methylsulfonyl)-piperidin-l -yi]carbonyl} -hexyl)quinazolin-4(311) one; 3 -(3-chlo-rophenyl)-2-[2-(2,8-diazaspiro[5,5]-undec-2-ylcarbonyl)-hexy1] quinazolin-4(3H)-one; 3-(3-chlorophenyl)-2-(2- {[4-(2-fluorophenyl)-piperaz.in-1 -yl]carhonyl} -hexyl)quinazolin-4(3TH 20 one; 3-(3-chlorophenyl)-2-(2- {[4-(4-fluaorophenyl)-piperazin-1 -yl] carbonyl} -hexyl)quinazoln-4(311 one; 3 -(3--chlorophenyl)-2- f{2- [(4,4-diphenylpiperi din- 1 -yI)carbonyl]hexyl} -quinazolin-4(3H)-one; 3(3 -chlorophenyl)-2- (2-[(4-pyridin-4-ylpipericlin- 1-yl)carbonyl]hexyl}-quinazolin-4(3TH-one; 25 3-(3-chlorophenyl)-2- {2-[(3 ,3-diphenylpiperidin- 1-yl)carbonyflhexyl }-quinazolin-4(311)-one; 3-(3 -chlorophenyl)-2- ([1 -(2,8-diazaspiro[5.5J-uandec-2-ylcarbonyl)-pentyljthio} -7-(methyl sulfony1)quinazolin-4(3H)-one; methyl 3-(3 -chloro-phenyl)-2- ([1-(2,8-diazaspirolS Sjundec-2-ylcarbonyl)pentyl]thio} -4-oxo 3 ,4-dihydro-quinazoline-7-carboxylate; 30 3-(3-chilorophenyl)-2-( (3-methyl-i -[(4-pyridin-2-ylpiperazin-1I-yl) carbonyllbutyl }thio)quinazolin-4(3H)-one; 3-(3-chlorophenyl)-2- [(1- {[4-(2,3-dihydro-1I,4-benzodioxin-5-yl)piperazin- 1-yflcarbonyl }-3 methylbutyl)thio] qunazolin-4(3H)-one; 3-(3-chlorophenyl)-2- [(1- {[4-(4-fluorophenyl)-piperazin-1I-yflcarbonyl) -3--methylbutyl)thioj 35 quinazolin-4(311)-one; 3-(3-chlorophenyl)-2-[(1- {['7-(3-chlorophenyl)-2,7-diazaspiro[4.4]fnori-2-yl] carbonyl }-3 methylhutyl)thio]quinazolin-4(3R)-one; - 92 - WO 2009/102537 PCT/US2009/031633 3-(3 -chlorophenyl)-2-[( I- { [7-(2-chlorophenyl)-2,7-diazaspirO[4.4lOl-2-ylCabOlI }-3 methylbutyI)thio]quinazolin-4(3H)-one; 2-( { 1 -[j7-benz.yJ-2,7-diazaspiro[4.4]Ol-2-Y)CarbOflYl]- 3 -inethylbutyl }thio)-3-(3 chlorophenyl)q-uinazolin-4(3TH-one; 5 3-(3-chlorophenyl)-2-[1 - { [7-(3-methoxyphenyl)-2,7-diazaspiro4.4]non-2-YlJCabOll}-3 methylbutyl)thioquinazolin-4(3H-oflC; 3-(3-chlorophenyl)-2-(l 1- { [7-(4-methoxyphenyl)-2,7-diazaspiro[4.4]lof-2-yl] carbonyl )-3 methylbutyl)thioquinazolin-4(311)-ofle; 1 -(2- { [3-(3-chlorophenyl)-4-oxo-3 ,4-dihydroquinazolin-2-yflthiolhexanoyl)piperidifle-4 10 carboxamide; 1 -[ f [3-(3 -chlorophenyl)-4-oxo-3 ,4-dihydroquinazoin-2-yI~thio} (phenyl)acetyl]piperidine-4 carhoxamicle; 1 -(2- { [3-(3-chlorophenyl)-4-oxo-3 ,4-dihydroq-uinazolin-2-yllthio }-4-methylpenitanoyl) piperidine-4-carboxamide; 15 3-(3-chlorophenyl)-2-[( 1- { [7-(2-chlorophenyl)-2,7-diazaspiro[4.4nOl-2-YI] carbonyl } pentyl)thio] -7-(methylsulfonyl)-quinazolini-4(3R)-ofle; 3-(3-chlorophenyl)-2-I( 1- {[7-(4-chlorophenyl)-2,7-diazaspiro [4.4I]non-2-yl] carbonyl 4penltyl) thio] -7-(methylsulfonyl)-quinazolin-4(3TH-ole; 3-(3-chlorophenyl)-2- 1- [7-(3-chlorophenyl)-2,7-diaaspiro4.4nOl-2-y1] carbonyl 4pentyl) 20 thio] -7-(methylsulfonyl-quinazolin-4(3fD-ofle; 3-(3-chlorophenyl)-7-(rnethylsulfonyl)-2-( {I-[(7-pheny1-2,7-diazaspiro[I4.4non-2-y)carbonyl] pentyl 4thio)quinazolin-4(3H)-one; 3 -(3-chlorophenyl)-2-[R1 -{ [7-(3-methoxyphenyl)-2,7-diazaspirO4]Ol-2-Y] carbonyl 4 pentyl)thioj-7-(methylsulfonyl)-qinaZolin- 4 ( 3 TH-flC 25 methyl 3-(3-chloroph-enyl)r2-( (1-[(3 -cyano-3-phenylazetidin-1I-yl)carbonyljpentylj -thio)-4-oxo 3 ,4-dihydroquinazoline-7-carboxyl ate; methyl 3 -(3-chlorophenyl)-2- {[1 -(2,6-diazaspirol3.3]hept-2-ylcarbonyl)pentylljthioj -4-oxo-3 ,4 dihydro-quinazoline-7-carboxylate; 3-(3-chlorophenyl)-2-[( - {[(2R)-2-(trifluoro-methyl) pyrrolidin-1 -y1]-carhonyl4 pentyl)thio] 30 quinazolin-4(311)-one; 3-(3-chlorophenyl)-2-I- {[3-(4-fhiorobenzyl)-2-oxo-lI-oxa-8-azaspirol4.Sldcc-8-yAlcarbonyl I pentyl)thio]quinazolin-4(3H)-one; 3 -(3-chlorophenyl)-2-f( 1- {[5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1 Jhept-2-yl] carbonyl 4 pentyl)-.thio]quinazolin-4(3H)-one; 35 3-(3-chlorophenyl)-2-[f{1-{[5-(3-fluorophenyl)-2,5-diaZabiCyClO[2.2.1 ]hept-2-yl]carbonyl 4 pentyl)-thio]quinazolin-4(3t1)-one; 3-(3-chlorophenyl)-2-I(1 - {[4-(4-methyl- 1,2 ,5-oxadiazol-3-yl)piperazin- l-yJ] carbonyl 4pentyl) tbio~quinazolin-4(3H)-one; - 93 - WO 2009/102537 PCT/US2009/031633 3-(3-chlorophcnyl)-2-f -([3-(4-fluoropheny- 1-oxa-8-azaspiro[4. 5]dec-8-yljcarbonyl }pentyl) thiol quinazolin-4(31)-one; 3 -(3-chlorophenyl)-2-[( 1- { [(3S ,4S)-3 ,4-difluoropyrrolidin- 1-yI] carbonyl }pentyl)thiolquinazolin 4(31-)-one; 5 3-(3-chlorophenyl)-2- { [2-(2,S-diazaspirol5.Sjundec-2-yl)- 1 -(1 H-imidazol-4-ylmethyl)-2 oxoethyl]thio I quinazolin-4(3E0-one; 2-(3-( IH-irnidazo1-4-y)- I-oxo- 1-(2 ,8-diazaspirol5.5jundecan-2-yl)propan-2-ylthio)-3-(3 chlorophenyl)-8-methylquinazolin-4(3H)-ofle; 3-(3 -chlorophenyl)-S-methyl-2-( 1 -oxo- I -(4-phenylpiperazin- I -yl)hexan-2-ylthio)quinazolin 10 4(311)-one; 3-(3 -chlorophenyl)-2-( 1-(7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]nOlal- 2 -Yl)-lI-oxohexan-2 ylthio)-3-methylquinazolin-4(3H)-one; 3-(3-chlorophenyl)-2-(1 -(7-(4-chlorophenyl)-2,7-diazaspiro[4.4nonan-2-Yl)-1I -oxohexan-2 ylthio)-8-methylquinazolin-4(311)-one; 15 3-(3-chlorophenyl)-2-( 1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nal-2-Yl)- I -oxohexan-2 ylthio)-8-methylquinazolin-4(3fl)-one; 3'-[2-[(l 1- { [4-(Amino-carbonyl)pipecri din- I1-yl] carbonyl }pentyl)thiol -4-oxoquinazolin-3(4H) yl]biphenyl-4-carboxylic acid; 1-(2- f 3(-hooilinl3y)4oo34dhdrqunzln2ylhohxny~ie'ie4 20 carboxamide; 1-(- [3('clroibnl3 l--x ,4-dihydroquinazolini-2-yllhiolhexanoyl)piperidifle-4 carboxamide; 1-(2- [-4-hooihnl3y)4-x-,-iyrqiaoi--y~bohxny~ieiie4 carboxanfide; 25 1 -(2- {[3-(2'-aminobipheniyl--3-yl)-4-oxo-3 ,4-dihydroquinazolin--2-ylltbio} hexanoyl)piperidine-4 carboxamide; 1-2 [-3-mnbpey--l--x-,-iyrounzln2y~hohxny~ieiie4 carboxamide; 1 (-[-4-mioibnl3 l--x-,4-dihydroquinazolin--2-ylJthiolhexanoyl)pipcridifle-4 30 carboxamide; 1 -(2- f [4-oxo-3 -(3-pyridin-3-ylphenyl)-3 ,4-dihydroquinazolin-2-yljthio }hexanoyl)piperidine-4 cart oxamide; 1 (-[-x--3prdn4yphnl-,-iyrqiaoi--yjhohxny~ieiie4 carboxamide; 35 3'-[2-[( 1- { [4-(an-inocarbonyl)piperidin- 1-yl] carbonyl }pentyl)thio] -4-oxoquinazolin-3 (41']) yljbiphenyl-3-carboxylic acid; I -[2-( {3-[3-(3-fury1)pheny1II-4-oxo-3 ,4-dihydroquinazolin-2-yl} thio)hexanoyl]piperidinc-4 carboxamide; - 94 - WO 2009/102537 PCT/US2009/031633 I-[2-(({3 -[3-(l1 -mnethyl- iH-pyrazol-4-yI)phenyl] -4-oxo-3 ,4-dihydroquinazolin-2-yl } thio) hexanoyljpiperidine-4-earboxamide; 1 -(2- { [3 -(3 -eychohex- 1 -en-I -ylphenyl)-4-oxo-3 ,4-dihydroquinazolin-2-yllthio }hexanoyl) piperidine-4-oarboxamicie; 5 1-[2-(f 4-x--3(-hey~hnl-,-iyrqinzln2y~liohxnyjieiie carhoxamuide; 2- f [7-(3-chiorophenyl)-2,7-diazaspiro[4.4]fon-2-Yfl carbonyl }-3-methylbutyl)thiio]-3 -13-(3 furyl)phenyl]quinazolin-4(3TH-one; 2-+[1- { [7-(3 -chlorophenyi)-2,7-diazaspiro[4.4jnon- 2 -yl] earbonyl }-3 -methylbutyl)thio]-3-(3 10 cyciohex- 1 -en- I -ylphenyl)q-uinazolin-4(3H)-one; 1 -(2- { -4-l-oo2-mtybphnl3y -- x-,4-dihydroquinazolin-2-yljthiolhexanoyl) piperidine-4-carboxamide; 2+1( 1- { [5-(4-fluorophienyl)-2,5-diazabicyelol2 .2.1 ]hept-2-yl]carbonyl }pentyl)thiol-3-[3-(3 fhryl)phenyl] quinazolin-4(311)-o-ne;, 15 2-[( 1- { [3-(4-fhiaorobenzyl)-2-oxo- I -oxa-8-azaspiro[4. 5] dee-8-yi] carbonyl }penty)thio]-3-13-(3 furyl)phenyl] quinazolin-4(3TH-one; 3 -(3-cyciohex- 1-en-i -ylphenyl)-2-[(I - { [5-(4-fluorophenyl)-2,5-diazabicyclo[2 .2.1I ]hept-2 yi]carbonyl 4pentyl)thiojquinazolin-4(3TH-one; 3 -(3 -cyclohex- 1-en-I -ylphenyl)-2-[I 1- { [3-(4-fluorobenzyl)-2-oxo- 1 -oxa-8-azaspiro[4.5]dee-8 20 yljcarbonyl 4pentyi)thiolquinazolin-4(3Lb-one; 3-(3 -cyclohex- 1-en-i -yiphenyi)-2- [(I-f{ [4-(2 ,4-difluorophenyl)piperazin- I1 yl]carbonyl 4pentyl)thio]quinazolini-4(3TH-one; 2-[( 1- { [4-(2,4-difluorophenyl)piperazin- i-yJ] carbonyl }pentyi)thio] -3-j3 -(3-friryl) phenyl] quinazoiin-4(3TH-one; 25 3 -(3 -cyciohex- 1-en- I -ylphenyi)-2- {[1 -(2 ,8-diazaspiro[5 .5}undee-2-ylcarboinyl)pentyl] thio I quainazolin-4(3H)-one; 2- { [3-amino-i -(2 ,8-diazaspiro[5 .5)undec-2-ylcarbonyl)propyllthio) -3-(3-chlorophenyi) quinazoiin-4(3H)-one; 2- { [4-amino-i -(2 ,8-diazaspiro[5 .5]undec-2-ylcarbonyl)butyllthio 4-3 -(3 -chlorophenyl)-8 30 methylquainazohin-4(3TH-one;, 2- {[5-amnino-i -(2 ,8-di azaspiro[5 .5]undec-2-ylcarb onyi)pentyljthio} -3-(3-chlorophenyl) quinazoiin-4(311)-one; 2-[4-amino- I- ([7-(3 -chlorophenyi)-2,7-diazaspiro[4. 4 ]non- 2 -yl] carbonyl 4butyIthio]I-3-(3 chlorophenyl)quinazolin-4(3TH-one; 35 2-(5-amino- 1 -(7-(3-chlorophenyl)-2 ,7-diazaspiro[4.4]nonan-2-yl)- I -oxopentan-2-ylthio)-3-(3 ehiorophenyi)-8-metbylquinazolin-4(34)-one; 2-[5-amino-I -([7-(3-ehiorophenyi)-2,7-diazaspiro[4.4]nOn-2-yl] carbonyl 4 pentyi)thioll-3-(3 chlorophenyi)quinazolin-4(3TH-one; and - 95 - WO 2009/102537 PCT/US2009/031633 2- { [5-amino-i -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]thio}-3-(3-chlorophenyl)-8 methylquinazolin-4(3H)-one; or a stereoisomer thereof. 5
8. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of Claim 1.
9. The use of the compound according to Claim I for the preparation of a 10 medicament useful in the treatment or prevention of cancer in a mammal in need of such treatment. - 96 -
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| Application Number | Priority Date | Filing Date | Title |
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| US6597408P | 2008-02-14 | 2008-02-14 | |
| US61/065,974 | 2008-02-14 | ||
| PCT/US2009/031633 WO2009102537A1 (en) | 2008-02-14 | 2009-01-22 | Inhibitors of checkpoint kinases |
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| CN113354592A (en) * | 2021-06-30 | 2021-09-07 | 赣南师范大学 | 2-thioquinazolinone compound and preparation method and application thereof |
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| EP2890696A1 (en) | 2012-08-29 | 2015-07-08 | Amgen, Inc. | Quinazolinone compounds and derivatives thereof |
| CN107033091B (en) * | 2017-05-16 | 2020-12-29 | 山东理工大学 | Piperazine derivative and preparation method and application thereof |
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| KR20060127413A (en) * | 2003-11-25 | 2006-12-12 | 카이론 코포레이션 | Quinazolinone compounds as anticancer agents |
| TW200806670A (en) * | 2006-04-25 | 2008-02-01 | Merck & Co Inc | Inhibitors of checkpoint kinases |
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| CN113354592A (en) * | 2021-06-30 | 2021-09-07 | 赣南师范大学 | 2-thioquinazolinone compound and preparation method and application thereof |
| CN113354592B (en) * | 2021-06-30 | 2022-06-21 | 赣南师范大学 | 2-thioquinazolinone compound and preparation method and application thereof |
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