WO2010151735A2 - Heterocyclic compounds and their uses - Google Patents

Heterocyclic compounds and their uses Download PDF

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WO2010151735A2
WO2010151735A2 PCT/US2010/039931 US2010039931W WO2010151735A2 WO 2010151735 A2 WO2010151735 A2 WO 2010151735A2 US 2010039931 W US2010039931 W US 2010039931W WO 2010151735 A2 WO2010151735 A2 WO 2010151735A2
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amino
pyrido
pyrimidin
alk
ethyl
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PCT/US2010/039931
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French (fr)
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WO2010151735A3 (en
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Timothy D. Cushing
Xiaolin Hao
Julia Winslow Lohman
Youngsook Shin
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Amgen Inc.
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Priority to US13/377,229 priority Critical patent/US8754089B2/en
Priority to CA2765817A priority patent/CA2765817A1/en
Priority to JP2012517758A priority patent/JP2012531435A/en
Priority to EP10728551A priority patent/EP2445898A2/en
Priority to AU2010266064A priority patent/AU2010266064A1/en
Priority to MX2011013901A priority patent/MX2011013901A/en
Publication of WO2010151735A2 publication Critical patent/WO2010151735A2/en
Publication of WO2010151735A3 publication Critical patent/WO2010151735A3/en

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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—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
    • 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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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—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
    • 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
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/36—Sulfur atom
    • C07D473/38—Sulfur atom attached in position 6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates generally to phosphatidylinositol 3 -kinase (PBK) enzymes, and more particularly to selective inhibitors of PBK activity and to methods of using such materials.
  • PBK phosphatidylinositol 3 -kinase
  • PI 3-kinase The enzyme responsible for generating these phosphorylated signaling products, phosphatidylinositol 3-kinase (PI 3-kinase; PBK), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol 2:358-60 (1992)).
  • PIP3 phosphatidylinositol-3,4,5-triphosphate
  • PI 3-kinase activation therefore, is involved in a wide range of cellular responses including cell growth, migration, differentiation, and apoptosis (Parker et al., Current Biology, 5:577-99 (1995); Yao et al., Science, 267:2003-05 (1995)).
  • Tec family members that are regulated by PBK include the phosphoinositide-dependent kinase (PDKl), AKT (also termed PKB) and certain iso forms of protein kinase C (PKC) and S6 kinase.
  • PDKl phosphoinositide- dependent kinase
  • AKT also termed PKB
  • PKC protein kinase C
  • AKT protein kinase C
  • Class I PBKs can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidyl- inositol-4,5-biphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5- triphosphate, respectively.
  • Class II PBKs phosphorylate PI and phosphatidyl- inositol-4-phosphate
  • Class III PBKs can only phosphorylate PI.
  • PBK ⁇ , ⁇ , ⁇ , and ⁇ each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit. More specifically, three of the catalytic subunits, i.e., pi 10a, pi lO ⁇ and pi lO ⁇ , each interact with the same regulatory subunit, p85; whereas pi lO ⁇ interacts with a distinct regulatory subunit, plOl.
  • the patterns of expression of each of these PBKs in human cells and tissues are also distinct.
  • bovine pi 10a Cloning of bovine pi 10a has been described. This protein was identified as related to the Saccharomyces cerevisiae protein: Vps34p, a protein involved in vacuolar protein processing. The recombinant pi 10a product was also shown to associate with p85 ⁇ , to yield a PI3K activity in transfected COS-I cells. See Hiles et al., Cell, 70, 419-29 (1992).
  • pi lO ⁇ The cloning of a second human pi 10 isoform, designated pi lO ⁇ , is described in Hu et al., MoI Cell Biol, 13:7677-88 (1993).
  • This isoform is said to associate with p85 in cells, and to be ubiquitously expressed, as pi lO ⁇ mRNA has been found in numerous human and mouse tissues as well as in human umbilical vein endothelial cells, Jurkat human leukemic T cells, 293 human embryonic kidney cells, mouse 3T3 fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wide expression suggests that this isoform is broadly important in signaling pathways.
  • Pl lO ⁇ has also been shown to be expressed at lower levels in breast cells, melanocytes and endothelial cells (Vogt et al. Virology, 344: 131-138 (2006) and has since been implicated in conferring selective migratory properties to breast cancer cells (Sawyer et al. Cancer Res. 63:1667-1675 (2003)). Details concerning the Pl lO ⁇ isoform also can be found in U.S. Pat. Nos. 5,858,753; 5,822,910; and 5,985,589. See also, Vanhaesebroeck et al., Proc Nat. Acad Sci USA, 94:4330-5 (1997), and international publication WO 97/46688.
  • the p85 subunit acts to localize PI 3 -kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins (Rameh et al., Cell, 83:821-30 (1995)).
  • Five isoforms of p85 have been identified (p85 ⁇ , p85 ⁇ , p55 ⁇ , p55 ⁇ and p50 ⁇ ) encoded by three genes.
  • Alternative transcripts of Pik3rl gene encode the p85 ⁇ , p55 ⁇ and p50 ⁇ proteins (Deane and Fruman, Annu.Rev.Immunol.
  • p85 ⁇ is ubiquitously expressed while p85 ⁇ , is primarily found in the brain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)). Association of the p85 subunit to the PI 3 -kinase pi 10a, ⁇ , or ⁇ catalytic subunits appears to be required for the catalytic activity and stability of these enzymes. In addition, the binding of Ras proteins also upregulates PI 3 -kinase activity.
  • pi lO ⁇ The cloning of pi lO ⁇ revealed still further complexity within the PI3K family of enzymes (Stoyanov et al., Science, 269:690-93 (1995)).
  • the pi lO ⁇ isoform is closely related to pi 10a and pi lO ⁇ (45-48% identity in the catalytic domain), but as noted does not make use of p85 as a targeting subunit. Instead, pi lO ⁇ binds a plOl regulatory subunit that also binds to the ⁇ subunits of heterotrimeric G proteins.
  • the pi 01 regulatory subunit for PBKgamma was originally cloned in swine, and the human ortholog identified subsequently
  • p87 PIKAP is homologous to plOl in areas that bind pi lO ⁇ and G ⁇ and also mediates activation of pi lO ⁇ downstream of G-protein-coupled receptors.
  • pgy PiKAP s J 1 JgJ 1 Jy e ⁇ p resse d in the heart and may be crucial to PBK ⁇ cardiac function.
  • a constitutively active PBK polypeptide is described in international publication WO 96/25488. This publication discloses preparation of a chimeric fusion protein in which a 102-residue fragment of p85 known as the inter-SH2 (iSH2) region is fused through a linker region to the N-terminus of murine pi 10.
  • iSH2 inter-SH2
  • PI 3 -kinases can be defined by their amino acid identity or by their activity. Additional members of this growing gene family include more distantly related lipid and protein kinases including Vps34 TORI, and TOR2 of Saccharo- myces cerevisiae (and their mammalian homologs such as FRAP and mTOR), the ataxia telangiectasia gene product (ATR) and the catalytic subunit of DNA- dependent protein kinase (DNA-PK). See generally, Hunter, Cell, 83: 1-4 (1995).
  • PI 3 -kinase is also involved in a number of aspects of leukocyte activation.
  • a p85-associated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, which is an important costimulatory molecule for the activation of T-cells in response to antigen (Pages et al., Nature, 369:327- 29 (1994); Rudd, Immunity, 4:527-34 (1996)).
  • Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response.
  • IL2 interleukin-2
  • T cell growth factor an important T cell growth factor
  • PI 3-kinase inhibitors Two compounds, LY294002 and wortmannin, have been widely used as PI 3-kinase inhibitors. These compounds, however, are nonspecific PI3K inhibitors, as they do not distinguish among the four members of Class I PI 3 -kinases.
  • the IC50 values of wortmannin against each of the various Class I PI 3 -kinases are in the range of 1-1OnM.
  • the IC50 values for LY294002 against each of these PI 3-kinases is about l ⁇ M (Fruman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited.
  • pi 10a kinase dead knock in mice were generated with a single point mutation in the DFG motif of the ATP binding pocket (pi 10 ⁇ D 933A ) that impairs kinase activity but preserves mutant pi 10a kinase expression.
  • the knockin approach preserves signaling complex stoichiometry, scaffold functions and mimics small molecule approaches more realistically than knock out mice.
  • pi 10 ⁇ D 933A homozygous mice are embryonic lethal.
  • heterozygous mice are viable and fertile but display severely blunted signaling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin- like growth factor- 1 and leptin action.
  • IFS insulin-receptor substrate
  • Pl lO ⁇ knock out and kinase-dead knock in mice have both been generated and overall show similar and mild phenotypes with primary defects in migration of cells of the innate immune system and a defect in thymic development of T cells (Li et al. Science, 287: 1046-1049 (2000), Sasaki et al. Science, 287: 1040-1046 (2000), Patrucco et al. Cell, 118: 375-387 (2004)).
  • mice Similar to pi lO ⁇ , PI3K delta knock out and kinase-dead knock-in mice have been made and are viable with mild and like phenotypes.
  • the pi 10 ⁇ D910A mutant knock in mice demonstrated an important role for delta in B cell development and function, with marginal zone B cells and CD5+ Bl cells nearly undetectable, and B- and T cell antigen receptor signaling (Clayton et al. J.Exp.Med. 196:753-763 (2002); Okkenhaug et al. Science, 297: 1031-1034 (2002)).
  • the pi 10 ⁇ D910A mice have been studied extensively and have elucidated the diverse role that delta plays in the immune system.
  • T cell dependent and T cell independent immune responses are severely attenuated in pi 10 ⁇ D910A and secretion of THl (INF - ⁇ ) and TH2 cytokine (IL-4, IL-5) are impaired (Okkenhaug et al. J.Immunol. 177: 5122-5128 (2006)).
  • a human patient with a mutation in pi lO ⁇ has also recently been described.
  • This mutation resulted in a mis-sense amino acid substitution (E to K) at codon 1021, which is located in the highly conserved catalytic domain of pi lO ⁇ protein.
  • the patient has no other identified mutations and his phenotype is consistent with pi lO ⁇ deficiency in mice as far as studied.
  • Iso form-selective small molecule compounds have been developed with varying success to all Class I PI3 kinase isoforms (Ito et al. J. Pharm. Exp. Therapeut, 321 :1-8 (2007)).
  • Inhibitors to alpha are desirable because mutations in pi 10a have been identified in several solid tumors; for example, an amplification mutation of alpha is associated with 50% of ovarian, cervical, lung and breast cancer and an activation mutation has been described in more than 50% of bowel and 25% of breast cancers (Hennessy et al. Nature Reviews, 4: 988-1004 (2005)).
  • Yamanouchi has developed a compound YM-024 that inhibits alpha and delta equi-potently and is 8- and 28-fold selective over beta and gamma respectively (Ito et al. J.Pharm.Exp.Therapeut., 321 : 1-8 (2007)).
  • Pl lO ⁇ is involved in thrombus formation (Jackson et al. Nature Med. 11 : 507-514 (2005)) and small molecule inhibitors specific for this isoform are thought after for indication involving clotting disorders (TGX-221 : 0.007uM on beta; 14-fold selective over delta, and more than 500-fold selective over gamma and alpha) (Ito et al. J.Pharm.Exp.Therapeut., 321 : 1-8 (2007)).
  • IC87114 inhibits pi lO ⁇ in the high nanomolar range (triple digit) and has greater than 100-fold selectivity against pi 10a, is 52 fold selective against pi lO ⁇ but lacks selectivity against pi lO ⁇ (approx. 8-fold). It shows no activity against any protein kinases tested (Knight et al. Cell, 125: 733-747 (2006)).
  • delta-selective compounds or genetically manipulated mice pi 10 ⁇ D910A . It was shown that in addition to playing a key role in B and T cell activation, delta is also partially involved in neutrophil migration and primed neutrophil respiratory burst and leads to a partial block of antigen-IgE mediated mast cell degranulation (Condliffe et al. Blood, 106: 1432-1440 (2005); AIi et al. Nature, 431 : 1007-1011 (2002)).
  • pi lO ⁇ is emerging as an important mediator of many key inflammatory responses that are also known to participate in aberrant inflammatory conditions, including but not limited to autoimmune disease and allergy.
  • PI3K ⁇ function in inflammatory and auto-immune settings. Furthermore, our understanding of PI3K ⁇ requires further elaboration of the structural interactions of pi lO ⁇ , both with its regulatory subunit and with other proteins in the cell. There also remains a need for more potent and selective or specific inhibitors of PI3K delta, in order to avoid potential toxicology associated with activity on isozymes pi 10 alpha (insulin signaling) and beta (platelet activation). In particular, selective or specific inhibitors of PI3K ⁇ are desirable for exploring the role of this isozyme further and for development of superior pharmaceuticals to modulate the activity of the isozyme.
  • the present invention comprises a new class of compounds having the general formula which are useful to inhibit the biological activity of human PBK ⁇ . Another aspect of the invention is to provide compounds that inhibit PBK ⁇ selectively while having relatively low inhibitory potency against the other PBK isoforms. Another aspect of the invention is to provide methods of characterizing the function of human PBK ⁇ . Another aspect of the invention is to provide methods of selectively modulating human PBK ⁇ activity, and thereby promoting medical treatment of diseases mediated by PBK ⁇ dysfunction. Other aspects and advantages of the invention will be readily apparent to the artisan having ordinary skill in the art.
  • One aspect of the invention relates to compounds having the structure:
  • X 1 is C(R 10 ) or N;
  • X 2 is C or N;
  • X 3 is C or N
  • X 4 is C or N
  • X 5 is C or N; wherein at least two of X 2 , X 3 , X 4 and X 5 are C;
  • Y is N(R 8 ), O or S; n is O, 1, 2 or 3;
  • R 4 is, independently, in each instance, halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk, Ci_ 4 haloalk or an unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, substituted by 0, 1 , 2 or 3 substituents selected from halo, Ci_ 4 alk, Ci_ 3 haloalk, -OCi_ 4 alk, -NH 2 , -NHCi_ 4 alk and -N(Ci_ 4 alk)Ci_ 4 alk;
  • R 5 is, independently, in each instance, H, halo, Ci_ 6 alk, Ci_ 4 haloalk or Ci_6alk substituted by 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_ 4 alk, Ci_ 4 alk, Ci_ 3 haloalk, OCi_ 4 alk, NH 2 , NHCi_ 4 alk and N(Ci_ 4 alk)Ci_ 4 alk; or both R 5 groups together form a C 3 _ 6 spiroalk substituted by 0, 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_ 4 alk, Ci_ 4 alk, Ci_ 3 haloalk, OC i_ 4 alk, NH 2 , NHCi_ 4 alk and N(Ci. 4 alk)Ci. 4 alk;
  • 6 alkNR a R a and -NR a C 2 . 6 alk0R a , and the C 1-6 alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk and Ci_ 4 haloalk; or R 7 and R 8 together form a -C N- bridge wherein the carbon atom is substituted by H, halo, cyano
  • R 9 is H, Ci_ 6 alk or Ci_ 4 haloalk
  • R 10 is H, halo, Ci_ 3 alk, Ci_ 3 haloalk or cyano;
  • R a is independently, at each instance, H or R b ;
  • R b is independently, at each instance, phenyl, benzyl or Ci_ 6 alk, the phenyl, benzyl and Ci_6alk being substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_ 4 alk, Ci_ 3 haloalk, -OCi_ 4 alk, -NH 2 , -NHCi_ 4 alk and -N(Ci_ 4 alk)Ci_ 4 alk.
  • Another aspect of the invention relates to compounds having the structure:
  • X 1 is C(R 10 ) or N;
  • X 2 is C or N
  • X 3 is C or N
  • X 4 is C or N
  • X 5 is C or N; wherein at least two of X 2 , X 3 , X 4 and X 5 are C;
  • Y is N(R 8 ), O or S; n is 0, 1, 2 or 3;
  • R is selected from H, halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(C M alk)C M alk or Ci_ 4 haloalk; R 4 is, independently, in each instance, halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk,
  • OCi_ 4 haloalk NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk, Ci_ 4 haloalk or an unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_ 4 alk, Ci_ 3 haloalk, -OCi_ 4 alk, -NH 2 , -NHCi_ 4 alk and -N(Ci_ 4 alk)Ci_ 4 alk;
  • R 5 is, independently, in each instance, H, halo, Ci_ 6 alk, Ci_ 4 haloalk, or Ci_6alk substituted by 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_ 4 alk, Ci_ 4 alk, Ci_ 3 haloalk, OC 1-4 alk, NH 2 , NHCi_ 4 alk and N(Ci_ 4 alk)Ci_ 4 alk; or both R 5 groups together form a C 3 _ 6 spiroalk substituted by 0, 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_ 4 alk, Ci_ 4 alk, Ci_ 3 haloalk, OC i_
  • R 6 is H, halo, NHR 9 or OH
  • 6 alkNR a R a and -NR a C 2 . 6 alk0R a , and the Ci_ 6 alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk and Ci_ 4 haloalk; or R 7 and R 8 together form a -C N- bridge wherein the carbon atom is substituted by H, halo, cyano
  • R 9 is H, Ci_ 6 alk or Ci_ 4 haloalk
  • R , 10 is H, halo, Ci_ 3 alk, Ci_ 3 haloalk or cyano
  • R a is independently, at each instance, H or R b ;
  • R b is independently, at each instance, phenyl, benzyl or Ci_ 6 alk, the phenyl, benzyl and Ci_6alk being substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_ 4 alk, Ci_ 3 haloalk, -OCi_ 4 alk, -NH 2 , -NHCi_ 4 alk and -N(Ci_ 4 alk)Ci_ 4 alk.
  • the compound in another embodiment, in conjunction with any of the above or below embodiments, has the structure
  • C 1-6 alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, C 1-4 alk, OC 1-4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk and Ci_ 4 haloalk.
  • the compound in another embodiment, in conjunction with any of the above or below embodiments, has the structure
  • the compound in another embodiment, in conjunction with any of the above or below embodiments, has the structure
  • X 1 is N. In another embodiment, in conjunction with any of the above or below embodiments, X 1 is C(R 10 ).
  • X 2 , X 3 , X 4 and X 5 are each C.
  • X 2 is N.
  • X 3 is N.
  • X 4 is N.
  • X 5 is N.
  • Y is N(R 8 ).
  • R 1 is a direct-bonded, carbon-linked or oxygen-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is additionally substituted by 0 or 1 R 2 substituents, and the ring is additionally substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk and Ci_ 4 haloalk.
  • R 1 is a direct-bonded unsaturated 6-membered monocyclic ring containing 0, 1 or 2 N atoms, substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_ 4 alk, OCi_ 4 alk, OCi_ 4 haloalk, NHCi_ 4 alk, N(Ci_ 4 alk)Ci_ 4 alk and C 1-4 haloalk.
  • R 2 is H
  • R 6 is NHR 9 . In another embodiment, in conjunction with any of the above or below embodiments, R 6 is NH 2 . Another aspect of the invention relates to a method of treating PBK- mediated conditions or disorders.
  • the PI3K-mediated condition or disorder is selected from rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases.
  • the PBK- mediated condition or disorder is selected from cardiovascular diseases, atherosclerosis, hypertension, deep venous thrombosis, stroke, myocardial infarction, unstable angina, thromboembolism, pulmonary embolism, thrombolytic diseases, acute arterial ischemia, peripheral thrombotic occlusions, and coronary artery disease.
  • the PBK- mediated condition or disorder is selected from cancer, colon cancer, glioblastoma, endometrial carcinoma, hepatocellular cancer, lung cancer, melanoma, renal cell carcinoma, thyroid carcinoma, cell lymphoma, lymphoproliferative disorders, small cell lung cancer, squamous cell lung carcinoma, glioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and leukemia.
  • the PBK- mediated condition or disorder is selected from type II diabetes.
  • the PBK- mediated condition or disorder is selected from respiratory diseases, bronchitis, asthma, and chronic obstructive pulmonary disease.
  • the subject is a human.
  • Another aspect of the invention relates to the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases or autoimmune diseases comprising the step of administering a compound according to any of the above embodiments.
  • Another aspect of the invention relates to the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases and autoimmune diseases, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, skin complaints with inflammatory components, chronic inflammatory conditions, autoimmune diseases, systemic lupus erythematosis (SLE), myestenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome and autoimmune hemolytic anemia, allergic conditions and hypersensitivity, comprising the step of administering a compound according to any of the above or below embodiments.
  • Another aspect of the invention relates to the treatment of cancers that are mediated, dependent on or associated with pi lO ⁇ activity, comprising the step of administering a compound according to any of the above or below embodiments.
  • Another aspect of the invention relates to the treatment of cancers are selected from acute myeloid leukaemia, myelo-dysplastic syndrome, myeloproliferative diseases, chronic myeloid leukaemia, T-cell acute lymphoblastic leukaemia, B-cell acute lymphoblastic leukaemia, non-hodgkins lymphoma, B- cell lymphoma, solid tumors and breast cancer, comprising the step of administering a compound according to any of the above or below embodiments.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to any of the above embodiments and a pharmaceutically-acceptable diluent or carrier.
  • Another aspect of the invention relates to the use of a compound according to any of the above embodiments as a medicament.
  • Another aspect of the invention relates to the use of a compound according to any of the above embodiments in the manufacture of a medicament for the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases.
  • the compounds of this invention may have in general several asymmetric centers and are typically depicted in the form of racemic mixtures. This invention is intended to encompass racemic mixtures, partially racemic mixtures and separate enantiomers and diasteromers.
  • Ci_6alk means an alk group comprising a minimum of ⁇ and a maximum of ⁇ carbon atoms in a branched, cyclical or linear relationship or any combination of the three, wherein ⁇ and ⁇ represent integers.
  • the alk groups described in this section may also contain one or two double or triple bonds. Examples of Ci_6alk include, but are not limited to the following:
  • Halo or "halogen” means a halogen atoms selected from F, Cl, Br and I.
  • Cv-whaloalk means an alk group, as described above, wherein any number—at least one— of the hydrogen atoms attached to the alk chain are replaced by F, Cl,
  • Heterocycle means a ring comprising at least one carbon atom and at least one other atom selected from N, O and S. Examples of heterocycles that may be found in the claims include, but are not limited to, the following:
  • “Available nitrogen atoms” are those nitrogen atoms that are part of a heterocycle and are joined by two single bonds (e.g. piperidine), leaving an external bond available for substitution by, for example, H or CH 3 .
  • “Pharmaceutically-acceptable salt” means a salt prepared by conventional means, and are well known by those skilled in the art.
  • the “pharmacologically acceptable salts” include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like.
  • suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like.
  • pharmaceutically acceptable salts see infra and Berge et al., J. Pharm. Sci. 66: 1 (1977).
  • “Saturated, partially saturated or unsaturated” includes substituents saturated with hydrogens, substituents completely unsaturated with hydrogens and substituents partially saturated with hydrogens.
  • “Leaving group” generally refers to groups readily displaceable by a nucleophile, such as an amine, a thiol or an alcohol nucleophile. Such leaving groups are well known in the art. Examples of such leaving groups include, but are not limited to, N-hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates, tosylates and the like. Preferred leaving groups are indicated herein where appropriate.
  • Protecting group generally refers to groups well known in the art which are used to prevent selected reactive groups, such as carboxy, amino, hydroxy, mercapto and the like, from undergoing undesired reactions, such as nucleophilic, electrophilic, oxidation, reduction and the like. Preferred protecting groups are indicated herein where appropriate. Examples of amino protecting groups include, but are not limited to, aralk, substituted aralk, cycloalkenylalk and substituted cycloalkenyl alk, allyl, substituted allyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, silyl and the like.
  • aralk examples include, but are not limited to, benzyl, ortho-methylbenzyl, trityl and benzhydryl, which can be optionally substituted with halogen, alk, alkoxy, hydroxy, nitro, acylamino, acyl and the like, and salts, such as phosphonium and ammonium salts.
  • aryl groups include phenyl, naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl), phenanthrenyl, durenyl and the like.
  • cycloalkenylalk or substituted cycloalkenylalk radicals preferably have 6-10 carbon atoms, include, but are not limited to, cyclohexenyl methyl and the like.
  • Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups include benzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl, substituted benzoyl, butyryl, acetyl, trifluoroacetyl, trichloro acetyl, phthaloyl and the like.
  • a mixture of protecting groups can be used to protect the same amino group, such as a primary amino group can be protected by both an aralk group and an aralkoxycarbonyl group.
  • Amino protecting groups can also form a heterocyclic ring with the nitrogen to which they are attached, for example, 1 ,2-bis(methylene)benzene, phthalimidyl, succinimidyl, maleimidyl and the like and where these heterocyclic groups can further include adjoining aryl and cycloalk rings.
  • the heterocyclic groups can be mono-, di- or tri-substituted, such as nitrophthalimidyl.
  • Amino groups may also be protected against undesired reactions, such as oxidation, through the formation of an addition salt, such as hydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.
  • an addition salt such as hydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.
  • Many of the amino protecting groups are also suitable for protecting carboxy, hydroxy and mercapto groups.
  • aralk groups aralk groups.
  • AIk groups are also suitable groups for protecting hydroxy and mercapto groups, such as tert-butyl.
  • Silyl protecting groups are silicon atoms optionally substituted by one or more alk, aryl and aralk groups.
  • Suitable silyl protecting groups include, but are not limited to, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl, 1 ,2-bis(dimethylsilyl)benzene, 1 ,2-bis(dimethylsilyl)ethane and diphenylmethylsilyl.
  • Silylation of an amino groups provide mono- or di- silylamino groups. Silylation of aminoalcohol compounds can lead to a N 5 N 5 O- trisilyl derivative.
  • silyl function from a silyl ether function is readily accomplished by treatment with, for example, a metal hydroxide or ammonium fluoride reagent, either as a discrete reaction step or in situ during a reaction with the alcohol group.
  • Suitable silylating agents are, for example, trimethylsilyl chloride, tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride, diphenylmethyl silyl chloride or their combination products with imidazole or DMF.
  • Methods for silylation of amines and removal of silyl protecting groups are well known to those skilled in the art.
  • Methods of preparation of these amine derivatives from corresponding amino acids, amino acid amides or amino acid esters are also well known to those skilled in the art of organic chemistry including amino acid/amino acid ester or aminoalcohol chemistry.
  • Protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like.
  • a preferred method involves removal of a protecting group, such as removal of a benzyloxycarbonyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof.
  • a t- butoxycarbonyl protecting group can be removed utilizing an inorganic or organic acid, such as HCl or trifluoroacetic acid, in a suitable solvent system, such as dioxane or methylene chloride.
  • the resulting amino salt can readily be neutralized to yield the free amine.
  • Carboxy protecting group such as methyl, ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can be removed under hydrolysis and hydrogenolysis conditions well known to those skilled in the art.
  • Prodrugs of the compounds of this invention are also contemplated by this invention.
  • a prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the 5 prodrug to a patient.
  • the suitability and techniques involved in making and using prodrugs are well known by those skilled in the art.
  • For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).
  • Examples of a masked carboxylate anion include a variety of esters, such as alk (for example, O methyl, ethyl), cycloalk (for example, cyclohexyl), aralk (for example, benzyl, p- methoxybenzyl), and alkcarbonyloxyalk (for example, pivaloyloxymethyl).
  • esters such as alk (for example, O methyl, ethyl), cycloalk (for example, cyclohexyl), aralk (for example, benzyl, p- methoxybenzyl), and alkcarbonyloxyalk (for example, pivaloyloxymethyl).
  • Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)).
  • drugs containing an acidic NH group such as imidazole, imide, indole and the like, have been masked with N- acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).
  • the present invention also includes isotopically-labelled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 16 O, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 Cl.
  • isotopically-labelled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detection.
  • Isotopically labelled compounds of this invention can generally be prepared by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.
  • Electrospray ionization (ESI) mass spectrometry analysis was conducted on a Agilent 1100 series LC/MSD electrospray mass spectrometer. All compounds could be analyzed in the positive ESI mode using acetonitrile: water with 0.1% formic acid as the delivery solvent.
  • Reverse phase analytical HPLC was carried out using a Agilent 1200 series on Agilent Eclipse XDB-C 18 5 ⁇ m column (4.6 x 150 mm) as the stationary phase and eluting with acetonitrile: water with 0.1% TFA.
  • Reverse phase Semi-Prep HPLC was carried out using a Agilent 1100 Series on a Phenomenex GeminiTM 10 ⁇ m Cl 8 column (250 x 21.20 mm) as the stationary phase and eluting with acetonitrile: water with 0.1% TFA.
  • the light yellow solid was purified by column chromatography on a 40 g Redi-SepTM column using 0% to 100% gradient of DCM:MeOH:NH 4 OH (89:9:1) in DCM over 14 min and then 100% isocratic of DCM:MeOH:NH 4 OH (89:9:1) for 6 min as eluent to give the desired product as a light yellow solid.
  • Example 3 Preparation of 3-(3-fluorophenyl)-6-methyl-2-((lS)-l-(9H-purin- 6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one and 3-(3-fluorophenyl)-6- methyl-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4- one 3-(3-Fluorophenyl)-6-methyl-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidine-2-carb- aldehyde
  • the mixture was then cooled to 0 0 C and to the cooled homogenous mixture was added dropwise over 3 min to diisopropyl azodicarboxylate (0.5148 mL, 2.615 mmol) at 0 0 C.
  • the reaction mixture was allowed to warm to rt and stirred at rt. After 5 h, the mixture was coned under reduced pressure and partitioned between EtOAc (100 mL) and brine (100 mL). The organic layer was dried over Na 2 SO 4 , filtered, and coned under reduced pressure.
  • the organic layer was dried over Na 2 SO 4 , filtered, and coned under reduced pressure to give a yellow liquid.
  • the yellow liquid was purified by column chromatography on a 80 g of Redi-SepTM column using 0 to 50% gradient of DCM:MeOH:NH 4 OH (89:9:1) in DCM over 25 min and then 50% isocratic of DCM:MeOH:NH 4 OH (89:9:1) in DCM for 25 min as eluent to give a yellow solid.
  • Example 4 Preparation of 7-fluoro-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-3- (2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one and 7-fluoro-2-((lR)-l-(9H- purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one 2-(Chloromethyl)-7-fluoro-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
  • the brown syrup was purified by column chromatography on a 40 g Redi-SepTM column using 0 to 50% gradient of DCM:MeOH:NH 4 OH (89:9:1) in DCM over 14 min, then 50% isocratic of DCM:MeOH:NH 4 OH (89:9:1) in DCM for 14 min, then 50 to 100% gradient of DCM:MeOH:NH 4 OH (89:9:1) in DCM over 14 min, and then 100% isocratic of DCM:MeOH:NH 4 OH (89:9:1) for 14 min as eluent to give a yellow solid.
  • the aq acidic mixture (pH ⁇ 1.5) was washed with DCM (50 mL x 2) to remove organic impurities. The aq mixture was then treated with satd aq NaHCO 3 solution (50 mL) and extracted with DCM (50 mL x 3).
  • the combined organic layers were washed with water (100 mL x 1), brine (100 mL x 1), and dried over MgSO ⁇ filtered, and coned under reduced pressure to give the desired product as a yellow syrupy solid.
  • the combined aq layers still contained the desired product.
  • the combined aq layers were satd with NaCl and extracted with DCM (100 mL x 2).
  • the syrup was purified by column chromatography on a 40 g Redi-SepTM column using 0 to 50% gradient of DCM:MeOH:NH 4 OH (89:9:1) in DCM over 14 min, then 50% isocratic of DCM:MeOH:NH 4 OH (89:9:1) in DCM for 14 min, then 50 to 100% gradient of DCM:MeOH:NH 4 OH (89:9: 1) in DCM over 14 min, and then 100% isocratic of DCM:MeOH:NH 4 OH (89:9:1) for 14 min as eluent to give 6-methyl-2-(l-(9H-purin-6-ylamino)ethyl)-3-
  • 4,6-Dichloropyrimidine-5-carbaldehyde oxime (8g) was dissolved in CHCl 3 (40 mL) and treated with SOCl 2 (6 mL) for 2 h at rt. The solvent was removed and redissolved in DCM (5 niL). The solid was filtered and washed with DCM (5 rnL). The filtrate was coned and purified by column chromatography on silica gel (dry loading, DCM/hexane, 3/1) to give 4,6-dichloropyrimidine-5-carbonitrile as a white solid. 4-Amino-6-chloropyrimidine-5-carbonitrile
  • the white solid, 4,6-dichloropyrimidine-5-carbonitrile (5.82 g, 33.5 mmol) was dissolved in THF (66.9 mL) in a 500 mL round-bottom flask and to the mixture was bubbled through ammonia gas (0.570 g, 33.5 mmol) for 3 min in 10 min intervals with stirring. After 50 min, a white precipitate (ammmonium chloride) was filtered and the solid was washed with THF (100 mL). To the filtrate was added silica gel and coned under reduced pressure.
  • the mixture was purified by silica gel column chromatography on a 12O g of Redi-SepTM column using 0 to 100% gradient of EtOAc in hexane over 27 min and then 100% isocratic of EtOAc in hexane for 20 min as eluent to give 4-amino-6-chloropyrimidine-5- carbonitrile as an off-white solid.
  • Example 10 Preparation of 4-amino-6-((l-(3-(2-(methylsulfonyl)phenyl)-4- oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6-(((lR)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2- a] pyrimidin-2-yl)ethyl)amino)-5-py rimidinecarbonitrile and 4-amino-6- (((lS)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile 2-(Chloromethyl)-4H-pyrido[
  • Example 11 Preparation of 4-amino-6-((l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6-(((lS)-l-(4- oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidine- carbonitrile, and 4-amino-6-(((lR)-l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile
  • Example 12 Preparation of 4-amino-6-((l-(3-(3-fluorophenyl)-4-oxo-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lR)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((l S)-l-(3-(3-fluorophenyl)- 4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile 4-Amino-6-((l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[
  • the racemic mixture was purified by chiral separation using SFC to give 2 fractions: First peak on SFC OJ column and Second peak on ChiralpakTM
  • Example 13 Preparation of 4-amino-6-((l-(4-oxo-3-(2-pyridinyl)-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lR)-l-(4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)- 5-pyrimidinecarbonitrile, and 4-amino-6-(((lS)-l-(4-oxo-3-(2-pyridinyl)-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile 4-Amino-6-((l-(4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyr
  • the racemic mixture was purified by chiral separation using SFC to give 2 fractions: First peak on SFC OJ column and Second peak on ChiralpakTM AD-H column: 4-amino-6-((( IR)-I -(4-oxo-3-(2-pyridinyl)-4H-pyrido[ 1,2- a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid:
  • Example 14 Preparation of 4-amino-6-((l-(3-(3,5-difluorophenyl)-4-oxo-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino- 6-(((lS)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)eth- yl)amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lR)-l-(3-(3,5-difluoro- phenyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidine- carbonitrile
  • Example 15 Preparation of 4-amino-6-((l-(3-(4-methyl-2-pyridinyl)-4-oxo- 4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4- amino-6-(((lR)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lS)-l- (3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl).
  • Example 16 Preparation of 4-amino-6-((l-(6-methyl-4-oxo-3-phenyl-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lS)-l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lR)-l-(6-methyl-4-oxo-3- phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbo- nitrile
  • Example 17 Preparation of 4-amino-6-((l-(3-(3,5-difluorophenyl)-6-fluoro-l- methyl-4-oxo-l,4-dihydro-2-quinolinyl)ethyl)amino)-5-pyrimidinecarbo- nitrile
  • a PBK Alphascreen® assay (PerkinElmer, Waltham, MA) was used to measure the activity of a panel of four phosphoinositide 3-kinases: PI3K ⁇ , PI3K ⁇ , PI3K ⁇ , and PI3K ⁇ .
  • Enzyme reaction buffer was prepared using sterile water (Baxter, Deerfield, IL) and 5OmM Tris HCl pH 7, 14mM MgCl 2 , 2mM sodium cholate, and 10OmM NaCl. 2mM DTT was added fresh the day of the experiment.
  • the Alphascreen buffer was made using sterile water and 1OmM Tris HCl pH 7.5, 15OmM NaCl, 0.10% Tween 20, and 3OmM EDTA. ImM DTT was added fresh the day of the experiment.
  • Compound source plates used for this assay were 384- well Greiner clear polypropylene plates containing test compounds at 5mM and diluted 1 :2 over 22 concentrations. Columns 23 and 24 contained only DMSO as these wells comprised the positive and negative controls, respectively. Source plates were replicated by transferring 0.5 uL per well into 384-well Optiplates (PerkinElmer, Waltham, MA). Each PI3K isoform was diluted in enzyme reaction buffer to 2X working stocks.
  • PI3K ⁇ was diluted to 1.6nM
  • PI3K ⁇ was diluted to 0.8nM
  • PI3K ⁇ was diluted to 15nM
  • PI3K ⁇ was diluted to 1.6nM
  • PI(4,5)P2 (Echelon Biosciences, Salt Lake City, UT) was diluted to lO ⁇ M and ATP was diluted to 20 ⁇ M. This 2x stock was used in the assays for PBK ⁇ and PBK ⁇ .
  • PI(4,5)P2 was diluted to lO ⁇ M and ATP was diluted to 8 ⁇ M to prepare a similar 2x working stock.
  • Alphascreen reaction solutions were made using beads from the anti-GST Alphascreen kit (PerkinElmer, Waltham, MA). Two 4X working stocks of the Alphascreen reagents were made in Alphascreen reaction buffer. In one stock, biotinylated-IP4 (Echelon Biosciences, Salt Lake City, UT) was diluted to 4OnM and streptavadin-donor beads were diluted to 80 ⁇ g/mL. In the second stock, PIP 3 -binding protein (Echelon Biosciences, Salt Lake City, UT) was diluted to 4OnM and anti-GST-acceptor beads were diluted to 80 ⁇ g/mL. As a negative control, a reference inhibitor at a concentration » Ki (40 uM) was included in column 24 as a negative (100% inhibition) control.
  • Ki 40 uM
  • lO ⁇ L/well of 2X enzyme stock was added to columns 1-24 of the assay plates for each isoform.
  • lO ⁇ L/well of the appropriate substrate2x stock (containing 20 ⁇ M ATP for the PBK ⁇ and ⁇ assays and containing 8 ⁇ M ATP for the PBK ⁇ and ⁇ assays) was then added to Columns 1-24 of all plates. Plates were then incubated at room temperature for 20 minutes. In the dark, lO ⁇ L/well of the donor bead solution was added to columns 1-24 of the plates to quench the enzyme reaction. The plates were incubated at room temperature for 30 minutes.
  • the ATPase activity of the alpha and gamma isozymes was not greatly stimulated by PtdIns(4,5)P2 under these conditions and was therefore omitted from the assay of these isozymes.
  • Test compounds were dissolved in dimethyl sulfoxide and diluted with three-fold serial dilutions. The compound in DMSO (1 ⁇ L) was added per test well, and the inhibition relative to reactions containing no compound, with and without enzyme was determined. After assay incubation at rt, the reaction was stopped and residual ATP determined by addition of an equal volume of a commercial ATP bio luminescence kit (Perkin Elmer EasyLite) according to the manufacturer's instructions, and detected using a AnalystGT luminometer.
  • a commercial ATP bio luminescence kit Perkin Elmer EasyLite
  • Isolate PBMCs from Leukopac or from human fresh blood Isolate human B cells by using Miltenyi protocol and B cell isolation kit II. -human B cells were Purified by using AutoMacsTM column. Activation of human B cells
  • B cell proliferation medium DMEM + 5% FCS, 10 mM Hepes, 50 ⁇ M 2-mercaptoethanol
  • 150 ⁇ L medium contain 250 ng/mL CD40L -LZ recombinant protein (Amgen) and 2 ⁇ g/mL anti-Human IgM antibody (Jackson ImmunoReseach Lab. #109- 006-129), mixed with 50 ⁇ L B cell medium containing PBK inhibitors and incubate 72 h at 37 0 C incubator. After 72h, pulse labeling B cells with 0.5-1 uCi /well 3 H thymidine for overnight ⁇ 18 h, and harvest cell using TOM harvester.
  • B cell proliferation medium (DMEM + 5% FCS, 50 ⁇ M 2-mercaptoethanol, 1OmM Hepes).
  • the medium 150 ⁇ L
  • the medium contain 250 ng/mL CD40L -LZ recombinant protein (Amgen) and 10 ng/mL IL-4 ( R&D system # 204-IL-025), mixed with 50 150 ⁇ L B cell medium containing compounds and incubate 72 h at 37 0 C incubator.
  • Human PBMC are prepared from frozen stocks or they are purified from fresh human blood using a Ficoll gradient. Use 96 well round-bottom plate and plate 2xlO 5 PBMC/well with culture medium (RPMI1640 + 10% FCS, 5OuM 2-
  • PBK inhibitors was tested from 10 ⁇ M to 0.001 ⁇ M, in half log increments and in triplicate.
  • Tetanus toxoid ,T cell specific antigen University of Massachusetts Lab
  • Supernatants are collected after 6 days for IL2 ELISA assay , then cells are pulsed with 3 H-thymidine for ⁇ 18 h to measure proliferation.
  • GFP assays for detecting inhibition of Class Ia and Class III PI3K AKTl is regulated by Class Ia PBK activated by mitogenic factors (IGF- 1, PDGF, insulin, thrombin, NGF, etc.). In response to mitogenic stimuli, AKTl translocates from the cytosol to the plasma membrane
  • FKHRLl Forkhead
  • AKT membrane ruffling assay (CHO-IR-AKTl -EGFP cells/GE Healthcare) Wash cells with assay buffer. Treat with compounds in assay buffer 1 h. Add 10 ng/mL insulin. Fix after 10 min at room temp and image Forkhead translocation assay (MDA MB468 Forkhead-DiversaGFP cells) Treat cells with compound in growth medium 1 h. Fix and image.
  • Class IIIPI(3)P assay U2OS EGFP-2XFYVE cells/GE Healthcare
  • AKT is cytoplasmic
  • PI(3)P depleted from endosomes Biomarker assay B-cell receptor stimulation of CD69 or B7.2 (CD86) expression
  • Heparinized human whole blood was stimulated with 10 ⁇ g/mL anti-IgD (Southern Biotech, #9030-01). 90 ⁇ L of the stimulated blood was then aliquoted per well of a 96-well plate and treated with 10 ⁇ L of various concentrations of blocking compound (from 10-0.0003 ⁇ M) diluted in IMDM + 10% FBS (Gibco). Samples were incubated together for 4 h (for CD69 expression) to 6 h (for B7.2 expression) at 37 0 C.
  • Treated blood 50 ⁇ L was transferred to a 96-well, deep well plate (Nunc) for antibody staining with 10 ⁇ L each of CD45-PerCP (BD Biosciences, #347464), CD 19-FITC (BD Biosciences, #340719), and CD69-PE (BD Biosciences, #341652).
  • the second 50 ⁇ L of the treated blood was transferred to a second 96-well, deep well plate for antibody staining with 10 ⁇ L each of CDl 9-FITC (BD Biosciences, #340719) and CD86-PeCy5 (BD Biosciences, #555666). All stains were performed for 15-30 min in the dark at rt.
  • a human monocyte cell line, THP-I was maintained in RPMI + 10% FBS (Gibco).
  • FBS 10% FBS
  • cells were counted using trypan blue exclusion on a hemocytometer and suspended at a concentration of 1 x 10 6 cells per mL of media. 100 ⁇ L of cells plus media (1 x 10 5 cells) was then aliquoted per well of 4-96-well, deep well dishes (Nunc) to test eight different compounds.
  • FACS Phosflow Lyse/Fix buffer (1 mL of 37 0 C) (BD Biosciences, #558049) was added to each well. Plates were then incubated at 37 0 C for an additional 10-15 min. Plates were spun at 1500 rpm for 10 min, supernatant was aspirated off, and 1 mL of ice cold 90% MeOH was added to each well with vigorous shaking. Plates were then incubated either overnight at -70 0 C or on ice for 30 min before antibody staining. Plates were spun and washed 2X in PBS + 2% FBS (Gibco). Wash was aspirated and cells were suspended in remaining buffer.
  • Rabbit pAKT (50 ⁇ L, Cell Signaling, #4058L) at 1 : 100, was added to each sample for 1 h at rt with shaking. Cells were washed and spun at 1500 rpm for 10 min. Supernatant was aspirated and cells were suspended in remaining buffer. Secondary antibody, goat anti-rabbit Alexa 647 (50 ⁇ L, Invitrogen, #A21245) at 1 :500, was added for 30 min at rt with shaking. Cells were then washed IX in buffer and suspended in 150 ⁇ L of buffer for FACS analysis. Cells need to be dispersed very well by pipetting before running on flow cytometer.
  • Mouse femurs were dissected from five female BALB/c mice (Charles River Labs.) and collected into RPMI + 10% FBS media (Gibco).
  • Mouse bone marrow was removed by cutting the ends of the femur and by flushing with 1 mL of media using a 25 gauge needle. Bone marrow was then dispersed in media using a 21 gauge needle. Media volume was increased to 20 mL and cells were counted using trypan blue exclusion on a hemocytometer. The cell suspension was then increased to 7.5 x 10 6 cells per 1 mL of media and 100 ⁇ L (7.5 x 10 5 cells) was aliquoted per well into 4-96-well, deep well dishes (Nunc) to test eight different compounds.
  • mice Transgenic Line 3751, female, 10-12 wks Amgen Inc, Thousand Oaks, CA
  • mice Transgenic Line 3751, female, 10-12 wks Amgen Inc, Thousand Oaks, CA
  • mice Transgenic Line 3751, female, 10-12 wks Amgen Inc, Thousand Oaks, CA
  • i.v 0.2 mLs
  • anti-IgM FITC 50 ug/mouse
  • mice are sacrificed within a CO 2 chamber.
  • Blood is drawn via cardiac puncture (0.3 mL) (Ice 25 g Syringes, Sherwood, St. Louis, MO) and transferred into a 15 mL conical vial (Nalge/Nunc International, Denmark). Blood is immediately fixed with 6.0 mL of BD Phosflow Lyse/Fix
  • Buffer (BD Bioscience, San Jose, CA), inverted 3X's and placed in 37 0 C water bath.
  • Half of the spleen is removed and transferred to an eppendorf tube containing 0.5 mL of PBS (Invitrogen Corp, Grand Island, NY).
  • the spleen is crushed using a tissue grinder (Pellet Pestle, Kimble/Kontes, Vineland, NJ) and immediately fixed with 6.0 mL of BD Phosflow Lyse/Fix buffer, inverted 3X's and placed in 37 0 C water bath. Once tissues have been collected the mouse is cervically-dislocated and carcass to disposed.
  • the 15 mL conical vials are removed from the 37 0 C water bath and placed on ice until tissues are further processed. Crushed spleens are filtered through a 70 ⁇ m cell strainer (BD Bioscience, Bedford, MA) into another 15 mL conical vial and washed with 9 mL of PBS. Splenocytes and blood are spun @ 2,000 rpms for 10 min (cold) and buffer is aspirated. Cells are resuspended in 2.0 mL of cold (-20 0 C) 90% MeOH (Mallinckrodt Chemicals, Phillipsburg, NJ). MeOH is slowly added while conical vial is rapidly vortexed.
  • Tissues are then stored at -20 0 C until cells can be stained for FACS analysis.
  • Multi-dose TNP immunization Blood was collected by retro-orbital eye bleeds from 7-8 week old BALB/c female mice (Charles River Labs.) at day 0 before immunization. Blood was allowed to clot for 30 min and spun at 10,000 rpm in serum microtainer tubes (Becton Dickinson) for 10 min. Sera were collected, aliquoted in Matrix tubes (Matrix Tech. Corp.) and stored at -70 0 C until ELISA was performed. Mice were given compound orally before immunization and at subsequent time periods based on the life of the molecule.
  • mice were then immunized with either 50 ⁇ g of TNP- LPS (Biosearch Tech., #T-5065), 50 ⁇ g of TNP-Ficoll (Biosearch Tech., #F- 1300), or 100 ⁇ g of TNP-KLH (Biosearch Tech., #T-5060) plus 1% alum (Brenntag, #3501) in PBS.
  • TNP-KLH plus alum solution was prepared by gently inverting the mixture 3-5 times every 10 min for 1 h before immunization.
  • mice were CO 2 sacrificed and cardiac punctured. Blood was allowed to clot for 30 min and spun at 10,000 rpm in serum microtainer tubes for 10 min.
  • TNP-BSA 10 ⁇ g/mL
  • KPL 10% BSA ELISA Block solution
  • Ig-HRP conjugated secondary antibodies (goat anti- mouse IgGl, Southern Biotech #1070-05, goat anti-mouse IgG2a, Southern Biotech #1080-05, goat anti-mouse IgM, Southern Biotech #1020-05, goat anti- mouse IgG3, Southern Biotech #1100-05) were diluted at 1 :5000 and incubated on the plates for 1 h.
  • TMB peroxidase solution (SureBlue Reserve TMB from KPL) was used to visualize the antibodies. Plates were washed and samples were allowed to develop in the TMB solution approximately 5-20 min depending on the Ig analyzed. The reaction was stopped with 2M sulfuric acid and plates were read at an OD of 45 O nm.
  • the compounds of the present invention may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • Treatment of diseases and disorders herein is intended to also include the prophylactic administration of a compound of the invention, a pharmaceutical salt thereof, or a pharmaceutical composition of either to a subject (i.e., an animal, preferably a mammal, most preferably a human) believed to be in need of preventative treatment, such as, for example, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases and the like.
  • a subject i.e., an animal, preferably a mammal, most preferably a human
  • preventative treatment such as, for example, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases and the like.
  • the dosage regimen for treating PBK ⁇ -mediated diseases, cancer, and/or hyperglycemia with the compounds of this invention and/or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Dosage levels of the order from about 0.01 mg to 30 mg per kilogram of body weight per day, preferably from about 0.1 mg to 10 mg/kg, more preferably from about 0.25 mg to 1 mg/kg are useful for all methods of use disclosed herein.
  • the pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.
  • the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid.
  • the pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient.
  • these may contain an amount of active ingredient from about 1 to 2000 mg, preferably from about 1 to 500 mg, more preferably from about 5 to 150 mg.
  • a suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
  • the active ingredient may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water.
  • suitable carriers including saline, dextrose, or water.
  • the daily parenteral dosage regimen will be from about 0.1 to about 30 mg/kg of total body weight, preferably from about 0.1 to about 10 mg/kg, and more preferably from about 0.25 mg to 1 mg/kg.
  • Injectable preparations such as sterile injectable aq or oleaginous suspensions, may be formulated according to the known are using suitable dispersing or wetting agents and suspending agents.
  • 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- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • a suitable topical dose of active ingredient of a compound of the invention is 0.1 mg to 150 mg administered one to four, preferably one or two times daily.
  • the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
  • Formulations suitable for topical administration include liquid or semi- liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • the compounds of this invention are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • the pharmaceutical compositions may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsif ⁇ ers, buffers etc.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents.
  • Compounds of the present invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof.
  • the optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, e.g., by formation of diastereoisomeric salts, by treatment with an optically active acid or base.
  • Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts.
  • a different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers.
  • Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate.
  • the synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound.
  • the optically active compounds of the invention can likewise be obtained by using active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.
  • the compounds of this invention may exist as isomers, that is compounds of the same molecular formula but in which the atoms, relative to one another, are arranged differently.
  • the alkylene substituents of the compounds of this invention are normally and preferably arranged and inserted into the molecules as indicated in the definitions for each of these groups, being read from left to right.
  • substituents are reversed in orientation relative to the other atoms in the molecule. That is, the substituent to be inserted may be the same as that noted above except that it is inserted into the molecule in the reverse orientation.
  • the compounds of the present invention can be used in the form of salts derived from inorganic or organic acids.
  • the salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methansulfonate, nicotinate
  • the basic nitrogen- containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
  • long chain halides such as
  • organic acids such as oxalic acid, maleic acid, succinic acid and citric acid.
  • Other examples include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic bases.
  • esters of a carboxylic acid or hydroxyl containing group including a metabolically labile ester or a prodrug form of a compound of this invention.
  • a metabolically labile ester is one which may produce, for example, an increase in blood levels and prolong the efficacy of the corresponding non-esterified form of the compound.
  • a prodrug form is one which is not in an active form of the molecule as administered but which becomes therapeutically active after some in vivo activity or biotransformation, such as metabolism, for example, enzymatic or hydro lytic cleavage.
  • esters for example, methyl, ethyl
  • cycloalkyl for example, cyclohexyl
  • aralkyl for example, benzyl, p- methoxybenzyl
  • alkylcarbonyloxyalkyl for example, pivaloyloxymethyl
  • Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N- acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)). Hydroxy groups have been masked as esters and ethers.
  • EP 039,051 (Sloan and Little, 4/11/81) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.
  • Esters of a compound of this invention may include, for example, the methyl, ethyl, propyl, and butyl esters, as well as other suitable esters formed between an acidic moiety and a hydroxyl containing moiety.
  • Metabolically labile esters may include, for example, methoxymethyl, ethoxymethyl, iso-propoxymethyl, ⁇ -methoxyethyl, groups such as ⁇ -((Ci-C 4 )- alkyloxy)ethyl, for example, methoxyethyl, ethoxyethyl, propoxyethyl, iso- propoxyethyl, etc.; 2-oxo-l,3-dioxolen-4-ylmethyl groups, such as 5-methyl-2- oxo-l,3,dioxolen-4-ylmethyl, etc.; C 1 -C 3 alkylthiomethyl groups, for example, methylthiomethyl, ethylthi
  • the compounds of the invention may exist as crystalline solids which can be crystallized from common solvents such as ethanol, N,N-dimethyl- formamide, water, or the like.
  • crystalline forms of the compounds of the invention may exist as polymorphs, solvates and/or hydrates of the parent compounds or their pharmaceutically acceptable salts. All of such forms likewise are to be construed as falling within the scope of the invention.
  • the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

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Abstract

Substituted bicyclic heteroaryls and compositions containing them, for the treatment of general inflammation, arthritis, rheumatic diseases, osteoarthritis, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, chronic inflammatory conditions, including but not restricted to autoimmune diseases such as systemic lupus erythematosis (SLE), myestenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome and autoimmune hemolytic anemia, allergic conditions including all forms of hypersensitivity, The present invention also enables methods for treating cancers that are mediated, dependent on or associated with p110 activity, including but not restricted to leukemias, such as Acute Myeloid leukaemia (AML) Myelo-dysplastic syndrome (MDS) myelo-proliferative diseases (MPD) Chronic Myeloid Leukemia (CML) T-cell Acute Lymphoblastic leukaemia ( T-ALL) B-cell Acute Lymphoblastic leukaemia (B-ALL) Non Hodgkins Lymphoma (NHL) B-cell lymphoma and solid tumors, such as breast cancer.

Description

HETEROCYCLIC COMPOUNDS AND THEIR USES
This application claims the benefit of U.S. Provisional Application No. 61/220,484, filed June 25, 2009, which is hereby incorporated by reference.
The present invention relates generally to phosphatidylinositol 3 -kinase (PBK) enzymes, and more particularly to selective inhibitors of PBK activity and to methods of using such materials. BACKGROUND OF THE INVENTION
Cell signaling via 3 '-phosphorylated phosphoinositides has been implicated in a variety of cellular processes, e.g., malignant transformation, growth factor signaling, inflammation, and immunity (see Rameh et al., J. Biol Chem, 274:8347-8350 (1999) for a review). The enzyme responsible for generating these phosphorylated signaling products, phosphatidylinositol 3-kinase (PI 3-kinase; PBK), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol 2:358-60 (1992)).
The levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primary product of PI 3-kinase activation, increase upon treatment of cells with a variety of stimuli. This includes signaling through receptors for the majority of growth factors and many inflammatory stimuli, hormones, neurotransmitters and antigens, and thus the activation of PBKs represents one, if not the most prevalent, signal transduction events associated with mammalian cell surface receptor activation (Cantley, Science 296:1655-1657 (2002); Vanhaesebroeck et al. Annu.Rev.Biochem, 70: 535-602 (2001)). PI 3-kinase activation, therefore, is involved in a wide range of cellular responses including cell growth, migration, differentiation, and apoptosis (Parker et al., Current Biology, 5:577-99 (1995); Yao et al., Science, 267:2003-05 (1995)). Though the downstream targets of phosphorylated lipids generated following PI 3-kinase activation have not been fully characterized, it is known that pleckstrin-homology (PH) domain- and FYVE-fmger domain-containing proteins are activated when binding to various phosphatidylinositol lipids (Sternmark et al., J Cell Sci, 112:4175-83 (1999); .Lemmon et al., Trends Cell Biol, 7:237-42 (1997)). Two groups of PH-domain containing PBK effectors have been studied in the context of immune cell signaling, members of the tyrosine kinase TEC family and the serine/threonine kinases of the AGC family. Members of the Tec family containing PH domains with apparent selectivity for Ptdlns (3,4,5)P3 include Tec, Btk, Itk and Etk. Binding of PH to PIP3 is critical for tyrosine kinase activity of the Tec family members (Schaeffer and Schwartzberg, Curr.Opin.Immunol. 12: 282-288 (2000)) AGC family members that are regulated by PBK include the phosphoinositide- dependent kinase (PDKl), AKT (also termed PKB) and certain iso forms of protein kinase C (PKC) and S6 kinase. There are three isoforms of AKT and activation of AKT is strongly associated with PBK- dependent proliferation and survival signals. Activation of AKT depends on phosphorylation by PDKl, which also has a 3-phosphoinositide-selective PH domain to recruit it to the membrane where it interacts with AKT. Other important PDKl substrates are PKC and S6 kinase (Deane and Fruman, Annu.Rev.Immunol. 22 563-598 (2004)). In vitro, some isoforms of protein kinase C (PKC) are directly activated by PIP3. (Burgering et al, Nature, 376:599-602 (1995)).
Presently, the PI 3 -kinase enzyme family has been divided into three classes based on their substrate specificities. Class I PBKs can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidyl- inositol-4,5-biphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5- triphosphate, respectively. Class II PBKs phosphorylate PI and phosphatidyl- inositol-4-phosphate, whereas Class III PBKs can only phosphorylate PI.
The initial purification and molecular cloning of PI 3 -kinase revealed that it was a heterodimer consisting of p85 and pi 10 subunits (Otsu et al., Cell, 65:91-
104 (1991); Hiles et al., Cell, 70:419-29 (1992)). Since then, four distinct Class I PBKs have been identified, designated PBK α, β, δ, and γ, each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit. More specifically, three of the catalytic subunits, i.e., pi 10a, pi lOβ and pi lOδ, each interact with the same regulatory subunit, p85; whereas pi lOγ interacts with a distinct regulatory subunit, plOl. As described below, the patterns of expression of each of these PBKs in human cells and tissues are also distinct. Though a wealth of information has been accumulated in recent past on the cellular functions of PI 3-kinases in general, the roles played by the individual isoforms are not fully understood.
Cloning of bovine pi 10a has been described. This protein was identified as related to the Saccharomyces cerevisiae protein: Vps34p, a protein involved in vacuolar protein processing. The recombinant pi 10a product was also shown to associate with p85α, to yield a PI3K activity in transfected COS-I cells. See Hiles et al., Cell, 70, 419-29 (1992).
The cloning of a second human pi 10 isoform, designated pi lOβ, is described in Hu et al., MoI Cell Biol, 13:7677-88 (1993). This isoform is said to associate with p85 in cells, and to be ubiquitously expressed, as pi lOβ mRNA has been found in numerous human and mouse tissues as well as in human umbilical vein endothelial cells, Jurkat human leukemic T cells, 293 human embryonic kidney cells, mouse 3T3 fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wide expression suggests that this isoform is broadly important in signaling pathways.
Identification of the pi lOδ isoform of PI 3 -kinase is described in Chantry et al., J Biol Chem, 272:19236-41 (1997). It was observed that the human pi lOδ isoform is expressed in a tissue-restricted fashion. It is expressed at high levels in lymphocytes and lymphoid tissues and has been shown to play a key role in PI 3- kinase-mediated signaling in the immune system (Al-Alwan etl al. JI 178: 2328- 2335 (2007); Okkenhaug et al JI, 177: 5122-5128 (2006); Lee et al. PNAS, 103: 1289-1294 (2006)). Pl lOδ has also been shown to be expressed at lower levels in breast cells, melanocytes and endothelial cells (Vogt et al. Virology, 344: 131-138 (2006) and has since been implicated in conferring selective migratory properties to breast cancer cells (Sawyer et al. Cancer Res. 63:1667-1675 (2003)). Details concerning the Pl lOδ isoform also can be found in U.S. Pat. Nos. 5,858,753; 5,822,910; and 5,985,589. See also, Vanhaesebroeck et al., Proc Nat. Acad Sci USA, 94:4330-5 (1997), and international publication WO 97/46688.
In each of the PI3Kα, β, and δ subtypes, the p85 subunit acts to localize PI 3 -kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins (Rameh et al., Cell, 83:821-30 (1995)). Five isoforms of p85 have been identified (p85α, p85β, p55γ, p55α and p50α) encoded by three genes. Alternative transcripts of Pik3rl gene encode the p85 α, p55 α and p50α proteins (Deane and Fruman, Annu.Rev.Immunol. 22: 563-598 (2004)). p85α is ubiquitously expressed while p85β, is primarily found in the brain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)). Association of the p85 subunit to the PI 3 -kinase pi 10a, β, or δ catalytic subunits appears to be required for the catalytic activity and stability of these enzymes. In addition, the binding of Ras proteins also upregulates PI 3 -kinase activity.
The cloning of pi lOγ revealed still further complexity within the PI3K family of enzymes (Stoyanov et al., Science, 269:690-93 (1995)). The pi lOγ isoform is closely related to pi 10a and pi lOβ (45-48% identity in the catalytic domain), but as noted does not make use of p85 as a targeting subunit. Instead, pi lOγ binds a plOl regulatory subunit that also binds to the βγ subunits of heterotrimeric G proteins. The pi 01 regulatory subunit for PBKgamma was originally cloned in swine, and the human ortholog identified subsequently
(Krugmann et al., J Biol Chem, 274:17152-8 (1999)). Interaction between the N- terminal region of plOl with the N-terminal region of pi lOγ is known to activate PBKγ through Gβγ. Recently, a plOl-homologue has been identified, p84 or p8y piκAP (PBKγ adapter protein of 87 kDa) that binds pi lOγ (Voigt et al. JBC, 281 : 9977-9986 (2006), Suire et al. Curr.Biol. 15: 566-570 (2005)). p87PIKAP is homologous to plOl in areas that bind pi lOγ and Gβγ and also mediates activation of pi lOγ downstream of G-protein-coupled receptors. Unlike plOl, pgyPiKAP |s J1JgJ1Jy eχpressed in the heart and may be crucial to PBKγ cardiac function. A constitutively active PBK polypeptide is described in international publication WO 96/25488. This publication discloses preparation of a chimeric fusion protein in which a 102-residue fragment of p85 known as the inter-SH2 (iSH2) region is fused through a linker region to the N-terminus of murine pi 10. The p85 iSH2 domain apparently is able to activate PBK activity in a manner comparable to intact p85 (Klippel et al., MoI Cell Biol, 14:2675-85 (1994)). Thus, PI 3 -kinases can be defined by their amino acid identity or by their activity. Additional members of this growing gene family include more distantly related lipid and protein kinases including Vps34 TORI, and TOR2 of Saccharo- myces cerevisiae (and their mammalian homologs such as FRAP and mTOR), the ataxia telangiectasia gene product (ATR) and the catalytic subunit of DNA- dependent protein kinase (DNA-PK). See generally, Hunter, Cell, 83: 1-4 (1995).
PI 3 -kinase is also involved in a number of aspects of leukocyte activation. A p85-associated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, which is an important costimulatory molecule for the activation of T-cells in response to antigen (Pages et al., Nature, 369:327- 29 (1994); Rudd, Immunity, 4:527-34 (1996)). Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are linked to increases in the transcription of a number of genes including interleukin-2 (IL2), an important T cell growth factor (Fraser et al., Science, 251 :313-16 (1991)). Mutation of CD28 such that it can no longer interact with PI 3-kinase leads to a failure to initiate IL2 production, suggesting a critical role for PI 3-kinase in T cell activation.
Specific inhibitors against individual members of a family of enzymes provide invaluable tools for deciphering functions of each enzyme. Two compounds, LY294002 and wortmannin, have been widely used as PI 3-kinase inhibitors. These compounds, however, are nonspecific PI3K inhibitors, as they do not distinguish among the four members of Class I PI 3 -kinases. For example, the IC50 values of wortmannin against each of the various Class I PI 3 -kinases are in the range of 1-1OnM. Similarly, the IC50 values for LY294002 against each of these PI 3-kinases is about lμM (Fruman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited.
Based on studies using Wortmannin, there is evidence that PI 3-kinase function also is required for some aspects of leukocyte signaling through G- protein coupled receptors (Thelen et al., Proc Natl Acad Sci USA, 91 :4960-64 (1994)). Moreover, it has been shown that Wortmannin and LY294002 block neutrophil migration and superoxide release. However, inasmuch as these compounds do not distinguish among the various iso forms of PBK, it remains unclear from these studies which particular PBK isoform or isoforms are involved in these phenomena and what functions the different Class I PBK enzymes perform in both normal and diseased tissues in general. The co-expression of several PBK isoforms in most tissues has confounded efforts to segregate the activities of each enzyme until recently.
The separation of the activities of the various PBK isozymes has been advanced recently with the development of genetically manipulated mice that allowed the study of isoform-specifϊc knock-out and kinase dead knock-in mice and the development of more selective inhibitors for some of the different isoforms. Pl 10a and pi lOβ knockout mice have been generated and are both embryonic lethal and little information can be obtained from these mice regarding the expression and function of pi 10 alpha and beta (Bi et al. Mamm.Genome, 13:169-172 (2002); Bi et al. J.Biol.Chem. 274:10963-10968 (1999)). More recently, pi 10a kinase dead knock in mice were generated with a single point mutation in the DFG motif of the ATP binding pocket (pi 10αD933A) that impairs kinase activity but preserves mutant pi 10a kinase expression. In contrast to knock out mice, the knockin approach preserves signaling complex stoichiometry, scaffold functions and mimics small molecule approaches more realistically than knock out mice. Similar to the pi 10a KO mice, pi 10αD933A homozygous mice are embryonic lethal. However, heterozygous mice are viable and fertile but display severely blunted signaling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin- like growth factor- 1 and leptin action. Defective responsiveness to these hormones leads to hyperinsulinaemia, glucose intolerance, hyperphagia, increase adiposity and reduced overall growth in heterozygotes (Foukas, et al. Nature, 441 : 366-370 (2006)). These studies revealed a defined, non-redundant role for pi 10a as an intermediate in IGF-I, insulin and leptin signaling that is not substituted for by other isoforms. We will have to await the description of the p 11 Oβ kinase-dead knock in mice to further understand the function of this isoform (mice have been made but not yet published; Vanhaesebroeck).
Pl lOγ knock out and kinase-dead knock in mice have both been generated and overall show similar and mild phenotypes with primary defects in migration of cells of the innate immune system and a defect in thymic development of T cells (Li et al. Science, 287: 1046-1049 (2000), Sasaki et al. Science, 287: 1040-1046 (2000), Patrucco et al. Cell, 118: 375-387 (2004)).
Similar to pi lOγ, PI3K delta knock out and kinase-dead knock-in mice have been made and are viable with mild and like phenotypes. The pi 10δD910A mutant knock in mice demonstrated an important role for delta in B cell development and function, with marginal zone B cells and CD5+ Bl cells nearly undetectable, and B- and T cell antigen receptor signaling (Clayton et al. J.Exp.Med. 196:753-763 (2002); Okkenhaug et al. Science, 297: 1031-1034 (2002)). The pi 10δD910A mice have been studied extensively and have elucidated the diverse role that delta plays in the immune system. T cell dependent and T cell independent immune responses are severely attenuated in pi 10δD910A and secretion of THl (INF -γ) and TH2 cytokine (IL-4, IL-5) are impaired (Okkenhaug et al. J.Immunol. 177: 5122-5128 (2006)). A human patient with a mutation in pi lOδ has also recently been described. A taiwanese boy with a primary B cell immunodeficiency and a gamma-hypoglobulinemia of previously unknown aetiology presented with a single base-pair substitution, m.3256G to A in codon 1021 in exon 24 of pi lOδ. This mutation resulted in a mis-sense amino acid substitution (E to K) at codon 1021, which is located in the highly conserved catalytic domain of pi lOδ protein. The patient has no other identified mutations and his phenotype is consistent with pi lOδ deficiency in mice as far as studied.
(Jou et al. Int.J.Immunogenet. 33: 361-369 (2006)).
Iso form-selective small molecule compounds have been developed with varying success to all Class I PI3 kinase isoforms (Ito et al. J. Pharm. Exp. Therapeut, 321 :1-8 (2007)). Inhibitors to alpha are desirable because mutations in pi 10a have been identified in several solid tumors; for example, an amplification mutation of alpha is associated with 50% of ovarian, cervical, lung and breast cancer and an activation mutation has been described in more than 50% of bowel and 25% of breast cancers (Hennessy et al. Nature Reviews, 4: 988-1004 (2005)). Yamanouchi has developed a compound YM-024 that inhibits alpha and delta equi-potently and is 8- and 28-fold selective over beta and gamma respectively (Ito et al. J.Pharm.Exp.Therapeut., 321 : 1-8 (2007)).
Pl lOβ is involved in thrombus formation (Jackson et al. Nature Med. 11 : 507-514 (2005)) and small molecule inhibitors specific for this isoform are thought after for indication involving clotting disorders (TGX-221 : 0.007uM on beta; 14-fold selective over delta, and more than 500-fold selective over gamma and alpha) (Ito et al. J.Pharm.Exp.Therapeut., 321 : 1-8 (2007)).
Selective compounds to pi lOγ are being developed by several groups as immunosuppressive agents for autoimmune disease (Rueckle et al. Nature Reviews, 5: 903-918 (2006)). Of note, AS 605240 has been shown to be efficacious in a mouse model of rheumatoid arthritis (Camps et al. Nature Medicine, 11 : 936-943 (2005)) and to delay onset of disease in a model of systemic lupus erythematosis (Barber et al. Nature Medicine, 11 : 933-935 (205)).
Delta-selective inhibitors have also been described recently. The most selective compounds include the quinazolinone purine inhibitors (PIK39 and IC87114). IC87114 inhibits pi lOδ in the high nanomolar range (triple digit) and has greater than 100-fold selectivity against pi 10a, is 52 fold selective against pi lOβ but lacks selectivity against pi lOγ (approx. 8-fold). It shows no activity against any protein kinases tested (Knight et al. Cell, 125: 733-747 (2006)). Using delta-selective compounds or genetically manipulated mice (pi 10δD910A) it was shown that in addition to playing a key role in B and T cell activation, delta is also partially involved in neutrophil migration and primed neutrophil respiratory burst and leads to a partial block of antigen-IgE mediated mast cell degranulation (Condliffe et al. Blood, 106: 1432-1440 (2005); AIi et al. Nature, 431 : 1007-1011 (2002)). Hence pi lOδ is emerging as an important mediator of many key inflammatory responses that are also known to participate in aberrant inflammatory conditions, including but not limited to autoimmune disease and allergy. To support this notion, there is a growing body of pi lOδ target validation data derived from studies using both genetic tools and pharmacologic agents. Thus, using the delta-selective compound IC 87114 and the pi 10δD910A mice, AIi et al. (Nature, 431 : 1007-1011 (2002)) have demonstrated that delta plays a critical role in a murine model of allergic disease. In the absence of functional delta, passive cutaneous anaphylaxis (PCA) is significantly reduced and can be attributed to a reduction in allergen-IgE induced mast cell activation and degranulation. In addition, inhibition of delta with IC 87114 has been shown to significantly ameliorate inflammation and disease in a murine model of asthma using ovalbumin-induced airway inflammation (Lee et al. FASEB, 20: 455-465 (2006). These data utilizing compound were corroborated in pi 10δD910A mutant mice using the same model of allergic airway inflammation by a different group (Nashed et al. Eur.J.Immunol. 37:416-424 (2007)).
There exists a need for further characterization of PI3Kδ function in inflammatory and auto-immune settings. Furthermore, our understanding of PI3Kδ requires further elaboration of the structural interactions of pi lOδ, both with its regulatory subunit and with other proteins in the cell. There also remains a need for more potent and selective or specific inhibitors of PI3K delta, in order to avoid potential toxicology associated with activity on isozymes pi 10 alpha (insulin signaling) and beta (platelet activation). In particular, selective or specific inhibitors of PI3Kδ are desirable for exploring the role of this isozyme further and for development of superior pharmaceuticals to modulate the activity of the isozyme.
Summary The present invention comprises a new class of compounds having the general formula
Figure imgf000011_0001
which are useful to inhibit the biological activity of human PBKδ. Another aspect of the invention is to provide compounds that inhibit PBKδ selectively while having relatively low inhibitory potency against the other PBK isoforms. Another aspect of the invention is to provide methods of characterizing the function of human PBKδ. Another aspect of the invention is to provide methods of selectively modulating human PBKδ activity, and thereby promoting medical treatment of diseases mediated by PBKδ dysfunction. Other aspects and advantages of the invention will be readily apparent to the artisan having ordinary skill in the art.
Detailed Description
One aspect of the invention relates to compounds having the structure:
Figure imgf000012_0001
or any pharmaceutically-acceptable salt thereof, wherein:
X1 is C(R10) or N; X2 is C or N;
X3 is C or N;
X4 is C or N;
X5 is C or N; wherein at least two of X2, X3, X4 and X5 are C;
Y is N(R8), O or S; n is O, 1, 2 or 3;
R1 is a direct-bonded, Ci_4alk-linked, OC i_2alk- linked, Ci_2alkO-linked, N(Ra)-linked or O-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S atom, substituted by 0, 1, 2 or 3 substituents independently selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra, wherein the available carbon atoms of the ring are additionally substituted by 0, 1 or 2 oxo or thioxo groups, and wherein the ring is additionally substituted by 0 or 1 directly bonded, SO2 linked, C(=O) linked or CH2 linked group selected from phenyl, pyridyl, pyrimidyl, morpholino, piperazinyl, piperadinyl, cyclopentyl, cyclohexyl all of which are further substituted by 0, 1 , 2 or 3 independent Rb groups;
R2 is selected from H, halo, d_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa; R is selected from H, halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk,
NHCi_4alk, N(Ci_4alk)Ci_4alk or Ci_4haloalk;
R4 is, independently, in each instance, halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk, Ci_4haloalk or an unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, substituted by 0, 1 , 2 or 3 substituents selected from halo, Ci_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_4alk;
R5 is, independently, in each instance, H, halo, Ci_6alk, Ci_4haloalk or Ci_6alk substituted by 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, Ci_4alk, Ci_3haloalk, OCi_4alk, NH2, NHCi_4alk and N(Ci_4alk)Ci_4alk; or both R5 groups together form a C3_6spiroalk substituted by 0, 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, Ci_4alk, Ci_3haloalk, OC i_ 4alk, NH2, NHCi_4alk and N(Ci.4alk)Ci.4alk;
R6 is H, halo, NHR9 or OH, cyano, OCi_4alk, Ci_4alk, Ci_3haloalk, OCi_4alk, -C(=O)ORa, -C(=O)N(Ra)Ra, -N(Ra)C(=O)Rb;
R7 is selected from H, halo, d_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa, -NRaC2.6alk0Ra and d_6alk, wherein the Ci_6alk is substituted by 0, 1 2 or 3 substituents selected from halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2.6alk0Ra, and the C1-6alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and Ci_4haloalk; or R7 and R8 together form a -C=N- bridge wherein the carbon atom is substituted by H, halo, cyano, or a saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2, 3 or 4 substituents selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa,
-ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa, -OC2_6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2.6alk0Ra; or R7 and R9 together form a -N=C- bridge wherein the carbon atom is substituted by H, halo, Ci_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra or -S(=O)2NRaRa;
Figure imgf000015_0001
R9 is H, Ci_6alk or Ci_4haloalk;
R10 is H, halo, Ci_3alk, Ci_3haloalk or cyano;
Ra is independently, at each instance, H or Rb; and
Rb is independently, at each instance, phenyl, benzyl or Ci_6alk, the phenyl, benzyl and Ci_6alk being substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_ 4alk.
Another aspect of the invention relates to compounds having the structure:
Figure imgf000015_0002
or any pharmaceutically-acceptable salt thereof, wherein:
X1 is C(R10) or N;
X2 is C or N;
X3 is C or N;
X4 is C or N;
X5 is C or N; wherein at least two of X2, X3, X4 and X5 are C;
Y is N(R8), O or S; n is 0, 1, 2 or 3;
R1 is a direct-bonded, Ci_4alk-linked, OC i_2alk- linked, Ci_2alkO-linked or O-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S atom, substituted by 0, 1, 2 or 3 substituents independently selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=0)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2.6alk0Ra, wherein the available carbon atoms of the ring are additionally substituted by 0, 1 or 2 oxo or thioxo groups;
R2 is selected from H, halo, d_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa;
R is selected from H, halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(CMalk)CMalk or Ci_4haloalk; R4 is, independently, in each instance, halo, nitro, cyano, Ci_4alk, OCi_4alk,
OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk, Ci_4haloalk or an unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_4alk;
R5 is, independently, in each instance, H, halo, Ci_6alk, Ci_4haloalk, or Ci_6alk substituted by 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, Ci_4alk, Ci_3haloalk, OC1-4alk, NH2, NHCi_4alk and N(Ci_4alk)Ci_4alk; or both R5 groups together form a C3_6spiroalk substituted by 0, 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, Ci_4alk, Ci_3haloalk, OC i_
4alk, NH2, NHCi_4alk and N(Ci_4alk)Ci_4alk;
R6 is H, halo, NHR9 or OH;
R7 is selected from H, halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra,
-S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2-6alkNRaRa, -NRaC2.6alk0Ra and d_6alk, wherein the Ci_6alk is substituted by 0, 1 2 or 3 substituents selected from halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa,
-OC2_6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2.6alk0Ra, and the Ci_6alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and Ci_4haloalk; or R7 and R8 together form a -C=N- bridge wherein the carbon atom is substituted by H, halo, cyano, or a saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2, 3 or 4 substituents selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa, -OC2_6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra,
-N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra; or R7 and R9 together form a -N=C- bridge wherein the carbon atom is substituted by H, halo, Ci_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra or -S(=O)2NRaRa;
Figure imgf000017_0001
R9 is H, Ci_6alk or Ci_4haloalk; R , 10 is H, halo, Ci_3alk, Ci_3haloalk or cyano;
Ra is independently, at each instance, H or Rb; and
Rb is independently, at each instance, phenyl, benzyl or Ci_6alk, the phenyl, benzyl and Ci_6alk being substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_ 4alk.
In another embodiment, in conjunction with any of the above or below embodiments, the compound has the structure
Figure imgf000018_0001
wherein R7 is selected from H, halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra,
-C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra,
-N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa, -NRaC2.6alk0Ra and C1^aIk, wherein the Ci_6alk is substituted by 0, 1 2 or 3 substituents selected from halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra, and the C1-6alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, C1-4alk, OC1-4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and Ci_4haloalk.
In another embodiment, in conjunction with any of the above or below embodiments, the compound has the structure
Figure imgf000019_0001
wherein R12 is selected from H, halo, Ci_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa.
In another embodiment, in conjunction with any of the above or below embodiments, the compound has the structure
Figure imgf000019_0002
wherein R13 is H, halo, cyano, or a saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2, 3 or 4 substituents selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2.6alk0Ra; or R7 and R9 together form a -N=C- bridge wherein the carbon atom is substituted by H, halo, Ci_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa.
In another embodiment, in conjunction with any of the above or below embodiments, X1 is N. In another embodiment, in conjunction with any of the above or below embodiments, X1 is C(R10).
In another embodiment, in conjunction with any of the above or below embodiments, X2, X3, X4 and X5 are each C.
In another embodiment, in conjunction with any of the above or below embodiments, X2 is N.
In another embodiment, in conjunction with any of the above or below embodiments, X3 is N.
In another embodiment, in conjunction with any of the above or below embodiments, X4 is N. In another embodiment, in conjunction with any of the above or below embodiments, X5 is N.
In another embodiment, in conjunction with any of the above or below embodiments, Y is N(R8).
In another embodiment, in conjunction with any of the above or below embodiments, R1 is a direct-bonded, Ci_4alk-linked, OC i_2alk- linked, Ci_2alkO- linked or O-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S atom, substituted by 0, 1, 2 or 3 substituents independently selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra, wherein the available carbon atoms of the ring are additionally substituted by 0, 1 or 2 oxo or thioxo groups
In another embodiment, in conjunction with any of the above or below embodiments, R1 is a direct-bonded, carbon-linked or oxygen-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is additionally substituted by 0 or 1 R2 substituents, and the ring is additionally substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and Ci_4haloalk.
In another embodiment, in conjunction with any of the above or below embodiments, R1 is a direct-bonded unsaturated 6-membered monocyclic ring containing 0, 1 or 2 N atoms, substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and C1-4haloalk.
In another embodiment, in conjunction with any of the above or below embodiments, R2 is H.
In another embodiment, in conjunction with any of the above or below embodiments, R6 is NHR9. In another embodiment, in conjunction with any of the above or below embodiments, R6 is NH2. Another aspect of the invention relates to a method of treating PBK- mediated conditions or disorders.
In certain embodiments, the PI3K-mediated condition or disorder is selected from rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases. In other embodiments, the PBK- mediated condition or disorder is selected from cardiovascular diseases, atherosclerosis, hypertension, deep venous thrombosis, stroke, myocardial infarction, unstable angina, thromboembolism, pulmonary embolism, thrombolytic diseases, acute arterial ischemia, peripheral thrombotic occlusions, and coronary artery disease. In still other embodiments, the PBK- mediated condition or disorder is selected from cancer, colon cancer, glioblastoma, endometrial carcinoma, hepatocellular cancer, lung cancer, melanoma, renal cell carcinoma, thyroid carcinoma, cell lymphoma, lymphoproliferative disorders, small cell lung cancer, squamous cell lung carcinoma, glioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, and leukemia. In yet another embodiment, the PBK- mediated condition or disorder is selected from type II diabetes. In still other embodiments, the PBK- mediated condition or disorder is selected from respiratory diseases, bronchitis, asthma, and chronic obstructive pulmonary disease. In certain embodiments, the subject is a human.
Another aspect of the invention relates to the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases or autoimmune diseases comprising the step of administering a compound according to any of the above embodiments. Another aspect of the invention relates to the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases and autoimmune diseases, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, skin complaints with inflammatory components, chronic inflammatory conditions, autoimmune diseases, systemic lupus erythematosis (SLE), myestenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome and autoimmune hemolytic anemia, allergic conditions and hypersensitivity, comprising the step of administering a compound according to any of the above or below embodiments.
Another aspect of the invention relates to the treatment of cancers that are mediated, dependent on or associated with pi lOδ activity, comprising the step of administering a compound according to any of the above or below embodiments.
Another aspect of the invention relates to the treatment of cancers are selected from acute myeloid leukaemia, myelo-dysplastic syndrome, myeloproliferative diseases, chronic myeloid leukaemia, T-cell acute lymphoblastic leukaemia, B-cell acute lymphoblastic leukaemia, non-hodgkins lymphoma, B- cell lymphoma, solid tumors and breast cancer, comprising the step of administering a compound according to any of the above or below embodiments.
Another aspect of the invention relates to a pharmaceutical composition comprising a compound according to any of the above embodiments and a pharmaceutically-acceptable diluent or carrier.
Another aspect of the invention relates to the use of a compound according to any of the above embodiments as a medicament.
Another aspect of the invention relates to the use of a compound according to any of the above embodiments in the manufacture of a medicament for the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases.
The compounds of this invention may have in general several asymmetric centers and are typically depicted in the form of racemic mixtures. This invention is intended to encompass racemic mixtures, partially racemic mixtures and separate enantiomers and diasteromers.
Unless otherwise specified, the following definitions apply to terms found in the specification and claims:
"Cα-βalk" means an alk group comprising a minimum of α and a maximum of β carbon atoms in a branched, cyclical or linear relationship or any combination of the three, wherein α and β represent integers. The alk groups described in this section may also contain one or two double or triple bonds. Examples of Ci_6alk include, but are not limited to the following:
Figure imgf000024_0001
"Benzo group", alone or in combination, means the divalent radical C4H4=, one representation of which is -CH=CH-CH=CH-, that when vicinally attached to another ring forms a benzene-like ring—for example tetrahydronaphthylene, indole and the like.
The terms "oxo" and "thioxo" represent the groups =0 (as in carbonyl) and =S (as in thiocarbonyl), respectively.
"Halo" or "halogen" means a halogen atoms selected from F, Cl, Br and I.
"Cv-whaloalk" means an alk group, as described above, wherein any number—at least one— of the hydrogen atoms attached to the alk chain are replaced by F, Cl,
Br or I.
"Heterocycle" means a ring comprising at least one carbon atom and at least one other atom selected from N, O and S. Examples of heterocycles that may be found in the claims include, but are not limited to, the following:
Figure imgf000024_0002
Figure imgf000025_0001
"Available nitrogen atoms" are those nitrogen atoms that are part of a heterocycle and are joined by two single bonds (e.g. piperidine), leaving an external bond available for substitution by, for example, H or CH3.
"Pharmaceutically-acceptable salt" means a salt prepared by conventional means, and are well known by those skilled in the art. The "pharmacologically acceptable salts" include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. When compounds of the invention include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. For additional examples of "pharmacologically acceptable salts," see infra and Berge et al., J. Pharm. Sci. 66: 1 (1977).
"Saturated, partially saturated or unsaturated" includes substituents saturated with hydrogens, substituents completely unsaturated with hydrogens and substituents partially saturated with hydrogens. "Leaving group" generally refers to groups readily displaceable by a nucleophile, such as an amine, a thiol or an alcohol nucleophile. Such leaving groups are well known in the art. Examples of such leaving groups include, but are not limited to, N-hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates, tosylates and the like. Preferred leaving groups are indicated herein where appropriate.
"Protecting group" generally refers to groups well known in the art which are used to prevent selected reactive groups, such as carboxy, amino, hydroxy, mercapto and the like, from undergoing undesired reactions, such as nucleophilic, electrophilic, oxidation, reduction and the like. Preferred protecting groups are indicated herein where appropriate. Examples of amino protecting groups include, but are not limited to, aralk, substituted aralk, cycloalkenylalk and substituted cycloalkenyl alk, allyl, substituted allyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, silyl and the like. Examples of aralk include, but are not limited to, benzyl, ortho-methylbenzyl, trityl and benzhydryl, which can be optionally substituted with halogen, alk, alkoxy, hydroxy, nitro, acylamino, acyl and the like, and salts, such as phosphonium and ammonium salts. Examples of aryl groups include phenyl, naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl), phenanthrenyl, durenyl and the like. Examples of cycloalkenylalk or substituted cycloalkenylalk radicals, preferably have 6-10 carbon atoms, include, but are not limited to, cyclohexenyl methyl and the like. Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups include benzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl, substituted benzoyl, butyryl, acetyl, trifluoroacetyl, trichloro acetyl, phthaloyl and the like. A mixture of protecting groups can be used to protect the same amino group, such as a primary amino group can be protected by both an aralk group and an aralkoxycarbonyl group. Amino protecting groups can also form a heterocyclic ring with the nitrogen to which they are attached, for example, 1 ,2-bis(methylene)benzene, phthalimidyl, succinimidyl, maleimidyl and the like and where these heterocyclic groups can further include adjoining aryl and cycloalk rings. In addition, the heterocyclic groups can be mono-, di- or tri-substituted, such as nitrophthalimidyl. Amino groups may also be protected against undesired reactions, such as oxidation, through the formation of an addition salt, such as hydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like. Many of the amino protecting groups are also suitable for protecting carboxy, hydroxy and mercapto groups. For example, aralk groups. AIk groups are also suitable groups for protecting hydroxy and mercapto groups, such as tert-butyl. Silyl protecting groups are silicon atoms optionally substituted by one or more alk, aryl and aralk groups. Suitable silyl protecting groups include, but are not limited to, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl, 1 ,2-bis(dimethylsilyl)benzene, 1 ,2-bis(dimethylsilyl)ethane and diphenylmethylsilyl. Silylation of an amino groups provide mono- or di- silylamino groups. Silylation of aminoalcohol compounds can lead to a N5N5O- trisilyl derivative. Removal of the silyl function from a silyl ether function is readily accomplished by treatment with, for example, a metal hydroxide or ammonium fluoride reagent, either as a discrete reaction step or in situ during a reaction with the alcohol group. Suitable silylating agents are, for example, trimethylsilyl chloride, tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride, diphenylmethyl silyl chloride or their combination products with imidazole or DMF. Methods for silylation of amines and removal of silyl protecting groups are well known to those skilled in the art. Methods of preparation of these amine derivatives from corresponding amino acids, amino acid amides or amino acid esters are also well known to those skilled in the art of organic chemistry including amino acid/amino acid ester or aminoalcohol chemistry.
Protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like. A preferred method involves removal of a protecting group, such as removal of a benzyloxycarbonyl group by hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof. A t- butoxycarbonyl protecting group can be removed utilizing an inorganic or organic acid, such as HCl or trifluoroacetic acid, in a suitable solvent system, such as dioxane or methylene chloride. The resulting amino salt can readily be neutralized to yield the free amine. Carboxy protecting group, such as methyl, ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can be removed under hydrolysis and hydrogenolysis conditions well known to those skilled in the art.
It should be noted that compounds of the invention may contain groups that may exist in tautomeric forms, such as cyclic and acyclic amidine and guanidine groups, heteroatom substituted heteroaryl groups (Y' = O, S, NR), and the like, which are illustrated in the following examples:
Figure imgf000028_0001
and though one form is named, described, displayed and/or claimed herein, all the tautomeric forms are intended to be inherently included in such name, description, 0 display and/or claim.
Prodrugs of the compounds of this invention are also contemplated by this invention. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the 5 prodrug to a patient. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examples of a masked carboxylate anion include a variety of esters, such as alk (for example, O methyl, ethyl), cycloalk (for example, cyclohexyl), aralk (for example, benzyl, p- methoxybenzyl), and alkcarbonyloxyalk (for example, pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N- acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).
Hydroxy groups have been masked as esters and ethers. EP 039,051 (Sloan and Little, 4/11/81) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.
The specification and claims contain listing of species using the language "selected from . . . and . . ." and "is . . . or . . ." (sometimes referred to as Markush groups). When this language is used in this application, unless otherwise stated it is meant to include the group as a whole, or any single members thereof, or any subgroups thereof. The use of this language is merely for shorthand purposes and is not meant in any way to limit the removal of individual elements or subgroups as needed.
The present invention also includes isotopically-labelled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 16O, 170, 31P, 32P, 35S, 18F, and 36Cl.
Compounds of the present invention that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detection. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of this invention can generally be prepared by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.
Experimental
The following abbreviations are used: aq - aqueous
BINAP - 2,2 ' -bis(diphenylphosphino)- 1,1 ' -binaphthyl coned - concentrated
DCM- dichloromethane
DIEA - N,N-diisopropylethylamine
DMF - N,N-dimethylformamide
Et2O - diethyl ether
EtOAc - ethyl acetate
EtOH - ethyl alcohol h - hour(s) min - minutes
MeOH - methyl alcohol
MsCl methanesulfonyl chloride rt - room temperature satd - saturated
TFA - trifluoroacetic acid
THF - tetrahydrofuran
General
Reagents and solvents used below can be obtained from commercial sources. 1H- NMR spectra were recorded on a Bruker 400 MHz and 500 MHz NMR spectrometer. Significant peaks are tabulated in the order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet) , coupling constant(s) in hertz (Hz) and number of protons. Mass spectrometry results are reported as the ratio of mass over charge, followed by the relative abundance of each ion (in parentheses. Electrospray ionization (ESI) mass spectrometry analysis was conducted on a Agilent 1100 series LC/MSD electrospray mass spectrometer. All compounds could be analyzed in the positive ESI mode using acetonitrile: water with 0.1% formic acid as the delivery solvent. Reverse phase analytical HPLC was carried out using a Agilent 1200 series on Agilent Eclipse XDB-C 18 5 μm column (4.6 x 150 mm) as the stationary phase and eluting with acetonitrile: water with 0.1% TFA. Reverse phase Semi-Prep HPLC was carried out using a Agilent 1100 Series on a Phenomenex Gemini™ 10 μm Cl 8 column (250 x 21.20 mm) as the stationary phase and eluting with acetonitrile: water with 0.1% TFA.
Example 1: Preparation of 3-(3-fluorophenyl)-6-methyl-2-((9H-purin-6-yl- amino)methyl)-4H-pyrido[l,2-a]pyrimidin-4-one 2-(Chloromethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000032_0001
A mixture of 2-amino-6-methylpyridine (10.00 g, 92.47 mmol), ethyl 4-chloro- acetoacetate (16.24 mL, 120.2 mmol), and polyphosphoric acid (50.00 g) was stirred at 125 0C. After 5.5 h, the mixture was removed from the heat. To the cooled mixture was added ice-water (200 mL) and neutralized with 2 N NaOH (400 mL) to pH 6-7. The resulting precipitate was collected by filtration, washed with water (~ 400 mL), and dried to give 2-(chloromethyl)-6-methyl-4H-pyrido- [l,2-a]pyrimidin-4-one as a dark brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.68 (1 H, dd, J=9.0, 7.0 Hz), 7.40 (1 H, dd, J=9.0, 0.8 Hz), 6.93 (1 H, d, J=6.7 Hz), 6.36 (1 H, s), 4.58 (2 H, s), 2.93 (3 H, s); Mass Spectrum (ESI) m/e = 208.9 (M + 1). 3-Bromo-2-(chloromethyl)-6-methyl-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000032_0002
A mixture of 2-(chloromethyl)-6-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4-one (3.5648 g, 17.09 mmol), N-bromosuccinimide (3.35 g, 18.79 mmol), and acetic acid (48.2 mL, 843 mmol) was stirred at rt. After 4.5 h, the mixture was poured into water (200 mL) and the resulting precipitate was collected by filtration, washed with water (200 mL), and dried to give an orange solid. The orange solid was dissolved in DCM (100 mL), dried over Na2SOφ filtered, and coned under reduced pressure to give 3-bromo-2-(chloromethyl)-6-methyl-4H-pyrido[l,2-a]- pyrimidin-4-one as an orange solid: H NMR (400 MHz, DMSO-dβ) δ ppm 7.76 (1 H, dd, J=9.0, 7.0 Hz), 7.47 - 7.52 (1 H, m), 7.04 - 7.09 (1 H, m), 4.71 (2 H, s), 2.95 (3 H, s); Mass Spectrum (ESI) m/e = 288.9 (M + 1).
(3-Bromo-6-methyl-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)methyl acetate
Figure imgf000033_0001
A mixture of 3-bromo-2-(chloromethyl)-6-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (4.5322 g, 15.76 mmol), potassium acetate (2.320 g, 23.64 mmol), and DMF (60.0 mL) was stirred at 40 0C. After 3.5 h, the mixture was coned under reduced pressure. To the residue was added water (100 mL) and the resulting precipitate was collected by filtration, washed with water (100 mL), and dried to give (3- bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)methyl acetate as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.75 (1 H, dd, J=9.0, 7.0 Hz), 7.45 (I H, dd, J=9.0, 0.8 Hz), 7.02 - 7.08 (1 H, m), 5.12 (2 H, s), 2.95 (3 H, s), 2.14 (3 H, s); Mass Spectrum (ESI) m/e = 310.9 [M + 1 (79Br)] and 313.0 [M + 1 (81Br)]. 3-Bromo-2-(hydr oxymethyl)-6-methyl-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000033_0002
A heterogeneous mixture of (3-bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrim- idin-2-yl)methyl acetate (4.2444 g, 13.64 mmol), coned. HCl (3.87 mL, 46.4 mmol), and 1,4-dioxane (39.0 mL) was heated with stirring at 70 0C. After 3 h, the mixture was cooled to rt and the mixture was coned under reduced pressure. The residue was diluted with water (100 mL) and the pH adjusted to 10 with 28% ammonium hydroxide (10 mL). The precipitate was filtered, washed with water (200 mL), and dried under high vacuum to give 3-bromo-2-(hydroxymethyl)-6- methyl-4H-pyrido[l,2-a]pyrimidin-4-one as a tan solid: ^H NMR (400 MHz, DMSO-dβ) δ ppm 7.74 (1 H, dd, J=8.8, 6.8 Hz), 7.49 (1 H, dd, J=9.0, 0.8 Hz), 7.01 - 7.06 (1 H, m), 5.24 (1 H, t, J=6.1 Hz), 4.52 (2 H, d, J=6.3 Hz), 2.96 (3 H, s); Mass Spectrum (ESI) m/e = 268.9 [M + 1 (79Br)] and 271.0 [M + 1 (81Br)]. 3-(3-Fluorophenyl)-2-(hydroxymethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin- 4-one
Figure imgf000034_0001
A mixture of 3-bromo-2-(hydroxymethyl)-6-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (0.09450 g, 0.3512 mmol), 3-fluorophenylboronic acid (0.09827 g, 0.7024 mmol), tetrakis(triphenylphosphine)palladium (0.02029 g, 0.01756 mmol), and sodium carbonate anhydrous (0.1861 g, 1.756 mmol) in acetonitrile-water (3:1) (4 mL) was stirred at 85 0C. After 2 h, the mixture was cooled to rt and partitioned between EtOAc (50 mL) and water (50 mL). The organic layer was washed with brine (50 mL x 2), dried over Na2SO4, filtered, and coned under reduced pressure. The residue was purified by silica gel column chromatography on a 40 g Redi-
Sep™ column using 0 to 100% gradient of EtOAc in hexane over 14 min and then 100% isocratic of EtOAc for 20 min as eluent to give 3-(3-fluorophenyl)-2- (hydroxymethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one as a yellow solid: Mass Spectrum (ESI) m/e = 285.1 (M + 1). 2-((3-(3-Fluorophenyl)-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)- methyl)isoindoline-l,3-dione
Figure imgf000034_0002
To a solution of 3-(3-fluorophenyl)-2-(hydroxymethyl)-6-methyl-4H-pyrido[l,2- a]pyrimidin-4-one (0.05110 g, 0.180 mmol) in THF (3.00 mL) was added tri- phenylphosphine (0.141 g, 0.539 mmol), phthalimide (0.0793 g, 0.539 mmol), and diisopropyl azodicarboxylate (0.106 mL, 0.539 mmol). The reaction mixture was stirred at rt. After 3 h, the mixture was coned under reduced pressure and partitioned between EtOAc (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, filtered, and coned under reduced pressure. The residue was purified by silica gel column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 14 min and 50% isocratic of EtOAc for 15 min as eluent to give 2-((3-(3-fluorophenyl)-6-methyl-4-oxo-4H-pyrido- [l,2-a]pyrimidin-2-yl)methyl)isoindoline-l,3-dione as a solid: Mass Spectrum (ESI) m/e = 414.1 (M + 1).
2-(Aminomethyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4- one
Figure imgf000035_0001
To a suspension of 2-((3-(3-fluorophenyl)-6-methyl-4-oxo-4H-pyrido[l,2-a]- pyrimidin-2-yl)methyl)isoindoline-l,3-dione (0.07430 g, 0.180 mmol) in EtOH (3.59 mL) was added hydrazine, anhydrous (0.0564 mL, 1.80 mmol), and the mixture was stirred under reflux. After 30 min, the mixture was coned under reduced pressure. The residue was purified by column chromatography on a 40 g Redi-Sep™ column using 0% to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) for 15 min as eluent to give 2-(aminomethyl)-3-(3-fluorophenyl)-6-methyl-4H- pyrido[l,2-a]pyrimidin-4-one as a brown syrupy solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 7.68 (1 H, dd, J=9.0, 6.7 Hz), 7.41 - 7.50 (2 H, m), 7.15 - 7.26 (3 H, m), 6.91 - 6.96 (1 H, m), 3.44 (2 H, s), 2.90 (3 H, s), 1.99 (2 H, br. s.); Mass Spectrum (ESI) m/e = 284.2 (M + 1). 3-(3-Fluorophenyl)-6-methyl-2-((9H-purin-6-ylamino)methyl)-4H-pyrido [ 1 ,2- a] pyrimidin-4-one
Figure imgf000036_0001
A mixture of 6-bromopurine (0.02040 g, 0.1025 mmol), 2-(aminomethyl)-3-(3- fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one (0.02420 g, 0.08542 mmol), and DIEA (0.04464 mL, 0.2563 mmol) in 1-butanol (2.000 mL) was stirred at 110 0C. After 18 h, the mixture was removed from the heat and coned under reduced pressure. The crude mixture was purified by revered-phase semi- prep HPLC using 20-70% gradient of CH3CN (0.1% of TFA) in water (0.1% of TFA) over 40 min as eluent. The acetonitrile was coned under reduced pressure and to the remaining acidic aq layer was added satd NaHCO3 to neutralize the TFA salt. The resulting precipitate was collected by filtration and washed with water to give 3-(3-fluorophenyl)-6-methyl-2-((9H-purin-6-ylamino)methyl)-4H- pyrido[l,2-a]pyrimidin-4-one as a light yellow solid: 1H NMR (400 MHz, DMSO- dβ) δ ppm 12.87 (1 H, s), 8.07 - 8.16 (2 H, m), 7.68 (1 H, dd, J=9.0, 7.0 Hz), 7.55 (1 H, s), 7.39 - 7.51 (2 H, m), 7.24 - 7.31 (2 H, m), 7.15 - 7.23 (1 H, m), 6.92 - 7.00 (1 H, m), 4.51 (2 H, br. s.), 2.92 (3 H, s); Mass Spectrum (ESI) m/e = 402.1 (M + 1). Example 2: Preparation of 2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin- l-yl)methyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one 2-(Chloromethyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4- one hydrochloride
Figure imgf000037_0001
A solution of 3-(3-fluorophenyl)-2-(hydroxymethyl)-6-methyl-4H-pyrido[l ,2-a]- pyrimidin-4-one (0.7141 g, 2.512 mmol, Prepared in Example 1) in chloroform (8.373 mL) was treated with SOCl2 (0.9139 mL, 12.56 mmol) dropwise at 0 °C, and the reaction mixture was allowed to warm to rt with stirring. After 1 h, the mixture was coned under reduced pressure, co-evaporated three times with DCM, and dried under high vacuum to give 2-(chloromethyl)-3-(3-fluorophenyl)-6- methyl-4H-pyrido[l,2-a]pyrimidin-4-one hydrochloride as a brown solid: Mass Spectrum (ESI) m/e = 303.0 (M + 1).
2-((4-Amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-l-yl)methyl)-3-(3-fluoro- phenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000037_0002
To a solution of 3-iodo-lH-pyrazolo[3,4-d]pyrimidin-4-amine (0.6560 g, 2.513 mmol) in 10 mL of DMF was added sodium hydride, 60% dispersion in mineral oil (0.2010 g, 5.026 mmol) at 0 0C and the mixture was stirred at rt. After 10 min the mixture was added to a solution of 2-(chloromethyl)-3-(3-fluorophenyl)-6- methyl-4H-pyrido[l,2-a]pyrimidin-4-one hydrochloride (0.8524 g, 2.513 mmol) in 5 mL of DMF and the resulting mixture was stirred at rt. After 3 h, the mixture was poured into ice-water (100 mL), the resulting precipitate was collected by filtration and washed with water (100 mL) to give a light yellow solid. The light yellow solid was purified by column chromatography on a 40 g Redi-Sep™ column using 0% to 100% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 100% isocratic of DCM:MeOH:NH4OH (89:9:1) for 6 min as eluent to give the desired product as a light yellow solid. The light yellow solid was suspended in EtOAc and filtered to give 2-((4-amino-3-iodo-lH-pyrazolo- [3,4-d]pyrimidin-l-yl)methyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2-a]- pyrimidin-4-one: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.12 (1 H, s), 7.63 (1 H, dd, J=8.8, 6.8 Hz), 7.27 - 7.37 (1 H, m), 7.22 (1 H, dd, J=9.0, 0.8 Hz), 7.08 - 7.16 (2 H, m), 7.00 - 7.07 (1 H, m), 6.92 - 6.98 (1 H, m), 5.34 (2 H, s), 2.90 (3 H, s); Mass Spectrum (ESI) m/e = 527.8 (M + 1).
Example 3: Preparation of 3-(3-fluorophenyl)-6-methyl-2-((lS)-l-(9H-purin- 6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one and 3-(3-fluorophenyl)-6- methyl-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4- one 3-(3-Fluorophenyl)-6-methyl-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidine-2-carb- aldehyde
Figure imgf000038_0001
A mixture of 3-(3-fluorophenyl)-2-(hydroxymethyl)-6-methyl-4H-pyrido[l ,2-a]- pyrimidin-4-one (2.0005 g, 7.037 mmol, Prepared in Example 1) and mang- anese(IV) oxide (6.118 g, 70.37 mmol) in toluene (46.91 mL) was heated to reflux. After 3 h, the mixture was cooled to rt and filtered through a pad of Celite™. The pad was rinsed with DCM (200 mL). The filtrate was coned under reduced pressure to give an orange solid. The orange solid was suspended in hexane (50 mL), sonicated, and filtered to give 3-(3-fluorophenyl)-6-methyl-4- oxo-4H-pyrido[l,2-a]pyrimidine-2-carbaldehyde as a yellow solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 9.77 (1 H, s), 7.78 (1 H, dd, J=8.6, 7.0 Hz), 7.18 - 7.68 (5 H, m), 7.06 (1 H, d, J=6.7 Hz), 2.92 (3 H, s); Mass Spectrum (ESI) m/e = 283.0 (M + 1). 3-(3-Fluorophenyl)-2-(l-hydroxyethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin- 4-one
Figure imgf000039_0001
To a stirring suspension of 3-(3-fluorophenyl)-6-methyl-4-oxo-4H-pyrido[l,2-a]- pyrimidine-2-carbaldehyde (1.3567 g, 4.806 mmol) in THF (48.06 rnL) was added methylmagnesium bromide 3 M in Et2O (2.403 rnL, 7.210 mmol) dropwise at 0 0C and the mixture was allowed to warm to rt and stirred at rt. After 5 h, the reaction was quenched with satd aq NH4Cl (50 mL) and water (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with water (50 mL x 1), brine (50 mL x 1), dried over Na2SO4, filtered, and coned under reduced pressure to give a dark red syrup. The dark red syrup was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 25 min, then 50% isocratic of EtOAc in hexane for 10 min, 50 to 100% gradient of EtOAc in hexane over 10 min, and then 100% isocratic of EtOAc for 10 min as eluent to give 3-(3-fluorophenyl)-2- (l-hydroxyethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one as a solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 7.66 (1 H, dd, J=8.8, 6.8 Hz), 7.41 - 7.52 (2 H, m), 7.12 - 7.24 (3 H, m), 6.88 - 6.96 (1 H, m), 4.98 (1 H, d, J=6.3 Hz), 4.42 - 4.51 (1 H, m), 2.89 (3 H, s), 1.26 (3 H, d, J=6.3 Hz); Mass Spectrum (ESI) m/e = 298.9 (M + 1).
2-(l-(3-(3-Fluorophenyl)-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)isoindoline- 1 ,3-dione
Figure imgf000039_0002
A solution of 3 -(3 -fluorophenyl)-2-( 1 -hydroxy ethyl)-6-methyl-4H-pyrido [ 1 ,2-a] - pyrimidin-4-one (0.6500 g, 2.179 mmol), triphenylphosphine (0.6858 g, 2.615 mmol), phthalimide (0.3847 g, 2.615 mmol), and THF (14.53 mL) was stirred at rt for 5 min to dissolve all reactants. The mixture was then cooled to 0 0C and to the cooled homogenous mixture was added dropwise over 3 min to diisopropyl azodicarboxylate (0.5148 mL, 2.615 mmol) at 0 0C. The reaction mixture was allowed to warm to rt and stirred at rt. After 5 h, the mixture was coned under reduced pressure and partitioned between EtOAc (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, filtered, and coned under reduced pressure. The residue was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 25 min and 50% isocratic of EtOAc for 10 min as eluent to give 2-(l-(3-(3-fluorophenyl)- 6-methyl-4-oxo-4H-pyrido[ 1 ,2-a]pyrimidin-2-yl)ethyl)isoindoline- 1 ,3-dione as a bright yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.75 - 7.82 (2 H, m), 7.65 - 7.74 (3 H, m), 7.35 - 7.42 (1 H, m), 7.13 - 7.24 (1 H, m), 6.90 - 7.01 (4 H, m), 5.43 (1 H, q, J=7.0 Hz), 2.89 (3 H, s), 1.62 (3 H, d, J=7.4 Hz); Mass Spectrum (ESI) m/e = 427.9 (M + 1).
2-(l-Aminoethyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4- one
Figure imgf000040_0001
To a suspension of 2-(l-(3-(3-fluorophenyl)-6-methyl-4-oxo-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)isoindoline-l,3-dione (0.6764 g, 1.582 mmol) in EtOH (31.65 mL) was added hydrazine monohydrate (0.7676 mL, 15.82 mmol), and the mixture was stirred under reflux. After 1 h, the mixture was filtered and washed with MeOH and DCM. The filtrate was coned under reduced pressure. The residue was purified by column chromatography on a 40 g Redi-Sep™ column using 0% to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 25 min as eluent to give 2-(l-aminoethyl)-3-(3-fluorophenyl)-6-methyl-4H-pyrido[l,2- a]pyrimidin-4-one as a brown syrupy solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 7.64 (1 H, dd, J=9.0, 6.7 Hz), 7.38 - 7.52 (2 H, m), 7.13 - 7.24 (3 H, m), 6.86 - 6.93 (1 H, m), 3.66 (1 H, q, J=6.4 Hz), 2.88 (3 H, s), 1.82 (2 H, s), 1.15 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 298.0 (M + 1). 3-(3-Fluorophenyl)-6-methyl-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-4H- pyrido[l,2-a]pyrimidin-4-one and 3-(3-fluorophenyl)-6-methyl-2-((lR)-l- (9H-purin-6-ylamino)ethyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000041_0001
Figure imgf000041_0002
A mixture of 6-bromopurine (0.3164 g, 1.590 mmol), 2-(l-aminoethyl)-3-(3- fluorophenyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one (0.4297 g, 1.445 mmol), and DIEA (0.7552 mL, 4.336 mmol) in 1-butanol (14.45 mL) was stirred at 110 0C. After 17 h, the mixture was removed from the heat and coned under reduced pressure. The residue was dissolved in DCM (100 mL) and washed with water (50 mL x 1). The organic layer was dried over Na2SO4, filtered, and coned under reduced pressure to give a yellow liquid. The yellow liquid was purified by column chromatography on a 80 g of Redi-Sep™ column using 0 to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 25 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 25 min as eluent to give a yellow solid. The yellow solid was suspended in EtOAc-hexane (1 :1) and filtered to give 3-(3-fluorophenyl)-6-methyl-2-(l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2- a]pyrimidin-4-one as a tan solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.85 (1 H, s), 8.13 (2 H, s), 7.68 (1 H, dd, J=9.0, 7.0 Hz), 7.42 - 7.54 (2 H, m), 7.18 - 7.33 (4 H, m), 6.94 (1 H, d, J=7.0 Hz), 5.22 (1 H, s), 2.89 (3 H, s), 1.38 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 415.9 (M + 1). The racemic mixture was separated by chiral separation using SFC to give two fractions: First-eluting enantiomer on AD-H column: 3-(3-fluorophenyl)-6-methyl-2- ((lS)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.21 (1 H, s), 8.07 - 8.16 (2 H, m), 7.68 (1 H, dd, J=9.0, 7.0 Hz), 7.43 - 7.55 (2 H, m), 7.17 - 7.33 (4 H, m), 6.94 (1 H, d, J=7.0 Hz), 5.23 (1 H, s), 2.89 (3 H, s), 1.38 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 415.9 (M + 1). Second-eluting enantiomer on AD-H column: 3-(3-fluorophenyl)-6-methyl-2- ((lR)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.39 (1 H, s), 8.04 - 8.17 (2 H, m), 7.67 (1 H, dd, J=9.0, 7.0 Hz), 7.41 - 7.55 (2 H, m), 7.17 - 7.34 (4 H, m), 6.94 (1 H, d, J=7.0 Hz), 5.23 (1 H, s), 2.89 (3 H, s), 1.37 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 415.9 (M + 1).
Example 4: Preparation of 7-fluoro-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-3- (2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one and 7-fluoro-2-((lR)-l-(9H- purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one 2-(Chloromethyl)-7-fluoro-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000042_0001
A mixture of 2-amino-5-fluoropyridine (5.1673 g, 46.09 mmol), ethyl 4-chloro- acetoacetate (8.097 mL, 59.92 mmol), and polyphosphoric acid (80.00 g) was stirred at 110 0C. After 4 h, the mixture was removed from the heat. The cooled mixture was suspended in water (100 mL) and the mixture was neutralized with 2 N NaOH (550 mL) until the pH 7. The resulting precipitate was collected by filtration, washed with water (1 L), and air-dried overnight to give 2-(chloro- methyl)-7-fluoro-4H-pyrido[l,2-a]pyrimidin-4-one as a brown solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 8.94 (1 H, dd, J=4.9, 2.9 Hz), 8.14 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.79 - 7.86 (1 H, m), 6.59 (1 H, s), 4.68 (2 H, s); Mass Spectrum (ESI) m/e = 212.9 (M + 1).
3-Bromo-2-(chloromethyl)-7-fluoro-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000043_0001
A mixture of 2-(chloromethyl)-7-fluoro-4H-pyrido[l,2-a]pyrimidin-4-one (8.310 g, 39.09 mmol),N-bromosuccinimide (8.055 g, 42.99 mmol), and acetic acid
(110.3 mL) was stirred at rt. After 6 h, the mixture was poured into water (300 mL) and the resulting precipitate was collected by filtration, washed with water (400 mL), and dried to give 3-bromo-2-(chloromethyl)-7-fluoro-4H-pyrido[l,2- a]pyrimidin-4-one as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.97 (I H, td), 8.19 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.86 - 7.94 (1 H, m), 4.81 (2 H, s); Mass Spectrum (ESI) m/e = 292.9 (M + 1).
(3-Bromo-7-fluoro-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)methyl acetate
Figure imgf000043_0002
A mixture of 3-bromo-2-(chloromethyl)-7-fluoro-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (10.60 g, 36.36 mmol), potassium acetate (4.283 g, 43.64 mmol), and DMF (138.5 mL) was stirred at 40 0C. After 3 h, the mixture was coned under reduced pressure. To the residue was added water (200 mL) and the resulting precipitate was collected by filtration, washed with water (300 mL), and dried to give (3- bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)methyl acetate as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (1 H, dd, J=4.9, 2.7 Hz), 8.18 (1 H, ddd, J=9.8, 7.1, 2.8 Hz), 7.86 (1 H, dd, J=9.7, 5.4 Hz), 5.21 (2 H, s), 2.16 (3 H, s); Mass Spectrum (ESI) m/e = 315.0 [M + 1 (79Br)] and 316.9 [M + 1 (81Br)]. 3-Bromo-7-fluoro-2-(hydroxymethyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000044_0001
A heterogeneous mixture of (3-bromo-7-fluoro-4-oxo-4H-pyrido[ 1 ,2-a]pyrimidin- 2-yl)methyl acetate (7.7539 g, 24.61 mmol), HCl (6.972 mL, 83.67 mmol), and 1,4-dioxane (70.31 mL) was heated under stirring at 70 0C. After 4 h, the mixture was cooled to rt and the mixture was coned under reduced pressure. The residue was diluted with water (100 mL) and treated with 28% ammonium hydroxide (10 mL) until pH neutral. The precipitate was filtered, washed with water (300 mL), and dried under high vacuum overnight to give 3-bromo-7-fluoro-2-(hydroxyl- methyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a brown solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.97 (1 H, ddd, J=4.9, 2.8, 0.7 Hz), 8.17 (1 H, ddd, J=9.8, 7.1, 2.8 Hz), 7.88 (1 H, ddd, J=9.8, 5.4, 0.7 Hz), 5.35 (1 H, br. s.), 4.60 (2 H, s); Mass Spectrum (ESI) m/e = 273.0 [M + 1 (79Br)] and 274.9 [M + 1 (81Br)].
3-Bromo-7-fluor o-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidine-2-carbaldehyde
Figure imgf000044_0002
A mixture of 3-bromo-7-fluoro-2-(hydroxymethyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (5.6359 g, 20.64 mmol) and manganese(IV) oxide (17.94 g, 206.4 mmol) in toluene (137.6 mL) was heated to reflux. After 3 h, the mixture was cooled to rt and filtered through a pad of Celite™. The pad was rinsed with DCM (1 L). The filtrate was coned under reduced pressure to give 3-bromo-7-fluoro-4-oxo-4H- pyrido[l,2-a]pyrimidine-2-carbaldehyde (2.4747 g, 44.24% yield) as a bright yellow solid: 1H NMR (500 MHz, DMSO-d6) δ ppm 10.13 (1 H, s), 9.03 - 9.06 (1 H, m), 8.24 (1 H, ddd, J=9.8, 7.1, 2.7 Hz), 8.00 - 8.05 (1 H, m); Mass Spectrum (ESI) m/e = 270.9 [M + 1 (79Br)] and 273.0 [M + 1 (81Br)]. 3-Bromo-7-fluoro-2-(l-hydroxyethyl)-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000045_0001
To a stirring suspension of 3-bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidine- 2-carbaldehyde (2.3555 g, 8.691 mmol) in THF (86.91 niL) was added methyl- magnesium bromide 3 M in Et2O (4.345 mL, 13.04 mmol) dropwise at 0 0C and the mixture was allowed to warm to 9 0C over 1.5 h. After 1.5 h, the reaction was quenched with satd aq NH4Cl (50 mL) and water (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with water (100 mL x 1), brine (100 mL x 1), dried over Na2SO4, filtered, and coned under reduced pressure to give a red syrup. The red syrup was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 100% gradient of EtOAc in hexane over 25 min and 100% isocratic of EtOAc in hexane for 4 min as eluent to give 3 -bromo-7-fluoro-2-(l -hydroxy ethyl)-4H-pyrido[ 1,2- a]pyrimidin-4-one as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.92 - 8.97 (1 H, m), 8.14 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.83 - 7.90 (1 H, m), 5.26 (1 H, d, J=6.7 Hz), 5.09 (1 H, qd, J=6.5, 6.3 Hz), 1.38 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 286.9 [M + 1 (79Br)] and 289.0 [M + 1 (81Br)]. 2-(l-(3-Bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- isoindoline-l,3-dione
Figure imgf000045_0002
A solution of 3 -bromo-7-fluoro-2-(l -hydroxy ethyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (1.238 g, 4.31 mmol), triphenylphosphine (1.357 g, 5.17 mmol), phthalimide (0.761 g, 5.17 mmol), and THF (28.7 mL) was stirred at rt for 5 min to dissolve all reactants. The mixture was then cooled to 0 0C and to the cooled homogenous mixture was added dropwise over 3 min diisopropyl azodicarboxylate (1.019 rnL, 5.17 mmol) at 0 0C. After stirring at 0 0C for 30 min, the cooling bath was removed and the mixture was stirred at rt. After 1.5 h, the mixture was partitioned between EtOAc (50 mL) and water (50 mL). The insoluble solid was filtered, washed with water (50 mL) and EtOAc (50 mL), and dried under vacuum to give the desired product 2-(l-(3-bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)isoindoline-l,3-dione as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.92 - 9.00 (1 H, m), 8.12 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.82 - 7.92 (4 H, m), 7.75 (1 H, dd, J=9.8, 5.5 Hz), 5.56 - 5.66 (1 H, m), 1.85 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 416.0 [M + 1 (79Br)] and 418.0 [M + 1 (81Br)]. To the filtrate was added brine (50 mL) and the organic layer was separated, dried over Na2SO4 filtered and coned under reduced pressure to give the crude material as a yellow solid. The yellow solid was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 25 min and 50% isocratic of EtOAc for 10 min as eluent to give the desired product 2-(l -(3 -bromo-7-fluoro-4-oxo-4H-pyrido [1, 2-a]pyrimidin-2 -yl)- ethyl)isoindoline-l,3-dione as a light yellow solid: 1H NMR (400 MHz, DMSO- d6) δ ppm 8.95 (1 H, td, J=3.1, 1.6 Hz), 8.12 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.83 - 7.93 (4 H, m), 7.71 - 7.79 (1 H, m), 5.57 - 5.65 (1 H, m), 1.85 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 416.0 [M + 1 (79Br)] and 418.0 [M + 1 (81Br)]. 2-(l-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)isoindoline-l,3-dione
Figure imgf000046_0001
A solution of 2-(l -(3 -bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)isoindoline-l,3-dione (0.587 g, 1.410 mmol), 2-(tributylstannyl)pyridine (0.761 mL, 2.115 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.163 g, 0.141 mmol) in 1,4-dioxane (11.75 mL) was stirred at 110 0C. After 22 h, the mixture was cooled to rt and coned under reduced pressure to give a black liquid. The black liquid was purified by column chromatography on a 40 g Redi-Sep™ column using 0 to 100% gradient of EtOAc in hexane over 14 min and then 100% isocratic of EtOAc for 14 min as eluent to give 2-(l-(6-fluoro-3-(pyridin-2-yl)- quinoxalin-2-yl)ethyl)isoindoline-l,3-dione as a yellow solid: Mass Spectrum (ESI) m/e = 415.1 (M + 1). 2-(l-Aminoethyl)-7-fluoro-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000047_0001
To a suspension of 2-(l-(7-fluoro-4-oxo-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)isoindoline-l,3-dione (0.584 g, 1.409 mmol) in EtOH (28.2 mL) was added hydrazine, monohydrate (0.684 mL, 14.09 mmol), and the mixture was stirred under reflux. After 1 h, the mixture was cooled to rt and the precipitate was filtered and washed with EtOAc (50 mL x 2). The filtrate was coned under reduced pressure and then it was redissolved in EtOAc (50 mL) and water (50 mL). The aq layer was extracted with EtOAc (50 mL x 1). The combined organic layers were treated with 3N aq HCl (50 mL). The separated aq layer was washed with DCM (50 mL x 2) to remove organic impurities, basified to ~ pH 13 with ION NaOH (80 mL), and extracted with EtOAc (100 mL x 3). The combined organic layers were dried over MgSOφ filtered, and coned under reduced pressure to give 2-(l-aminoethyl)-7-fluoro-3-(pyridin-2-yl)-4H-pyrido[l,2-a]- pyrimidin-4-one as a yellow syrupy solid: H NMR (400 MHz, DMSO-dβ) δ ppm 8.93 - 8.98 (1 H, m), 8.65 - 8.71 (1 H, m), 8.13 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.81 - 7.93 (2 H, m), 7.59 (1 H, dt, J=7.9, 1.1 Hz), 7.39 (1 H, dd, J=6.3, 4.7 Hz), 3.85 (1 H, q, J=6.7 Hz), 1.23 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 285.1 (M + 1). 7-Fluoro-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido- [l,2-a]pyrimidin-4-one and 7-fluoro-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-3- (2-pyridinyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000048_0001
Figure imgf000048_0002
A mixture of 6-chloropurine (0.063 g, 0.408 mmol), 2-(l-aminoethyl)-7-fluoro-3- (pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-4-one (0.116 g, 0.408 mmol), and DIEA (0.213 mL, 1.223 mmol) in butan-1-ol (4.08 mL) was stirred at 110 0C. After 17 h, the mixture was removed from the heat and coned under reduced pressure to give a brown syrup. The brown syrup was purified by column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min, then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min, then 50 to 100% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min, and then 100% isocratic of DCM:MeOH:NH4OH (89:9:1) for 14 min as eluent to give a yellow solid. The yellow solid was suspended in EtOAc-hexane (1 :1) and filtered to give 7-fluoro- 2-(l-(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4- one as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.86 (1 H, br. s.), 8.97 (1 H, td, J=3.1, 1.6 Hz), 8.71 (1 H, dd, J=2.7, 1.6 Hz), 8.07 - 8.23 (2 H, m), 8.05 (1 H, s), 7.91 (2 H, td, J=7.7, 1.4 Hz), 7.64 (1 H, d, J=7.8 Hz), 7.19 - 7.49 (2 H, m), 5.45 (1 H, br. s.), 1.50 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e =
403.1 (M + 1). The racemic mixture was separated by chiral separation using
SFC to give two fractions:
First-eluting enantiomer on AD-H column: 7-fluoro-2-(( IS)-I -(9H-purin-6- ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.90 - 9.03 (1 H, m), 8.71 (1 H, br. s.), 7.98 - 8.24 (3 H, m), 7.84 - 7.98 (2 H, m), 7.64 (1 H, d, J=8.2 Hz), 7.37 - 7.49
(1 H, m), 7.30 (1 H, br. s.), 5.44 (1 H, br. s.), 1.49 (3 H, d, J=6.7 Hz); Mass
Spectrum (ESI) m/e = 403.1 (M + 1).
Second-eluting enantiomer on AD-H column: 7-fluoro-2-(( IR)-I -(9H-purin-6- ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.98 (1 H, dd, J=4.5, 2.9 Hz), 8.66 -
8.76 (1 H, m), 8.00 - 8.22 (3 H, m), 7.84 - 7.97 (2 H, m), 7.64 (1 H, d, J=8.2 Hz),
7.37 - 7.46 (1 H, m), 7.30 (1 H, br. s.), 5.43 (1 H, br. s.), 1.49 (3 H, d, J=6.7 Hz);
Mass Spectrum (ESI) m/e = 403.1 (M + 1).
Example 5: Preparation of 7-fluoro-3-(3-fluorophenyl)-2-((lS)-l-(9H-purin-
6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one and 7-fluoro-3-(3- fluorophenyl)-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2- a] pyrimidin-4-one
2-(l-(7-Fluoro-3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethylcarbamoyl)benzoic acid
Figure imgf000049_0001
A mixture of 2-(l-(3-bromo-7-fluoro-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)isoindoline-l,3-dione (0.4061 g, 0.976 mmol, Prepared in Example 4), 3- fluorophenyl boronic acid (0.205 g, 1.464 mmol), tetrakis(triphenylphosphine)- palladium(O) (0.056 g, 0.049 mmol), and sodium carbonate (0.517 g, 4.88 mmol) in a mixture of acetonitrile (6.10 mL) and water (2.033 mL) was stirred at 85 0C. After 20 h, the mixture was cooled to rt. The mixture was coned under reduced pressure to remove acetonitrile. The mixture was partitioned between DCM (50 mL) and water (50 mL). The water layer (pH 10-11) was washed with DCM (50 mL x 2) to remove byproducts. The aq layer was treated with 2 N HCl (50 mL) and extracted with DCM (50 mL x 2). The combined organic layers were washed with water (50 mL x 2), brine (50 mL x 1), dried over Na2SO4, filtered, and coned under reduced pressure to give 2-(l-(7-fluoro-3-(3-fluorophenyl)-4-oxo-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethylcarbamoyl)benzoic acid as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.74 (1 H, br. s.), 8.93 (1 H, dd, J=4.3, 2.7 Hz), 8.57 (1 H, d, J=7.0 Hz), 8.10 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.80 (1 H, dd, J=9.8, 5.5 Hz), 7.72 (1 H, dd, J=7.6, 1.0 Hz), 7.41 - 7.60 (4 H, m), 7.22 - 7.35 (3 H, m), 4.96 (1 H, quin, J=6.9 Hz), 1.31 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 450.1 (M + 1).
2-(l-Aminoethyl)-7-fluoro-3-(3-fluorophenyl)-4H-pyrido[l,2-a]pyrimidin-4- one
Figure imgf000050_0001
To a suspension of 2-(l-(7-fluoro-3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethylcarbamoyl)benzoic acid (0.2462 g, 0.548 mmol) in EtOH (5.48 mL, 0.548 mmol) was added coned. HCl (0.457 mL, 5.48 mmol), and the mixture was stirred under reflux. After 25 h, the mixture was cooled to rt. To the mixture was added ice water (50 mL). The aq acidic mixture (pH~1.5) was washed with DCM (50 mL x 2) to remove organic impurities. The aq mixture was then treated with satd aq NaHCO3 solution (50 mL) and extracted with DCM (50 mL x 3). The combined organic layers were washed with water (50 mL x 1) and brine (50 mL x 1), dried over Na2SO4, filtered, and coned under reduced pressure to give 2-(l- aminoethyl)-7-fluoro-3-(3-fluorophenyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4-one as a yellow foamy solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.88 - 8.93 (1 H, m), 8.10 (1 H, ddd, J=9.9, 7.1, 2.9 Hz), 7.83 (1 H, dd, J=9.6, 5.7 Hz), 7.47 - 7.55 (1 H, m), 7.17 - 7.28 (3 H, m), 3.77 (1 H, q, J=6.7 Hz), 1.93 (2 H, br. s.), 1.18 (3 H, d, J=6.3 Hz); Mass Spectrum (ESI) m/e = 302.0 (M + 1). 7-Fluoro-3-(3-fluorophenyl)-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-4H- pyrido[l,2-a]pyrimidin-4-one and 7-fluoro-3-(3-fluorophenyl)-2-((lR)-l-(9H- purin-6-ylamino)ethyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000051_0001
Figure imgf000051_0002
A mixture of 6-chloropurine (0.071 g, 0.459 mmol), 2-(l-aminoethyl)-7-fluoro-3- (3-fluorophenyl)-4H-pyrido[l,2-a]pyrimidin-4-one (0.1383 g, 0.459 mmol), and DIEA (0.240 mL, 1.377 mmol) in butan-1-ol (4.59 mL) was stirred at 110 0C.
After 20 h, the mixture was removed from the heat and left at rt. The mixture was coned under reduced pressure to give a brown syrup. The residue was dissolved in DCM (50 mL). The solution was washed with water (30 mL x 2). The organic layer was dried over Na2SOφ filtered, and coned under reduced pressure to give a brown syrup. The brown syrup was purified by column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min as eluent to give a light yellow solid. The light yellow solid was co-evaporated with EtOAc-Hexane (1 :4), then suspended in EtOAc-Hexane (1 :4), and filtered to give 7-fluoro-3-(3-fluorophenyl)-2-(l-(9H-purin-6-ylamino)- ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one as an off-white solid: 1H NMR] (400 MHz, DMSO-Ct6) δ ppm 12.88 (1 H, br. s.), 8.93 (1 H, dd, J=A.1, 2.7 Hz), 8.05 - 8.19 (3 H, m), 7.85 (1 H, dd, J=9.6, 5.3 Hz), 7.54 (1 H, q, J=7.4 Hz), 7.22 - 7.45 (4 H, m), 5.31 (1 H, br. s.), 1.41 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 420.1 (M + 1). The racemic mixture was separated by chiral separation using SFC to give two fractions: First-eluting enantiomer on AD-H column: 7- fluoro-3-(3-fluorophenyl)-2-((l S)- 1 -(9H-purin-6-ylamino)ethyl)-4H-pyrido[ 1 ,2- a]pyrimidin-4-one as a tan solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 12.78 (1 H, br. s.), 8.93 (1 H, dd, J=A.1, 2.7 Hz), 8.06 - 8.18 (3 H, m), 7.85 (1 H, dd, J=10.0, 5.3 Hz), 7.49 - 7.59 (1 H, m), 7.20 - 7.46 (4 H, m), 5.31 (1 H, br. s.), 1.41 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 420.1 (M + 1). Second-eluting enantiomer on AD-H column: 7-fluoro-3-(3-fluorophenyl)-2-((lR)-l-(9H-purin-
6-ylamino)ethyl)-4H-pyrido[l,2-a]pyrimidin-4-one as an off-white solid: ^H
NMR (400 MHz, DMSO-d6) δ ppm 12.74 (1 H, br. s.), 8.89 - 8.96 (1 H, m), 8.05 - 8.19 (3 H, m), 7.85 (1 H, dd, J=10.0, 5.3 Hz), 7.48 - 7.58 (1 H, m), 7.21 - 7.45 (4
H, m), 5.30 (1 H, br. s.), 1.41 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e =
420.1 (M + 1).
Example 6: Preparation of 6-methyl-2-(l-(9H-purin-6-ylamino)ethyl)-3-(2- pyridinyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one 3-Bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidine-2-carbaldehyde
Figure imgf000052_0001
A mixture of 3-bromo-2-(hydroxymethyl)-6-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4- one (2.7421 g, 10.19 mmol, Prepared in Example 1) and manganese(IV) oxide (8.86 g, 102 mmol) in toluene (67.9 mL) was heated to reflux. After 3 h, the mixture was cooled to rt and filtered through a pad of Celite™, The filtrate was coned under reduced pressure to give 3-bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]- pyrimidine-2-carbaldehyde as a bright yellow solid: 1H NMR (400 MHz, DMSO- d6) δ ppm 10.07 (1 H, s), 7.81 (1 H, dd, J=9.0, 7.0 Hz), 7.60 (1 H, dd, J=9.0, 0.8 Hz), 7.10 - 7.15 (1 H, m), 2.96 (3 H, s); Mass Spectrum (ESI) m/e = 267.0 [M + 1 (79Br)] and 268.9 [M + 1 (81Br)]. 3-Bromo-2-(l-hydroxyethyl)-6-methyl-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000053_0001
To a stirred suspension of 3-bromo-6-methyl-4-oxo-4H-pyrido[ 1 ,2-a]pyrimidine- 2-carbaldehyde (1.4162 g, 5.30 mmol) in THF (53.0 rnL) was added methyl- magnesium bromide 3 M in Et2O (2.65 mL, 7.95 mmol) dropwise at 0 0C and the mixture was allowed to warm to 9 0C over 2 h. After 2 h, the reaction was quenched with satd aq NH4Cl (50 mL) and water (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with water (50 mL x 1), brine (50 mL x 1), dried over Na2SO4, filtered, and coned under reduced pressure to give an orange solid. The orange solid was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 100% gradient of EtOAc in hexane over 25 min and 100% isocratic of EtOAc in hexane for 4 min as eluent to give 3 -bromo-2-( 1 -hydroxy ethyl)-6-methyl-4H-pyrido [1,2- a]pyrimidin-4-one as a bright yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.72 (1 H, dd, J=8.6, 7.0 Hz), 7.48 (1 H, d, J=9.4 Hz), 7.01 (1 H, d, J=7.0 Hz), 5.15 (1 H, d, J=6.7 Hz), 4.99 (1 H, qd, J=6.5, 6.3 Hz), 2.94 (3 H, s), 1.35 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 283.0 [M + 1 (79Br)] and 285.0 [M + 1 (81Br)].
2-(l-(3-Bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- isoindoline-l,3-dione
Figure imgf000053_0002
A solution of 3 -bromo-2-( 1 -hydroxy ethyl)-6-methyl-4H-pyrido [ 1 ,2-a]pyrimidin- 4-one (0.909 g, 3.21 mmol), triphenylphosphine (1.010 g, 3.85 mmol), phthal- imide (0.567 g, 3.85 mmol), and THF (21.39 mL) was stirred at rt for 5 min to dissolve all reactants. The mixture was then cooled to 0 0C and to the cooled homogenous mixture was added dropwise over 3 min to diisopropyl azodi- carboxylate (0.758 mL, 3.85 mmol) at 0 0C. After stirring at 0 0C for 30 min, the cooling bath was removed and the mixture was stirred at rt. After 1.5 h, the mixture was partitioned between EtOAc (50 mL) and water (50 mL) and the organic layer was separated, dried over Na2SC>4 filtered and coned under reduced pressure to give the crude material as a yellow solid. The yellow solid was purified by silica gel column chromatography on a 80 g of Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 25 min and 50% isocratic of EtOAc in hexane for 25 min to give the desired product 2-(l-(3-bromo-6-methyl- 4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)isoindoline-l,3-dione as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 7.82 - 7.92 (4 H, m), 7.70 (1 H, dd, J=9.0, 7.0 Hz), 7.32 (1 H, dd, J=S.6, 0.8 Hz), 7.04 (1 H, ddd, J=6.8, 1.4, 1.2 Hz), 5.47 - 5.57 (1 H, m), 2.94 (3 H, s), 1.82 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) m/e = 412.0 [M + 1 (79Br)] and 414.0 [M + 1 (81Br)]. 2-(l-(6-Methyl-4-oxo-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- isoindoline-l,3-dione
Figure imgf000054_0001
A solution of 2-(l -(3 -bromo-6-methyl-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)isoindoline-l,3-dione (0.9386 g, 2.277 mmol), 2-(tributylstannyl)pyridine (1.098 mL, 3.05 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.263 g, 0.228 mmol) in 1,4-dioxane (18.97 mL) was stirred using an overhead stirrer at 110 0C. After 20 h, the mixture was cooled to rt and coned under reduced pressure to give a black liquid. The black liquid was purified by column chromatography on a 80 g of Redi-Sep™ column using 0 to 100% gradient of EtOAc in hexane over 25 min and then 100% isocratic of EtOAc for 25 min to give 2-(l-(6- methyl-4-oxo-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)isoindoline- 1,3-dione as a yellow solid: Mass Spectrum (ESI) m/e = 411.1 (M + 1).
2-(l-Aminoethyl)-6-methyl-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000055_0001
To a suspension of 2-(l-(6-methyl-4-oxo-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)isoindoline- 1,3-dione (0.934 g, 2.276 mmol) in EtOH (45.5 mL) was added hydrazine, monohydrate (1.104 mL, 22.76 mmol), and the mixture was stirred under reflux. After 1 h, the mixture was cooled to rt and the precipitate was filtered and washed with EtOAc (50 mL x 2). The filtrate was coned under reduced pressure, redissolved in EtOAc (50 mL) and water (50 mL). The aq layer was extracted with EtOAc (50 mL x 1). The combined organic layers were treated with 2M aq HCl (50 mL). The separated aq layer was washed with EtOAc (50 mL x 2) to remove organic impurities and then basified to ~ pH 13 with IO N NaOH (20 mL), and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with water (100 mL x 1), brine (100 mL x 1), and dried over MgSOφ filtered, and coned under reduced pressure to give the desired product as a yellow syrupy solid. The combined aq layers still contained the desired product. The combined aq layers were satd with NaCl and extracted with DCM (100 mL x 2). The organic layers were combined with the above yellow syrupy solid, dried over MgSOφ filtered, and coned under reduced pressure to give 2-(l-amino- ethyl)-6-methyl-3-(pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-4-one as a yellow foamy solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.64 (1 H, ddd, J=4.9, 2.0,
1.0 Hz), 7.85 (1 H, td, J=7.7, 1.9 Hz), 7.67 (1 H, dd, J=9.0, 6.8 Hz), 7.54 (1 H, dt, J=7.8, 1.1 Hz), 7.44 (I H, ddd, J=8.9, 1.4, 0.7 Hz), 7.35 (1 H, ddd, J=7.6, 4.9, 1.2 Hz), 6.89 - 6.95 (1 H, m), 3.70 (1 H, q, J=6.7 Hz), 2.91 (3 H, s), 1.88 (2 H, br. s.), 1.19 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 281.0 (M + 1). 6-Methyl-2-(l-(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2- a]pyrimidin-4-one
Figure imgf000056_0001
A mixture of 6-chloropurine (0.046 g, 0.300 mmol), 2-(l-aminoethyl)-6-methyl-3- (pyridin-2-yl)-4H-pyrido[l,2-a]pyrimidin-4-one (0.084 g, 0.300 mmol), and DIEA (0.157 mL, 0.901 mmol) in butan-1-ol (3.00 mL) was stirred at 110 0C. After 21 h, the mixture was removed from the heat and coned under reduced pressure to give a syrup. The syrup was purified by column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min, then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min, then 50 to 100% gradient of DCM:MeOH:NH4OH (89:9: 1) in DCM over 14 min, and then 100% isocratic of DCM:MeOH:NH4OH (89:9:1) for 14 min as eluent to give 6-methyl-2-(l-(9H-purin-6-ylamino)ethyl)-3-
(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-4-one as an orange solid: H NMR (400 MHz, DMSO-dβ) δ ppm 12.90 (1 H, s), 8.68 (1 H, br. s.), 8.07 (2 H, s), 7.80 - 7.93 (1 H, m), 7.73 (1 H, dd, J=8.6, 7.2 Hz), 7.49 - 7.64 (2 H, m), 7.32 - 7.44 (1 H, m), 7.23 (1 H, br. s.), 6.98 (1 H, d, J=6.7 Hz), 5.35 (1 H, br. s.), 2.92 (3 H, s), 1.44 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e = 399.1 (M + 1). Example 7: Preparation of 4-amino-6-(((lS)-l-(6-methyl-4-oxo-3-(2-pyr- idinyl)-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbo- nitrile and 4-amino-6-(((lR)-l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido- [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile 4,6-Dichloropyrimidine-5-carbaldehyde
Figure imgf000057_0001
A mixture of DMF (64 niL) and POCl3 (200 mL) at 0 0C was stirred for 1 h, treated with 4,6-dihydroxypyrimidine (50.0 g, 446 mmol), and stirred for 0.5 h at rt, and then the heterogeneous mixture was refluxed for 3 h. The volatiles were removed under reduced pressure, and the residue was poured into ice water and extracted six times with Et2O. The organic phase was washed with aq NaHCO3 and water, dried over Na2SO4, coned, and crystallized (EtO Ac-petroleum ether) to give 4,6-dichloropyrimidine-5-carbaldehyde. Mass Spectrum (ESI) m/e = 177 (M+l).
4,6-Dichloropyrimidine-5-carbaldehyde oxime
Figure imgf000057_0002
A mixture of 4,6-dichloropyrimidine-5-carbaldehyde (8.00 g, 44.8 mmol), NaOAc (3.7 g, 1.0 eq) and NH2OKHCl (3.1 g, 1.0 eq) in EtOH (320 mL) was stirred at rt for 2 h. The reaction mixture was filtered, coned and purified by column chromatography on silica gel (dry loading, first DCM then DCM/EtOAc, 1/9) to give 4,6-dichloropyrimidine-5-carbaldehyde oxime as a white solid. 4,6-Dichloropyrimidine-5-carbonitrile
Figure imgf000057_0003
4,6-Dichloropyrimidine-5-carbaldehyde oxime (8g) was dissolved in CHCl3 (40 mL) and treated with SOCl2 (6 mL) for 2 h at rt. The solvent was removed and redissolved in DCM (5 niL). The solid was filtered and washed with DCM (5 rnL). The filtrate was coned and purified by column chromatography on silica gel (dry loading, DCM/hexane, 3/1) to give 4,6-dichloropyrimidine-5-carbonitrile as a white solid. 4-Amino-6-chloropyrimidine-5-carbonitrile
Figure imgf000058_0001
The white solid, 4,6-dichloropyrimidine-5-carbonitrile (5.82 g, 33.5 mmol) was dissolved in THF (66.9 mL) in a 500 mL round-bottom flask and to the mixture was bubbled through ammonia gas (0.570 g, 33.5 mmol) for 3 min in 10 min intervals with stirring. After 50 min, a white precipitate (ammmonium chloride) was filtered and the solid was washed with THF (100 mL). To the filtrate was added silica gel and coned under reduced pressure. The mixture was purified by silica gel column chromatography on a 12O g of Redi-Sep™ column using 0 to 100% gradient of EtOAc in hexane over 27 min and then 100% isocratic of EtOAc in hexane for 20 min as eluent to give 4-amino-6-chloropyrimidine-5- carbonitrile as an off-white solid. The off-white solid was suspended in EtOAc- hexane (1 :1, 20 mL), filtered, washed with EtOAc-hexane (1 :1, 30 mL), and dried to give 4-amino-6-chloropyrimidine-5-carbonitrile as a white solid: ^H NMR (500 MHz, DMSO-d6) δ ppm 7.91 - 8.77 (3 H, m); Mass Spectrum (ESI) m/e = 154.9 (M + 1).
4- Amino-6-(((l S)- l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido [ 1 ,2-a] pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((lR)-l- (6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile
Figure imgf000059_0001
A mixture of 4-amino-6-chloropyrimidine-5-carbonitrile (0.149 g, 0.966 mmol), 2-(l -aminoethyl)-6-methyl-3-(pyridin-2-yl)-4H-pyrido[ 1 ,2-a]pyrimidin-4-one (0.2707 g, 0.966 mmol, Prepared in Example 6), and DIEA (0.505 mL, 2.90 mmol) in butan-1-ol (9.66 mL) was stirred at 120 0C. After 3.5 h, the mixture was cooled to rt and coned under reduced pressure to give a yellow solid. The yellow solid was purified by column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min as eluent to give 4-amino-6-((l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido- [l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.67 (1 H, ddd, J=4.9, 1.8, 0.9 Hz), 7.93 (1 H, s), 7.87 (1 H, td, J=7.7, 1.8 Hz), 7.76 (1 H, dd, J=8.9, 6.9 Hz), 7.59 (1 H, dt, J=7.9, 1.1 Hz), 7.45 (1 H, dd, J=8.7, 0.7 Hz), 7.38 (1 H, ddd, J=7.6, 4.9, 1.2 Hz), 7.30 (2 H, br. s.), 7.24 (1 H, d, J=7.6 Hz), 7.01 (1 H, dt, J=6.9, 1.1 Hz), 5.23 - 5.33 (1 H, m, J=7.0, 7.0, 6.8, 6.6 Hz), 2.93 (3 H, s), 1.31 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 399.1 (M + 1). The racemic mixture was separated by chiral separation using SFC to give two fractions:
First-eluting enantiomer on AD-H column: 4-amino-6-((( IS)-I -(6-methyl-4- oxo-3 -(2-pyridinyl)-4H-pyrido [ 1 ,2-a]pyrimidin-2-y l)ethyl)amino)-5 -pyrimidine- carbonitrile as a yellow solid: 1H NMR (500 MHz, DMSO-d6) δ ppm 8.67 (1 H, ddd, J=4.9, 1.7, 1.0 Hz), 7.93 (1 H, s), 7.87 (1 H, td, J=7.8, 1.8 Hz), 7.76 (1 H, dd, J=8.9, 7.0 Hz), 7.59 (1 H, dt, J=7.8, 1.0 Hz), 7.45 (1 H, d, J=9.0 Hz), 7.38 (1 H, ddd, J=7.6, 4.9, 1.2 Hz), 7.31 (2 H, br. s.), 7.24 (1 H, d, J=7.6 Hz), 6.99 - 7.04 (1 H, m), 5.23 - 5.32 (1 H, m, J=7.0, 7.0, 6.8, 6.6 Hz), 2.93 (3 H, s), 1.31 (3 H, d, J=6.6 Hz); Mass Spectrum (ESI) m/e = 399.1 (M + 1).
Second-eluting enantiomer on AD-H column: 4-amino-6-((( IR)-I -(6-methyl-4- oxo-3 -(2-pyridinyl)-4H-pyrido [ 1 ,2-a]pyrimidin-2-y l)ethyl)amino)-5 -pyrimidine- carbonitrile as a yellow solid: 1U NMR (500 MHz, DMSO-d6) δ ppm 8.67 (1 H, ddd, J=4.9, 1.7, 1.0 Hz), 7.93 (1 H, s), 7.87 (1 H, td, J=7.7, 1.7 Hz), 7.76 (1 H, dd, J=8.9, 7.0 Hz), 7.59 (1 H, dt, J=7.8, 1.0 Hz), 7.45 (1 H, dd, J=8.8, 0.7 Hz), 7.38 (1 H, ddd, J=7.5, 5.0, 1.2 Hz), 7.31 (2 H, br. s.), 7.24 (1 H, d, J=7.6 Hz), 7.01 (1 H, dt, J=6.9, 1.2 Hz), 5.28 (1 H, qd, J=7.0, 6.7 Hz), 2.93 (3 H, s), 1.31 (3 H, d, J=6.6 Hz); Mass Spectrum (ESI) m/e = 399.1 (M + 1). Example 8: Preparation of 6-methyl-3-(2-methylphenyl)-2-((9H-purin-6-yl- sulfanyl)methyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
(6-Methyl-4-oxo-3-o-tolyl-4H-pyrido[l,2-a]pyrimidin-2-yl)methyl methanesulfonate
Figure imgf000060_0001
To a solution of 2-(hydroxymethyl)-6-methyl-3-o-tolyl-4H-pyrido[l,2-a]pyr- imidin-4-one (280 mg, 1 mmol) (prepared from 3-bromo-2-(hydroxymethyl)-6- methyl-4H-pyrido[l,2-a]pyrimidin-4-one according to the procedure for the preparation of 3-(3-fluorophenyl)-2-(hydroxymethyl)-6-methyl-4H-pyrido[ 1 ,2-a]- pyrimidin-4-one) in 6ml DCM at 0 0C was added Et3N (0.30 mL, 2.2eq) followed by the addition of MsCl (239 mg, 2.1eq) and the resulting mixture was stirred at room temp for 1 h. Aqueous work up was done and crude (6-methyl-4-oxo-3-o- tolyl-4H-pyrido[l,2-a]pyrimidin-2-yl)methyl methanesulfonate was used for the next step.
2-((9H-Purin-6-yl)methyl)-6-methyl-3-o-tolyl-4H-pyrido [ 1 ,2-a] pyrimidin-4- one
Figure imgf000061_0001
A mixture of (6-methyl-4-oxo-3-o-tolyl-4H-pyrido[ 1 ,2-a]pyrimidin-2-yl)methyl methanesulfonate (100 mg, 0.28 mmol), 9H-purine-6-thiol (51 mg, 1.2 eq) and K2CO3 (46 mg, 1.2 eq) in 2 mL DMF was stirred at rt overnight. Water was added and the resulting solid was washed with water and dried in the open air. 6- Methyl-3 -(2-methylphenyl)-2-((9H-purin-6-ylsulfanyl)methyl)-4H-pyrido [ 1 ,2-a] - pyrimidin-4-one was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.49 (1 H, s), 8.47 (1 H, s), 8.40 (1 H, s), 7.68 (1 H, t, J=8.0 Hz), 7.43 (1 H, d, J=8.0 Hz), 7.23 - 7.19 (4 H, m), 6.95 (1 H, d, J=8.0 Hz), 4.45 (1 H, d, ./=12.0 Hz), 4.29 (1 H, d, J=12.0 Hz), 2.90 (3 H, s), 2.10 (3 H, s); Mass Spectrum (ESI) m/e = 415 (M + 1).
Example 9: Preparation of 2-((6-amino-9H-purin-9-yl)methyl)-6-methyl-3-(2- methylphenyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000061_0002
2-((6-Amino-9H-purin-9-yl)methyl)-6-methyl-3-(2-methylphenyl)-4H-pyrido[l,2- a]pyrimidin-4-one was prepared according to the above procedure. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.36 (1 H, s), 8.33 (1 H, s), 7.65 (1 H, dd, J=8.0, 4.0 Hz), 7.32 - 7.26 (4 H, m), 7.21 (1 H, d, J=8.0 Hz), 6.97 (1 H, d, J=8.0 Hz), 5.22 (1 H, d, J=16.0 Hz), 5.08 (1 H, d, J=16.0 Hz), 2.90 (3 H, s), 2.16 (3 H, s); Mass Spectrum (ESI) m/e = 398 (M + 1). General Procedures
General Procedure A for Suzuki Coupling
A mixture of 2-(l-(3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)iso- indoline-l,3-dione (Prepared in Example 10, 1.0 equiv.), corresponding boronic acids (1.2 equiv.), PdCl2(PPh3) (0.1 equiv.) and K2CO3 (2.0 equiv.) in 1,4- dioxane-water (4:1) was stirred at 110 0C overnight. The reaction was monitored by TLC, after completion of reaction, the reaction mass was cooled to rt. The insoluble solid was filtered off and washed with EtOAc. The filtrate was cond under reduced pressure, redissolved in EtOAc, washed with brine twice, dried over Na2SO4, filtered and cond under reduced pressure. The residue was purified by silica gel column chromatography using 0-60% EtOAc in hexane as eluent to give the corresponding product. General Procedure B for Stille Coupling
A mixture of the 2-(l-(3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- isoindoline-l,3-dione (1 equiv.), 2-(tributylstannyl) pyridine (1.2 equiv.), Pd (PPh3)4 (0.1 equiv.) in 1 ,4-dioxane was stirred at 110 0C overnight. The reaction was monitored by TLC, when complete the reaction, reaction mass was cooled to rt and cond under reduced pressure to give black oil. The black oil was purified by silica gel column chromatography using 0-40 % EtOAc in hexane as eluent to give the corresponding product. General Procedure C for Hydrazinolysis
To a suspension of the corresponding phthalimide protected reactant (1 equiv.) in EtOH was added hydrazine monohydrate (5.0 equiv.). The mixture was stirred at reflux for 3 h at which point TLC showed the reaction was complete. After concentrating under reduced pressure, the residue was redissolved in DCM-Et2O (3:7). The insoluble solid was filtered off and the filtrate was cond under reduced pressure to give the corresponding product. General Procedure D for Final coupling step with 4-amino-6-chloropyrimid- ine-5-carbonitrile
A mixture of 4-amino-6-chloropyrimidine-5-carbonitrile (1 equiv.), corresponding 2-(l-aminoethyl)-4H-pyrido[l,2-a]pyrimidin-4-one (1 equiv.) and DIEA (3.0 equiv.) in 1-butanol was stirred at 110 0C overnight. After completion of the reaction, it was cooled to rt and diluted with hexane and stirred. The precipitated solid was filtered off and washed with a mixture of DCM-ether (0.1 : 1 ) to give a corresponding product. Example 10: Preparation of 4-amino-6-((l-(3-(2-(methylsulfonyl)phenyl)-4- oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6-(((lR)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2- a] pyrimidin-2-yl)ethyl)amino)-5-py rimidinecarbonitrile and 4-amino-6- (((lS)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile 2-(Chloromethyl)-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000063_0001
To a mixture of 2-aminopyridine (10.0 g, 1.0 equiv.) and polyphosphoric acid (50 g) was added slowly ethyl-4-chloroacetate (1.0 equiv.) at rt while stirring, after completion of addition, the reaction mixture was stirred at 125 0C for 5 h. TLC showed mostly product. The mixture was cooled to rt and 200 mL of ice water added to it. The mixture was neutralized with 2 N NaOH (400 mL) to pH 6-7, then the resulting precipitate was collected by filtration, washed with water (200 mL) and dried to give a brown solid, the solid was dissolved in DCM (500 mL), dried over Na2SO4, filtered and cond under reduced pressure to give the desired product 2-(chloromethyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4-one as a brown solid:
1HNMR (400 MHz, DMSO-d6): δ 8.95 (d,lH), 7.9-8.0 (m,lH), 7.72 (d,lH), 7.2- 7.3 (m,lH), 6.5 (s,lH), 4.6 (s,2H). 3-Bromo-2-(chloromethyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000064_0001
A mixture of 2-(chloromethyl)-4H-pyrido[l,2-a]pyrimidin-4-one (3.564 g, 1.0 equiv.), JV-bromosuccinamide (1.0 equiv.) and acetic acid (48.2 mL) was stirred at rt for 4.5 h, at this point LCMS showed reaction was complete. The mixture was poured into water (200 mL) and the resulting precipitate was collected by filtration, washed with water (200 mL) and dried to give an orange solid. The solid was dissolved in DCM (100 mL), dried over Na2SO4, filtered and cond under reduced pressure to give desired 3-bromo-2-(chloromethyl)-4Η-pyrido[l,2- a]pyrimidin-4-one as an orange solid which was carried forward without any further purification: 1HNMR (400MHz, DMSO-d6): δ 8.94-8.97 (dd,lH), 8.03- 8.08 (m,lH), 7.76-7.80 (dd, IH), 7.43-7.48 (m,lH), 4.7 (s,2H). 3-Bromo-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)methyl acetate
Figure imgf000064_0002
A mixture of 3-bromo-2-(chloromethyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4-one (4.53 g, 15.76 mmol), potassium acetate (1.5 equiv.) and DMF (60 mL) was stirred at 40 0C for 3.5 h, at this point the reaction was completed by LCMS. The mixture was cond under reduced pressure. To the residue was added water (100 mL) and the resulting precipitate was collected by filtration, washed with water (100 mL) and dried to give the (3-bromo-4-oxo-4H-pyrido[l ,2-a]pyrimidin-2-yl)methyl acetate as a brown solid, which was carried forward without further purification: 1HNMR (400 MHz, DMSO-d6): δ 8.96-8.98 (dd,lH), 8.03-8.08 (m,lH), 7.74- 7.76 (dd,lH), 7.43-7.48 (m,lH), 5.2 (s,2H), 2.1 (s,3H). 3-Bromo-2-(hydr oxymethyl)-4H-pyrido [ 1 ,2-a] pyrimidin-4-one
Figure imgf000065_0001
A heterogeneous mixture of the (3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)methyl acetate (4.24 g, 1.0 equiv.), cone. HCl (8.0 equiv.) and 1, 4-dioxane (39 rnL) was heated with stirring at 70 0C for 3 h. LCMS showed the completion of reaction. The residue was diluted with water (100 mL) and treated with 28% ammonium hydroxide (10 mL) to pH 10. The precipitate was filtered, washed with water (200 mL) and dried under high vacuum to give 3-bromo-2-(hydroxyl- methyl)-4H-pyrido[l,2-a]pyrimidin-4-one as tan solid. Used without further purification: 1HNMR (400MHz, DMSO-d6): δ 8.96-8.98 (dd, IH), 8.03-8.08 (m,lH), 7.74-7.76 (dd, IH), 7.43-7.48 (m,lH), 5.3 (t,lH), 4.6 (d,2H). 3-Bromo-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidine-2-carbaldehyde
Figure imgf000065_0002
To a suspension of 3-bromo-2-(hydroxymethyl)-4H-pyrido[l,2-a]pyrimidin-4-one (1 equiv) and NaHCO3 (6 equiv) in DCM was added Dess-Martin periodinane (1.5 equiv) at rt with stirring. The reaction mixture was stirred at rt for 3 h, monitered by MS and TLC. The mixture was diluted with DCM and filtered off through Celite™, and the filtrate was cond under vacuum. The crude was purified by silica gel column chromatography using 80% EtOAc in hexane as eluent to give the 3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidine-2-carbaldehyde as a yellowish solid: 1HNMR (400MHz, DMSO-d6): £10.12 (s,lH), 9.00-9.01 (dd,lH), 8.03-8.12 (m,lH), 7.89-7.91 (dd,lH), 7.50-7.54 (m,lH). 3-Bromo-2-(l-hydroxyethyl)-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000065_0003
To a stirred solution of 3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidine-2-carbalde- hyde (2.14 g, 8.46 mmol) in THF (85 mL) was added methylmagnesium bromide (3M in Et2O, 5.64 mL, 16.92 mmol) drop wise at 0 0C. The mixture was allowed to warm to 9 0C over 4.5 h, when the reaction was quenched with sat. aq NH4Cl (50 mL), water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extract were washed with water(50 mL), brine(50 mL), dried over Na2Sθ4,fϊltered and cond under reduced pressure to give a dark brown solid, which was purified by silica gel column chromatography using 0-60 % EtOAc in Hexane to give the desired product 3 -bromo-2-(l -hydroxy ethyl)-4H-pyrido[ 1,2- a]pyrimidin-4-one as a yellow solid: 1HNMR (400MHz, DMSO-d6): δ 8.94-8.99 (dd,lH), 8.00-8.06 (m,lH), 7.76-7.78 (dd,lH), 7.40-7.47 (m,lH), 5.3(s,lH), 5.0 (m,lH).
2-(l-(3-Bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)isoindoline-l,3- dione
Figure imgf000066_0001
A 500 mL of round-bottom flask was charged with 3 -bromo-2-(l -hydroxy ethyl)- 4H-pyrido[l,2-a]pyrimidin-4-one (0.76 g, 2.86 mmol), phthalimide (0.504 g, 3.43 mmol) and PPh3 (0.899 g, 3.43 mmol) in THF (20 mL) followed by the dropwise addition of diisopropylazodicarboxylate (0.675 g, 3.43 mmol) in THF (3 mL). The reaction mixture was stirred overnight at rt. After the completion of the reaction, monitored by TLC, the reaction mass was cond under vacuum and purified by silica gel column chromatography using 5 to 35 % EtOAc in hexane to provide 2-(l-(3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)isoindoline- 1,3-dione as light yellow solid: 1HNMR (400MHz, DMSO-d6): £8.95-8.96 (dd,lH), 8.00-8.06 (m,lH), 7.8 (s,4H), 7.5-7.6 (m,2H), 7.4 (d,lH), 5.6 (q,lH), 1.84 (d,3H). 2-(l-(3-(2-(Methylthio)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)isoindoline- 1 ,3-dione
Figure imgf000067_0001
A mixture of 2-(l-(3-bromo-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)iso- indoline-l,3-dione (0.9836 g, 2.470 mmol), 2-(methylthio)phenylboronic acid (0.623 g, 3.71 mmol), tetrakis(triphenylphosphine)palladium(0) (0.143 g, 0.124 mmol), and potassium carbonate (1.024 g, 7.41 mmol) in DME (12.35 mL) was stirred at 85 0C. After 25.5 h, to the mixture were added 2-(methylthio)phenyl- boronic acid (0.623 g, 3.71 mmol), tetrakis(triphenylphosphine)palladium(0) (0.143 g, 0.124 mmol), and potassium carbonate (1.024 g, 7.41 mmol) and the mixture was stirred at 85 0C. After 4 days 21 h, the mixture was cooled to rt. The insoluble solid was filtered off and the solid was washed with DCM (50 mL). The filtrate was cond under reduced pressure. The residue was dissolved in DCM (50 mL), washed with brine (50 mL x 2), dried over Na2SO4, filtered, and cond under reduced pressure. The residue was purified by silica gel column chromatography on a 40 g Redi-Sep™ column using 0 to 50% gradient of EtOAc in hexane over 14 min and then 50% isocratic of EtOAc for 20 min as eluent to give 2-(l-(3-(2- (methylthio)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)isoindoline-l,3- dione as a yellow syrup: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.96 (1 H, ddd, J=7.2, 1.6, 0.8 Hz), 7.98 - 8.05 (1 H, m), 7.77 - 7.84 (2 H, m), 7.68 - 7.75 (3 H, m), 7.43 (1 H, td, J=6.9, 1.5 Hz), 7.31 (1 H, dd, J=8.1, 0.9 Hz), 7.18 - 7.24 (1 H, m), 6.79 - 6.85 (1 H, m), 6.70 - 6.76 (1 H, m), 5.36 - 5.44 (1 H, m), 2.41 (3 H, s), 1.64 (3 H, d, J=7.2 Hz); Mass Spectrum (ESI) m/e = 442.1 (M+l). 2-(l-(3-(2-(Methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)isoindoline- 1 ,3-dione
Figure imgf000068_0001
To a mixture of 2-(l-(3-(2-(methylthio)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin- 2-yl)ethyl)isoindoline-l,3-dione (0.4278 g, 0.969 mmol) in THF (7.27 niL) and water (2.422 rnL) was added oxone (1.489 g, 2.422 mmol) and the mixture was stirred at rt. After 24 h, LC-MS (ESI) and HPLC showed that the reaction was complete and no reactant remained. After 26 h, to the mixture was added water (50 mL) and the resulting precipitate was filtered and washed with water (50 mL) to give 2-(l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)isoindoline-l,3-dione as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 (1 H, ddd, J=7.0, 1.6, 0.8 Hz), 8.01 - 8.11 (2 H, m), 7.69 - 7.86 (5 H, m), 7.56 (1 H, td, J=7.8, 1.3 Hz), 7.46 (1 H, td, J=6.9, 1.4 Hz), 7.29 (1 H, td, J=7.5, 1.4 Hz), 7.09 (1 H, dd, J=7.6, 1.2 Hz), 5.37 - 5.46 (1 H, m), 3.03 (3 H, s), 1.60 (3 H, d, J=7.0 Hz); Mass Spectrum (ESI) a major peak of m/e = 474.1 (M+ 1).
2-(l-Aminoethyl)-3-(2-(methylsulfonyl)phenyl)-4H-pyrido[l,2-a]pyrimidin-4- one
Figure imgf000068_0002
To a suspension of 2-(l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)isoindoline-l,3-dione (0.296 g, 0.626 mmol) in EtOH (12.52 niL) was added hydrazine, monohydrate (0.152 rnL, 3.13 mmol) and the mixture was stirred under reflux. After 1 h, LC-MS (ESI) showed the reaction was complete. After 1.5 h, the mixture was cond under reduced pressure. The residue was purified by column chromatography on a 40 g Redi-Sep™ column using 0% to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min, then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min, then 50% to 100% gradient of DCM:MeOH:NH4OH (89:9: 1) in DCM over 14 min, and then 100% isocratic of DCM:MeOH:NH4OH (89:9:1) for 5 min as eluent to give 2-(l-amino- ethyl)-3-(2-(methylsulfonyl)phenyl)-4H-pyrido[l,2-a]pyrimidin-4-one as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.94 (1 H, ddd, J=7.1, 1.5, 0.8 Hz), 8.10 (1 H, dd, J=8.0, 1.2 Hz), 7.98 (1 H, ddd, J=9.0, 6.7, 1.6 Hz), 7.68 - 7.85 (3 H, m), 7.55 (1 H, dd, J=7.5, 1.1 Hz), 7.36 (1 H, td, J=6.9, 1.4 Hz), 3.49 (1 H, q, J=6.5 Hz), 3.13 (3 H, s), 1.81 (2 H, br. s.), 1.13 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 344.0 (M+l).
4-Amino-6-((l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000069_0001
A mixture of 4-amino-6-chloropyrimidine-5-carbonitrile (0.090 g, 0.582 mmol), 2-(l -aminoethyl)-3-(2-(methylsulfonyl)phenyl)-4H-pyrido[ 1 ,2-a]pyrimidin-4-one (0.1999 g, 0.582 mmol), and DIEA (0.304 mL, 1.746 mmol) in Butan-1-ol (5.82 mL) was stirred at 120 0C. After 3 h, LC-MS (ESI) showed that the reaction was almost complete. After 4 h, the mixture was cooled to rt. The precipitated solid was filtered and washed with a mixture of EtOH-ether (1 :1, 25 mL) to give a white solid. The white solid (0.2001 g) was purified by column chromatography on a 40 g Redi-Sep™ column using 0% to 50% gradient of DCM:MeOH:NH4OH (89:9:1) in DCM over 14 min and then 50% isocratic of DCM:MeOH:NH4OH (89:9:1) in DCM for 14 min as eluent to give 4-amino-6-((l-(3-(2-(methyl- sulfonyl)phenyl)-4-oxo-4H-pyrido [ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5 -pyr- imidinecarbonitrile as a white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 (1 H, ddd, J=7.1, 1.5, 0.8 Hz), 8.12 (1 H, dd, J=7.8, 1.4 Hz), 8.00 - 8.08 (1 H, m), 7.95 (1 H, s), 7.75 - 7.81 (2 H, m), 7.68 - 7.75 (1 H, m), 7.60 (1 H, dd, J=7.5, 1.5 Hz), 7.43 (1 H, td, J=6.9, 1.4 Hz), 7.29 (2 H, br. s.), 7.08 (1 H, d, J=7.2 Hz), 5.06 (1 H, qd, J=6.8, 6.7 Hz), 3.19 (3 H, s), 1.25 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 462.0 (M+l).
4-Amino-6-(((lR)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2- a] pyrimidin-2-yl)ethyl)amino)-5-py rimidinecarbonitrile and 4-amino-6- (((lS)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000070_0001
The racemic mixture was separated by chiral separation using SFC to give 2 fractions: First peak on Chiralpak™ AS-H and AD-H column: 4-amino-6- (((lR)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 8.99 (1 H, ddd, J=I 2, 1.6, 0.8 Hz), 8.12 (1 H, dd, J=7.8, 1.4 Hz), 8.04 (1 H, ddd, J=8.9, 6.7, 1.6 Hz), 7.95 (1 H, s), 7.75 - 7.82 (2 H, m), 7.68 - 7.75 (1 H, m), 7.60 (1 H, dd, J=I.5, 1.5 Hz), 7.43 (1 H, td, J=6.9, 1.4 Hz), 7.29 (2 H, br. s.), 7.08 (1 H, d, J=7.4 Hz), 5.06 (1 H, quin, J=6.9 Hz), 3.19 (3 H, s), 1.25 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e = 462.1 (M+l). Second peak on Chiralpak™ AS-H and AD-H column: 4-amino-6-(((lS)-l-(3-(2- (methylsulfonyl)phenyl)-4-oxo-4H-pyrido [ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5 - pyrimidinecarbonitrile as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 (1 H, ddd, J=IA, 1.5, 0.8 Hz), 8.12 (1 H, dd, J=7.8, 1.4 Hz), 8.04 (1 H, ddd, J=8.9, 6.7, 1.6 Hz), 7.95 (1 H, s), 7.75 - 7.81 (2 H, m), 7.69 - 7.75 (1 H, m), 7.60 (1 H, dd, J=7.5, 1.5 Hz), 7.43 (1 H, td, J=6.9, 1.4 Hz), 7.29 (2 H, br. s.), 7.08
(1 H, d, J=7.2 Hz), 5.06 (1 H, quin, J=6.9 Hz), 3.19 (3 H, s), 1.25 (3 H, d, J=6.7
Hz); Mass Spectrum (ESI) m/e 462.1 (M+ 1).
Example 11: Preparation of 4-amino-6-((l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6-(((lS)-l-(4- oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidine- carbonitrile, and 4-amino-6-(((lR)-l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile
4-Amino-6-((l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-
5-pyrimidinecarbonitrile
Figure imgf000071_0001
Prepared according to General Procedures A through D to give 4-amino-6-((l- (4-0X0-3 -phenyl-4H-pyrido [ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5 -pyrimidine- carbonitrile: 1H-NMR (400 MHz, DMSO-d6): δ 8.9(d, IH), 8.0(m, IH), 7.9 (s, IH), 7.70(d, IH), 7.46(m, 2H), 7.3-7.4(m, 4H), 7.30(bs, 2H), 7.08-7.10(d, IH), 5.15-5.19(m, IH), 1.27-1.29(d, 3H).
4- Amino-6-(((l S)- l-(4-oxo-3-phenyl-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((lR)-l-(4-oxo-3-phenyl- 4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000071_0002
The racemic mixture was purified by chiral separation using SFC to give 2 fractions: First peak on Chiralpak™ AD-H column: 4-amino-6-(((lS)-l-(4- oxo-3 -phenyl-4H-pyrido [ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5 -pyrimidinecarbo- nitrile as an off-white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.96 (1 H, d, J=7.0 Hz), 7.90 - 8.06 (2 H, m), 7.72 (1 H, d, J=8.8 Hz), 7.22 - 7.55 (8 H, m), 7.09 (1 H, d, J=7.0 Hz), 5.18 (1 H, quin, J=6.5 Hz), 1.28 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI)] m/e = 384.1 (M+ 1). Second peak on AD-H column: 4-amino- 6-((( IR)-I -(4-0X0-3 -phenyl-4H-pyrido[ l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile as an off-white solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.96 (1 H, d, J=6.8 Hz), 7.93 - 8.05 (2 H, m), 7.72 (1 H, d, J=9.0 Hz), 7.25 - 7.53 (8 H, m), 7.09 (1 H, d, J=7.2 Hz), 5.18 (1 H, quin, J=6.7 Hz), 1.28 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 384.1 (M+ 1).
Example 12: Preparation of 4-amino-6-((l-(3-(3-fluorophenyl)-4-oxo-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lR)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((l S)-l-(3-(3-fluorophenyl)- 4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile 4-Amino-6-((l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000072_0001
Prepared according to General Procedures A through D to give 4-amino-6-((l- (3-(3-fluorophenyl)-4-oxo-4H-pyrido[ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyr- imidinecarbonitrile: 1H-NMR (400 MHz, DMSO-d6): δ 8.97 (d,lH),8.0 (m,lH), 7.9 (s,lH),7.72 (d, IH), 7.5 (m, IH), 7.3-7.4 (m, IH), 7.30 (bs, 2H), 7.2 (m, 3H),7.1 (d, 1H),5.15-5.19 (m, IH), 1.3 (d, 3H). 4-Amino-6-(((lR)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((lS)-l-(3-(3- fluor ophenyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile
Figure imgf000073_0001
The racemic mixture was purified by chiral separation using SFC to give 2 fractions: First peak on SFC OJ column and Second peak on Chiralpak™
AD-H column: 4-Amino-6-(((lR)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a tan solid: ^H NMR (400 MHz, DMSO-dβ) δ ppm 8.97 (1 H, d, J=6.8 Hz), 8.02 (1 H, ddd, J=8.8, 7.0,
1.5 Hz), 7.96 (1 H, s), 7.73 (1 H, d, J=9.0 Hz), 7.48 - 7.56 (1 H, m), 7.40 (1 H, td, J=6.9, 1.3 Hz), 7.20 - 7.36 (5 H, m), 7.11 (1 H, d, J=7.2 Hz), 5.17 (1 H, quin, J=6.8 Hz), 1.31 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) (ESI) m/e = 402.1 (M+l). Second peak on SFC OJ column and First peak on Chiralpak™ AD- H column: 4-amino-6-(((lS)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitril as a tan solid: 1H NMR (400 MHz, DMSO-dβ) δ ppm 8.97 (1 H, dd, J=7.0, 0.6 Hz), 8.02 (1 H, ddd, J=8.8, 6.9,
1.6 Hz), 7.96 (1 H, s), 7.73 (1 H, d, J=8.8 Hz), 7.47 - 7.56 (1 H, m), 7.40 (1 H, td, J=6.9, 1.3 Hz), 7.20 - 7.36 (5 H, m), 7.11 (1 H, d, J=7.2 Hz), 5.17 (1 H, quin, J=6.8 Hz), 1.31 (3 H, d, J=6.7 Hz); Mass Spectrum (ESI) m/e = 402.1 (M+l). Example 13: Preparation of 4-amino-6-((l-(4-oxo-3-(2-pyridinyl)-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lR)-l-(4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)- 5-pyrimidinecarbonitrile, and 4-amino-6-(((lS)-l-(4-oxo-3-(2-pyridinyl)-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile 4-Amino-6-((l-(4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000074_0001
Prepared according to General Procedures A through D to give 4-amino-6-((l- (4-0X0-3 -(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)arnino)-5-pyr- imidinecarbonitrile: 1H-NMR (400 MHz, DMSO-U6): δ 9.02 (d,lH),8.6 (s,lH), 8.0 (m,lH),7.8-7.9 (m, 2H), 7.7 (d, IH), 7.6 (d, IH), 7.4 (m, 2H), 7.1 (m, 3H), 5.3 (m, lH),1.3 (d, 3H).
4-Amino-6-(((lR)-l-(4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((lS)-l-(4-oxo-3-(2- pyridinyl)-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidine- carbonitrile
Figure imgf000074_0002
The racemic mixture was purified by chiral separation using SFC to give 2 fractions: First peak on SFC OJ column and Second peak on Chiralpak™ AD-H column: 4-amino-6-((( IR)-I -(4-oxo-3-(2-pyridinyl)-4H-pyrido[ 1,2- a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid:
1H NMR (400 MHz, DMSO-d6) δ ppm 9.00 - 9.04 (1 H, m), 8.70 (1 H, dt, J=4.1, 0.8 Hz), 8.06 (1 H, ddd, J=8.8, 6.9, 1.4 Hz), 7.93 (1 H, s), 7.87 - 7.91 (1 H, m), 7.75 (1 H, d, J=8.8 Hz), 7.63 (1 H, d, J=7.8 Hz), 7.37 - 7.47 (2 H, m), 7.24 - 7.34 (3 H, m), 5.38 (1 H, quin, J=6.8 Hz), 1.35 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e = 385.1 (M+l). Second peak on SFC OJ column and First peak on Chiralpak™ AD-H column: 4-amino-6-(((lS)-l-(4-oxo-3-(2-pyridinyl)-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.02 (1 H, d, J=6.7 Hz), 8.70
(1 H, dd, J=4.9, 0.6 Hz), 8.06 (1 H, ddd, J=8.8, 6.9, 1.4 Hz), 7.93 (1 H, s), 7.87 - 7.91 (1 H, m), 7.75 (1 H, d, J=8.8 Hz), 7.63 (1 H, d, J=7.8 Hz), 7.37 - 7.47 (2 H, m), 7.24 - 7.34 (3 H, m), 5.38 (1 H, quin, J=6.8 Hz), 1.35 (3 H, d, J=6.7 Hz);
Mass Spectrum (ESI) m/e = 385.0 (M+l).
Example 14: Preparation of 4-amino-6-((l-(3-(3,5-difluorophenyl)-4-oxo-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino- 6-(((lS)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)eth- yl)amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lR)-l-(3-(3,5-difluoro- phenyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidine- carbonitrile
4-Amino-6-((l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000075_0001
Prepared according to General Procedures A through D to give 4-Amino-6-((l- (3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile: 1H-NMR (400 MHz, DMSO-d6): δ 8.97 (d,lH),8.0 (m,lH), 7.9 (s,lH), 7.73(d, IH), 7.40 (m, IH), 7.24-7.29(m, 3H), 7.1 (m, 3H), 5.15-5.19(m, lH),1.2(d, 3H). 4-Amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin- 2-yl)ethyl)amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((lR)-l-(3-(3,5- difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile
Figure imgf000076_0001
The racemic mixture was puified by chiral separation using SFC to give 2 fractions: First peak on Chiralpak™ AD-H column: 4-amino-6-(((lS)-l-(3- (3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile as an off-white solid: ^H NMR (400 MHz, DMSO-dβ) δ ppm 8.94 - 8.99 (1 H, m), 8.03 (1 H, ddd, J=8.8, 6.9, 1.7 Hz), 7.96 (1 H, s), 7.71 - 7.77 (1 H, m), 7.42 (1 H, td, J=6.9, 1.2 Hz), 7.22 - 7.35 (3 H, m), 7.11 - 7.20 (3 H, m), 5.18 (1 H, quin, J=6.8 Hz), 1.34 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e = 420.1 (M+ 1). Second peak on Chiralpak™ AD-H column: 4-amino-6- (((lR)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile as an off-white solid: 1H NMR (400 MHz,
DMSO-dβ) δ ppm 8.94 - 9.00 (1 H, m), 8.03 (1 H, ddd, J=8.8, 6.9, 1.6 Hz), 7.95 (1 H, s), 7.74 (1 H, d, J=8.8 Hz), 7.42 (1 H, td, J=6.9, 1.3 Hz), 7.22 - 7.36 (3 H, m), 7.10 - 7.21 (3 H, m), 5.18 (1 H, quin, J=6.8 Hz), 1.34 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI)] m/e = 420.1 (M+l). Example 15: Preparation of 4-amino-6-((l-(3-(4-methyl-2-pyridinyl)-4-oxo- 4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4- amino-6-(((lR)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lS)-l- (3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl). amino)-5-pyrimidinecarbonitrile 4-Amino-6-((l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000077_0001
Prepared according to General Procedures A through D to give 4-amino-6-((l- (3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile: 1H-NMR (400 MHz, DMSO-d6): δ 9.02 (d,lH), 8.6 (d,lH), 8.0 (m,lH), 7.9 (s, IH), 7.7 (d, IH), 7.4 (m, 2H), 7.2 (m, 4H), 5.3 (m, IH), 2.3 (s, 3H), 1.3 (d, 3H).
4-Amino-6-(((lR)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile and 4-amino-6-(((l S)-l-(3- (4-methyl-2-pyridinyl)-4-oxo-4H-pyrido [ 1 ,2-a] pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile
Figure imgf000077_0002
The racemic mixture (0.13 g) was separated by chiral separation using SFC to give 2 fractions: First peak on SFC OJ column and Second peak on Chiral- pak™ AD-H column: 4-amino-6-(((lR)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H- pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5 -pyrimidinecarbonitrile as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 - 9.03 (1 H, m), 8.53 (1 H, dd, J=5.1, 0.4 Hz), 8.06 (1 H, ddd, J=8.8, 6.9, 1.6 Hz), 7.93 (1 H, s), 7.75 (1 H, dt, J=8.8, 1.1 Hz), 7.41 - 7.47 (2 H, m), 7.20 - 7.34 (4 H, m), 5.36 (1 H, quin, J=6.9 Hz), 2.38 (3 H, s), 1.35 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e = 399.1 (M+l). Second peak on SFC OJ column and First peak on Chiralpak™ AD-H column: 4-amino-6-(((lS)-l-(3-(4-methyl-2-pyridinyl)-4- oxo-4H-pyrido[ 1 ,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ ppm 8.99 - 9.03 (1 H, m),
8.53 (1 H, dd, J=5.1, 0.4 Hz), 8.06 (1 H, ddd, J=8.8, 6.9, 1.6 Hz), 7.93 (1 H, s), 7.75 (I H, dt, J=8.8, 1.1 Hz), 7.41 - 7.47 (2 H, m), 7.21 - 7.34 (4 H, m), 5.36 (1 H, quin, J=6.9 Hz), 2.38 (3 H, s), 1.35 (3 H, d, J=6.8 Hz); Mass Spectrum (ESI) m/e
= 399.1 (M+l).
Example 16: Preparation of 4-amino-6-((l-(6-methyl-4-oxo-3-phenyl-4H-pyr- ido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile, 4-amino-6- (((lS)-l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- amino)-5-pyrimidinecarbonitrile, and 4-amino-6-(((lR)-l-(6-methyl-4-oxo-3- phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbo- nitrile
2-(l-(6-Methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)ethyl)- isoindoline-l,3-dione
Figure imgf000078_0001
To a solution of phenylboronic acid (0.177 g, 1.45 mmol), 2-(l-(3-bromo-6- methyl-4-oxo-4H-pyrido[ 1 ,2-a]pyrimidin-2-yl)ethyl)isoindoline- 1 ,3-dione (Prepared in Example 6, 0.40 g, 0.97 mmol) and potassium carbonate (0.402 g, 2.91 mmol) in a mixture of water (0.5 mL) and dioxane (9 mL) was added dichloro 1 , r-bis(diphenylphosphino)ferrocene palladium (II) (0.040 g, 0.049 mmol) under an argon atmosphere. The mixture was stirred at 90 0C for 3 h then was loaded onto silica gel and purified by MPLC (eluted with a gradient of 0-3% MeOH in DCM) to afford 2-(l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]- pyrimidin-2-yl)ethyl)isoindoline-l,3-dione as a yellow solid. Mass Spectrum (ESI) m/e = 410.1 (M + l). 2-(l-Aminoethyl)-6-methyl-3-phenyl-4H-pyrido[l,2-a]pyrimidin-4-one
Figure imgf000079_0001
A solution of 2-(l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2-yl)- ethyl)isoindoline-l,3-dione (0.3Og, 0.73 mmol) and hydrazine monohydrate (0.36 rnL, 7.3 mmol) in EtOH (14.7 mL) was stirred at 90 0C for 1 h. The mixture was loaded onto silica gel and purified by MPLC (eluted with a gradient of 0-100% (1 :10:90 NH4OH:MeOH:DCM solution) in DCM) to afford 2-(l-aminoethyl)-6- methyl-3-phenyl-4H-pyrido[l,2-a]pyrimidin-4-one as a light yellow foam. Mass Spectrum (ESI) m/e = 280.3 (M + 1).
4-Amino-6-((l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000079_0002
To a solution of 2-(l-aminoethyl)-6-methyl-3-phenyl-4H-pyrido[l,2-a]pyrimidin- 4-one (0.178 g, 0.637 mmol) and 4,6-diaminopyrimidine-5-carbonitrile (0.090 g, 0.67 mmol) in butan-1-ol (4 mL) was added diisopropylethyl amine (0.33 mL, 1.91 mmol). After stirring at 120 0C for 3 h, the solution was loaded onto silica gel and purified by MPLC (eluted with a gradient of 0-8% MeOH in DCM) to afford 4-amino-6-(l -(6-methyl-4-oxo-3-phenyl-4H-pyrido[ 1 ,2-a]pyrimidin-2- yl)ethylamino)pyrimidine-5-carbonitrile as an off-white solid. IH NMR (400 MHz, DMSO-J6) δ ppm 1.27 (d, J=6.65 Hz, 3 H) 2.91 (s, 3 H) 5.09 (quin, J=6.85 Hz, 1 H) 6.97 (d, J=7.04 Hz, 1 H) 7.03 (d, J=7.24 Hz, 1 H) 7.32 (br. s., 2 H) 7.35 - 7.50 (m, 6 H) 7.72 (dd, J=8.80, 6.85 Hz, 1 H) 7.98 (s, 1 H)Mass Spectrum (ESI) m/e = 398.1 (M + 1). The racemic mixture (0.210 g) was separated by chiral separation using SFC to give 2 fractions. First-eluting enantiomer on AD-H column: 4-amino-6-(((lS)-l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyr- imidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid. IH NMR (500 MHz, DMSO-J6) δ ppm 1.26 (d, 3 H) 2.90 (s, 3 H) 5.09 (qd, J=6.89, 6.72 Hz, 1 H) 6.97 (d, J=6.85 Hz, 1 H) 7.04 (d, J=7.34 Hz, 1 H) 7.33 (br. s., 2 H) 7.35 - 7.50 (m, 6 H) 7.71 (dd, J=8.93, 6.97 Hz, 1 H) 7.97 (s, 1 H) Mass Spectrum (ESI) m/e = 398.1 (M + 1). Second-eluting enantiomer on AD-H column: Concentration of product fractions gave a solid that was triturated in water and filtered to afford 4-amino-6-((( IR)-I -(6-methyl-4-oxo-3-phenyl-4H-pyrido[ 1,2- a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile as a light yellow solid. Mass Spectrum (ESI) m/e = 398.1 (M + 1).
Example 17: Preparation of 4-amino-6-((l-(3-(3,5-difluorophenyl)-6-fluoro-l- methyl-4-oxo-l,4-dihydro-2-quinolinyl)ethyl)amino)-5-pyrimidinecarbo- nitrile
3-(3,5-Difluorophenyl)-2-ethyl-6-fluoroquinolin-4(lH)-one
Figure imgf000080_0001
A stirred mixture of 2-ethyl-6-fluoroquinolin-4(lH)-one (4.00 g, 21 mmol), I2
(10.62 g, 2.0 eq) and Na2CO3 (3.33 g, 1.5 eq) in THF (100 mL) was stirred at rt overnight. To the reaction mixture was added Na2S2O3 solution, after 2 min the resulted mixture was filtered, washed with water and dried in the air to give a white solid as 2-ethyl-6-fiuoro-3-iodoquinolin-4(lH)-one. Mass Spectrum (ESI) m/e = 318 (M + 1). A mixture of 2-ethyl-6-fluoro-3-iodoquinolin-4(lH)-one (400 mg, 1.3 mmol), 3,5-difiuorophenylboronic acid (398 mg, 2.0 eq), Na2CO3 (401 mg, 3.0 eq) and tetrakis(triphenylphosphine)palladium(0) (73 mg, 0.05 eq) in acetonitrile/water (15 mL/5 mL) was purged with N2 and heated to reflux. After overnight, the reaction mixture was cooled at rt, partitioned between water and EtOAc. The layers were separated and the aq layer was extracted with EtOAc (10 niL x 2). The combined organic layers were washed with water (10 mL x 2), brine (10 mL), dried over Na2SO4, filtered, and coned under reduced pressure. The residue was purified by combiflash on silica gel (EtOAc/DCM, 1 :2) to give 3- (3,5-difluorophenyl)-2-ethyl-6-fluoroquinolin-4(lH)-one as a white solid. Mass Spectrum (ESI) m/e = 304 (M + 1). 3-(3,5-Difluorophenyl)-2-ethyl-6-fluoro-l-methylquinolin-4(lH)-one
Figure imgf000081_0001
A suspension of 3-(3,5-difluorophenyl)-2-ethyl-6-fluoroquinolin-4(lH)-one (280 mg, 0.9 mmol) in DMF (5 mL) was treated with NaH (60%, 1.5 eq, 55.3 mg). After 30 min, MeI (0.12 mL, 2.0 eq) was added dropwise and the reaction mixture was stirred at rt overnight before quenching with water. The reaction mixture was extracted with EtOAc (5 mL x X). The organic layers were combined washed with water, brine, dried, coned and purified by column chromatography on silica gel (EtOAc/hexane, 1/2) to give 3-(3,5-difluorophenyl)-2-ethyl-6-fluoro-l-methyl- quinolin-4(lH)-one as a white solid. Mass Spectrum (ESI) m/e = 318 (M + 1). 2-(l-(3-(3,5-Difluorophenyl)-6-fluoro-l-methyl-4-oxo-l,4-dihydroquinolin-2- yl)ethyl)isoindoline- 1 ,3-dione
Figure imgf000081_0002
3 -(3 ,5 -Difluorophenyl)-2-ethyl-6-fluoro- 1 -methylquinolin-4( 1 H)-one (120 mg, 0.38 mmol) and l,3-dibromo-5,5-dimethylhydantoin (76 mg, 0.7 eq) were suspended in carbon tetrachloride (5 mL). To the mixture was added benzoyl peroxide (9.2 mg, 0.1 eq) and the mixture was heated at reflux for 3 h. After cooling to rt, satd. aq sodium bicarbonate solution (5 mL) was added. The layers were separated and the aq layer was extracted with DCM (3 mL x 2). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and coned under reduced pressure to give 2-(l-bromoethyl)-3-(3,5-difluorophenyl)-6- fluoro-l-methylquinolin-4(lH)-one as a yellow solid. Mass Spectrum (ESI) m/e = 397 (M + 1). The yellow solid was dissolved in DMF (5 mL) and treated with phthalimide potassium salt (140 mg, 2.0 eq) at 60 0C for 4h. The reaction mixture was partitioned between water and EtOAc, The layers were separated and the aq layer was extracted with EtOAc (10 mL x 2). The combined organic layers were washed with water (10 mL x 2), brine (10 mL), dried over Na2SO4, filtered, and coned under reduced pressure. The residue was purified by combiflash on silica gel (DCM/hexane, 0/1 to 1/1) to give 2-(l-(3-(3,5-difluorophenyl)-6-fiuoro-l- methyl-4-oxo-l,4-dihydroquinolin-2-yl)ethyl)isoindoline-l,3-dione as a white solid. Mass Spectrum (ESI) m/e = 463 (M + 1).
4-Amino-6-(l-(3-(3,5-difluorophenyl)-6-fluoro-l-methyl-4-oxo-l,4-dihydro- quinolin-2-yl)ethylamino)pyrimidine-5-carbonitrile
Figure imgf000082_0001
A suspension of 2-( 1 -(3 -(3 ,5 -difluorophenyl)-6-fluoro- 1 -methyl-4-oxo- 1 ,4-di- hydroquinolin-2-yl)ethyl)isoindoline-l,3-dione (80 mg, 0.17 mmol) in EtOH (3 mL) was treated with 0.2 mL hydrazine at 90 0C overnight. After cooling to rt, the reaction mixture was partitioned between water (5 mL) and EtOAc (5 mL). The organic layer was separated, washed with water, brine, dried and coned to give a white solid, which was treated with 4-amino-6-chloropyrimidine-5-carbonitrile (26.7 mg, 1.0 eq) and Hunig's base (36 μL, 1.2 eq) in n-BuOH (2 mL) at 130 0C overnight. After cooling to rt, the reaction mixture was coned and purified by reverse phase HPLC (10-50%, MeCN/water, 0.1% TFA) to give 4-amino-6-(l-(3- (3, 5 -difluorophenyl)-6-fluoro-l -methyl-4-oxo- 1,4-dihy droquinolin-2-yl)- ethylamino)pyrimidine-5-carbonitrile as TFA salt. 1H-NMR (400 Hz, CD3OD) δ 8.06 (s, IH), 7.98 (dd, J = 8.0, 4.0 Hz, IH), 7.94 (dd, J = 8.0, 4.0 Hz, IH), 7.63
(td, J = 8.0, 4.0 Hz, IH), 7.04 (d, J = 8.0 Hz, IH), 7.01 (t, J = 8.0 Hz, IH), 6.73 (d,
J = 8.0 Hz, IH), 5.43 (q, J = 8.0 Hz, IH), 4.14 (s, 3H), 1.78 (d, J = 8.0 Hz, 3H).
Mass Spectrum (ESI) m/e = 451 (M + 1).
Example 18: Preparation of 4-amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-6- fluoro-4-oxo-l,4-dihydro-2-quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrile
4-Amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-6-fluoro-4-oxo-l,4-dihydro-2- quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrile
Figure imgf000083_0001
4-amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-6-fluoro-4-oxo-l,4-dihydro-2- quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrile was synthesized from (S)-2-(l- (3-(3,5-difluorophenyl)-6-fluoro-4-oxo-l,4-dihydroquinolin-2-yl)ethyl)- isoindoline-l,3-dione in a similar manner as the above compound 4-amino-6-(l- (3-(3,5-difluorophenyl)-6-fluoro-l-methyl-4-oxo-l,4-dihydroquinolin-2-yl)- ethylamino)pyrimidine-5-carbonitrile. 1H-NMR (400 Hz, DMSO-d6) δ 11.50 (s, IH), 7.97 (s, IH), 7.69-7.73 (m, 2H), 7.59 (td, J = 8.0, 4.0 Hz, IH), 7.46 (s, br, IH), 7.17-7.22 (m, 2H), 7.05 (s, br, IH), 5.01-5.06 (m, IH), 1.48 (t, J = 8.0 Hz, 3H). Mass Spectrum (ESI) m/e = 437 (M + 1). Biological Assays Recombinant expression of PI3Ks
Full length pi 10 subunits of PI3k α, β and δ, N-terminally labeled with polyHis tag, were coexpressed with p85 with Baculo virus expression vectors in sf9 insect cells. Pl 10/p85 heterodimers were purified by sequential Ni-NTA, Q-HP, Superdex-100 chromatography. Purified α, β and δ isozymes were stored at -20 0C in 2OmM Tris, pH 8, 0.2M NaCl, 50% glycerol, 5mM DTT, 2mM Na cholate. Truncated PI3Kγ, residues 114-1102, N-terminally labeled with polyHis tag, was expessed with Baculo virus in Hi5 insect cells. The γ isozyme was purified by sequential Ni-NTA, Superdex-200, Q-HP chromatography. The γ isozyme was stored frozen at -80 0C in NaH2PO4, pH 8, 0.2M NaCl, 1% ethylene glycol, 2mM β-mercaptoethanol.
Figure imgf000084_0001
In vitro PI3K enzyme assays
A PBK Alphascreen® assay (PerkinElmer, Waltham, MA) was used to measure the activity of a panel of four phosphoinositide 3-kinases: PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ. Enzyme reaction buffer was prepared using sterile water (Baxter, Deerfield, IL) and 5OmM Tris HCl pH 7, 14mM MgCl2, 2mM sodium cholate, and 10OmM NaCl. 2mM DTT was added fresh the day of the experiment. The Alphascreen buffer was made using sterile water and 1OmM Tris HCl pH 7.5, 15OmM NaCl, 0.10% Tween 20, and 3OmM EDTA. ImM DTT was added fresh the day of the experiment. Compound source plates used for this assay were 384- well Greiner clear polypropylene plates containing test compounds at 5mM and diluted 1 :2 over 22 concentrations. Columns 23 and 24 contained only DMSO as these wells comprised the positive and negative controls, respectively. Source plates were replicated by transferring 0.5 uL per well into 384-well Optiplates (PerkinElmer, Waltham, MA). Each PI3K isoform was diluted in enzyme reaction buffer to 2X working stocks. PI3Kα was diluted to 1.6nM, PI3Kβ was diluted to 0.8nM, PI3Kγ was diluted to 15nM, and PI3Kδ was diluted to 1.6nM. PI(4,5)P2 (Echelon Biosciences, Salt Lake City, UT) was diluted to lOμM and ATP was diluted to 20μM. This 2x stock was used in the assays for PBKα and PBKβ. For assay of PBKγ and PBKδ, PI(4,5)P2 was diluted to lOμM and ATP was diluted to 8μM to prepare a similar 2x working stock. Alphascreen reaction solutions were made using beads from the anti-GST Alphascreen kit (PerkinElmer, Waltham, MA). Two 4X working stocks of the Alphascreen reagents were made in Alphascreen reaction buffer. In one stock, biotinylated-IP4 (Echelon Biosciences, Salt Lake City, UT) was diluted to 4OnM and streptavadin-donor beads were diluted to 80μg/mL. In the second stock, PIP3-binding protein (Echelon Biosciences, Salt Lake City, UT) was diluted to 4OnM and anti-GST-acceptor beads were diluted to 80μg/mL. As a negative control, a reference inhibitor at a concentration » Ki (40 uM) was included in column 24 as a negative (100% inhibition) control.
Using a 384-well Multidrop (Titertek, Huntsville, AL), lOμL/well of 2X enzyme stock was added to columns 1-24 of the assay plates for each isoform. lOμL/well of the appropriate substrate2x stock (containing 20μM ATP for the PBKα and β assays and containing 8μM ATP for the PBKγ and δ assays) was then added to Columns 1-24 of all plates. Plates were then incubated at room temperature for 20 minutes. In the dark, lOμL/well of the donor bead solution was added to columns 1-24 of the plates to quench the enzyme reaction. The plates were incubated at room temperature for 30 minutes. Still in the dark, lOμL/well of the acceptor bead solution was added to columns 1-24 of the plates. The plates were then incubated in the dark for 1.5 hours. The plates were read on an Envision multimode Plate Reader (PerkinElmer, Waltham, MA) using a 680nm excitation filter and a 520-620nm emission filter. Alternative in vitro enzyme assays. Assays were performed in 25 μL with the above final concentrations of components in white polyproplyene plates (Costar 3355). Phospatidyl inositol phosphoacceptor, PtdIns(4,5)P2 P4508, was from Echelon Biosciences. The ATPase activity of the alpha and gamma isozymes was not greatly stimulated by PtdIns(4,5)P2 under these conditions and was therefore omitted from the assay of these isozymes. Test compounds were dissolved in dimethyl sulfoxide and diluted with three-fold serial dilutions. The compound in DMSO (1 μL) was added per test well, and the inhibition relative to reactions containing no compound, with and without enzyme was determined. After assay incubation at rt, the reaction was stopped and residual ATP determined by addition of an equal volume of a commercial ATP bio luminescence kit (Perkin Elmer EasyLite) according to the manufacturer's instructions, and detected using a AnalystGT luminometer.
Human B Cells Proliferation stimulate by anti-IgM
Isolate human B Cells:
Isolate PBMCs from Leukopac or from human fresh blood. Isolate human B cells by using Miltenyi protocol and B cell isolation kit II. -human B cells were Purified by using AutoMacs™ column. Activation of human B cells
Use 96 well Flat bottom plate, plate 50000/well purified B cells in B cell proliferation medium (DMEM + 5% FCS, 10 mM Hepes, 50 μM 2-mercaptoethanol); 150 μL medium contain 250 ng/mL CD40L -LZ recombinant protein (Amgen) and 2 μg/mL anti-Human IgM antibody (Jackson ImmunoReseach Lab. #109- 006-129), mixed with 50 μL B cell medium containing PBK inhibitors and incubate 72 h at 37 0C incubator. After 72h, pulse labeling B cells with 0.5-1 uCi /well 3H thymidine for overnight ~18 h, and harvest cell using TOM harvester. Human B Cells Proliferation stimulate by IL-4 Isolate human B Cells:
Isolate human PBMCs from Leukopac or from human fresh blood. Isolate human B cells using Miltenyi protocol - B cell isolation kit. Human B cells were purified by AutoMacs. column. Activation of human B cells Use 96-well flat bottom plate, plate 50000/well purified B cells in B cell proliferation medium (DMEM + 5% FCS, 50 μM 2-mercaptoethanol, 1OmM Hepes). The medium (150 μL) contain 250 ng/mL CD40L -LZ recombinant protein (Amgen) and 10 ng/mL IL-4 ( R&D system # 204-IL-025), mixed with 50 150 μL B cell medium containing compounds and incubate 72 h at 37 0C incubator. After 72 h, pulse labeling B cells with 0.5-1 uCi /well JH thymidine for overnight ~18 h, and harvest cell using TOM harvester. Specific T antigen (Tetanus toxoid) induced human PBMC proliferation assays
Human PBMC are prepared from frozen stocks or they are purified from fresh human blood using a Ficoll gradient. Use 96 well round-bottom plate and plate 2xlO5 PBMC/well with culture medium (RPMI1640 + 10% FCS, 5OuM 2-
Mercaptoethanol,10 mM Hepes). For IC50 determinations, PBK inhibitors was tested from 10 μM to 0.001 μM, in half log increments and in triplicate. Tetanus toxoid ,T cell specific antigen ( University of Massachusetts Lab) was added at 1 μg/mL and incubated 6 days at 37 0C incubator. Supernatants are collected after 6 days for IL2 ELISA assay , then cells are pulsed with 3H-thymidine for ~18 h to measure proliferation.
GFP assays for detecting inhibition of Class Ia and Class III PI3K AKTl (PKBa) is regulated by Class Ia PBK activated by mitogenic factors (IGF- 1, PDGF, insulin, thrombin, NGF, etc.). In response to mitogenic stimuli, AKTl translocates from the cytosol to the plasma membrane
Forkhead (FKHRLl) is a substrate for AKTl . It is cytoplasmic when phosphorylated by AKT (survival/growth). Inhibition of AKT (stasis/apoptosis) - forkhead translocation to the nucleus FYVE domains bind to PI(3)P. the majority is generated by constitutive action of PBK Class III
AKT membrane ruffling assay (CHO-IR-AKTl -EGFP cells/GE Healthcare) Wash cells with assay buffer. Treat with compounds in assay buffer 1 h. Add 10 ng/mL insulin. Fix after 10 min at room temp and image Forkhead translocation assay (MDA MB468 Forkhead-DiversaGFP cells) Treat cells with compound in growth medium 1 h. Fix and image.
Class IIIPI(3)P assay (U2OS EGFP-2XFYVE cells/GE Healthcare)
Wash cells with assay buffer. Treat with compounds in assay buffer 1 h. Fix and image.
Control for all 3 assays is lOuM Wortmannin: AKT is cytoplasmic
Forkhead is nuclear
PI(3)P depleted from endosomes Biomarker assay: B-cell receptor stimulation of CD69 or B7.2 (CD86) expression
Heparinized human whole blood was stimulated with 10 μg/mL anti-IgD (Southern Biotech, #9030-01). 90 μL of the stimulated blood was then aliquoted per well of a 96-well plate and treated with 10 μL of various concentrations of blocking compound (from 10-0.0003 μM) diluted in IMDM + 10% FBS (Gibco). Samples were incubated together for 4 h (for CD69 expression) to 6 h (for B7.2 expression) at 37 0C. Treated blood (50 μL) was transferred to a 96-well, deep well plate (Nunc) for antibody staining with 10 μL each of CD45-PerCP (BD Biosciences, #347464), CD 19-FITC (BD Biosciences, #340719), and CD69-PE (BD Biosciences, #341652). The second 50 μL of the treated blood was transferred to a second 96-well, deep well plate for antibody staining with 10 μL each of CDl 9-FITC (BD Biosciences, #340719) and CD86-PeCy5 (BD Biosciences, #555666). All stains were performed for 15-30 min in the dark at rt. The blood was then lysed and fixed using 450 μL of FACS lysing solution (BD Biosciences, #349202) for 15 min at rt. Samples were then washed 2X in PBS + 2% FBS before FACS analysis. Samples were gated on either CD45/CD19 double positive cells for CD69 staining, or CD 19 positive cells for CD86 staining. Gamma Counterscreen: Stimulation of human monocytes for phospho-AKT expression
A human monocyte cell line, THP-I, was maintained in RPMI + 10% FBS (Gibco). One day before stimulation, cells were counted using trypan blue exclusion on a hemocytometer and suspended at a concentration of 1 x 106 cells per mL of media. 100 μL of cells plus media (1 x 105 cells) was then aliquoted per well of 4-96-well, deep well dishes (Nunc) to test eight different compounds.
Cells were rested overnight before treatment with various concentrations (from 10-0.0003μM) of blocking compound. The compound diluted in media (12 μL) was added to the cells for 10 min at 37 0C. Human MCP-I (12 μL, R&D Diagnostics, #279-MC) was diluted in media and added to each well at a final concentration of 50 ng/mL. Stimulation lasted for 2 min at rt. Pre-warmed
FACS Phosflow Lyse/Fix buffer (1 mL of 37 0C) (BD Biosciences, #558049) was added to each well. Plates were then incubated at 37 0C for an additional 10-15 min. Plates were spun at 1500 rpm for 10 min, supernatant was aspirated off, and 1 mL of ice cold 90% MeOH was added to each well with vigorous shaking. Plates were then incubated either overnight at -70 0C or on ice for 30 min before antibody staining. Plates were spun and washed 2X in PBS + 2% FBS (Gibco). Wash was aspirated and cells were suspended in remaining buffer. Rabbit pAKT (50 μL, Cell Signaling, #4058L) at 1 : 100, was added to each sample for 1 h at rt with shaking. Cells were washed and spun at 1500 rpm for 10 min. Supernatant was aspirated and cells were suspended in remaining buffer. Secondary antibody, goat anti-rabbit Alexa 647 (50 μL, Invitrogen, #A21245) at 1 :500, was added for 30 min at rt with shaking. Cells were then washed IX in buffer and suspended in 150 μL of buffer for FACS analysis. Cells need to be dispersed very well by pipetting before running on flow cytometer. Cells were run on an LSR II (Becton Dickinson) and gated on forward and side scatter to determine expression levels of p AKT in the monocyte population. Gamma Counterscreen: Stimulation of monocytes for phospho-AKT expression in mouse bone marrow
Mouse femurs were dissected from five female BALB/c mice (Charles River Labs.) and collected into RPMI + 10% FBS media (Gibco). Mouse bone marrow was removed by cutting the ends of the femur and by flushing with 1 mL of media using a 25 gauge needle. Bone marrow was then dispersed in media using a 21 gauge needle. Media volume was increased to 20 mL and cells were counted using trypan blue exclusion on a hemocytometer. The cell suspension was then increased to 7.5 x 106 cells per 1 mL of media and 100 μL (7.5 x 105 cells) was aliquoted per well into 4-96-well, deep well dishes (Nunc) to test eight different compounds. Cells were rested at 37 0C for 2 h before treatment with various concentrations (from 10-0.0003 μM) of blocking compound. Compound diluted in media (12 μL) was added to bone marrow cells for 10 min at 37 0C. Mouse MCP- 1 (12 μL, R&D Diagnostics, #479- JE) was diluted in media and added to each well at a final concentration of 50 ng/mL. Stimulation lasted for 2 min at rt. 1 mL of 37 0C pre -warmed FACS Phosflow Lyse/Fix buffer (BD Biosciences,
#558049) was added to each well. Plates were then incubated at 37°C for an additional 10-15 min. Plates were spun at 1500 rpm for 10 min. Supernatant was aspirated off and 1 niL of ice cold 90% MEOH was added to each well with vigorous shaking. Plates were then incubated either overnight at -70 0C or on ice for 30 min before antibody staining. Plates were spun and washed 2X in PBS + 2% FBS (Gibco). Wash was aspirated and cells were suspended in remaining buffer. Fc block (2 μL, BD Pharmingen, #553140) was then added per well for 10 min at rt. After block, 50 μL of primary antibodies diluted in buffer; CDl Ib- Alexa488 (BD Biosciences, #557672) at 1 :50, CD64-PE (BD Biosciences, #558455) at 1 :50, and rabbit pAKT (Cell Signaling, #4058L) at 1 :100, were added to each sample for 1 h at rt with shaking. Wash buffer was added to cells and spun at 1500 rpm for 10 min. Supernatant was aspirated and cells were suspended in remaining buffer. Secondary antibody; goat anti-rabbit Alexa 647 (50 μL, Invitrogen, #A21245) at 1 :500, was added for 30 min at rt with shaking. Cells were then washed IX in buffer and suspended in 100 μL of buffer for FACS analysis. Cells were run on an LSR II (Becton Dickinson) and gated on CD 1 lb/CD64 double positive cells to determine expression levels of pAKT in the monocyte population. pAKT in vivo Assay
Vehicle and compounds are administered p.o. (0.2 mL) by gavage (Oral Gavage Needles Popper & Sons, New Hyde Park, NY) to mice (Transgenic Line 3751, female, 10-12 wks Amgen Inc, Thousand Oaks, CA) 15 min prior to the injection i.v (0.2 mLs) of anti-IgM FITC (50 ug/mouse) (Jackson Immuno Research, West Grove, PA). After 45 min the mice are sacrificed within a CO2 chamber. Blood is drawn via cardiac puncture (0.3 mL) (Ice 25 g Syringes, Sherwood, St. Louis, MO) and transferred into a 15 mL conical vial (Nalge/Nunc International, Denmark). Blood is immediately fixed with 6.0 mL of BD Phosflow Lyse/Fix
Buffer (BD Bioscience, San Jose, CA), inverted 3X's and placed in 37 0C water bath. Half of the spleen is removed and transferred to an eppendorf tube containing 0.5 mL of PBS (Invitrogen Corp, Grand Island, NY). The spleen is crushed using a tissue grinder (Pellet Pestle, Kimble/Kontes, Vineland, NJ) and immediately fixed with 6.0 mL of BD Phosflow Lyse/Fix buffer, inverted 3X's and placed in 37 0C water bath. Once tissues have been collected the mouse is cervically-dislocated and carcass to disposed. After 15 min, the 15 mL conical vials are removed from the 37 0C water bath and placed on ice until tissues are further processed. Crushed spleens are filtered through a 70 μm cell strainer (BD Bioscience, Bedford, MA) into another 15 mL conical vial and washed with 9 mL of PBS. Splenocytes and blood are spun @ 2,000 rpms for 10 min (cold) and buffer is aspirated. Cells are resuspended in 2.0 mL of cold (-20 0C) 90% MeOH (Mallinckrodt Chemicals, Phillipsburg, NJ). MeOH is slowly added while conical vial is rapidly vortexed. Tissues are then stored at -20 0C until cells can be stained for FACS analysis. Multi-dose TNP immunization Blood was collected by retro-orbital eye bleeds from 7-8 week old BALB/c female mice (Charles River Labs.) at day 0 before immunization. Blood was allowed to clot for 30 min and spun at 10,000 rpm in serum microtainer tubes (Becton Dickinson) for 10 min. Sera were collected, aliquoted in Matrix tubes (Matrix Tech. Corp.) and stored at -70 0C until ELISA was performed. Mice were given compound orally before immunization and at subsequent time periods based on the life of the molecule. Mice were then immunized with either 50 μg of TNP- LPS (Biosearch Tech., #T-5065), 50 μg of TNP-Ficoll (Biosearch Tech., #F- 1300), or 100 μg of TNP-KLH (Biosearch Tech., #T-5060) plus 1% alum (Brenntag, #3501) in PBS. TNP-KLH plus alum solution was prepared by gently inverting the mixture 3-5 times every 10 min for 1 h before immunization. On day 5, post-last treatment, mice were CO2 sacrificed and cardiac punctured. Blood was allowed to clot for 30 min and spun at 10,000 rpm in serum microtainer tubes for 10 min. Sera were collected, aliquoted in Matrix tubes, and stored at -70 0C until further analysis was performed. TNP-specifϊc IgGl, IgG2a, IgG3 and IgM levels in the sera were then measured via ELISA. TNP-BSA
(Biosearch Tech., #T-5050) was used to capture the TNP-specific antibodies. TNP-BSA (10 μg/mL) was used to coat 384-well ELISA plates (Corning Costar) overnight. Plates were then washed and blocked for 1 h using 10% BSA ELISA Block solution (KPL). After blocking, ELISA plates were washed and sera samples/standards were serially diluted and allowed to bind to the plates for 1 h.
Plates were washed and Ig-HRP conjugated secondary antibodies (goat anti- mouse IgGl, Southern Biotech #1070-05, goat anti-mouse IgG2a, Southern Biotech #1080-05, goat anti-mouse IgM, Southern Biotech #1020-05, goat anti- mouse IgG3, Southern Biotech #1100-05) were diluted at 1 :5000 and incubated on the plates for 1 h. TMB peroxidase solution (SureBlue Reserve TMB from KPL) was used to visualize the antibodies. Plates were washed and samples were allowed to develop in the TMB solution approximately 5-20 min depending on the Ig analyzed. The reaction was stopped with 2M sulfuric acid and plates were read at an OD of 45 O nm.
The compounds below exhibit the associated data from the PBKδ Alphascreen™ assay:
Figure imgf000092_0001
Figure imgf000093_0001
For the treatment of PBKδ-mediated-diseases, such as rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases, the compounds of the present invention may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
Treatment of diseases and disorders herein is intended to also include the prophylactic administration of a compound of the invention, a pharmaceutical salt thereof, or a pharmaceutical composition of either to a subject (i.e., an animal, preferably a mammal, most preferably a human) believed to be in need of preventative treatment, such as, for example, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmune diseases and the like.
The dosage regimen for treating PBKδ-mediated diseases, cancer, and/or hyperglycemia with the compounds of this invention and/or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Dosage levels of the order from about 0.01 mg to 30 mg per kilogram of body weight per day, preferably from about 0.1 mg to 10 mg/kg, more preferably from about 0.25 mg to 1 mg/kg are useful for all methods of use disclosed herein.
The pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.
For oral administration, the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient. For example, these may contain an amount of active ingredient from about 1 to 2000 mg, preferably from about 1 to 500 mg, more preferably from about 5 to 150 mg. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
The active ingredient may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water. The daily parenteral dosage regimen will be from about 0.1 to about 30 mg/kg of total body weight, preferably from about 0.1 to about 10 mg/kg, and more preferably from about 0.25 mg to 1 mg/kg.
Injectable preparations, such as sterile injectable aq or oleaginous suspensions, may be formulated according to the known are using suitable dispersing or wetting agents and suspending agents. 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- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, 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.
Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
A suitable topical dose of active ingredient of a compound of the invention is 0.1 mg to 150 mg administered one to four, preferably one or two times daily. For topical administration, the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi- liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose. For administration, the compounds of this invention are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
The pharmaceutical compositions may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifϊers, buffers etc.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents. Compounds of the present invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures thereof. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, e.g., by formation of diastereoisomeric salts, by treatment with an optically active acid or base. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts. A different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound. The optically active compounds of the invention can likewise be obtained by using active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.
Likewise, the compounds of this invention may exist as isomers, that is compounds of the same molecular formula but in which the atoms, relative to one another, are arranged differently. In particular, the alkylene substituents of the compounds of this invention, are normally and preferably arranged and inserted into the molecules as indicated in the definitions for each of these groups, being read from left to right. However, in certain cases, one skilled in the art will appreciate that it is possible to prepare compounds of this invention in which these substituents are reversed in orientation relative to the other atoms in the molecule. That is, the substituent to be inserted may be the same as that noted above except that it is inserted into the molecule in the reverse orientation. One skilled in the art will appreciate that these isomeric forms of the compounds of this invention are to be construed as encompassed within the scope of the present invention. The compounds of the present invention can be used in the form of salts derived from inorganic or organic acids. The salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methansulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 2-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate. Also, the basic nitrogen- containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.
Examples of acids that may be employed to from pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Other examples include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic bases.
Also encompassed in the scope of the present invention are pharmaceutically acceptable esters of a carboxylic acid or hydroxyl containing group, including a metabolically labile ester or a prodrug form of a compound of this invention. A metabolically labile ester is one which may produce, for example, an increase in blood levels and prolong the efficacy of the corresponding non-esterified form of the compound. A prodrug form is one which is not in an active form of the molecule as administered but which becomes therapeutically active after some in vivo activity or biotransformation, such as metabolism, for example, enzymatic or hydro lytic cleavage. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examples of a masked carboxylate anion include a variety of esters, such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p- methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N- acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)). Hydroxy groups have been masked as esters and ethers. EP 039,051 (Sloan and Little, 4/11/81) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use. Esters of a compound of this invention, may include, for example, the methyl, ethyl, propyl, and butyl esters, as well as other suitable esters formed between an acidic moiety and a hydroxyl containing moiety. Metabolically labile esters, may include, for example, methoxymethyl, ethoxymethyl, iso-propoxymethyl, α-methoxyethyl, groups such as α-((Ci-C4)- alkyloxy)ethyl, for example, methoxyethyl, ethoxyethyl, propoxyethyl, iso- propoxyethyl, etc.; 2-oxo-l,3-dioxolen-4-ylmethyl groups, such as 5-methyl-2- oxo-l,3,dioxolen-4-ylmethyl, etc.; C1-C3 alkylthiomethyl groups, for example, methylthiomethyl, ethylthiomethyl, isopropylthiomethyl, etc.; acyloxymethyl groups, for example, pivaloyloxymethyl, α-acetoxymethyl, etc.; ethoxycarbonyl-
1 -methyl; or α-acyloxy-α-substituted methyl groups, for example α-acetoxyethyl.
Further, the compounds of the invention may exist as crystalline solids which can be crystallized from common solvents such as ethanol, N,N-dimethyl- formamide, water, or the like. Thus, crystalline forms of the compounds of the invention may exist as polymorphs, solvates and/or hydrates of the parent compounds or their pharmaceutically acceptable salts. All of such forms likewise are to be construed as falling within the scope of the invention. While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosed compounds. Variations and changes which are obvious to one skilled in the art are intended to be within the scope and nature of the invention which are defined in the appended claims.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

We Claim:
1. A compound having the structure:
Figure imgf000101_0001
or any pharmaceutically-acceptable salt thereof, wherein:
X1 is C(R10) or N;
X2 is C or N; X3 is C or N;
X4 is C or N;
X5 is C or N; wherein at least two of X2, X3, X4 and X5 are C;
Y is N(R8), O or S; n is 0, 1, 2 or 3; R1 is a direct-bonded, Ci_4alk-linked, OC i_2alk- linked, Ci_2alkO-linked,
N(Ra)-linked or O-linked saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic or 8-, 9-, 10- or 11-membered bicyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S atom, substituted by 0, 1, 2 or 3 substituents independently selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra, wherein the available carbon atoms of the ring are additionally substituted by 0, 1 or 2 oxo or thioxo groups, and wherein the ring is additionally substituted by 0 or 1 directly bonded, SO2 linked, C(=O) linked or CH2 linked group selected from phenyl, pyridyl, pyrimidyl, morpholino, piperazinyl, piperadinyl, cyclopentyl, cyclohexyl all of which are further substituted by 0, 1, 2 or 3 independent Rb groups; R2 is selected from H, halo, Ci_6alk, C1-4haloalk, cyano, nitro, ORa, NRaRa,
-C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa;
R3 is selected from H, halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk or Ci_4haloalk;
R4 is, independently, in each instance, halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk, Ci_4haloalk or an unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, substituted by 0, 1, 2 or 3 substituents selected from halo, Ci_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_4alk;
R5 is, independently, in each instance, H, halo, Ci_6alk, Ci_4haloalk or Ci_6alk substituted by 1, 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, Ci_4alk, C1-3haloalk, OCi_4alk, NH2, NHC1-4alk and N(CMalk)CMalk; or both R5 groups together form a C3_6spiroalk substituted by 0, 1 , 2 or 3 substituents selected from halo, cyano, OH, OCi_4alk, d_4alk, d_3haloalk, OC i_ 4alk, NH2, NHCi_4alk and N(CMalk)CMalk; R6 is H, halo, NHR9 or OH, cyano, OCi_4alk, Ci_4alk, Ci_3haloalk, OCi_4alk, -C(=O)ORa, -C(=O)N(Ra)Ra, -N(Ra)C(=O)Rb;
R7 is selected from H, halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa, -NRaC2_6alk0Ra and C1-6alk, wherein the Ci_6alk is substituted by 0, 1 2 or 3 substituents selected from halo, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2.6alkNRaRa, -OC2.6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2.6alkNRaRa and -NRaC2_6alk0Ra, and the Ci_6alk is additionally substituted by 0 or 1 saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic rings containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2 or 3 substituents independently selected from halo, nitro, cyano, Ci_4alk, OCi_4alk, OCi_4haloalk, NHCi_4alk, N(Ci_4alk)Ci_4alk and Ci_4haloalk; or R7 and R8 together form a -C=N- bridge wherein the carbon atom is substituted by H, halo, cyano, or a saturated, partially-saturated or unsaturated 5-, 6- or 7-membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, O and S, but containing no more than one O or S, wherein the available carbon atoms of the ring are substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by 0, 1, 2, 3 or 4 substituents selected from halo, Ci_6alk, Ci_4haloalk, cyano, nitro, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -ORa, -OC(=O)Ra, -OC(=O)NRaRa, -OC(=O)N(Ra)S(=O)2Ra, -OC2_6alkNRaRa,
-OC2_6alkORa, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -S(=O)2N(Ra)C(=O)Ra, -S(=O)2N(Ra)C(=O)ORa, -S(=O)2N(Ra)C(=O)NRaRa, -NRaRa, -N(Ra)C(=O)Ra, -N(Ra)C(=O)ORa, -N(Ra)C(=O)NRaRa, -N(Ra)C(=NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2-6alkNRaRa and -NRaC2_6alk0Ra; or R7 and R9 together form a -N=C- bridge wherein the carbon atom is substituted by H, halo, Ci_6alk, Ci_4haloalk, cyano, nitro, ORa, NRaRa, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRa, -C(=NRa)NRaRa, -S(=O)Ra, -S(=O)2Ra or -S(=O)2NRaRa;
Figure imgf000104_0001
R9 is H, Ci_6alk or Ci_4haloalk;
R10 is H, halo, Ci_3alk, Ci_3haloalk or cyano;
Ra is independently, at each instance, H or Rb; and Rb is independently, at each instance, phenyl, benzyl or Ci_6alk, the phenyl, benzyl and Ci_6alk being substituted by 0, 1, 2 or 3 substituents selected from halo, d_4alk, Ci_3haloalk, -OCi_4alk, -NH2, -NHCi_4alk and -N(Ci_4alk)Ci_ 4alk.
2. A compound according to Claim 1 , wherein the compound is:
2-((4-amino-3-iodo-lH-pyrazolo[3,4-d]pyrimidin-l-yl)methyl)-3-(3- fluorophenyl)-6-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4-one;
2-((6-amino-9H-purin-9-yl)methyl)-6-methyl-3-(2-methylphenyl)-4H-pyrido[l,2- a]pyrimidin-4-one; 3-(3-fluorophenyl)-6-methyl-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-4H- pyrido[l ,2-a]pyrimidin-4-one;
3-(3-fluorophenyl)-6-methyl-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-4H- pyrido[l ,2-a]pyrimidin-4-one;
3 -(3 -fluorophenyl)-6-methyl-2-((9H-purin-6-ylamino)methyl)-4H-pyrido [ 1 ,2- a]pyrimidin-4-one;
3-(3-fluorophenyl)-6-methyl-2-(l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2- a]pyrimidin-4-one;
4-amino-6-(((lR)-l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2- a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-(((lR)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-(((lR)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lR)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-
2-yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-((( IR)-I -(4-0X0-3 -(2 -pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lR)-l-(4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lR)-l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin- 2-yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((( IR)-I -(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((l S)- 1 -(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[ 1 ,2- a]pyrimidin-2-yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lS)-l-(3-(3,5-difluorophenyl)-6-fluoro-4-oxo-l,4-dihydro-2- quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lS)-l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lS)-l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-
2-yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((l S)- 1 -(4-oxo-3-(2-pyridinyl)-4H-pyrido[ 1 ,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-((( IS)-I -(4-0X0-3 -phenyl-4H-pyrido[ l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-(((lS)-l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-
2-yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((( IS)-I -(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((l-(3-(2-(methylsulfonyl)phenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin- 2-yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-((l-(3-(3,5-difluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((l-(3-(3,5-difluorophenyl)-6-fluoro-l-methyl-4-oxo-l,4-dihydro-2- quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-((l-(3-(3-fluorophenyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((l-(3-(4-methyl-2-pyridinyl)-4-oxo-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((l -(4-0X0-3 -(2 -pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
4-amino-6-((l -(4-0X0-3 -phenyl-4H-pyrido[ l,2-a]pyrimidin-2-yl)ethyl)amino)-5- pyrimidinecarbonitrile;
4-amino-6-((l-(6-methyl-4-oxo-3-(2-pyridinyl)-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile; 4-amino-6-((l-(6-methyl-4-oxo-3-phenyl-4H-pyrido[l,2-a]pyrimidin-2- yl)ethyl)amino)-5-pyrimidinecarbonitrile;
6-methyl-2-(l -(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[ 1 ,2- a]pyrimidin-4-one;
6-methy 1-3 -(2-methylphenyl)-2-((9H-purin-6-ylsulfanyl)methyl)-4H-pyrido [ 1 ,2- a]pyrimidin-4-one;
7-fluoro-2-(( IR)-I -(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[ 1,2- a]pyrimidin-4-one;
7-fluoro-2-((l S)- 1 -(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[ 1 ,2- a]pyrimidin-4-one; 7-fluoro-2-(l-(9H-purin-6-ylamino)ethyl)-3-(2-pyridinyl)-4H-pyrido[l,2- a]pyrimidin-4-one;
7-fluoro-3-(3-fluorophenyl)-2-((lR)-l-(9H-purin-6-ylamino)ethyl)-4H- pyrido[l ,2-a]pyrimidin-4-one; 7-fluoro-3-(3-fluorophenyl)-2-((lS)-l-(9H-purin-6-ylamino)ethyl)-4H-pyrido[l,2- a]pyrimidin-4-one; or
7-fluoro-3-(3-fluorophenyl)-2-(l -(9H-purin-6-ylamino)ethyl)-4H-pyrido[ 1 ,2- a]pyrimidin-4-one; or a pharmaceutically-acceptable salt thereof.
3. A method of treating rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases and autoimmune diseases, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, skin complaints with inflammatory components, chronic inflammatory conditions, autoimmune diseases, systemic lupus erythematosis (SLE), myestenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome and autoimmune hemolytic anemia, allergic conditions and hypersensitivity, comprising the step of administering a compound according to Claim 1.
4. A method of treating cancers, which are mediated, dependent on or associated with pi lOδ activity, comprising the step of administering a compound according to Claim 1.
5. A pharmaceutical composition comprising a compound according to Claim 1 and a pharmaceutically-acceptable diluent or carrier.
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