Novel Sphinqosine 1 -Phosphate Receptor Antagonists

Cross- Reference to Related Applications

This application is a continuation-in-part of PCT/US13/33289 filed on March 21 , 2013 which claims the benefit of priority of U.S. Provisional Appln. No. 61 /615454 filed on March 26, 2012.

Field of the Invention

The present invention relates to sphingosine-1 -phosphate (S1 P) receptors and compounds used in the treatment and prevention of conditions associated with such receptors. More specifically, the present invention relates to the synthesis and use of new derivatives of JTE013, a known sphingosine 1 -phosphate receptor 2 (S1 P ₂ ) antagonist. S1 P ₂ antagonists are useful in the treatment of cancer, in atherosclerosis, diabetic retinopathy, and other inflammatory diseases. Among these inflammatory diseases that could be treated with an S1 P ₂ antagonist are those characterized by fibrosis including chronic lung disease, chronic kidney and liver disease, chronic heart disease, and skin diseases such as sclerosis/scleroderma. The S1 P ₂ antagonists can also be used in the treatment of glioblastoma multiforme (brain cancer), pediatric neuroblastoma, and other cancers.

Background of the Invention

Receptor antagonists are chemical compounds that act as cellular receptor ligands that do not provoke a biological response upon binding to a receptor, but rather they block or dampen agonist-mediated responses. In pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding thereto will disrupt the interaction and inhibit the function of an agonist or inverse agonist at the receptor site. Antagonists mediate their effects by binding to the active site or to allosteric sites on receptors, or they may interact at unique binding sites not normally involved in the biological regulation of the cellular receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of antagonist receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally-defined binding sites on receptors. Angiogenesis is directly involved in a number of pathological conditions such as tumor growth, inflammation and diabetic retinopathy. Current approaches to the treatment of abnormal angiogenesis in the eye include laser therapy, which destroys some retinal tissue in order to preserve some vision, and the administration of anti-VEGF antibody and/or anti-VEGF RNA aptomer. There remains a clear need for improved methods and agents for prevention and treatment of conditions involving abnormal angiogenesis and harmful pathological angiogenesis in the tissues of the eye

Vascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate.

Serum concentration of VEGF is high in bronchial asthma and diabetes mellitus. VEGF's normal function is to create new blood vessels

during embryonic development, new blood vessels after injury, muscle following exercise, and new vessels (collateral circulation) to bypass blocked vessels. When VEGF is over-expressed, it can contribute to disease. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize. Over-expression of VEGF can cause vascular disease in the retina of the eye and other parts of the body. Drugs such as bevacizumab can inhibit VEGF and control or slow those diseases.

Sphingosine 1 -phosphate (S1 P) is a lipid mediator that regulates various biological processes, such as cell proliferation, migration, survival and

differentiation. S1 P which is generated by the phosphorylation of sphingosine by sphingosine kinase 1 (Sphkl ) and sphingosine kinase 2 (Sphk2), is degraded by S1 P-specific phosphatases and a lyase. It is a ligand with high affinity for five (5) G- protein coupled S1 P receptors on the cell-surface, S1 P-m, S1 P ₂ R, S1 P ₂ R, S1 P ₄ R and S1 P ₅ R, that regulate distinct intracellular signaling pathways. S1 P-i , S1 P ₂ and S1 P ₃ receptors are widely expressed, whereas S1 P ₄ and S1 P ₅ expression is prominent in cells of the immune and nervous systems,

respectively. The S1 Pi receptor couples exclusively to G, signaling pathway, whereas S1 P ₂ and S1 P ₃ receptors couple to Gi as well as to the Gq and G-12/13 pathways. However, S1 P ₂ activates G12/13 potently, whereas S1 P ₃ activates Gq preferentially.

FTY720 is a potent immuno-modulator that has a mechanism of action which includes phosphorylation into FTY720-P, which is an agonist for four of the five S1 P receptors in T lymphocytes. It was shown previously that FTY720 is a potent modulator of lymphocyte trafficking, however, the effect of FTY720 on vascular elements was previously unknown.

It has been demonstrated that vascular endothelial cells contain enzyme systems that "activate" FTY720 and its analogs. Cultured endothelial cells such as human umbilical vein endothelial cells (HUVEC) are accepted in in vitro model systems for studying angiogenesis. Upon incubation with HUVEC conditioned medium or cell extracts, FTY720 is phosphorylated and is able to activate the endothelial cells to migrate in a pertussis - toxin sensitive manner, suggesting that it is activating the Gi-coupled S1 P receptors. It is shown herein that endothelial cell-derived sphingosine kinase-2 (SK2) is involved in the activation of FTY720 into FTY720-P.

S1 P receptors also regulate important physiological functions of the vascular system, such as vascular morphogenesis and maturation, cardiac function, vascular permeability and tumor angiogenesis. Indeed, S1 Pi null embryos die due to massive hemorrhage at E 12.5-14.5 days of gestation since the S1 Pi receptor is essential for proper stabilization of the embryonic vascular system by promoting the formation of strong N-cadherin-based junctions between

endothelial and vascular smooth muscle cells. However, mice that lack either the S1 P ₂ or the S1 P ₃ receptor are viable and fertile.

Interestingly, S1 P S1 P ₂ double null embryos showed a more severe phenotype than S1 Pi single null embryos, suggesting that S1 P ₂ receptor is also significant during embryonic vascular development. In addition, S1 P ₂ null mice are profoundly deaf due to vascular abnormalities in the stria vascularis of the inner ear and degeneration of sensory hair cells of the organ of Corti. Moreover, a mutation in the zebrafish gene miles-apart (Mil), an S1 P ₂ related receptor, results in cardiac developmental defects (cardia bifida) due to the defective migration of cardiomyocyte precursors, underscoring the significance of this receptor for the fish cardiac development. However, the role of the S1 P ₂ receptor in vascular development and pathology is an active area of study.

The process of forming new blood vessels is termed angiogenesis. During angiogenesis, vascular endothelial cells undergo orderly proliferation, migration, and morphogenesis to form new capillary networks. Under normal or non- pathologic conditions, angiogenesis occurs under well-defined conditions such as in wound healing, tissue and cellular response to ischemia, and during

embryonal and fetal development. However, persistent or uncontrolled

angiogenesis can lead to a variety of disease states or conditions and, in the case of solid tumors, may be a necessary condition to maintain the disease state. United States Patent No. 7,838,562 to Hla et. al. discloses and claims a number of agonist compounds of vascular endothelial sphingosine-1 -phosphate receptors that are asserted to be useful in the treatment of vascular permeability disorders, comprising the administration of a therapeutically effective amount of a

compound selected from the group comprising 2-amino-2-[2-(4- octaphenyl)ethyl]propane-1 ,3 diol, or 2-amino-2-methyl-4-[4-heptoxy- phenyl]butane-1 -ol

For example it is known that although blood platelets store high amounts of S1 P, the main sources of plasma S1 P are erythrocytes and endothelial cells, both of which exhibit high levels of SphK activity. The S1 P receptor mediates multiple cellular responses in many different cell types by activating the

endothelial differentiation gene family of G protein-coupled receptors (GPCR), renamed as S1 PR-i -5. Since endothelial cells express S1 PR-i , S1 PR ₂ and S1 PR ₃ activity, plasma S1 P can bind and activate these receptors in the endothelium. In adult mice and humans, the receptor is critical for the regulation of vascular permeability and lymphocyte trafficking and also couples to Gi and activates the phosphatidylinositol-3-kinase (PI3K) pathway Rac, cortical actin assembly and cell migration. Hence, this pathway is essential for vascular stabilization and inhibition of vascular permeability and therefore. The balance between S1 PR and S1 PR ₂ signaling in a specific vascular bed will determine the endothelial responses to S1 P See T. Sanchez et. al. The Critical Role of Sphingosine-1- phosphate receptor 2 (SiPR ₂ ) in Acute Vascular inflammation Blood 11 ;

467191 (2012)

The Sanchez article further indicates that during inflammation, the endothelium becomes activated with an increase in endothelial permeability and acquires a pro-adhesion and pro-coagulant phenotype that promotes the innate immune response and sustained activation results in endothelial dysfunction. Therefore, S1 PR ₂ expression in the stromal/endothelial compartment is critical for the induction of vascular permeability, and the sustained expression of pro-inflammatory and pro-coagulation markers in several vascular beds during endotoxemia. Vascular permeability disorders may be any that is associated with endothelial injury, thrombocytopenia, ischemic peripheral vascular disease, any one of a number of peripheral vascular disorders associated with diabetes, , acute respiratory distress syndrome, vascular leak syndrome, or a combination thereof, (signaling via the S1 P ₂ receptor may be involved with cytokine storm, which is an important component of the mortality associated with sepsis, and hemorrhagic fevers such as Dengue, Ebola and others). The afore-mentioned compounds can be phosphorylated by sphingosine kinase-2 into the

phosphorylated forms which serve as sphingosine-1 -phosphate receptor agonists.

PCTUS2007/024907 to Pelura et. al. broadly defines characteristics of fibrotic diseases and relates to the use of conjoint therapies for treating fibrotic and fibro- proliferative disorders, involving the administration of a combination of agents that suppress fibrocyte formation ("fibrocyte suppressors") together with agents that inhibit activation of resident collagen - producing cells such as fibroblasts, myofibroblasts, or myofibrocytes, such as antagonists of TGF-β and other pro fibrotic factors (collectively "profibrotic factor 30 antagonists"). The claimed method and compositions can be practiced using such fibrocyte suppressors as serum amyloid P (SAP), IL-12, Laminin-I, anti-Fcvk antibodies that are able to cross-link FcyR, aggregated IgG, cross-linked IgG and/or combinations thereof Designations for "SAP", "IL-12", "Laminin-I ", IgG and anti-FcyR antibodies as well as functional fragments thereof.

The pro fibrotic factor antagonists may be selected from antagonists of peptide growth factors, cytokines, chemokines, and the like. Examples of such factors that may be antagonized by the subject profibrotic factor antagonists include 1 0 - transforming growth factor type beta (TGF-β), VEGF, EGF, PDGF, IGF,

RANTES, members of the interleukin family (e.g., IL-I, IL-4, IL-5, IL-6, IL-8 and IL-13), tumor necrosis factor \type alpha (TNF-a), platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF), monocyte chemo-attractant protein type 1 (MCP-I), macrophage inflammatory protein (e.g., MIP-la, MIP-2), connective tissue growth factor (CTGF), endothelin angiotensin-ll, leptin, chemokines (e.g., CCL2, CCLI2, CXCLI2, CXCR4, CCR3, CCR5, CCR7,

SLC/CCL21 ), integrins (e.g., ull31 , u2131 uvl36, uvl33), tissue inhibitors of matrix metallo-proteinases (e.g., TIMP-I, TIMP-2) and other factors known to promote or be related to the formation, growth, or maintenance of fibrotic tissue. In certain embodiments, the profibrotic factor antagonists can be replaced with, or may be augmented with, a cytokine known to have anti-fibrotic effects itself, such as IFN- y, BMP-7, HGF or lL-IO.

Receptor tyrosine kinase inhibitors have also been shown to block growth factor signaling in tumor cells. These also display important and not yet fully characterized effects on liver non-parenchymal cells including hepatic

stellate cells (HSC) and liver endothelial cells (LEC) which modulate

fibrogenesis, angiogenesis, and portal hypertension. These then may regulate matrix and vascular changes associated with chronic liver injury.

See D. Thabut et. al. Hepatology. 54(2); 573-585 (201 1 ).

U.S. Patent No. 7,910,626 to Brinkman et. al. discloses compounds for treating chronic or congestive heart failure comprising administering to said subject a therapeutically effective amount of 2-amino-2-[2-(4- octylphenyl)ethyl]propane-1 ,3-diol in free form or a pharmaceutically acceptable salt or phosphate thereof. U.S. Patent No. 8,1 14,902 to Kiuchi et. al. discloses and claims compounds useful in the treatment or

prophylaxis of auto-immune diseases or acute and chronic rejection due to organ or tissue transplantation, graft vs host (GvH) disease due to bone marrow transplantation and the treatment or prophylaxis of allergic diseases. Particularly suitable compounds include the pharmaceutically acceptable acid addition salt , hydrate or a solvate of 2-amino-2-{2-[2'- fluoro-4'-(4- methylphenylthio)biphenyl-4-yl]ethyl}propane-1 ,3-diol and 2- amino-4-[2'-fluoro-4'-(4-methylphenylthio)biphenyl-4-yl]-2-( phosphorylo- xymethyl) butanol.

U.S. Patent Appln. No. 201 1 /0015159 also to Hla et. al. discloses and claims a number of novel agonists of vascular endothelial sphingosine-1 - phosphate receptors. Known compounds agonists such as FTY720 can be phosphorylated by sphingosine kinase-2 into the phosphorylated forms which serve as sphingosine-1 -phosphate receptor agonists. These vascular endothelial receptor agonists can be formulated into pharmaceutical compositions for treating vascular permeability disorders and unwanted vascular endothelial cell apoptosis. WO 01 /98301 to Kawasaki et. al.

relates to new pyrazolopyridine compounds having sphingosine-1 -phosphate receptor antagonistic activity and their use in pharmaceutical compositions as fibrosis remedies that contain Sph-1 -P receptor antagonistic activity or pharmaceutically acceptable salts as an active ingredient. Specifically, the invention relates to new compounds of the formula:

where in R ¹ , R ² and R ³ are each C _{1 -8} alkyl and the like; R ⁴ is hydrogen and the like; R ⁵ and R ⁶ are each independently hydrogen, Ci _-8 alkyl, Ci- ₆ alkoxy, halogen, ; X is NH-, -0-, -CH ₂ -, Y is NH- ; Z is CO- ; W is NH- ; and A is aryl or heteroaryl, These compounds are asserted to have therapeutic efficacy for liver, kidney, and lung fibrosis or arteriosclerosis caused by thickening of vascular smooth muscle.

European patent application EP 1 424 078 A1 to S. Nakade et. Al discloses and claims a number of S1 P antagonists as remedies for respiratory diseases comprising shingosine-1 -phosphate receptor controller. These compounds can be used to treat or prevent airway constriction, bronchial asthma and chronic obstructive pulmonary disease (COPD), pulmonary emphysema,

tracheostenosis, diffused panbronchialitis, or bronchitis with infection,

connective-tissues diseases or transplantation, lymphangioleiomyomatosis, adult respiratory distress syndrome (ARDS), interstitial pneumonia, lung cancer, hypersensitive pneumonitis or idiopathic interstitial pneumonia. Based on this research, other lung diseases characterized by fibrosis such as idiopathic pulmonary fibrosis (IPF) will also benefit by an S1 P ₂ R antagonist

WO 201 1 /048287 A1 to W. K. Fang et. al discloses and claims condensed ring pyridine compounds as subtype-selective modulators of sphingosine-1 - phosphate-2 (S1 P ₂ ) receptors. These compounds can be used to treat or prevent diseases and conditions consisting of ocular disease: cardiac diseases or conditions, fibrosis, pain and wounds.

U.S. Patent Appln. No. 2009/00004207 to Hla et. al. discloses and claims a number of novel agonists of vascular endothelial sphingosine-1 -phosphate receptors. Known compounds agonists such as FTY720 can be phosphorylated by sphingosine kinase-2 into the phosphorylated forms which serve as

sphingosine-1 -phosphate receptor agonists. These vascular endothelial receptor agonists can be formulated into pharmaceutical compositions for treating vascular permeability disorders and unwanted vascular endothelial cells. Known agonists such as FTY720 can be phosphorylated by sphingosine kinase-2 into the phosphorylated forms which serve as sphingosine-1 -phosphate receptor agonists. These vascular endothelial receptor agonists can be formulated into pharmaceutical compositions for treating vascular permeability disorders and unwanted vascular endothelial cell tumors.

S1 PR ₂ pathway is therefore a crucial step in the development of atherosclerotic disease. It has been reported that since sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque leading to the formation of sphingosine-1 -phosphate (S1 P), th e activation of G protein - coupled receptors enable the regulation of vascular and immune cells. S1 P receptors may therefore be involved in the control and treatment of

atherosclerosis. This occurs through S1 PR ₂ signaling in the plaque

macrophage cells and through the regulation of macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.

{Arterioscler Thromb Vase Biol. 31 ;81 -85 (2011).) Sphingolipid signaling then plays an important role in atherogenesis. In particular, enzymes in the sphingolipid metabolic cascade and the receptors for sphingolipid metabolites may regulate the complex process of atherosclerosis. S1 PR ₂ promotes

atherosclerosis. The effect of S1 P in atherosclerotic vascular disease might be different depending on the expression of different S1 P receptors in the cells of the vessel wall or infiltrating hematopoietic cells. In addition, local production of S1 P may also be important and specific inhibition of S1 PR ₂ function could lessen lesion formation and facilitate plaque stability. See A. Skoura et. al. Arterioscler Thromb Vase Biol. 31 ; 81 -85 (201 1 );U.S. Patent Appln. No. 201 1 /041287 also to Hla et al discloses a number of compounds that inhibit abnormal angiogenesis in the eye, particularly in the retina. The compounds are asserted to be effective inhibitors of the receptor activity of S1 P _2R . The compositions include the S1 P _2R antagonist and an ophthalmically acceptable excipient.

Fibrosis is scar tissue comprised of an accumulation of extracellular matrix molecules that make up, a common feature of chronic tissue injury. Pulmonary fibrosis, renal fibrosis, and hepatic cirrhosis are among the more common fibrotic diseases represent a huge unmet clinical need. New appreciation of the common features of fibrosis that are conserved among tissues has led to a clearer understanding of how epithelial injury provokes dysregulation of cell differentiation, signaling, and protein secretion. At the same time, discovery of tissue-specific features of fibrogenesis, combined with insights about genetic regulation of fibrosis, has laid the groundwork for biomarker discovery and validation, and the rational identification of mechanism-based anti-fibrotic drugs. Together, these advances herald an era of sustained focus on translating the biology of fibrosis into meaningful improvements in quality and length of life in patients with chronic fibrotic diseases.

A wide range of injuries stimulate and initiate several distinct cell ular pro- fibrotic reactions in skin cells. It has been reported for example that while injury to the skin in the most dire situations can cause cell death, this is sufficient to initiate fibrosis since i njured epithelial cells also undergo a dysregulated metabolism. This may leads to the production of reactive oxygen species (ROS) and ER stress. The cells may also be re-programmed toward a mesenchymal-like phenotype (pathologic EMT). In response to danger signals that are released or activated in response to injury, epithelial cells use cell-surface integrins to activate latent TGF , a well-characterized central mediator of tissue fibrosis. Finally, injured epithelial cells recruit and activate a variety of immune cells, which, in turn, release profibrotic cytokines, along with innate lymphoid cells, T cells and macrophages such as interleukin-13 (IL-13), IL-17, among many others. Each of these perturbations of epithelial cells ultimately leads to expansion, recruitment, and/or activation of tissue myofibroblasts, the principal source of the pathologic ECM that characterizes organ fibrosis. See S. Friedman et. al. Science 5, 167 (2013).

In other therapeutic applications of the present inventive compounds and compositions, fibrotic scar tissue may also result from exposure of healthy organ tissue, regardless of the organ type, to radiation, whether accidental,

environmental or collaterally through cancer treatment. Radiation therapy, like surgery and chemotherapy, is a mainstay of cancer treatment. The reason radiation is used to treat cancer is that it is usually toxic to the fast growing cancer cells while having little adverse effects on the slow growing and relatively radiation resistant normal body cells. Unfortunately, normal cells are often affected by radiation in a variety of ways, especially over time. One of these changes is the abnormal production of a protein fibrin, which accumulates in and damages the radiated tissue. This process is known as radiation fibrosis (RF) .

Any tissue within the radiation field can be affected including nerves, muscles, blood vessels, bones, tendons, ligaments, heart or lungs. The clinical

manifestations (i.e., signs and symptoms) that result from RF are called radiation fibrosis syndrome (RFS). RF can occur a few weeks or months after radiation treatment and continues for the duration of a cancer survivor's life. Radiation is another form of tissue injury that gives rise to fibrosis. However S1 P ₂ antagonists of the present invention may be used to pre-dose a patient before the radiation, which could provide protection from the induced fibrosis. A patent could then continue treatment with the S1 P ₂ antagonists of the present invention after the radiation to prevent the possible later development of fibrotic scar tissue.

In another therapeutic application the most common cause of failure after glaucoma surgery is scarring at the surgical site, and compounds of the present invention are also useful in the minimization and/or prevention of ocular scar formation. It is believed that vascular endothelial growth factor (VEGF) activates fibrosis, whereas VEGF inhibition results in reduced scar formation and better surgical results. Moreover prior research suggests that VEGF probably exerts its effects through induction of transforming growth factor (TGF)-pi , which may open up a new target for therapies to improve glaucoma surgical outcomes. See C. Park et. al The American Journal of Pathology. 182, 2147-2154 (June 2013). Moreover, the study shows that there is evidence that the cytokine TGF- β1 is a key mediator of wound healing and is critically involved in postoperative scarring. VEGF induces TGF-βΙ production, and inhibiting VEGF reduces TFG- β1 levels and decreases sub-conjunctival fibrosis after eye surgery wherein the conjunctival and sub-conjunctiva space is opened and the aqueous humor is driven from the anterior eye chamber, lowering the pressure within the eye. This and related studies suggest that VEGF has potential effects on the TGF- 1 β/Smad/Snail pathway involved in myoblast transformation.

In order to determine the role of the S1 P ₂ receptor in mammalian vascular development, the retinal vascular development of mice lacking the S1 P ₂ receptor was examined under physiological (normal retina development) and pathophysiological conditions (ischemia-driven retinopathy). Post-natal vascular development of the mouse retina provides an attractive model system to explore the mechanisms of angiogenesis and vascular stabilization. After birth, endothelial cells emerge from the optic disc and form the primary vasculature of the mouse retina. Vessels that grow with radial orientation are formed along the retina neuronal and astrocytic plexus. On the other hand, pathological retina angiogenesis produces abnormally growing and chaotically oriented

dysfunctional vessels that grow into the vitreous fluid as "vascular tufts" and eventually lead to vision loss. This phenotype is common in the pediatric retinopathy of prematurity (ROP) and in diabetic retinopathy of the adult.

It has been shown that the angiogenic process proceeds normally in S1 P./ _-2 R mice during normal retinal development. However, when mice were exposed to ischemic stress, S1 P ₂ R - _/ - retinas appear to have increased "physiological" intra- retinal angiogenesis and reduced "pathological" intra-vitreal neo-vascularization. It was further demonstrated that the S1 P ₂ receptor is required for inflammatory cell infiltration, induction of the pro-inflammatory and pro-angiogenic enzyme cyclo-oxygenase (COX)-2 and the suppression of the endothelial nitric oxide synthase (eNOS) which produces the vasodilator nitrous oxide (NO). This study identified S1 P signaling by the S1 P ₂ receptor as a novel target for the prevention and/or treatment of vision-threatening retinopathies.

In a paper by Schwalm Pfeilschifter and Huwiler (Biochimica et Biophysica Acta 1831 ; 239-250, (2013)) the general effects of extracellular and intracellular S1 P on the multi-step cascade of pathological fibrogenesis including tissue injury, inflammation and the action of pro-fibrotic cytokines that stimulate ECM production and deposition is examined, The current knowledge about the

involvement of S1 P is suggested as being involved in the control of signaling in the development of organ fibrosis of the lung, kidney, liver, heart and skin. It is further shown that targeting the sphingosine kinase-1 /S1 P signaling pathway offers therapeutic potential in the treatment of various fibrotic processes.

In the J. Am. Soc. Nephrol 23; 266-280 (2012), a paper by Park et al.

discloses that S1 P ₂ receptors modulate and heal renal IR injury by regulating renal proximal tubular synthesis via Rho kinase and HIF-1 a. Pre-treatment of a patient regulating renal proximal tubular SphKI synthesis via Rho kinase and HIF-la Clinically, pretreatment (before renal ischemia or hypoperfusion) or post- treatment is an excellent option for the frequent clinical scenarios where patients are subjected to renal injury during surgical procedures (e.g., kidney

transplantation, open heart surgery, aorto-vascular surgery, or liver

transplantation). A selective SI P ₂ R blockade with specific S1 P ₂ receptor antagonists provided significant renal protection when given immediately before renal ischemia or even 30 minutes after re- perfusion. The paper cited above by Park et. al. and all the previously delineated patents and references are hereby incorporated by reference .

S1 PR ₂ pathway is a crucial step in the development of atherosclerotic an d ot h e r h eart d i seases . It has been reported that since sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque leading to the formation of sphingosine-1 -phosphate (S1 P), th e activatation of G protein - coupled receptors enable the regulation of vascular and immune cells. S1 P receptors may therefore be involved in the control and treatment of

atherosclerosis. This occurs through S1 PR ₂ signaling in the plaque

macrophage cells and through the regulation of macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.

(Arterioscler Thromb Vase Biol. 31 :81 -85, (201 1 ).) Sphingolipid signaling then plays an important role in atherogenesis. In particular, enzymes in the sphingo-lipid metabolic cascade and the receptors for sphingolipid metabolites may regulate the complex process of atherosclerosis. S1 PR ₂ promotes atherosclerosis. The effect of S1 P in atherosclerotic vascular disease might be different depending on the expression of different S1 P receptors in the cells of the vessel wall or infiltrating hematopoietic cells. In addition, local production of S1 P may also be important and specific inhibition of S1 PR ₂ function could lessen lesion formation and facilitate plaque stability. See A. Skoura et. al. Arterioscler Thromb Vase Biol. 31 ; 81 -85 (201 1 ). In one embodiment of the present invention, a method of treating abnormal

angiogenesis in the eye comprises administering to an individual in need thereof an effective amount of an S1 P ₂ receptor antagonist. As used herein, the term "treating" includes the administration to an individual suffering from abnormal angiogenesis of the eye as well as the administration, both preventative^ or prophylactically to an individual at risk of abnormal angiogenesis of the eye. Administration of an S1 P ₂ receptor antagonist to an individual at risk for abnormal angiogenesis of the eye can prevent abnormal angiogenesis of the eye. In one embodiment, the individual is at risk of, or has been diagnosed with, abnormal angiogenesis of the eye.

In another embodiment of the invention, the method of treatment involves the pathological angiogenesis in the eye associated with an ocular neo-vascular disease. This type of disease is characterized by invasion of new blood vessels into the structures of the eye such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty (20) eye diseases. In age-related macular degeneration, the associated visual problems are caused by an in-growth of choroidal capillaries through defects in Bruch's membrane with proliferation of fibro-vascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, and retro-lental fibroplasia. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic kerato-conjunctivitis, Vitamin A deficiency, contact lens over-wear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens disease, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infection, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson's disease, pemphigoid, and radial keratotomy.

Other diseases associated with retinal/choroidal neo-vascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, Mycobacterial infections, lyme disease, systemic lupus erythematosis, infant retinopathy, Eales' disease, Behcet's disease, retinitis or choroiditis caused by bacterial or viral infection, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease, pars planatitis, chronic retinal

detachment, hyper-viscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other eye-related diseases include, but are not limited to, diseases associated with rubeosis (neo-vascularization of the eye) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue, including all forms of prolific vitreo-retinopathy and glaucoma.

In another embodiment, pathological angiogenesis of the eye is associated with a neoplastic eye disease. Neoplastic eye diseases include primary ocular tumors, such as uveal melanomas, melanocytomas, retinocytomas, retinal hematomas and choristomas, retinal angiomas, retinal gliomas and

astocytomas, choroidal hemangiomas, choroidal neurofibromas, choroidal hematomas and choristomas, ocular lymphomas and ocular phakomatoses; and metastatic ocular tumors related to choroidal and retinal neo-vascularization. Similar to the non-neoplastic diseases, the above tumors also share the retinal neovascularization as a key component.

Other diseases that are treatable with the compositions of the present invention include scleroderma which is a chronic systemic auto-immune disease. One of the most visible indicators of the disease is a thickening or scarring of the skin. There is a localized form which is relatively mild. It is found in only a small number of places on the skin or muscles and does not normally spread elsewhere. The patient's internal organs are usually not affected.

Systemic scleroderma occurs in two forms. Limited systemic scleroderma involves cutaneous scarring that affects mainly the hands, arms, and face. It can have complications such as: Raynaud's phenomenon, esophageal dysfunction, thickening of the skin and fingers and toes, and the presence of small dilated veins near the surface of the skin. Pulmonary arterial hypertension, the most serious complication for this form of scleroderma, can occur in up to one-third of those afflicted with a scleroderma pathology. Another form, severe diffuse systemic scleroderma, progresses quickly and, in addition to affecting large areas of the skin, often affects the kidneys, esophagus, heart, and/or lungs. Scleroderma is difficult to diagnose because it is often similar to other autoimmune diseases. There may be many mis-diagnosed or undiagnosed cases and it is currently estimated that scleroderma affects approximately 300,000 people in the US. Approximately, one third of those people have the systemic form of scleroderma and female patients outnumber male patients 4 to 1 .

Currently, there is no cure for scleroderma, but there are treatments available to help particular symptoms. Most of the compounds in development are aimed at the complications of systemic scleroderma such as Raynaud's phenomenon and pulmonary involvement. Ocular tissues are also susceptible to fibrotic diseases. Visual impairment can be the end result optical infection, inflammation or metabolic disease. This visual impairment can be caused by fibrosis in either the front or back of the eye. In the anterior part of the eye, visual loss can be caused by corneal opacification and glaucoma. In glaucoma, fibrosis of the tracts that carry intraocular fluid from the eye are believed to cause most of the damage. These tracks become scarred and fluid can build up causing an increase in intraocular pressure. The cornea is covered by a layer of cells both on the inside and outside. Fibro-vascular growths on the surface of the cornea secondary to an astigmatism or obstruction can lead to an total eye obstruction. In the back of the eye, fibrovascular scarring can contribute to macular degeneration and diabetic retinopathy. Estimates are that over 2.2 million Americans have glaucoma and of these U.S. estimates for diabetic retinopathy were approximately eight million and growing. Estimates for age related macular degeneration in Americans over 65 is 12-15 million. While many of these eye diseases have treatments, they are not always a cure and there is a continuing need for new therapies.

Cardiac fibrosis is one of the most interesting and potentially necessary areas for intervention. While the development of cardiac fibrosis is similar to that in other organs such as the lungs and kidneys, research in this area seems to be further back than fibrosis of these other organs. Cardiac fibrosis is defined as the overgrowth of the extracellular matrix (ECM) in the heart. This is present in most types of heart disease. This fibrosis causes increased rigidness which ultimately results in progressive or congestive heart failure (CHF), wherein the heart cannot pump enough blood to meet the body's needs. Over time, the fibrotic disease leaves the affected heart too weak or stiff to fill and pump efficiently.

Pathological angiogenesis and several types of inflammatory disease have been correlated with increased S1 P ₂ receptor levels. JTE013 is the only currently known and commercially available S1 P ₂ receptor selective antagonist compound. However, the anecdotal reports have confirmed that JTE013 has non-optimal in vivo characteristics. These characteristics limit the efficacy and usefulness of JTE013 in treating and preventing sphingosine-1 -phosphate- mediated diseases and disorders.

Ferrer and Hla have reported that neuroblastoma cell lines over - express S1 P ₂ receptors, and suggest that S1 P ₂ R antagonists could be used for this pediatric cancer. Van Brooklyn and Young, and others have shown the importance of S1 P ₂ receptors in the morphology of cellular invasiveness and its' proliferation in glioma cells (which are responsible for glioblastoma multiforme). JTE013 is a well known sphingosine 1 -phosphate receptor 2 (S1 P2R) class of antagonists that have the potential to be useful as anti-angiogenesis agents in cancer, atherosclerosis, and other inflammatory diseases. The present invention comprises the development of a group of sphingosine 1 -phosphate receptor 2 (S1 P2) receptor derivatives that function as S1 P ₂ R antagonists that are useful as anti-angiogenesis agents in cancer, atherosclerosis, and in other inflammatory disorders by blocking or inhibiting the signaling of the sphingosine 1 -phosphate 2 receptor (S1 P ₂ R). The S1 P ₂ R antagonists may also be useful in the treatment of these diseases characterized by fibrosis, which include cystic fibrosis, cirrhosis of the liver, endomyocardial fibrosis, Crohns disease, nephrogenic systemic fibrosis, myocardial infarctions and the like. See Trojanowska, Maria). "Mediators of Fibrosis". The Open Rheumatology Journal 6; 70-71 (2012)

Summary of the Invention

The present invention comprises the development of a specific group of compounds that function as S1 P ₂ R antagonists useful as anti-angiogenesis agents for the treatment of cancer, atherosclerosis, and in other inflammatory disorders by blocking or inhibiting a sphingosine 1 -phosphate 2 receptor (S1 P ₂ ). The S1 P ₂ antagonists are also useful in the treatment of a number of chronic fibrosis - related diseases such as renal fibrosis, cirrhosis, cardiovascular fibrosis and the like. Cancer such as glioblastoma multiforme, neuroblastoma and other cancers, are treatable as well. These compositions are effective in the inhibition of abnormal angiogenesis in the eye, particularly in the retina, based on improved in vivo characteristics such as improved pharmacokinetic parameters, increased blood concentration levels over time and greater receptor affinity, based at least in part, on its slower clearance. Also provided herein are methods for treating or preventing certain types of blindness through the administration of compositions comprising a S1 P ₂ receptor antagonist and an opthalamically - acceptable excipient. Detailed Description of the Invention

Pathological angiogenesis and several types of inflammatory disease have been correlated with increased S1 P ₂ receptor levels. In the prior art,

JTE013 is a known selective S1 P ₂ receptor antagonist compound. This compound has non-optimal in vivo characteristics,. These characteristics may limit the efficacy and usefulness of this compound in treating and preventing sphingosine-1 -phosphate-mediated diseases.

The present invention then, comprises compositions and methods for the inhibition of abnormal angiogenesis in the eye, particularly in the retina. Also provided herein are methods for treating or preventing certain types of blindness.

Further provided are compositions comprising a S1 P ₂ receptor antagonist and an opthalamically - acceptable excipient with improved in vivo characteristics. The present invention comprises compositions and methods for the inhibition of abnormal angiogenesis in the eye, particularly in the retina. Also provided herein are methods for treating or preventing certain types of blindness. Further provided are compositions comprising a S1 P ₂ receptor antagonist and an opthalamically - acceptable excipient, with improved in vivo characteristics, based on metabolic stability and/or increased duration of action.

Sphingosine-1 -phosphate (S1 P) is a multi-functional lipid mediator that signals via the S1 P family of G protein-coupled receptors (S1 PR). S1 P is known to regulate vascular maturation, permeability and angiogenesis. For example,

S1 P is known to be a stimulator of angiogenesis, i.e., new blood vessel growth.

As used herein, the terms S1 P ₂ R, S1 P ₂ R, S1 P ₂ receptor and S1 P ₂ receptor are used interchangeably to mean the sphingosine-1 -phosphate receptor 2.

A number of novel sphingosine 1 -phosphate receptor 2 (S1 P ₂ ) antagonists were prepared. Details for making antagonist compounds are provided in the examples. These compounds showed unexpectedly enhanced stability to liver microsomes using a well-established in vitro model for in vivo metabolism and metabolic stability. The compounds have utility as therapeutic drugs for the treatment and prevention of conditions or diseases mediated by S1 P receptors.

Such conditions and diseases include, but are not limited to a wide variety of inflammatory diseases and conditions as well as diseases and conditions mediated by angiogenesis processes.

In one embodiment of the present invention, the novel sphingosine-1 -phosphate receptor 2 antagonist compounds may be used to treat or prevent

atherosclerosis and conditions associated therewith, including cardiovascular and cerebral vascular diseases, such as, for example, myocardial infarction, stroke, angina and peripheral vascular disease. These compounds also may be used to treat or prevent cytokine storms, a hallmark of sepsis, septic shock and hemorrhagic fevers such as Dengue, Ebola, etc,, as well as a wide variety of other diseases including diabetes, liver cirrhosis, vascular diseases with increased permeability, and allergic reactions. The compounds may also have use in diseases characterized by fibrosis, including excess matrix deposition. The general method of synthesis of these compounds is as follows:

Scheme 1

Dioxane

The preferred compounds of the present invention comprise those of the general structure

Wherein each consists of the following: Compound No.

-allyl -H 4-substituted-2,6-dichloropyridine

2. -CH ₃ -CH ₂ CO ₂ H 4-substituted-2,6-dichloropyridine 3. -allyl -CH ₂ CO ₂ H 4-substituted-2,6-dichloropyridine

-CH; -allyl 4-substituted-2,6-dichloropyridine

5.

The following examples are provided to more specifically set forth and define the process of the present invention. It is recognized that changes may be made to the specific parameters and ranges disclosed herein and that there are a number of different ways known in the art to change the disclosed variables. And whereas it is understood that only the preferred embodiments of these elements are disclosed herein as set forth in the specification and drawings, the invention should not be so limited and should be construed in terms of the spirit and scope of the claims that follow herein.

Example 1

sis of 5-amino-1-allyl-3-methylpyrazole

β-Amino-crotononitrile (4.2g, 50 mmol) and allylhydrazine (3.6g, 50 mmol) were dissolved in isopropanol (20 mL) and the solution was gradually heated to reflux under nitrogen atmosphere for 5 h. The reaction mixture was concentrated and purified by column chromatography using 2.5% methanol in DCM to give 5- amino-1 -allyl-3- methylpyrazole (4.2 g) as a syrup. ¹ H 300 MHz NMR (CDCI ₃ ): δ 6.00-5.88 (1 H, m), 5.35 (1 H, s), 5.23-5.04 (2H, m), 4.56 (2H, dd, J=3.6 Hz, 1 .5Hz), 3.47 (2H, bs) 2.15 (3H, s).

b. Synthesis of 1 W-6-hydroxy-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-d] pyridine

Ethyl isobutyryl acetate (10.5g, 66.5 mmol) was added to a solution of 5-amino-1 - allyl-3-methylpyrazole (9.00g, 65.7 mmol) in propionic acid and heated to reflux for 20 h. After cooling, ethyl acetate (80 mL) was added and heated to reflux for 1 h. The solvent was evaporated and the residue was purified by column chromatography using 5% methanol in DCM to give 1 H-6-hydroxy-4-isopropyl-1 - allyl-3-methylpyrazolo[3,4-£>]pyridine as colorless crystals (1 .1 g, 7%). ¹ H 300 MHz NMR (CDCIs) δ 6.16 (1 H, s), 6.06-5.97 (1 H, m), 5.30-5.22 (2H, m), 4.95 (2H, dd, J=3.6 Hz, 1 .5Hz ), 3.31 -3.27 (1 H, m), 2.52 (3H, s), 1 .31 (6H, d, J=7 Hz). Example 2

Synthesis of 2, 6-dichloropyridyl-4-isocyanate

a) Synthesis of 2,6-dichloropyridine-4-carbonylazide

Diphenylphosphoryl azide (DPPA) (5 mL, 23.2 mmol) was added to a solution of 2,6-dichloroisonicotinic acid (4.05 g, 21 mmol) and triethylamine (3.8 mL, 27.5 mmol) in ethyl acetate (40 mL) at 0-5° C, and stirred for 20 h at room

temperature. Ethyl acetate was added for dilution and the organic layer was washed with water. The organic layer dried over Na ₂ S0 ₄ , filtered and

concentrated under vacuum to get crude product of 2, 6-dichloropyridine-4- carbonylazide (7.08 g). It was dissolved in ethyl acetate and treated with activated carbon. After filtration and evaporation, 2,6-dichloropyridine-4- carbonylazide (4.57g) was obtained as colorless crystals. The 2, 6- dichloropyridine-4-carbonylazide (4.21 g) that was obtained from the above procedure was dissolved into dry toluene (40 mL) and the solution was heated for 4 h at 100°C to give 2,6-dichloropyridyl-4-isocyanate. It was stored as a solution at 0°C.

Triethyl amine (2.17 mL, 3.0 eq, 15.57 mmol) was added to a solution of 1 H-6- hydroxy-4-isopropyl-1 -allyl-3-methylpyrazolo[3,4-£>] pyridine (1 .20 g, 5.19 mmol) in dichloromethane (30 mL) and cooled to -10°C. Trifluoromethanesulphonic anhydride (1 .30 mL, 7.78 mmol, 1 .5 eq) was added to this cold solution dropwise The solution was stirred for 45 minutes or until completion by TLC monitoring. The reaction was quenched with water and extracted with dichloromethane (3 x 10 mL). The mixture was concentrated, dried and purified by column

chromatography (10:1 Hexane/Ethyl Acetate) to give 1 .32 g of the desired product as a yellow oil in 70% yield. ¹ H 300 MHz NMR (CDCI ₃ ): δ 6.75 (1 H, s), 6.06-5.95 (1 H, m), 5.29-5.22 (2H, m), 4.95 (2H, dd, J=3.0 Hz, 1 .5Hz), 3.65-3.60

) and 1 .38 (6H, d, J=6.6 Hz).

The compound above (200 mg, 0.551 mmol), t-butyl carbazate (87 mg, 0.661 mmol, 1 .2 eq), oven dried cesium carbonate (431 mg, 1 .322 mmol, 2.4 eq), Xantphos (15% mol, 48 mg, 0.082 mmol) and Pd ₂ (dba) ₃ (5% mol, 26 mg, 0.0276 mmol) were all placed in an oven dried flask under nitrogen. This reaction mixture was dissolved in dry de-gassed dioxane and heated at 65°C for 12 h or until completion by TLC monitoring. Material was concentrated and subjected to column chromatography (3:1 Hexanes/Ethyl Acetate) to give 133 mg of the desired hydrazine derivative in 70% yield ¹ H 300 MHz NMR (CDCI ₃ ) δ 7.34 (1 H, s), 6.01 (1 H, m), 5.22 (1 H, m), 5.16 (1 H, m), 4.97 (2H, d, J= 6 Hz), 4.34 (2H, bs),

(9H, s), 1 .34 (3H, s) 1 .36 (3H, s).

A solution of 2,6-dichloro-pyridyl-4-isocyanate (2.0 eq, 1 .3 mL, 0.771 mmol) in toluene was added to the above hydrazine derivative (133 mg, 0.385 mmol) in THF (5 mL) and stirred for 12 h or until completion by TLC monitoring. The crude reaction was concentrated, purified by column chromatography (2:1 to 1 :1 Hexanes/ Ethyl Acetate) to afford the desired product as a yellow solid. (193 mg, 94% yield). ¹ H 300 MHz NMR (CDCI ₃ ) δ 9.56 (1 H, bs), 7.44 (2H, s), 7.25 (1 H, s), 7.10 (1 H, bs) 6.10-5.97 (1 H, m), 5.24(1 H, dd, J=7.5 Hz, 1 .5Hz), 5.01 (1 H, dd, J=7.5 Hz, 1 .5Hz), 4.98-4.95 (2H, m), 3.69-3.60 (1 H, m), 2.70 (3H, s), 1 .55 (9H, s), 1 .40 (6H, d, J=7Hz).

Hydrochloric acid (2M in ether, 5ml_, 10 mmol) was added to a solution of the above Boc-compound (500 mg, 0.94 mmol) in diethyl ether (10 mL) at 0° C, the reaction was stirred for 12 h at room temperature. The reaction mixture concentrated, diluted with DCM (20 mL), washed with sat NaHC0 ₃ solution (15 mL), dried and concentrated to afford crude product (424 mg). The pure product (Compound-1 ) was isolated after purification by using preparative TLC (30% EtOAc in hexanes) as a white solid (140 mg, yield 34%). ¹ H 300 MHz NMR (CDCI ₃ +CD ₃ OD) δ 7.41 (2H, s), 6.38 (1 H, s(1 H, m), 5.07-4.97 (2H, m), 4.77 (2H, d, J=6Hz), 3.41 -3.29 (1 H, m), 2.51 (3H, s), 1 .24 (6H, d, J=7Hz), MS (m/z MH+) 434.2.

N-(1 H-4-isopropyl-1 , 3-dimethylpyrazolo[3,4-b]pyridine-6-yl) amino-N'-(2,6- dichloropyridine-4-yl) urea (JTE013)

The above titled compound was prepared as reported in literature (WO

01 /98301 ) Synthesis of Compound-2

tert-Butyl bromoacetate (500 mg, 2.60 mmol) was added to a solution of N-(1 H- 4-isopropyl-1 , 3-dimethylpyrazolo[3,4-b]pyridine-6-yl) amino-N'-(2,6- dichloropyridine-4-yl) urea (125 mg, 0.306 mmol) in dry DME (1 ml_) and the reaction mixture heated overnight at 100° C. It was then diluted with DCM (20 ml_) and washed with aqueous NaHC0 ₃ (1 x 10 ml_) followed by water (2 x 10 ml_). The organic layer was dried over Na ₂ S0 ₄ , and concentrated under reduced pressure. The residue was purified by column chromatography (5% MeOH/ CH ₂ CI ₂ ) to give the desired pure product as a white solid (25 mg). ¹ H 300 MHz NMR (CD ₃ OD) δ 7.58 (2H, s), 6.78 (1 H, s), 4.87 (2H, bs), 3.99 (3H, s), 3.65 (1 H, m), 2.81 (3H, s), 1 .50 (9H, s), 1 .37 (6H, d, J=6.6Hz), MS (m/e) 523 (MH+).

The above t-butyl ester (15 mg, 0.028 mmol) dissolved in DCM (1 ml_) was added to a solution of trifluoromethanesulfonic acid (15 mg, 0.10 mmol) in dry DCM (3 ml_) at 0°C. The reaction mixture was stirred for 1 h at room

temperature. After completion of the reaction, as indicated by TLC, NaHC0 ₃ (9 mg) and MeOH (0.5 ml_) were added to the reaction mixture and stirred for 15 min. The reaction mixture was concentrated under vacuum, co-evaporated with DCM and triturated with hexanes to get the desired product (Compound-2) as a brown colored solid (25 mg, contains 34% compound and 66% sodium triflate). ¹ H 300 MHz NMR (CD ₃ OD) δ 7.58 (2H, s), 6.78 (1 H, s), 5.49 (2H, d, J= 7.5Hz), 4.00 (3H, s), 3.44-3.57 (1 H, m), 2.82 (3H, s), 1 .37 (6H, d, J=6.6Hz), MS (m/z MH+) 466.2.

Cesium carbonate (140 mg, 0.4 mmol) was added to a solution of compound 1 (180 mg, 0.328 mmol) and tert-butyl bromoacetate (80 mg, 0.41 mmol) in dry DMF (1 mL). The reaction mixture stirred for 3 h at room temperature. It was then diluted with DCM (20 mL), washed with water (3 x 10 mL), dried over Na ₂ S0 ₄ , and concentrated under reduced pressure. The desired product was isolated after purification using prep TLC (25% EtOAc/Hexanes) followed by trituration with isopropyl ether as colorless crystals (45 mg). ¹ H 300 MHz NMR (CDCIs): δ 8.49 (1 H, bs), 7.49 (2H, s), 7.01 (1 H, bs), 6.40 (1 H, s), 6.02-5.96 (1 H, m), 5.21 -5.15 (2H, m), 4.97 (1 H,d, J=18Hz), 4.96-4.88 (2H, m), 3.67(1 H,d, J=18Hz), 3.55-3.42 (1 H, m), 2.62 (3H, s), 1 .47 (9H, s), 1 .32 (6H, d, J=7Hz), MS (m/z MH+) 447.

The Boc-protected compound above (22 mg, 0.04 mmol) dissolved in DCM (1 mL) was added to Trifluoromethane sulfonic acid (22 mg, 0.14 mmol) in dry DCM (4 ml_) at 0°C. The reaction mixture was stirred for 1 h at room temperature. After completion of the reaction, as indicated by TLC, NaHC0 ₃ (1 1 mg) and MeOH (0.5 ml_) were added to the reaction mixture and stirred for 15 min.

Reaction mixture was concentrated under vacuum, co-evaporated with DCM and triturated with hexanes to get the desired product (Compound-3) as a light brown colored solid (36 mg, contains 34% compound and 66% sodium triflate). ¹ H 300 MHz NMR (CD ₃ OD) δ 9.69 (1 H, bs), 7.67 (2H, s), 6.64 (1 H, s), 5.97-5.91 (1 H, m), 5.09-4.85 (6H, m), 3.57-3.34 (1 H, m), 2.60 (3H, s), 1 .36 (6H, d, J=7Hz), MS (m/e MH+) 490.

Compound 4 was prepared as in the scheme above. MS (m/e MH+) 447. Synthesis of Compound-5

Synthesis of 1 /-/-6-hydroxy-4-trifluoromethyl-1 -allyl-3-methylpyrazolo[3,4-fo] pyridine

Ethyl-4,4,4-trifluoroacetoacetate (5.6g, 30 mmol) in propionic acid (10 ml_) was added to a solution of 5-amino-1 -allyl-3-methylpyrazole (4.2 g, 30 mmol) in propionic acid and heated (150° C) to reflux for 23 h. After cooling, ethyl acetate (40 ml_) was added and heated to reflux for 1 h. On slow cooling, crystals of the desired compound were deposited, which were filtered, washed ethyl acetate and dried under reduced pressure. Thus 1 H-6-hydroxy-4-trifluoromethyl-1 -allyl-3- methylpyrazolo[3,4-£>] pyridine (5.1 gm, 66%) was obtained as white crystals, ¹ H 300 MHz NMR (CDCI ₃ ) δ 6.63 (1 H, s), 6.07-5.94 (1 H, m), 5.29-5.23 (2H, m), 4.96 2H, dd, J=3.0 Hz, 1 .5Hz), 2.51 (3H, s) and 1 .85(1 H, bs).

Triethyl amine (1 .77g, 2.42 ml_, 3.0 eq, 17.52 mmol) was added to a solution of 1 /-/-6-hydroxy-4-trifluoromethyl-1 -allyl-3-methylpyrazolo[3,4-fo] pyridine (1 .50 g, 5.84 mmol) in dichloromethane (30 ml_) and cooled to -10°C.

Trifluoromethanesulfonic anhydride (1 .48 ml_, 2.47 g, 8.75 mmol, 3.0 eq) was added to this cold solution. The solution was stirred at this temperature for 45 minutes or until completion by TLC monitoring. The reaction was quenched with water and extracted with dichloromethane (3 x 10 ml_). The organic layer was concentrated, dried and purified by column chromatography (10:1 Hexane/Ethyl Acetate) to give 2.15 g of the desired product as yellow oil. 96% yield ¹ H 600 MHz NMR (CDCIs) δ 6.60 (1 H, s), 6.03 (1 H, m), 5.30 (2H, m), 4.98 (2H, dd, J=6.0 Hz, 1 .5 Hz), 2.49 (3H, s).

The above compound (215 mg, .553 mmol), t-butyl carbazate (88 mg, 0.664 mmol, 1 .2 eq), oven dried cesium carbonate (432 mg, 1 .327 mmol, 2.4 eq), Xantphos (15% mol, 48 mg, .083 mmol) and Pd ₂ (dba) ₃ (5% mol, 25 mg, 0.0276 mmol) were all placed in a flask dry under a nitrogen atmosphere. This reaction mixture was dissolved in dry degassed dioxane and heated at 65°C for 12 h or until completion by TLC monitoring. The reaction mixture was concentrated and the material was subjected directly to column chromatography (3:1

Hexanes/Ethyl Acetate) to give 85 mg of the desired product in 42% yield ¹ H 300 MHz NMR (CDCIs) δ 7.93 (1 H, s), 6.04 (1 H, m), 5.24 (2H, m), 5.02 (2H, dd, J=6.0

3H, s), 1 .58 (9H, s).

A solution of 2,6-dichloro-pyridyl-4-isocyanate (-2.0 eq, 1 .0 ml_, 0.458 mmol) in toluene was added to a solution of the above hydrazine derivative (85 mg, 0.229 mmol) in THF (5 ml_) at 0-5°C. The solution was stirred for 12 h or until completion by TLC monitoring. The reaction was concentrated and directly purified by column chromatography (2:1 to 1 :1 Hexanes/ Ethyl Acetate) to afford 122 mg of the desired Boc product in 95% yield ¹ H 600 MHz NMR (CDCI ₃ ) δ 8.75 (1 H, bs), 8.06 (1 H, bs),7.72 (1 H, s), 7.32 (2H, s), 6.01 (1 H, m), 5.24 (2H, d J=5.6 Hz, 2.5 Hz), 5.00 (2H, d,J=5.4Hz), 2.64 (3H, s), 1 .57 (9H, s).

Hydrochloric acid (2M sol in ether, 1 .0 ml_, 2 mmol) was added to a solution of the above Boc derivative (44 mg, 0.095 mmol) in dry dichloromethane (5 ml_) at 0° C. The reaction mixture was stirred for 12 h at room temperature. The reaction mixture was filtered and the crystalline compound thus obtained was washed with isopropyl ether and dried in a vacuum oven. Compound-5 was isolated as a colorless solid (26 mg, yield 72%). ¹ H 300 MHz NMR (CD ₃ OD) δ 7.60 (2H, s), 6.93 (1 H, s), 5.97-5.88 (1 H, m), 5.08-4.92 (2H, m), 4.91 -4.88 (2H, m), 2.49 (3H, s), MS (m/e MH+) 460. mpound-6

POBr ₃ (365 mg, 1 .29 mmol) was added to a solution of 1 H-6-hydroxy-4- isopropyl-1 -allyl-3-methylpyrazolo[3,4-£>] pyridine (200 mg, 0.86 mmol) in anisole (1 mL). The reaction mixture was heated for 3 h at 130° C. After completion of reaction as indicated by TLC, reaction mixture diluted with toluene (10 mL). It was washed with saturated NaHC0 ₃ (10 mL) followed by saturated aqueous NaCI (10 mL), dried over Na ₂ S0 ₄ and filtered. The filtrate was evaporated under reduced pressure and the resulting residue was purified by column

chromatography using 10% ethyl acetate/hexanes as eluent. The desired product (compound 6) was obtained as yellow oil (108 mg, Yield: 42%). ¹ H 300 MHz NMR (CDCI ₃ ) δ 7.03 (1 H, s), 6.15 (1 H, m), 5.22-5.16 (2H, m), 5.02 (2H, dd, J=6.0

-3.45 (1 H, m), 2.62 (3H, s), 1 .38 (6H, d, J=6.0 Hz).

Potassium - ter t- butoxide (1 M solution, 1 mL) was added to a solution of ethyl- /V-hydroxyacetimidate (105 mg, 102 mmol) in dry DMF (1 mL) at 0° C, and stirred for 2.5 h. The above bromo compound (100 mg, 0.34 mmol) dissolved in DMF (1 mL) at 0° C was carefully added and stirring continued for 12 h at room

temperature. The reaction mixture was quenched with water (5 mL) and extracted with ethyl acetate. The organic layer was dried over Na ₂ S0 ₄ and the solvent was removed under reduced pressure. The residue was purified by column chromatography (20% EtOAc/Hexanes) to give acetohydroxamate derivative as colorless oil (80 mg, Yield: 74%). ¹ H 300 MHz NMR (CDCI ₃ ) δ 6.91 (1 H, s), 5.99 (1 H, m), 5.23-5.17 (2H, m), 4.97 (2H, dd, J=6.0 Hz, 1 .5Hz),

H, m), 2.63 (3H, s), 2.19 (3H, s), 1 .39-1 .33 (9H, m).

Sulfuric acid (0.01 mL) was added to a solution of above acetohydroxamate compound (80 mg, 0.25 mmol) in methanol (4 mL). The reaction mixture was stirred for 2.5 h at room temperature. After completion of the reaction as indicated by TLC, it was neutralized with Na ₂ C0 ₃ (108 mg), added water (10 mL) and extracted with EtOAc (3 x 10 mL). The organic layers were combined, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The crude amine (60 mg, Yield: 96%) used for next reaction without further purification. ¹ H 300 MHz NMR (CDCIs) δ 6.55 (2H, bs), 6.49 (1 H, s), 6.08-5.97 (1 H, m), 5.23-5.16 (2H, m), 4.96 (2H, dd, J=6.0 Hz, 1 .5Hz), 3.52-3.47 (1 H, m), 2.62 (3H, s), 1 .33 (6H, d, J=7.0

Hz).

A solution of 2,6-dichloro-pyridyl-4-isocyanate (-2.0 eq, 1 .1 mL, 0.48 mmol) in toluene was added to the above crude amine above (60 mg, 0.24 mmol) in dry THF (4 mL) at 0°C. The solution was stirred for 12 h or until completion by TLC monitoring. The solvent was removed under reduced pressure and the desired compound. 6 was isolated by column chromatography (EtOAc/Hexane) as a white solid (70 mg, Yield: 66%). ¹ H 300 MHz NMR (CDCI ₃ ) δ 8.67 (1 H, s), 7.72 (1 H, s), 7.48(2H, s), 6.63 (1 H, s), 5.99-5.94 (1 H, m), 5.21 -5.13 (2H, m), 4.94 (2H, dd, J=5.0 Hz, 1 .5Hz), 3.60-3.56 (1 H, m), 2.65 (3H, s), 1 .38 (6H, d, J=7Hz), MS (m/e MH+) 435.

Using the above methods the following compounds can also be prepared The methods of synthesi shown below .

Compound Ri R ₂ R? No. 7 -CH ₂ C0 ₂ Et 4-substituted-2,6-dichloropyridine

No. 8 ^■ CH ₃ -CH ₂ CONH ₂ 4-substituted-2,6-dichloropyridine No.9 CH ₃ -CH ₂ CH ₂ OH 4-substituted-2,6-dichloropyridine No. 10 -CH ₃ -CH ₂ S0 ₃ H 4-substituted-2,6-dichloropyridine No. 11 -CH ₃ -CH ₂ S0 ₂ NH ₂ 4-substituted-2,6-dichloropyridine No. 12 -CH ₃ -CH ₂ P0 ₃ H ₂ 4-substituted-2,6-dichloropyridine

No.13 -CH ₂ -CH=CF ₂ -H 4-substituted-2,6-dichloropyridine No. 14 -CH ₂ CF=CH ₂ -H 4-substituted-2,6-dichloropyridine

No. 15 (E) -CH ₂ -CH=CFH -H 4-substituted-2,6-dichloropyridine No. 16 (Z) -CH ₂ CH=CFH -H 4-substituted-2,6-dichloropyridine No. 17 -CH ₂ -cyclopropyl -H 4-substituted-2,6-dichloropyridine No .18 -cyclopropyl -H 4-substituted-2,6-dichloropyridine No. 19 -allyl -H 4-substituted-3,5-bis-trifluoromethylphenyl No. 20 -CH ₃ -CH ₂ C0 ₂ H 1 -substituted-3,5-bis- trifluoromethylphenyl No. 21 -allyl -H 1 -substituted-2, 4-bis-trifluoromethylphenyl No. 22 -Me -CH ₂ C0 ₂ H 1 -substituted-2,4-bis- trifluoromethylphenyl

No. 23 -allyl -H 3-substituted-5-trifluoromethyl-2-pyridone

No. 24 -Me -CH ₂ C0 ₂ H 3-substituted-5-trifluoromethyl-2-pyridone

No.25 -allyl -H 1 -substituted-3, 5-dichlorophenyl

No.26 -Me -CH ₂ C0 ₂ H 1 -substituted-3, 5-dichlorophenyl

N-(2,6-Dichloropyridin-4-yl)-2-(2-hydroxyethyl)-2-(4-isop ropyl-1 ,3-dimethyl- 1 H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide (compound 9) :

2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6- yl)hydrazinyl)ethan-1 -ol :

2-Hydrazinoethanol (5.1 mL, 149.25 mmol) was added to a solution of 1 H-6- bromo-4-isopropyl-1 , 3-dimethylpyrazolo[3,4-b]pyridine (2.0 g, 7.46 mmol) in ethanol (10 mL), and heated under reflux overnight. After cooling, it was stirred for four hours in an ice bath. The crude material was filtered and the obtained solid was triturated with a 50% aqueous ethanol (5 mL), filtered and dried under reduced pressure at 60° C to give 2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4- b]pyridin-6-yl)hydrazinyl)ethan-1 -ol as white solid, 1 .3 g (4.94 mmol, 66 % yield). ¹ H NMR (300 MHz, CDCI ₃ ) δ 6.76 (s, 1 H), 4.92 (brs, 2H), 4.20-3.96 (m, 2H), 3.94-3.86 (m, 2H), 3.84 (s, 3H), 3.52- 3.45 (m, 2H), 2.58 (s, 3H), 1 .25 (d, 6H, J = 6.6 Hz).

2,6-Dichloropyridine-4-carbonylazide (1 .0 g, 4.61 mmol) was dissolved in toluene

(10 ml_) and the solution was stirred for 4 h at 100°C. After cooling to 0-5° C, a

solution of 2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6- yl)hydrazinyl)ethan-1 -ol (750 mg, 2.85 mmol) in THF (10 ml_) was added and

stirred for 18 hours at rt. After concentrating, the crude material was purified by

column chromatography using gradient elution (0-20% methanol/DCM) to give N- (2,6-dichloropyridin-4-yl)-2-(2-hydroxyethyl)-2-(4-isopropyl -1 ,3-dimethyl-1 H- pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1 -carboxamide as a white solid, 624 mg

(1 .38 mmol, 30% yield). ¹ H NMR (300 MHz, CDCI ₃ ) δ 9.28 (brs, 1 H), 7.47 (s, 2H),

6.70 (brs, 1 H), 6.58 (s, 1 H), 4.70-4.50 (m, 1 H), 4.30-4.20 (m, 1 H), 4.08-3.98 (m,

1 H), 3.91 (s, 3H), 3.53-3.46 (m, 2H), 2.92-2.70 (m, 1 H), 2.60 (s, 3H), 1 .32 (d, 6H,

J = 6.6 Hz). HRMS (ESI): mass calculated for CigHssClsNyOs [M+H] ⁺ , 452.1290

found, 452.1480.

Compounds 27-33 can be prepared as set forth and disclosed in published patent application. WO201 1041287A1 which is hereby incorporated by reference. The synthesis of compounds 27 and 33 are described below. These comprise the structure

wherein:

Compound R1 R2

No. 27 -allyl H No. 28 -CH ₃ -CH ₂ C0 ₂ H

No. 29 -CH3 -CH ₂ CONH ₂

No. 30 -CH3 -CH ₂ CH ₂ OH

No. 31 -CH ₂ CF=CH ₂ H

No. 32 -CH ₂ CH=CF ₂ H

No. 33 -CH ₂ CH ₂ CH ₂ OH H

Synthesis of compounds 27 and 33

Preparation of compound A-2

LDA (247 mL, 1 M) was slowly added to a solution of compound A-1 (20 g, 206 mmol) in THF (300 mL) at -78°C. The solution was stirred at -78 °C for 1 h, then acetone(100 mL) was added. The reaction was stirred for 1 h, then quenched with NH ₄ CI (aq.) and extracted with ethyl acetate. The organic layer was washed by brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give crude compound A-2.

Preparation of compound A- 3

Red-P (20 g), was added to a solution of compound A-2 (20 g, 129 mmol) in 40% HI (aq. 250ml_). The solution was heated to reflux at 140 °C for 2 days, the solution was quenched with 5N NaOH (aq.), and extracted with ethyl acetate. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ , concentrated and purified by flash chromatography (petroleum ether:ethyl acetate 20:1 ) to give compound A-3 (5 g, yield = 27%).

Preparation of compound A-4

n-BuLi (17 mL, 2.5M) was slowly added to a solution of compound A-3 (5 g,36 mmol) in THF (150 mL) at -78 °C. The reaction was stirred at -78 °C for 1 h, then comp B (7.5 g, 43.2 mmol) was added slowly, the reaction was stirred at -78 °C for 2h, then quenched with NH ₄ CI(aq), and extracted with ethyl acetate. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ and

concentrated to give crude compound A-4 (10 g)

Preparation of compound A-5

lodoxybenzoic acid (IBX, 18 g, 63 mmol) was added to a solution of compound A-4 (10g,31 .5mmol) in 40 ml DMSO. The solution was stirred at 60 °C for 3h ,then cooled 0 °C, quenched with NaHC0 ₃ (aq) , and extracted with ethyl aceate. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ and concentrated. The compound was purified by flash chromatography (petroleum ether:ethyl acetate=20:1 -10:1 ) and gave compound A-5 (6g, yield 60%).

Preparation of compound A-6

MeNHNH ₂ (10 mL) was added to a solution of compound A-5 (6 g,19.4 mmol) in glycol (3 mL). The solution was heated to reflux at 140 °C overnight. Water (50 ml) was added and the solution was extracted with dichloromethane. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ , concentrated, and purified by flash chromatography to give compound A-6 (3 g, yield 50% ). Preparation of compound Bn-A-6 6M HCI (10 ml_) was added to a solution of compound A-6 (3 g, 9.5 mmol) in THF (50 ml_). The reaction was stirred at RT for 1 h, quenched with

NaHC0 ₃ (aq.), extracted with ethyl acetate. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give comp A-6-1 . NaH (760 mg, 19 mmol) was added to compound A-6-1 in THF (50 ml_) , the solution was stirred at 60 °C for 2h. The solution was cooled to 0°C and BnBr( 3.23 g ,19 mmol). The solution was warmed to RT and stirred overnight. Water (20 ml_) was added, and the solution extracted with ethyl acetate. The organic layer was washed with brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give compound Bn-A-6 (2.7 g, yield 90%).

Preparation of compound A-7

m-Chloroperbenzoic acid (MCPBA, 3.7 g,16.7 mmol) was added to a solution of compound Bn-A-6 (2.7 g,8.4 mmol) in CHCI ₃ (30 ml_) at 0 °C. The solution was heated to reflux at 90 °C for 3h. The solution was quenched with NaHC0 ₃ (aq.) and extracted with DCM. The organic layer was washed by brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give crude compound A-7 (3g)

Preparation of compound A-8

POCI ₃ (5ml_) was added to a solution of compound A-7(3 g, 8.8 mmol) in toluene (30 ml_). The the solution was stirred at 90 °C for 3h, then concentrated and quenched with NaHC0 ₃ (aq.), extracted with ethyl acetate. The organic layer was washed by brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give compound A-8 (800 mg, yield 25%).

Preparation of compound A-9

To a solution of compound A-8 (250 mg, 0.7mmol), and NH ₂ NH ₂ (aq) in

EtOH(1 ml) was stirred at 120 °C for 12h in a CEM Microwave. The solution was cooled and extracted with ethyl acetate. The organic layer was washed by brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give crude compound A-9 (200mg).

Preparation of compound A-10

2, 6-Dichloro-4-isocyanate pyridine (compound C, 160 mg ,0.85 mmol) was added to a solution of compound A-9 (200 mg,0.56 mmol) in THF (5 ml_). The solution was stirred at RT for 30min. Methanol was added and the resulting solid was filtered. The filtrate was concentrated and compound A-10 was purified by preparative TLC (100 mg, yield 33%).

Preparation of compound 33

Trimethyl silyl iodide (0.5 mL) was slowly added to a solution of A-10 (100 mg,0.18 mmol) in dichloromethane. The solution was stirred at RT for 30min , quenched with NaHC0 ₃ (aq) , and extracted with DCM. The organic layer was washed by brine, dried with anhydride Na ₂ S0 ₄ and concentrated to give compound 33 (40mg, yield 48% ). ¹ HNMR (400MHz, CDCI3) δ: 8.68 (1 H, br), 7.55 (1 H, br), 7.40 (2H, s), 7.00 (1 H, s), 6.71 (1 H, s), 4.18 (3H, s), 3.62-3.55 (1 H, m), 3.56 (2H, J = 5.6 Hz, t), 3.04 (2H, J = 6.4 Hz, t), 2.00-1 .98 (2H, m), 1 .36 (6H, J = 6.4 Hz, d); MS(ESI): 452[M+H] ⁺ .

Preparation of compound 27

Burgess Reagent (80 mg, 0.33 mmol) was added to a solution of 33 (50 mg, 0.1 1 mmol) in THF (4 mL). The solution was stirred at RT overnight. The reaction was warmed 60 °C for 1 h and then the solution was concentrated and purified by prep-HPLC to give compound 27 (2 mg).

Using the above methods the following compounds can also be prepared

(methods below table).

42 CH ₂ CH ₂ OH 44 -CH ₂ CH ₂ OH

General methods for synthesis of compounds 34-45

General Procedure for Conversion of the Acid to Isocyanate.

Method A.

The appropriate acid (1 .0 eq, 0.5 mmol) was dissolved in freshly distilled Ethyl Acetate (5 mL) and the solution was cooled to 0° C. To this solution was added triethylamine (1 .3 eq, 0.65 mmol, 0.09 mL) followed by diphenylphosphoryl azide (1 .1 eq, 0.55mmol, 0.12 mL). The reaction was allowed to warm to room temperature and stirred overnight. It was then quenched with water and extracted (2 x 25 mL) with ethyl acetate. Organic layer was washed with water (20 mL), followed by brine (20 mL), dried with sodium sulfate, and concentrated to half the volume. Toluene (10 mL) was added and the remainder ethyl acetate was removed with the internal temperature of the water bath not exceeding 35°C. The toluene solution (10 mL, 0.5 mmol) was then heated under reflux for 3-4 h and monitored by TLC for completion. The solution was cooled to room temperature and used directly for the next reaction. Method B.

The appropriate acid (1 .0 eq, 0.5 mmol) was dissolved in dry THF (5 mL) with 2 drops of dry DMF. To this solution was added oxalyl chloride (1 .3eq, 0.65 mmol, 0.06 mL) dropwise. The solution was stirred for 1 h at rt and monitored by TLC for completion. Azidotrimethylsilane (2.0 eq, 1 .0 mmol, 0.13 mL) was added, stirred for 2 h and monitored by TLC for completion. The reaction was quenched with water, concentrated and diluted with ethyl acetate (20 mL). The organic layer was washed with water (20 mL) and brine (20 mL), dried and concentrated to half the volume. Toluene (10 mL) was added and the remaining ethyl acetate was removed with the internal temperature of the rotovapor water bath not exceeding 35°C. The Toluene solution (10 mL, 0.5 mmol) was then refluxed for 3-4 h and monitored by TLC for completion. The solution was cooled to room temperature and used directly for the next reaction.

Synthesis Preparation of Acids 1 to 5

Synthesis of Acid 1

The synthesis of acid 1 is reported in the literature (PCT Int. Appl., 2012042433, 05 Apr 2012, Didiuk, Mary Theresa et al. Preparation of pyrazolospiroketone acetyl-CoA carboxylase).

Synthesis of Acid 2

2a 2b 2

Preparation of Intermediate 2b:

To a solution of compound 2a (1 .5 g, 6.84 mmol) in n-propanol (10 mL) was added sodium ⁿ PrONa (3.4 mL, 2M) and stirred at 50°C for 48 h. The solvent was removed under reduced pressure, diluted with ice (10 g), and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS0 ₄ , filtered, and concentrated under reduced pressure provided (1 .4 g, 79 %) of compound 2b. ¹ H NMR (300 Hz, CDCI ₃ ) δ 7.41 (s, 1 H), 7.20 (s, 1 H), 4.22-4.35 (m, 4 H), 1 .75-1 .82 (m, 4 H), 0.99-1 .15 (m, 6 H).

Preparation of Acid 2:

To a solution of compound 2b (1 .48 g, 5.75 mmol) in methanol (15 mL), water (7 mL) was added potassium carbonate (1 .6 g, 1 1 .59 mmol) and stirred at rt for 16 h. The solvent was removed under reduced pressure, diluted with water (15 mL), acidified with KHS0 ₄ , filter the solid, and dried (2, 910 mg, 74 %). ¹ H NMR (300 Hz, CDCIs) δ 8.21 (br s, 1 H), 7.44 (s, 1 H), 7.24 (s, 1 H), 4.28 (t, J = 6.6 Hz, 2 H), 1 .76-1 .83 (m, 2 H), 1 .01 (t, J = 7.2 Hz, 3 H).

Preparation of Intermediate 3a:

To a solution of sodium hydride (1 15 mg, 4.79 mmol, 1 .05eq) in dry THF (10 mL) was added dropwise 2-methoxyethanol at 0°C and stirred for 30 minutes. This solution was added to ethyl 2,6- dichloroisonicotinate (1 .0g, 4.57 mmol, 1 .Oeq) in dry THF (5 mL) at room temperature and then heated at 50 °C overnight. The reaction was neutralized by addition of 2N HCI (2.5 mL, 5 mmol). The solvent was removed, added water (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS0 ₄ , filtered, and concentrated under reduced pressure provided 3a. (600 mg. 51 %) ¹ H NMR (MeOD): δ 7.42 (s, 1 H), 7.22 (s, 1 H), 4.46-4.35 (m, 4H), 3.75(m, 2H), 3.40 (s, 3H), 1 .40 (t, 3H).

Preparation of Acid 3:

To a solution of compound 3a (600 mg, 2.31 mmol) in methanol (10 mL), water (4 mL) was added potassium carbonate (635 mg, 4.6 mmol) and stirred at rt for 16 h. The solvent was removed under reduced pressure, diluted with water (10 mL), acidified with KHS0 ₄ , and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS0 ₄ , filtered, and concentrated under reduced pressure provided 3 (320 mg, 59%). ¹ H NMR (MeOD): δ 7.41 (s, 1 H), 7.22 (s, 1 H), 4.45 (t, 2H), 3.74(t, 2H), 3.40 (s, 3H).

Synthesis of Acid 4

Preparation of Intermediate 4a:

To a solution of compound 2a (2.5 g, 10.9 mmol), ethyl amine (5.45 mL, 2M), DIPEA (2 mL) in THF (20 mL) were heated in a sealed tube at 75 °C for 16 h. The solvent was removed under reduced pressure, diluted with water (15 mL), and extracted with ethyl acetate (3 x 15 mL). The combined extracts were dried over MgSO ₄ , filtered, and concentrated under reduced pressure provided the crude compound. Further, purification by column chromatography using a silica gel column provided the desired compound 4a (450 mg, 17 %) ¹ H NMR (300 Hz, CDCIs) δ 7.08 (s, 1 H), 6.80 (s, 1 H), 4.40 (q, J = 7.2, 14.3 Hz, 2 H), 3.31 (q, J = 7.2, 14.1 Hz, 2 H), 1 .39 (t, J = 6.9 Hz, 3 H), 1 .24 (t, J = 6.9 Hz, 3 H).

Preparation of Intermediate 4b:

To a solution of compound 4a (400 mg, 1 .68 mmol) in THF (5 mL) at -10°C was added LHMDS (2 mL, 1 M) slowly in drops. After 30 min, (Boc) ₂ 0 (440 mg, 2.01 mmol) in THF was added and slowly bring to rt and stirred for 30 min. The solvent was removed under reduced pressure, diluted with saturated NH ₄ CI (10 mL), and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS04, filtered, and concentrated under reduced pressure provided (1 .4 g, 79 %) of compound 4b. ¹ H NMR (300 Hz, CDCI ₃ ) δ 8.19 (s, 1 H), 7.52 (s, 1 H), 4.40 (q, J = 7.2, 14.4 Hz, 2 H), 4.00 (q, J = 7.2, 14.1 Hz, 2 H), 1 .54 (s, 9 H), 1 .37 (t, J = 6.9 Hz, 3 H), 1 .23 (t, J = 6.9 Hz, 3 H).

Preparation of Acid 4:

To a solution of compound 4b (1 .00 g, 2.95 mmol), in methanol (12 mL), water (3 mL) was added potassium carbonate (800 mg, 5.79 mmol) and stirred at rt for 16 h. The solvent was removed under reduced pressure, diluted with water (15 mL), acidified with KHS0 ₄ , filter the solid, and dried to obtain the desired compound 4 (910 mg, 74 %). ¹ H NMR (300 Hz, CDCI ₃ ) δ 8.31 (s, 1 H), 7.57 (s, 1 H), 4.02 (q, J = 7.2, 14.2 Hz, 2 H), 1 .55 (s, 9 H), 1 .25 (t, J = 7.2 Hz, 3 H).

Synthesis of Acid 5

2a 5

Preparation of Acid 5:

To a solution of compound 2a (1 .0 g, 5.20 mmol), /V-ethylmethylamine (1 .5 g, 25.42 mmol), in water (3 mL) and heated to reflux for 48 h. The solvent was removed under reduced pressure. Triturating with I PE and hexane provided the desired compound 5 (300 mg, 27 %). ¹ H NMR (300 Hz, CDCI ₃ ) δ 1 1 .90 (br s, 1 H), 7.04 (s, 1 H), 6.97 (s, 1 H), 3.59 (q, J = 6.9, 14.4 Hz, 2 H), 4.00 (s, 3 H), 1 .17 (t, J = 6.9 Hz, 3 H).

General Procedure for the reaction of pyrazolopyridine derivative below with isocyanates.

2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6- yl)hydrazinyl)ethan-1 -ol :

A solution of hydrazine derivative (100 mg, .38 mmol) in dry THF (10 mL) was stirred at room temperature in which a solution of the appropriate isocyanate (~1 .3 eq, .5 mmol) in toluene (10 mL) was added drop wise and stirred for 12 h or until completion by TLC monitoring. The crude reaction was concentrated and purified by column chromatography using 1 :1 dichloromethane/ethyl acetate as the eluent to give the desired product.

Synthesis of compound 34

N-(2,6-Dichloropyridin-4-yl)-2-(2-hydroxypropyl)-2-(4-iso propyl-1 ,3- dimethyl-1 H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide

(Compound 34):

2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6- yl)hydrazinyl)propan-1 -ol

2-Hydrazinopropanol (3.35g, 37.2 mmol) was added to a solution of 1 H-6-bromo- 4-isopropyl-1 , 3-dimethylpyrazolo [3, 4-b] pyridine (500 mg, 1 .87 mmol) in ethanol (2.5 mL) and heated under reflux overnight. After cooling, it was stirred for 4 h in an ice bath. The crude material was filtered and the obtained solid was triturated with a 50% aqueous ethanol (5 mL), filtered and dried under reduced pressure at 60°C to give 2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin- 6-yl)hydrazinyl)propan-1 -ol as white solid, B-2 (300mg, 58% yield).

1H 300 MHz NMR (CDCI ₃ ) δ 6.89 (1 H, s), 4.95 (2H, bs), 4.28 (1 H, bs), 3.99 (2H, m), 3.85 (3H, s), 3.58 (2H,t),3.48 (1 H, m), 2.57 (3H, s), 1 .93 (2H,m), 1 .33 (3H, s), 1 .31 (3H,s).

Method A (80 mg, 45%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ + MeOD-4) δ 7.47 (2H, s), 6.52 (1 H, s), 4.06 (2H, bm) 3.96-3.80 (2H, bm), 3.84 (3H, s), 3.44 (2H, m), 3.39 (1 H,bs), 2.51 (3H, s), 1 .24 (6H, d).ESI+(M+H) m/z= 466.1

Method A (75mg, 43%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ ) δ 8.91 (1 H, s), 7.06 (1 H, s), 6.85 (1 H, s), 6.65( 1 H, s), 6.60 (1 H,s) 4.45 (1 H, bs), 4.30 (2H, qt), 4.16 (2H, m), 3.90 (3H, s) 3.51 (2H, m), 2.80 (1 H, bs) 2.60 (3H, s), 1 .31 (6H, m) 1 .29 (3H, m). ESI+(M+H) m/z= 462.1

Compound 36

300 MHz NMR (CDCI ₃ ) δ 8.99 (1 H, s), 7.05 (1 H, s), 6.87 (1 H, s), 6.81 ( 1 H, s), 6.60 (1 H,s) 4.45 (1 H, bs), 4.160 (2H, t), 4.10 (2H, m), 3.87 (3H, s) 3.48 (2H, m), 3.20 (1 H, bs) 2.59 (3H, s), 1 .75 (2H,m) 1 .30 (6H, d) .97 (3H, t). ESI+(M+H) m/z= 476.1

Method A (80 mg, 44%, 3 steps) ¹ H 400 MHz NMR (CDCI ₃ ) δ 8.91 (1 H, s), 7.1 1 (1 H, s), 6.89 (1 H, s), 6.62( 1 H, s), 6.58 (1 H,s) 4.55 (1 H, bs), 4.51 (2H, m) ,4.09 (2H, m), 3.91 (3H, s), 3.67 (2H,m) 3.49 (2H, m), 3.39 (3H,s), 2.71 (1 H, bs) 2.59 (3H, s), 1 .30 (6H, d). ESI+(M+H) m/z= 492.1

Method A (120 mg, 56%, 3 steps) ¹ H 400 MHz NMR (CDCI ₃ ) δ 8.96 (1 H, s), 7.58 (2H, d), 6.81 (1 H, bs), 6.62( 1 H, s), 4.49 (1 H, bs), 4.17-4.06 (2H, bm) 3.96 (2H, m), 3.91 (3H, s), 3.48 (2H, m), 3.03 (1 H,bs), 2.62 (3H, s), 1 .48 (9H,s), 1 .30 (6H, d), 1 .17 (3H,t). ESI+(M+H) m/z= 561 .2

The above Boc - derivative above was dissolved in dichloromethane ( 1 mL) cooled to 0-5°C in an ice bath and added 2M HCI solution in ether (1 mL) and stirred overnight. The solid obtained was filtered, dried in vacuum oven to yield 60 mg of the hydrochloride salt. ¹ H 400 MHz NMR (MeOD-4) δ 7.31 (1 H, s), 6.99 (1 H, s), 6.71 (1 H, bs), 4.49 (1 H, bs), 4.09-3.99 (2H, bm) 3.96 (2H, m), 3.93 (3H, s), 3.67 (1 H,m), 3.53 (2H, m), 3.38 (2H,m), 2.64 (3H, s), 1 .32 (6H, d) 1 .12 (3H,t). ESI+(M+H) m/z= 461 .1

Method A (1 10 mg, 61 %, 3 steps) ¹ H 400 MHz NMR (DMSO) δ 9.47 (1 H, s), 8.96 (1 H, bs), 6.88 (1 H, bs), 6.61 ( 1 H, s), 6.49 (1 H, s), 5.38,4.73 (1 H, bs), 4.17-3.86 (2H, bm) 3.80 (3H, s), 3.80-3.71 (2H, bm), 3.45 (2H, m), 2.89 (3H,s), 2.50 (3H, s), 1 .23, (6H, d), 1 .03 (3H,t). ESI+(M+H) m/z= 475.1

Method B (55 mg, 32%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ ) δ 9.08 (1H, s), 8.35 (1H, s), 8.23 (1H,s), 6.70 (1H, s), 6.61 ( 1H, s), 4.49 (1H, bs), 4.17-4.06 (2H, bm), 3.91 (3H, s), 3.49 (2H, m), 3.11 (1H,bs), 2.60 (3H, s), 1.30 (6H,d). ESI+(M+H) m/z= 452.1

Method A (95 mg, 55%, 3 steps) ¹ H 400 MHz NMR (CDCI ₃ ) δ 9.08 (1 H, s), 8.36 (1 H, d), 8.22(1 H,d), 6.71 (1 H, bs), 6.62( 1 H, s), 4.29 (1 H, bs), 4.2-4.17 (2H, bm) 3.96 (2H, m), 3.451 (2H, m), 3.12 (1H,bs), 2.60 (3H, s), 1.32 (6H, d). ESI+(M+H) m/z= 452.1

Method A (45 mg, 26%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ ) δ 9.54 (1H, s), 6.81 (1 H, bs), 6.60( 1 H, s), 6.25 (1 H,s), 4.45 (1 H, bs), 4.11 -4.08 (2H, bm) 3.84 (3H, s), 3.49 (2H, m), 2.89 (1H,bs), 2.60 (3H, s), 1.30 (6H, d).ESI+(M+H) m/z= 457.0

Method B (90 mg, 54%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ + MeOD-4) δ 6.49 (1H, bs), 6.21 (1H, s), 4.19-4.06 (2H, bm) 3.83 (3H, s), 3.48-3.38 (2H, bm) 3.35 (1H,m), 2.47 (3H, s), 2.16 (3H,s), 1.19 (6H,d). ESI+(M+H) m/z= 437.1

Method A (1 10 mg, 60%, 3 steps) ¹ H 300 MHz NMR (CDCI ₃ + MeOD-4) δ 9.18 (1 H, s), 7.76 (1 H, s), 7.61 (1 H,s), 7.19 (1 H, s), 6.59( 1 H, s), 4.43 (1 H, bs), 4.07- .92 (2H, bm), 3.85 (3H, s), 3.60-3.48 (2H, m), 2.54 (3H, s), 1 .27 (6H, d).

ESI+(M+H) m/z= 485.1

Method B (90 mg, 58%, 3 steps) ¹ H 400 MHz NMR (CDCI ₃ + MeOD-4) δ 6.50 (1 H, bs), 6.22(1 H, s), 4.34 (1 H, bs), 4.17-4.06 (2H, bm), 3.82 (3H, s), 3.43 (2H, m), 2.52 (3H, s), 1 .26 (6H, d). ESI+(M+H) m/z= 408.1

Using the above methods the following compounds can be prepared

Compoun 46 Compound 47 Compound 48

Compounds 46-48 were prepared by reacting the desired respective isocyanate of the corresponding carboxylic acids B1 , B2, B3 below and 2-(1-(4-lsopropyl- 1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6-yl)hydrazinyl)ethan-1-ol (decribed above):

The acid B3 was prepared in a three step procedure from ethyl-2,6-dichloro- isonicotinate.

Ethyl -2,6-dichloro-isonicotinate

Preparation of B3

Stepl Preparation of Ethyl-2-chloro-6-isopropylamino-isonicotinate:

A solution of Ethyl-2,6-dichloro-isonicotinate (1 .0 g, 4.36 mmol) and n- propylamine (772 mg, 13.08 mmol) in THF (5 mL) was heated in a sealed tube at 80° C for 16 h. The solvent was removed under reduced pressure, diluted with water (15 mL), and extracted with ethyl acetate (3 x 15 mL). The combined extracts were dried over MgS0 ₄ , filtered, and concentrated under reduced pressure provided the crude compound. Further, purification by column chromatography using a silica gel column provided the desired compound (170 mg, 15 %) ¹ H NMR (300 Hz, CDCI ₃ ) δ 7.05 (s, 1 H), 6.82 (s, 1 H), 4.84 (br s, 1 H), 4.35 (q, J = 7.1 , 14.2 Hz, 2 H), 3.24 (q, J = 6.9, 13.8 Hz, 2 H), 1 .58-1 .65 (m, 2H), 1 .37 (t, J = 7.1 1 Hz, 3 H), 0.98 (t, J = 7.3 Hz, 3 H).

Step 2 Preparation of Ethyl-2-chloro-6-isopropyl-t-butoxycarbonylamino- isonicotinate

Lithium hexamethyldisilazide (LHMDS) (1 .1 mL, 1 M) was slowly to a solution of Ethyl-2-chloro-6-isopropylamino-isonicotinate (170 mg, 0.67 mmol) in THF (4 mL) at -10°C. After 30 min, (Boc) ₂ 0 (162 mg, 1 .1 mmol) in THF was added, slowly brought to rt and stirred for 30 min. The solvent was removed under reduced pressure, diluted with saturated NH ₄ CI (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS04, filtered, and concentrated under reduced pressure provided (1 10 mg, 47 %) of Ethyl-2-chloro- 6-isopropyl-t-butoxycarbonylamino-isonicotinate. ¹ H NMR (300 Hz, CDCI ₃ ) δ 8.16 (s, 1 H), 7.52 (s, 1 H), 4.38 (q, J = 7.2, 14.4 Hz, 2 H), 3.91 (t, J = 7.2 Hz, 2 H), 1 .60-1 .65 (m, 2H), 1 .54 (s, 9 H), 1 .39 (t, J = 6.9 Hz, 3 H), 0.90 (t, J = 7.2 Hz, 3 H).

Preparation of Acid B3:

Potassium carbonate (165 mg, 1 .2mmol) was added to a solution of Ethyl-2- chloro-6-isopropyl-t-butoxycarbonylamino-isonicotinate (1 10 mg, .597mmol) in methanol (5 mL) and water (2 mL) and stirred at rt for 16 h. The solvent was removed under reduced pressure, diluted with water (10 mL), acidified with KHS0 ₄ , and extracted with ethyl acetate (3 x 10 mL). The combined extracts were dried over MgS04, filtered, and concentrated under reduced pressure provided B3 (80 mg, 86%). Ή 300 MHz NMR (CDCI ₃ ) δ 10.240 (1 H, s), 8.32 (1 H, s) 7.63 (1 H, s), 3.98 (2H, m), 1 .75(2H,m), 1 .59 (9H,s), 0.99 (3H, m).

General Procedure for the reaction of pyrazolopyridine derivative with isocyanates

A solution of 2-(1 -(4-lsopropyl-1 ,3-dimethyl-1 H-pyrazolo[3,4-b]pyridin-6- yl)hydrazinyl)ethan-1 -ol (100 mg, 0.38 mmol) in dry THF (10 mL) was stirred at room temperature in which a solution of the appropriate isocyanate (~1 .3 eq, 0.5 mmol) in toluene (10 mL) was added drop-wise and stirred for 12 hours or until completion by TLC monitoring. The crude reaction was concentrated and purified by column chromatography using 1 :1 dichloromethane/ethyl acetate as the eluent to give the desired products 46, 47, and Boc-derivative of 48.

Compound 46

(89 mg, 52%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ +MeOD) δ 6.44 (1 H, d), 6.32 (1 H, s) 6.14 (1 H, d), 3.70 (3H, s), 3.31 (1 H, m), 2.40 (3H,s) 1 .15 (6H, d, J = 6.6 Hz). ESI+(M+H) m/z= 379.0 Compound 47

(80mg, 47%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ ) δ 8.36 (1 H, s), 6.61 (2H, s) 6.45 (3H, m), 3.90 (3H, s), 3.54( 1 H,m), 2.60 (3H,s),1 .32 (6H, d, J= 6.6 Hz). ESI+(M+H) m/z= 379.2

Compound 48

The initial coupled reaction of the isocyanate of B3 gave the Boc-derivative of 48. (120 mg, 50%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ ) δ 8.12 (1 H, s), 7.60 (1 H, s) 7.54 (1 H, s), 6.73 (1 H, s), 6.62 (1 H, s), 6.44 (1 H,s) 3.89 (3H, s), 3.85(2H, m) 3.50 (1 H, m), 2.60 (3H, s) 1 .55 (2H, m), 1 .46 (9H,s), 1 .32 (6H, d, J= 6.8 Hz) 0.88 (3H, t, J= 7.4 Hz). ESI+(M+H) m/z= 531 .3

The above Boc-derivative (60 mg, 0.1 13 mmol) was dissolved in

dichloromethane (1 mL), cooled to 0-5°C in an ice bath, added 2M HCI solution in ether (1 mL) and stirred at rt overnight. The solid obtained was filtered and dried in vacuum oven to yield 44 mg of the hydrochloride salt in 92% yield of compound 48. Ή 300 MHz NMR (MeOD) δ 7.30 (1 H, s), 7.10 (1 H, s), 6.68 (1 H,s) 4.01 (3H, s), 3.55(2H, m) 3.47 (1 H, m), 2.70 (3H, s) 1 .70 (2H, m), 1 .37 (6H, m) 0.88 (3H, bs). ESI+(M+H) m/z= 431 .3

Using the above disclosed methods, the following compounds can be prepared with the Boc-hydrazine derivative and corresponding isocyanates as described above. After removal of the Boc-group with HCI in ether, compounds 49-52 can

d d

General method for compounds 49-52

A solution of the allyl hydrazine derivative (100 mg, .302 mmol) in dry THF (10 ml_) was stirred at room temperature in which a solution of the appropriate isocyanate (~1 .3 eq, .5 mmol) in toluene (10 ml_) was added drop wise and stirred for 12 hours or until completion by TLC monitoring. The crude reaction was concentrated and purified by column chromatography using 1 :1

dichloromethane/ethyl acetate as the eluent to give the desired product.

Compound 49

Preparation of Boc-derivative of compound 49

(80 mg, 47%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ ) δ 9.17 (1 H, s), 7.60 (1 H, bs), 7.14 (1 H, s), 6.99 (1 H, s), 6.81 (1 H, s) 5.98(1 H, m), 5.15 (2H, m), 4.94 (2H, m), 4.17 (2H, t, J= 6.9 Hz), 3.61 (1 H, m), 2.68 (3H, s), 1 .70 (2H, m), 1 .49 (9H, s), 1 .26 (6H, s), 1 .00 (3H, m). ESI+(M+H) m/z= 558.3

Preparation of compound 49

The above Boc derivative (40 mg, 0.072 mmol) was dissolved in

dichloromethane (1 mL) cooled to 0-5°C in an ice bath, added 2M HCI solution in ether (1 mL) and stirred overnight. The reaction mixture was concentrated and triturated with hexanes. The solid obtained was filtered, dried in vacuum oven to yield 28 mg of the hydrochloride salt in 84% yield.

Ή 300 MHz NMR (MeOD) δ 7.18 (1 H,s), 6.93 (1 H, s), 6.69 (1 H, s) 5.94(1 H, m), 5.20-5.12 (2H, m), 5.00 (2H, m), 4.18 (2H, t, J= 6.6 Hz), 3.55 (1 H, m), 2.69 (3H, s), 1 .76 (2H, m), 1 .38 (6H, d, J= 6.9 Hz), 0.99 (3H, t, J= 6.9 Hz). ESI+(M+H) m/z= 458.3

Compound 50

Preparation of Bis-Boc-derivative of compound 50

(89 mg, 46%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ ) δ 9.23 (1 H, s), 7.63 (1 H, s), 7.51 (1 H, s), 7.10 (2H, bs), 5.99 (1 H, m), 5.17-5.09 (2H, m), 4.99 (2H, m), 3.97 (2H, m), 3.65 (1 H, m), 2.69 (3H, s), 1 .49 (18H, s), 1 .39 (6H, d, J= 6.6 Hz), 1 .25 (3H, m). ESI+(M+H) m/z= 643.4

Preparation of compound 50

The above bis-Boc derivative (50 mg, .077 mmol) was dissolved in

dichloromethane (1 mL) cooled to 0-5°C in an ice bath, added 2M HCI solution in ether (1 mL) and stirred overnight. The reaction mixture was concentrated and triturated with hexanes. The solid obtained was filtered, dried in vacuum oven to yield 27 mg of the hydrochloride salt in 79% yield.

Ή 300 MHz NMR (MeOD) δ 7.30 (1 H, bs), 7.09 (1 H, bs), 6.68 (1 H, bs), 5.98(1 H, bs), 5.20-5.092 (4H, m), 3.54 (1 H, bs), 3.36 (2H, bs), 2.69 (3H, s), 1 .39 (6H, s), 1 .28 (3H, bs). ESI+(M+H) m/z= 443.3

Compound 51

Preparation of Boc-derivative of compound 51 .

(85 mg, 51 %, 3 steps) Ή 300 MHz NMR (CDCI ₃ ) δ 8.94 (1 H, s), 7.63 (1 H, s), 7.14 (1 H, s), 6.75 (1 H, s), 6.42(1 H,s), 5.96 (1 H, m), 5.17-5.1 1 (2H, m), 4.93 (2H, m), 3.58 (1 H, m), 3.50 (2H, m), 2.95 (3H, s), 2.67 (3H, s), 1 .49 (9H, s), 1 .35 (6H, d, J = 7.2 Hz), 1 .12 (3H, t, J= 7.2 Hz). ESI+(M+H) m/z = 557.4

Preparation of compound 51 .

The above Boc derivative (50 mg, .090 mmol) was dissolved in dichloromethane (1 mL), cooled to 0-5°C in an ice bath, added 2M HCI solution in ether (1 mL) and stirred at rt overnight. The reaction mixture was concentrated and triturated with hexanes. The solid obtained was filtered, dried in vacuum oven to yield 37 mg of the hydrochloride salt in 91 % yield. Ή 300 MHz NMR (MeOD) δ 7.29 (1 H, s), 7.15 (1 H, s), 6.67 (1 H,s), 5.96 (1 H, m), 5.21 - 5.18 (2H, m) 5.08 (2H, bs), 3.58 (3H, m) 3.17 (3H, s), 2.68 (3H, s), 1 .37 (6H, d, J= 5.7 Hz), 1 .22 (3H, m).

ESI+(M+H) m/z= 457.3

Compound 52

Preparation of the Bis-Boc derivative of compound 52.

(108 mg, 55%, 3 steps)

Ή 300 MHz NMR (CDCI ₃ ) δ 9.23 (1 H, bs), 7.59 (1 H, s), 7.48 (1 H, s), 7.26 (1 H, bs), 7.12 (1 H, s), 5.98 (1 H, m), 5.16-5.09 (2H, m), 4.97 (2H, m), 3.88 (2H, m), 3.62 (1 H, m), 2.68 (3H, s), 1 .65 (2H, m), 1 .57 (18H, s), 1 .39 (6H, d, J= 6.9 Hz), 0.89 (3H, t, J = 7.2 Hz). ESI+(M+H) m/z= 657.4

Preparation of compound 52.

The above Boc derivative (68 mg, 0.104 mmol) was dissolved in

dichloromethane (1 mL), cooled to 0-5°C in an ice bath, added 2M HCI solution in ether (1 mL) and stirred at rt overnight. The reaction mixture was concentrated and titrated with hexanes. The solid obtained was filtered, dried in vacuum oven to yield 42 mg of the hydrochloride salt in 88% yield. Ή 300 MHz NMR (MeOD) δ 7.30 (1 H, s), 7.10 (1 H, bs), 6.72 (1 H, s), 6.00 (1 H, m), 5.23-5.1 1 (4H, m), 3.55 (1 H, m), 3.32 (2H, m), 2.73 (3H, s), 1 .70 (2H, m), 1 .39 (6H, s), 1 .01 (3H, s). ESI+(M+H) m/z= 457.3

Using the methods described, above the compounds 53-56 can be prepared.

IN VITRO BINDING AFFINITIES

A number of compounds set forth above according to the present invention were tested to_determine the binding affinity for S1 P ₂ receptor (see below).

Antagonist percentage inhibition determinations were obtained by assaying sample compounds and referencing the control (EC ₈ o) wells for each profiled (GPCR) which evaluated binding to S1 P ₂ receptor. The samples were run using a single addition assay protocol. The protocol design is as follows.

I. Master stock solution

Unless specified otherwise, the sample compounds were diluted in 100% anhydrous DMSO including all dilutions. The compounds were tested as the citrate salt (1 equivalent per molecule). If the sample compound is provided in a different solvent all master stock dilutions are performed in the specified solvent. All control wells contained identical solvent final concentrations as the sample compound wells.

II. Compound plate assay

The sample compounds were transferred from a master stock solution into a daughter plate that was used in the assay. Each sample compound was diluted into an assay buffer (1 X HBSS with 20mM HEPES, 2.5mM Probenecid, and 0.4% Free Fatty Acid BSA) at an appropriate concentration to obtain final specified concentrations.

III. Antagonist Assay Format

Using the EC ₈ o values that were determined real-time, stimulated all pre- incubated sample compounds and reference antagonists (if applicable) were compared with the EC ₈ o values of reference agonist. These were read for 180 seconds using the FLIPR ^TETRA (This assay added reference agonist to respective wells-then fluorescences measurements were collected to calculate IC ₅ o values). All plates were subjected to appropriate baseline corrections. Once baseline corrections were processed, maximum fluorescence values were exported and data manipulated to calculate percentage activation, percentage inhibition and Z'. The results are in the table below, and show that these compounds bind to the S1 P ₂ receptor.

Compound Structure S1 P? IC

61

62 34. 19 nM

35. 29 nM

36. nM

37. 160 nM

38.

39. 19 nM

64

65

Pharmacokinetic profile of select compounds in normal mice

Blood concentration data for JTE013 and Compound 1

In order to determine the pharmacokinetics of JTE013 and compound 1 , 1 mg/kg of each compound was injected by IV and blood was withdrawn at the times in tables 1 -4. The amount of starting compound was quantified by a standard curve using MS/MS determinations. Blood concentrations of JTE013 versus time following 1 mg/kg i.v. administration of JTE013 to 3 mice (M01 -M03).

NP denotes no peak.

n/a denotes not applicable.

Value is greater than 50% of LLOQ (>0.5 ng/mL) and was included in calculations.

BLQ denotes below lower level of quantification (LLOQ = 1 ng/mL).

Blood concentrations of Compound 1 versus time following 1 mg/kg i.v. administration of Compound-1 to 3 mice (M01-M03).

a NP denotes no peak.

b n/a denotes not applicable. ^c BLQ denotes below lower level of quantification (LLOQ

3. Estimated pharmacokinetics parameters for JTE013 in blood following 1 mg/kg i.v. administration of JTE013 to 3 mice (M01

Terminal half-life was estimated from the last 2 measurable concentrations.

Estimated pharmacokinetics parameters for Compound 1 in blood following 1 mg/kg i.v. administration of Compound 1 to 3 mice (M01-M03).

MRTo-tlast h 0.236 2.10 2.39 1 .58 (n=2)

MRTo-inf h NC 6.53 46.9 26.7 (n=2)

Vss L/kg NC 70.7 244 157 (n=2) a Terminal half-life was estimated from the last 2 measurable concentrations.

b NC denotes not calculable due to truncated nature of PK curve.

Figure 1. Mean JTE013 and Compound 1 blood concentrations following 1 mg/kg i. v. administration to groups of 3 mice nd 1

Time (h)

The blood concentrations reported above that were measured for JTE013 and

Compoundl following 1 mg/kg i.v. administration to the mice shown above clearly demonstrates that since the mice that were administered Compound 1 had the highest drug blood levels over time, increases the likelihood the compound will have a biological effect based on antagonizing the S1 P ₂ receptor.

Additional pharmacokinetic studies by IV (1 mg/kg) and PO (10mg/kg) dosing.

Compound 33 and 36 were tested by IV bolus of 1 mg/kg and PO (10mg/kg) in groups of 3 mice (Tables 5-12).

Table 5. Plasma concentrations of compound 33 following bolus i.v.

administration to 3 mice.

†ns denotes no sample collected due to death of animal.

Table 6. Plasma concentrations of compound 33 following p.o. administration to 3 mice.

Table 7. Summary of PK parameters of compound 33 following i.v. administration to 3 mice.

Table 8. Summary of PK parameters of compound 33 following

.o. administration to 3 mice.

†nc denotes not

calculable.

φΑΙΙΟ, MRT and F are estimated between time 0 and ti _ast (10 h) instead of infinity. concentration at time sero (extrapolated)

terminal half-life

area under the concentration vs time curve from time 0 to infinity

systemic clearance

mean residence

time

steady-state volume of

distribution

time at which maximum concentration is observed following p.o. dosing

maximum observed concentration following p.o. dosing apparent terminal half-life

bioavailability (Dose ^iv* AUC ^po )/(Dose ^po* AUC ^iv )

time of last measurable concentration Figure 2. Plasma concentration of Compound 33 after (1 mg/kg) and PO (10mg/kg dosing)

Table 9. Plasma concentrations of Compound 36 following bolus i.v. administration to 3 mice.

Table 10. Plasma concentrations of Compound 36 following p.o. administration to 3 mice.

†ns denotes no sample collected due to death of animal.

Table 11. Summary of PK parameters of compound 36 following i.v. administration to 3 mice.

Table 12. Summary of PK parameters of compound 36 following p.o. adminstration to 3 mice.

^† nc denotes not calculable. Figure 3. Plasma concentration of Compound 36 after (1 mg/kg) and PO (10mg/kg) dosing

0 2 4 6 8 10 12

Time (h)

The PO pharmacokinetics of AB33 and AB36 showed that the compounds were more slowly cleared from the blood than JTE013 (Tables 13 and 14), which should indicate an improved potential of AB33 and AB36 for treating diseases by oral dosing.

Table 13. Plasma concentrations of JTE013 following p.o.

administration to 3 mice.

Table 14. Summary of PK parameters of JTE013

followin .o. administration to 3 mice. Cmax (ng/mL) 873 1650 175 899 738

Apparent t _V2

2.57 2.48 2.26 2.44 0.159

(h ^* ng/mL) 1870 1300 673 1281 599

MRT (h) 4.38 1 .54 6.08 4.00 2.29

F (%)† 175 120 53.9 1 16 60.5

†Based on the mean AUC ^IV (107 h ^* ng/ml_, n=3) determined for 1 mg/kg i.v. dose from NoAb study no. DH-A12001 -1 time at which maximum concentration is observed

t _max following p.o. dosing

Cmax maximum observed concentration following p.o. dosing

Apparent t _V2 apparent terminal half-life

area under the concentration vs time curve from time 0 to AUCo-inf infinity

mean residence

MRT time

bioavailability

F (Dose ^iv* AUC ^po )/(Dose ^po* AUC ^iv )

From these pharmacokinetic studies compound 1 by iv and compound 36 by both PO and IV have improved in vivo blood levels over JTE013.

In vitro ADME-Tox Summary

In order to get additional information on the protein binding and metabolism of JTE013 and compound 1 and compound 2, in vitro plasma protein binding and microsomal intrinsic clearance studies were completed. The results are set forth below (Tables 15 and 16).

Table 15. Plasma Protein Binding Summary

Compound Test concentration Species mean fraction bound (%)

Warfarin 5nM mouse 92.1

Propanolol 5nM mouse 87.4

Compound 1 5nM mouse 99.9

Compound 2 5nM mouse 77.0

JTE013 5nM mouse 99.7 Table 16. Microsomal Intrinsic Clearance Summary

Compound test species NADPH- NADPH- NADPH- NADPH

Cone. (μΜ) dependent dependent free free

CLinf T1/2 ^b Clint ³ T1/2 ^b

(μΙ min ^"1 ma ^"1 ) (min) (ul min ^"1 ma ^"1 ) (min)

Verapamil 1 mouse 398 5.8 9 >180

Warfarin 1 mouse 15 157 10 >180

Compound 1 1 mouse 1 172 2 48 48

Compound 2 1 mouse 20 1 13 9 >180

JTE013 1 mouse 770 3.0 22 105 aMicrosomal intrinsic clearance

bHalf-life

Verapamil is a metabolized control, while Warfarin is a non-metabolized control.

From the initial in vitro ADME toxicology study using plasma protein binding and microsomal intrinsical clearance, it's clear that the known control JTE013 and compound 1 behave very similarly in both assays, while compound 2 was much more stable in liver microsomes and had lower plasma protein binding. To further clarify the properties of these molecules the clearance of the compounds were studied following intravenous injection in mice. Compound 1 had substantially improved pharmacokinetics, over JTE013, which was better than compound 2 (see area under curves). With greater potency for receptor binding and higher drug plasma levels after 2h, one would expect greater in vivo efficacy for compound 1 . The differences between the in vivo metabolism and pharmacokinetics of the three compounds are noteworthy. The strong protein binding of compound 1 must offset the metabolism in the liver. The free carboxyl group of compound 2 may lead to glucoronylation and more rapid excretion than the in - vitro metabolism study might suggest, or that the decreased protein binding is deleterious

Additional liver microsome studies on other compounds is provided in table 17. Table 17. Microsomal Intrinsic Clearance Summary

Compound test species NADPH- NADPH- NADPH- NADPH

Cone. (μΜ) dependent dependent free free

CLinf T1/2 ^b Clint ³ T1/2 ^b min ^"1 mq ^" ) (min) (ul min ^"1 mq ^"1 ) (min)

9 1 mouse 235 9.8 12.8 180

33 1 mouse 147 15.7 4.1 >240

34 1 mouse 381 6.1 2.3 >240

35 1 mouse 516 4.5 0.0 >240

36 1 mouse 415 6 6.0 >240

38 1 mouse 958 2.4 4.7 >240

39 1 mouse 893 2.6 0.0 >240

48 1 mouse 996 2.3 0.0 >240

50 1 mouse 1309 1.8 2.7 >240

52 1 mouse 1029 2.2 4.1 >240

As can be seen in the table the stability of compounds 9, 33, 36, and 48 is improved over JTE013.

Pharmaceutical compositions comprising the above-listed S1 P receptor agonists may comprise additional pharmacological agents used in the treatment of disorders relating to vascular permeability and vascular endothelial cell apoptosis. Suitable additional pharmacological agents include, for example, cytotoxic agents,

chemotherapeutic agents, hormones, steroidal anti-inflammatory drugs (e.g., prednisone, corticosteroids, and the like), non-steroidal anti-inflammatory drugs (e.g., NSAIDs, aspirin, acetaminophen, and the like); and combinations comprising one or more of the foregoing additional pharmacological agents.

Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by means known in the art with

pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); wetting agents (e.g., sodium lauryl sulfate); and combinations comprising one or more of the foregoing excipients. The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid); and combinations comprising one or more of the foregoing additives. The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated by techniques known in the art. For administration by inhalation, the compounds are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, for example, di-chloro-difluoromethane, tri-chloro- fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may also be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.