HK1086005A - 2-(1h-indazole-6-ylamino)-benzamide compounds as protein kinases inhibitors useful for the treatment of ophthalmic diseases - Google Patents

Description

2- (1H-indazol-6-ylamino) -benzamide compounds as protein kinase inhibitors for the treatment of ocular diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

The benefit of U.S. provisional patent application No. US60/434,902, filed 12/19/2002, the entire contents of which are hereby incorporated by reference for all purposes.

Technical Field

The present invention relates to indazole compounds that mediate and/or inhibit the activity of ocular diseases and certain protein kinases, and pharmaceutical compositions containing such compounds. The invention is also directed to the therapeutic or prophylactic use of such compounds and compositions and to methods of treating ocular diseases and cancer, as well as other disease states associated with unwanted angiogenesis and/or cell proliferation, by administering effective amounts of such compounds.

Background

Several diseases and conditions of the posterior segment of the eye threaten vision. Age-related macular degeneration (ARMD or AMD), Choroidal Neovascularization (CNV), retinopathies (e.g., diabetic retinopathy, vitreoretinopathy, retinopathy of prematurity), retinitis (e.g., Cytomegalovirus (CMV) retinitis), uveitis, macular edema, and glaucoma are several examples.

Age-related macular degeneration (ARMD or AMD) is the leading cause of blindness in the elderly. ARMD attacks the center of vision and blurs it, making reading, driving, and other detailed tasks difficult or impossible. There are approximately 200,000 new cases of ARMD occurring each year in the united states alone. Current estimates show that approximately 40% of people over the age of 75 years have some degree of macular degeneration, and approximately 20% of people over the age of 60 years. "Wet" ARMD is the most common blinding type of ARMD. In wet ARMD, newly formed choroidal blood vessels (choroidal neovascularization (CNV)) leak fluid and cause progressive damage to the retina. In specific cases of CNV in ARMD, two major therapeutic approaches are currently being developed: (a) photocoagulation; and (b) the use of an angiogenesis inhibitor.

However, photocoagulation can be harmful to the retina and is impractical when the CNV is located near the fovea. In addition, photocoagulation often results in recurrent CNV over a period of time. Oral administration of anti-angiogenic compounds is also being tested as a systemic treatment for ARMD. However, systemic administration often provides inadequate levels of therapeutic drug to the eye due to drug-specific metabolic limitations. Thus, in order to achieve effective intraocular drug concentrations, unacceptably high doses or repeated conventional doses are required. Various implants have also been developed to deliver anti-angiogenic compounds locally to the eye. Examples of such implants are disclosed in Wong U.S. Pat. No. 5,824,072, Gwon et al U.S. Pat. No. 5,476,511, and Ashton et al U.S. Pat. No. 5,773,019, each of which is incorporated herein in its entirety by reference for all purposes.

Ocular neovascular disease

As noted above, the present invention also provides methods of treating ocular neovascular diseases, including: such as corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fbiroplasia, and macular degeneration.

Briefly, corneal neovascularization as a result of anterior segment injury is a major cause of vision loss and blindness and a major risk factor for rejection of corneal allografts. Just as Burger et al in Lab, invest.48: 169-180, 1983, corneal angiogenesis comprises three phases: the pre-vascular incubation period, the active neovascularization period, and the mature and degenerate phases of the blood vessels are incorporated herein by reference in their entirety for all purposes. The nature and mechanism of various angiogenic factors must also be revealed, including inflammatory response factors such as leukocytes, platelets, cytokines and eicosanoids or unidentified plasma constituents.

Currently, there is no clinically satisfactory therapy to inhibit corneal neovascularization or the degeneration of existing corneal neovessels. Topical corticosteroids appear to have some clinical utility, and are presumed to act by limiting interstitial inflammation.

Accordingly, the present invention provides, in one aspect, a method of treating ocular neovascular diseases such as corneal neovascularization, including corneal graft neovascularization, comprising the step of administering to the cornea of a patient a therapeutically effective amount of an anti-angiogenic composition (as described above) such that angiogenesis is inhibited. Briefly, the cornea is a tissue that is usually devoid of blood vessels. However, in certain pathological conditions, capillaries may extend from the peripheral vascular plexus of the limbus into the cornea. When the cornea vascularizes, it also blurs, resulting in a loss of vision for the patient. If the cornea is completely opaque, vision is completely lost.

Blood vessels can enter the cornea in different patterns and depths depending on the process of stimulating neovascularization. These patterns are traditionally defined by ophthalmologists as the following types: nebula, leprosy pannus, vesicular pannus (pannus phytenulosus), denatured pannus and glaucomatous pannus. The anterior ciliary artery branch can also be invaded in the corneal stroma (called interstitial vascularization), leading to several different clinical lesions: a "brush-like" pattern, an umbrella shape, a grid-shaped end ring, a stromal arch (from episcleral vessels), and abnormal irregular vessels.

A wide variety of conditions can lead to corneal neovascularization, including: such as corneal infections (e.g. trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immune processes (e.g. graft rejection and Stewart-Johnson syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states and complications of contact lens wear.

Although the cause of corneal neovascularization may vary, the corneal response to injury and subsequent vascular ingrowth is similar regardless of the cause. Briefly, the location of the lesion appears to be important because only those lesions that are within marginal critical distances stimulate the angiogenic response. This may be due to the fact that: angiogenic factors that cause vascular invasion are produced at the site of the lesion and must spread to the nearest vascular site (margin) to exert their effect. After a certain distance from the edge, this is no longer possible and does not induce the growth of the limbal endothelium into the cornea. Several angiogenic factors may be involved in this process, many of them being products of the inflammatory response. In fact, corneal neovascularization occurs only as a result of inflammatory cell infiltration and the degree of angiogenesis is directly proportional to the degree of inflammatory response. Corneal edema favors vascular ingrowth by relaxing the corneal stromal structure and providing a "minimal resistance" path through which capillaries can grow.

After the initial inflammatory response, capillary growth into the cornea continues in the same manner as it occurs in other tissues. The normal quiescent endothelial cells of the marginal capillaries and venules are stimulated to divide and migrate. Endothelial cells extend away from their vascular origin, digest the surrounding basement membrane and the tissues they pass through and migrate to the source of angiogenic stimuli. The blind terminal buds gain cavities and then anastomose to each other into capillary loops. The net result is the establishment of a vascular plexus within the corneal stroma.

The anti-angiogenic factors and compositions of the present invention act by blocking the stimulatory effects of angiogenesis promoters, reducing endothelial cell division, reducing endothelial cell migration, and attenuating endothelial secreted proteolytic enzyme activity.

Summary of The Invention

In a particularly preferred embodiment of the invention, the anti-angiogenic factor may be prepared for topical administration in saline (in combination with any preservatives and antimicrobials commonly used in ophthalmic formulations) and administered in the form of eye drops. Anti-angiogenic factor solutions or suspensions may be prepared in pure form and administered several times per day. Alternatively, the anti-angiogenic composition prepared as described above may also be administered directly to the cornea.

In a preferred embodiment, the anti-angiogenic composition is prepared using a mucoadhesive polymer that adheres to the cornea. In other embodiments, anti-angiogenic factors or anti-angiogenic compositions can be used as an adjunct to conventional steroid therapy.

Topical therapy may also be used prophylactically for corneal lesions known to have a high potential to induce angiogenic responses, such as chemical burns. In these cases, therapies may be established directly, possibly in combination with steroids, to help prevent subsequent complications.

In other embodiments, the ophthalmologist may inject the anti-angiogenic composition described above directly into the corneal stroma under microscopic guidance. The preferred injection site may vary depending on the morphology of the individual lesion, but the administration is intended to place the composition in front of the vasculature (i.e., interspersed between blood vessels and normal cornea). In most cases, this procedure may involve perilimbal injection to "prevent" the cornea from revascularizing. This method can also be used shortly after corneal injury to prophylactically prevent corneal neovascularization. In this case, the substance may be injected in the perilimbal space interspersed between the corneal lesion and its unwanted potential limbal blood supply. Such methods may also be used in a similar manner to prevent capillary invasion of the implanted cornea. For sustained release dosage forms, only 2-3 injections are required per year. Asteroid may also be added to the injection solution to reduce inflammation caused by the injection itself.

In another aspect, the invention provides a method of treating neovascular glaucoma comprising the step of administering to the eye of a subject a therapeutically effective amount of an anti-angiogenic composition such that angiogenesis is inhibited.

Briefly, neovascular glaucoma is a pathological condition in which new capillaries occur in the iris of the eye. Angiogenesis usually originates from blood vessels located at the pupillary edge and progresses through the iris root and into the trabecular meshwork. Fibroblasts and other connective tissue elements are involved in capillary growth and fibrovascular membranes develop extending across the anterior surface of the iris. Eventually the tissue reaches the anterior chamber angle where it forms an iris synechia. These adhesions in turn fuse, scar, and contract to eventually close the anterior chamber angle. Scarring prevents adequate drainage of aqueous humor through the anterior chamber angle and into the trabecular meshwork, causing increased intraocular pressure which can lead to blindness.

Neovascular glaucoma generally occurs as a complication of diseases in which retinal ischemia is significant. In particular, about one third of patients with this disorder have diabetic retinopathy and 28% have central retinal vein occlusion. Other causes include chronic retinal detachment, end-stage glaucoma, carotid obstructive disease, retrolental fibroplasia, sickle cell anemia, intraocular tumors, and carotid cavernous sinus fistula. Neovascular glaucoma can be diagnosed at its early stages by high magnification slit lamp biomicroscopy, where it shows small dilated, structurally disrupted capillaries on the iris surface (which leak fluorescein). Subsequent gonioscopy revealed incremental occlusion of the anterior chamber angle by the fibrovascular band. Although the anterior chamber angle remains open, conservative therapy may have an adjuvant effect. However, once the anterior chamber angle is closed, surgery is required to reduce the pressure.

Thus, in one embodiment of the invention, an anti-angiogenic factor (alone or in combination with an anti-angiogenic composition as described above) may be administered topically to the eye to treat early forms of neovascular glaucoma.

In other embodiments of the invention, the anti-angiogenic composition may be implanted by injecting the composition into the angular region of the anterior chamber. This allows the anti-angiogenic factor to continue to increase locally and prevents blood vessels from growing into the area. Implanted or injected anti-angiogenic compositions placed between the capillaries in the anterior iris and the anterior chamber angle can "shield" the open anterior chamber angle from neovascularization. The patency of the anterior chamber angle may be maintained when the capillaries do not grow within the effective radius of the anti-angiogenic composition. In other embodiments, the anti-angiogenic composition may also be placed at any location such that the anti-angiogenic factor is released into the aqueous humor on a sustained basis. This may increase the concentration of anti-angiogenic factors in the aqueous humor, which in turn bathes the iris surface and its abnormal capillaries, thereby providing another mechanism for drug delivery. These treatment modalities may also be prophylactically useful and may be used in conjunction with existing treatment methods.

In another aspect, the invention provides a method of treating proliferative diabetic retinopathy comprising the step of administering to the eye of a patient a therapeutically effective amount of an anti-angiogenic composition such that angiogenesis is inhibited.

Briefly, the pathology of diabetic retinopathy is believed to be similar to that described above for neovascular glaucoma. In particular, it is believed that background diabetic retinopathy converts to proliferative diabetic retinopathy under the influence of retinal hypoxia. In general, neovascular tissue sprouts from the optic nerve (usually within 10mm of the limbus) and from the retinal surface in areas where tissue perfusion is lacking. Initially capillaries grow between the inner limiting membrane of the retina and the posterior aspect of the vitreous body. Eventually the blood vessels grow into the vitreous and pass through the internal limiting membrane. When the vitreous contracts, traction is applied to the vessel, often resulting in shearing of the vessel and vitreous blurring due to bleeding. Fibrous traction of scarring in the retina can also produce retinal detachments.

The conventional therapy of choice is panretinal photocoagulation to reduce the retinal tissue and thereby reduce the oxygen demand of the retina. Although initially effective, there is a high rate of recurrence due to the formation of new lesions elsewhere in the retina. Complications of this therapy include peripheral vision loss, mechanical abrasion of the cornea, laser-induced cataract formation, acute glaucoma, and stimulation of subretinal neovascularization growth (which can lead to vision loss) in up to 50% of patients. Thus, this method is only performed when there are several hazards and the danger-benefit ratio is clearly advantageous for the surgery.

Thus, in a particularly preferred embodiment of the invention, proliferative diabetic retinopathy may be treated by injecting an anti-angiogenic factor (or anti-angiogenic composition) into the aqueous humor or vitreous to increase the local concentration of the anti-angiogenic factor within the retina. Preferably, this treatment should be initiated before a serious disease requiring photocoagulation. In other embodiments of the invention, the newly-damaged artery may be embolized (using an anti-angiogenic composition as described above).

In another aspect, the invention provides a method of treating retrolental fibroplasia (retrolental fibroplasia) comprising the step of administering to the eye of a patient a therapeutically effective amount of an anti-angiogenic factor (or anti-angiogenic composition) such that angiogenesis is inhibited.

Briefly, retrolental fibroplasia is a disease that occurs in premature infants receiving oxygen therapy. The peripheral retinal vasculature, particularly on the temporal side, is not fully formed until the end of fetal life. Excess oxygen (even at physiological levels when expired) and the formation of oxygen radicals are thought to be important because of the vascular destruction that leads to immature retinas. These vessels contract and then become structurally occluded when exposed to oxygen. As a result, peripheral retina is unable to vascularize and retinal ischemia ensues. In response to ischemia, neovascularization is induced at the junction of the normal and ischemic retina.

In 75% of cases, these vessels spontaneously regress. However, there is continued capillary growth, fibrovascular component contraction, and vessel and retinal tractions in the remaining 25%. This results in vitreous hemorrhage and/or retinal detachment, which can lead to blindness. Neovascular angle closure glaucoma is also a complication of this situation.

Because it is often impossible to determine that these cases may spontaneously resolve and become severe, conventional treatment (i.e., surgery) is typically initiated in patients with well-developed established diseases and conditions. This "waiting and observing" approach precludes early intervention and allows disease progression in 25% of patients with complicated processes. Thus, in one embodiment of the invention, an anti-angiogenic factor (or anti-angiogenic composition as described above) is administered locally in infants at high risk of developing this disease in an attempt to reduce the incidence of the development of retrolental fibroplasia. In other embodiments, intravitreal injections and/or intraocular implants of anti-angiogenic compositions may be used. Such methods are particularly preferred in cases with established disease to reduce the need for surgery.

Protein kinase

Protein kinases are a family of enzymes that catalyze the phosphorylation of the hydroxyl group of specific tyrosine, serine or threonine residues in proteins. In general, such phosphorylation significantly disrupts the function of the protein and thus protein kinases have important roles in regulating various cellular processes, including metabolism, cell proliferation, cell differentiation, and cell survival. Among the many different cellular functions known to require protein kinase activity, certain processes represent attractive targets for therapeutic intervention in certain disease states. Two examples are angiogenesis and cell cycle control, where protein kinases play a key role; these processes are essential for solid tumor growth as well as other diseases.

Angiogenesis is the mechanism by which new capillary blood vessels are formed from existing blood vessels. When needed, the vascular system has the ability to create a new network of capillary vessels in order to maintain proper function of tissues and organs. However, in adults, angiogenesis is rather limited, occurring only in the wound healing process and endometrial neovascularization during the menstrual period. See Merenmies et al, Cell Growth & Differentiation, 8, 3-10(1997), which is incorporated herein by reference for all purposes. On the other hand, unwanted angiogenesis is a hallmark of several diseases, such as retinopathy, psoriasis, rheumatoid arthritis, age-related macular degeneration (AMD), and cancer (solid tumors). Folkman, Nature Med., 1, 27-31(1995), which is incorporated herein by reference for all purposes. Protein kinases that have been shown to be involved in the angiogenic process include three members of the growth factor receptor tyrosine kinase family: VEGF-R2 (vascular endothelial growth factor receptor 2, also known as KDR (kinase insert domain receptor) and known as FLK-1); FGF-R (fibroblast growth factor receptor); and TEK (also known as Tie-2).

VEGF-R2, expressed only on endothelial cells, binds to the potent angiogenic growth factor VEGF and mediates subsequent signal transduction by activating its intracellular kinase activity. Thus, direct inhibition of the kinase activity of VEGF-R2 is expected to reduce angiogenesis even in the presence of exogenous VEGF (see Strawn et al Cancer Research, 56, 3540-3545(1996)) since the use of VEGF-R2 mutants has been shown to be unable to mediate signal transduction. Millauer et al, Cancer Research, 56, 1615-. Furthermore, it appears that the role of VEGF-R2 in adults does not outweigh its role in mediating VEGF angiogenic activity. Thus, selective inhibitors of VEGF-R2 kinase activity would be expected to exhibit little toxicity.

Similarly, FGF-R binds to the angiogenic growth factors aFGF and bFGF and mediates subsequent intracellular signaling. Recently, it has been suggested that growth factors such as bFGF may play a key role in inducing angiogenesis in solid tumors that have reached a certain size. Yoshiji et al, Cancer Research, 57, 3924-. However, unlike VEGF-R2, FGF-R is expressed in many different cell types throughout the body and may or may not play a significant role in other physiological processes in adults. Nevertheless, it has been reported that systemic administration of small molecule inhibitors of FGF-R kinase activity can block bFGF-induced angiogenesis in mice without significant toxicity. Mohammad et al, EMBO Journal, 17, 5996-.

TEK (also known as Tie-2) is another receptor tyrosine kinase expressed only on endothelial cells, which has been shown to play a role in angiogenesis. Binding of angiopoietin-1 results in autophosphorylation of the TEK kinase domain and the generation of a signal transduction process that appears to mediate the interaction of endothelial cells with surrounding endothelial supporting cells, thereby favoring the maturation of newly formed blood vessels. On the other hand, the factor angiopoietin-2 appears to antagonize the effects of angiopoietin-1 on TEK and disrupt angiogenesis. Maison pierre et al, Science, 277, 55-60 (1997).

As a result of the above developments, it has been proposed to treat angiogenesis by using compounds that inhibit the kinase activity of VEGF-R2, FGF-R and/or TEK. For example, WIPO international publication No. WO97/34876 discloses certain cinnoline derivatives that are VEGF-R2 inhibitors that are useful in the treatment of disease states associated with abnormal angiogenesis and/or increased vascular permeability, such as cancer, diabetes, psoriasis, rheumatoid arthritis, kaposi's sarcoma, hemangioma, acute and chronic kidney disease, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, and ocular diseases associated with retinal vascular proliferation.

Phosphorylase kinase activates glycogen phosphorylase, thereby increasing glycogenolysis and hepatic glucose release. Hepatic glucose production is deregulated in type 2 diabetes and is the leading cause of invariable hyperglycemia, which can produce many secondary complications in patients with these conditions. Thus, reducing glucose release from the liver can reduce high plasma glucose levels. Phosphorylase kinase inhibitors should therefore reduce phosphorylase activity and glycogenolysis and thus reduce hyperglycemia in patients.

Another physiological response to VEGF is vascular hyperpermeability, which has been proposed to play a role in the early stages of angiogenesis. In ischemic tissues such as those found in the brain of stroke victims, hypoxia triggers VEGF expression, leading to increased vascular permeability and ultimately edema in the surrounding tissues. In a rat model of stroke, van Bruggen et al have demonstrated that administration of monoclonal antibodies to VEGF reduces infarct volume in J.clinical Invest, 104, 1613-20 (1999). Therefore, VEGFR inhibitors are expected to be useful in the treatment of stroke.

In addition to roles in angiogenesis, protein kinases play a crucial role in cell cycle control. Uncontrolled cell proliferation is a hallmark of cancer. Cell proliferation in response to various stimuli is indicated by dysregulation of the cell division cycle during cell proliferation and division. Tumor cells generally have a damaging effect on genes that directly or indirectly regulate development through the cell division cycle.

Cyclin-dependent kinases (CDKs) are serine-threonine protein kinases that play a key role in regulating transitions between different stages of the cell cycle. See, for example, the paper compiled in Science, 274, 1643-. CDK complexes are formed by linking regulatory cyclin subunits (e.g., cyclin A, B1, B2, D1, D2, D3, and E) with catalytic kinase subunits (e.g., cdc2(CDK1), CDK2, CDK4, CDK5, and CDK 6). The term means that CDKs exhibit an absolute dependence on cyclin subunits in order to phosphorylate their target substrates and that different kinase/cyclin pairs act to regulate progression through specific phases of the cell cycle.

It is the CDK4 complexed with D cyclins that plays a partially critical role in initiating the cell division cycle from the resting or dormant stage to the stage where the cell undergoes cell division. This progression is through different growth regulatory mechanisms, including both positive and negative regulatory mechanisms. Abnormalities in this control system, particularly those affecting CDK4 function, involve the development of cells to a highly proliferative state characterized by malignancies, particularly familial melanoma, esophageal cancer, and pancreatic cancer. See, for example, Kamb, Trends in Genetics, 11, 136-140 (1995); kamb et al, Science, 264, 436-.

Numerous publications describe various chemical compounds for different therapeutic targets. For example, WIPO International publication Nos. WO 99/23077 and WO 99/23076 describe indazole-containing compounds that have type IV phosphodiesterase inhibitory activity resulting from the bioisosteric replacement of indazole-catechol. The use as alpha is disclosed in US 5,760,028_γβ₃Heterocycles comprising 3- [1- [3- (imidazolin-2-ylamino) propyl ] integrin antagonists and related cell surface fibronectin receptors]Indazol-5-ylcarbonylamino]-2- (benzyloxycarbonylamino) propionic acid. WIPO International publication No. WO 98/09961 discloses certain indazole derivatives and their use as inhibitors of phosphodiesterase type IV or as inhibitors of the production of Tumor Necrosis Factor (TNF) in mammals. Recent additions to the actual repertoire of known compounds include those compounds described as antiproliferative therapeutic agents that inhibit CDKs. For example, Xiong et al disclose nucleic acids encoding CDK6 inhibitors in US patent No. US 5,621,082, and european patent publication No. EP 0666270 a2 describes peptides and peptidomimetics that act as CDK1 and CDK2 inhibitors. WIPO International publication No. WO 97/16447 discloses certain chromone analogs belonging to cyclin dependent kinases, particularly CDK/cyclin complexes, such as CDK 4/cyclin D1, which may be useful in inhibiting excessive or abnormal cell proliferation and thus in the treatment of cancer. 4-aminothiazole derivatives useful as CDK inhibitors are described in WIPO International publication No. WO 99/21845. The entire contents of each of the above-listed patents and/or applications are incorporated herein by reference for all purposes.

However, there remains a need for small molecule compounds that are easily synthesized and that can effectively inhibit one or more CDKs or CDK/cyclin complexes. Because CDK4 may serve as a general activator of cell division in most cells and the complex of CDK4 and D-type cyclins controls the early G1 stage of the cell cycle, there is a need for effective inhibitors of CDK4 and its D-type cyclin complexes for the treatment of one or more types of tumors. Furthermore, G₂stage/S and G₂The key roles of cyclin E/CDK2 and cyclin B/CDK1 kinases in the/M transition each provide additional targets for therapeutic intervention in inhibiting dysregulated cell cycle progression in cancer.

The other protein kinase, CHK1, plays an important role as a checkpoint in cell cycle progression. Checkpoints are control systems that coordinate the progression of the cell cycle by affecting cyclin-dependent kinase formation, activation and subsequent inactivation. When the requirements of the checkpoint are not met, the checkpoint prevents cell cycle progression at inappropriate times, maintains the metabolic balance of the cell while arresting the cell, and can induce apoptosis (programmed cell death) in some cases. See, for example: o' Connor, Cancer Surveys, 29, 151-; nurse, Cell, 91, 865 and 867 (1997); hartwell et al, Science, 266, 1821-; hartwell et al, Science, 246,629-634 (1989).

A series of checkpoints monitor the integrity of the genome and when DNA damage is detected, these "DNA damage checkpoints" block G₁And G₂The metaphase cell cycle progresses and slows down progression through the S phase. O' Connor, Cancer Surveys, 29, 151-; hartwell et al, Science, 266, 1821-. This action enables the DNA repair process to complete its task, after which the genome is replicated and subsequent segregation of this genetic material into new daughter cells occurs. Importantly, the most common mutant gene in human cancers, p53, tumor suppressor gene production blocks G after DNA damage₁DNA-damaged checkpoint proteins that progress through the cell cycle in phase and/or induce apoptosis (programmed cell death). Hartwell et al, Science, 266, 1821-. It also demonstrates that the p53 tumor suppressor gene enhances cell cycle G₂The stage of DNA damage checkpoint. See, for example: bunz et al, Science, 28, 1497-1501 (1998); winters et al, Oncogene, 17, 673-; thompson, Oncogene, 15, 3025-3035 (1997).

Given the key nature of the p53 tumor suppressor gene pathway in human cancers, therapeutic interventions that exploit the vulnerability in p 53-deficient cancers are actively being sought. One emerging vulnerability is that G is present in p 53-deficient cancer cells₂And (5) operation of the level. Lack of G in cancer cells₁Checkpoint controls by eliminating the DNA active agent G which prevents them from being damaged₂The last remaining barrier to cancer killing by checkpoints is particularly vulnerable. G₂The checkpoint is regulated by a control system from yeast storage to human. At this pointImportant in this preservation system is the kinase CHK1, which transduces signals from the DNA-damaged sensory complex to inhibit activation of cyclin B/Cdc2 kinase that promotes mitotic entry. See, for example: peng et al, Science, 277, 1501-1505 (1997); sanche et al, Science, 277, 1497-1501 (1997). Inactivation of CHK1 has been shown to eliminate either anti-cancer agent-induced DNA damage or endogenous DNA damage-induced G₂Blocking, in turn, can lead to preferential killing of the resulting checkpoint defective cells. See, for example: nurse, Cell, 91, 865 and 867 (1997); weinert, Science, 277, 1450-1451 (1997); walworth et al, Nature, 363, 368-371 (1993); and Al-Khodairy et Al, mol. biol. cell, 5, 147-.

Selective control of checkpoint control in cancer cells can provide a broad range of applications in cancer chemotherapy and radiotherapy protocols and can also provide a general hallmark of human cancer "genomic instability" that is exploited as a selective basis for the destruction of cancer cells. CHK1 has been identified as a key target in the control of DNA-damage checkpoints for a number of factors. The description of inhibitors of this and functionally related kinases, such as Cds1/CHK2, a kinase recently found to act synergistically with CHK1 in regulating the development of phase S (see Zeng et al Nature 395, 507- & 510 (1998); Matsuoka, Science 282, 1893- & 1897(1998)) may provide valuable new therapeutic entities for the treatment of cancer.

Integrin receptors bound to the ECM initiate FAK (focal adhesion kinase) -mediated intracellular signals involved in cell motility, cell proliferation and survival. In human cancers, FAK overexpression is involved in tumorigenesis and metastatic potential through its role in integrin-mediated signaling pathways.

Tyrosine kinases can be of the receptor type (containing both the extracellular transmembrane and intracellular domains) or the non-receptor type (fully intracellular). At least one non-receptor protein tyrosine kinase, LCK, is thought to mediate transduction in T-cells from signals from the interaction of cell surface proteins (Cd4) with cross-linked anti-Cd 4 antibodies. A more detailed discussion of non-receptor tyrosine kinases is provided by Bolen in Oncogene, 8, 2025-one 2031(1993), the entire contents of which are incorporated herein by reference for all purposes.

In addition to the protein kinases identified above, many other protein kinases are considered therapeutic targets and inhibitors of kinase activity are disclosed in a number of publications, as reviewed in: US patent US6,534,524 granted on 3/18/2003; US patent US6,531,491 granted on 11/3/2003; PCT International patent application publication No. WO 00/38665 (published on 7/6/2001); PCT International patent application publication No. WO 97/49688 (published on 31/12/1997); PCT International patent application publication No. WO 98/23613 (published on 6/4/1998); US patent US6,071,935 granted 6/2000; PCT International patent application publication No. WO 96/30347 (published 3/10/1996); PCT International patent application publication No. WO 96/40142 (published on 12/19 1996); PCT International patent application publication No. WO 97/13771 (published 1997 on 17.4.1997); PCT International patent application publication No. WO 95/23141 (published on 31/8/1995); PCT International patent application publication No. WO 03/006059 (published on 23/1/2003); PCT International patent application publication No. WO 03/035047 (published 5/1/2003); PCT International patent application publication No. WO 02/064170 (published on 8/22 2002); PCT International patent application publication No. WO02/41882 (published 5/30 2002); PCT International patent application publication No. WO02/30453 (4/18/2002); PCT International patent application publication No. WO 01/85796 (published on 11/15/2001); PCT International patent application publication No. WO01/74360 (published 10/11/2001); PCT International patent application publication No. WO 01/74296 (published on 10/11/2001); PCT International patent application publication No. WO 01/70268 (published on 9/27/2001); european patent application publication No. EP 1086705 (published 3/28/2001); and PCT international patent application publication No. WO 98/51344 (published on 19/11/1998). The entire contents of each of the above-identified patents and applications are incorporated herein by reference for all purposes.

However, there remains a need for effective protein kinase inhibitors. Furthermore, as will be appreciated by those skilled in the art, there is a need for kinase inhibitors that have a high affinity for target kinases and a high selectivity compared to other protein kinases.

It is therefore an object of the present invention to find effective active agents for the treatment of the following eye diseases: such as age-related macular degeneration (ARMD), Choroidal Neovascularization (CNV), retinopathies (e.g., diabetic retinopathy, vitreoretinopathy, retinopathy of prematurity), retinitis (e.g., Cytomegalovirus (CMV) retinitis), uveitis, macular edema, and glaucoma.

It is another object of the present invention to find potent inhibitors of protein kinases.

It is another object of the present invention to find potent kinase inhibitors with a strong and selective affinity for one or more specific kinases.

These and other objects of the present invention, which will become apparent from the specification, are achieved by the discovery of indazole compounds, pharmaceutically acceptable prodrugs thereof, pharmaceutically active metabolites thereof, and pharmaceutically acceptable salts thereof (such compounds, prodrugs, metabolites and salts are collectively referred to as "active agents") that modulate and/or inhibit the activity of protein kinases as described below. Pharmaceutical compositions containing such agents are useful for treating diseases mediated by kinase activity, such as cancer and other disease states associated with: unwanted angiogenesis and/or cell proliferation such as diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis and psoriasis. Furthermore, these agents have advantageous properties relating to the modulation and/or inhibition of kinase activity associated with the VEGF-R, FGF-R, CDK complex, CHK1, LCK, TEK, FAK and/or phosphorylase kinases.

The present invention relates generally to compounds having the structure:

the invention also relates to methods of modulating and/or inhibiting VEGF-R, FGF-R, CDK complex, CHK1, LCK, TEK, FAK and/or phosphorylase kinase by administering a compound of formula I, II, III or IV or a pharmaceutically acceptable prodrug, pharmaceutically active metabolite or pharmaceutically acceptable salt thereof. Preferred compounds of the invention have selective kinase activity, i.e., they have significant activity against one or more specific kinases, while having less or minimal activity against one or more different kinases. In a preferred embodiment of the invention, the compounds of the invention are those of formula I which have substantially higher efficacy against VEGF receptor tyrosine kinase than against FGF-R1 receptor tyrosine kinase. The invention also relates to methods of modulating VEGF receptor tyrosine kinase activity without significantly modulating FGF receptor tyrosine kinase activity.

The compounds of the invention may be advantageously used in combination with other known therapeutic agents. For example, compounds of formula I, II, III or IV having anti-angiogenic activity may be co-administered with cytotoxic chemotherapeutic agents such as taxol, taxotere, vinblastine, cisplatin, doxorubicin and the like to produce enhanced anti-tumor effects. Additional or synergistic potentiation of therapeutic effects can also be obtained by co-administration of compounds of formula I, II, III or IV having anti-angiogenic activity with other anti-angiogenic agents such as combretastatin A-4, endostatin, prinostat, celecoxib, rofocoxib, EMD121974, IM862, anti-VEGF monoclonal antibodies and anti-KDR monoclonal antibodies. Other combinations are exemplified by the combinations in WO 0038716, WO 00387171, WO 0038715, WO0038730, WO 0038718, WO 0038665, WO 0037107, WO 0038786, WO0038719, all filed concurrently in 1999 on 12/22, the entire contents of which are incorporated herein by reference for all purposes.

The invention also relates to pharmaceutical compositions comprising an effective amount of an active agent selected from the group consisting of compounds of formula I and pharmaceutically acceptable salts, pharmaceutically active metabolites and pharmaceutically acceptable prodrugs thereof and a pharmaceutically acceptable carrier or excipient for such active agents. The invention further provides methods of treating diseases/conditions of the eye and cancer and other disease states associated with unwanted angiogenesis and/or cell proliferation comprising administering to a patient in need of such treatment an effective amount of such agents.

The compounds of the general formula I, II, III or IV of the invention are useful for treating ocular diseases and for mediating protein kinase activity. More particularly, the compounds are useful as antiangiogenic agents and as agents that modulate and/or inhibit the activity of protein kinases, thereby providing methods of treating ocular diseases and cancers or other diseases associated with protein kinase mediated cell proliferation.

The term "alkyl" as used herein refers to straight and branched chain alkyl groups having 1 to 12 carbon atoms. Typical alkyl groups include methyl (Me), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (t-Bu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. The term "lower alkyl" denotes an alkyl group (C) having 1-8 carbon atoms₁-C₈Alkyl groups). Suitable substituted alkyls include fluoromethyl, difluoromethylTrifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl and the like.

The term "alkylene" refers to a divalent group having 1 to 12 carbon atoms. Illustrative alkylene groups include CH₂、CHCH₃、(CH₃)₂And the like.

The term "alkenyl" refers to straight and branched chain alkenyl groups having 2 to 12 carbon atoms. Illustrative alkenyl groups include prop-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl and the like.

The term "alkynyl" refers to straight and branched chain alkynyl groups having 2 to 12 carbon atoms.

The term "cycloalkyl" refers to a saturated or partially unsaturated carbocyclic ring having 3 to 12 carbon atoms, including bicyclic and tricyclic cycloalkyl structures. Suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

"heterocycloalkyl" is intended to mean a saturated or partially unsaturated monocyclic radical containing carbon atoms, preferably 4 or 5 ring carbon atoms, and at least one heteroatom selected from nitrogen, oxygen and sulfur.

The terms "aryl" and "heteroaryl" refer to monocyclic and polycyclic unsaturated or aromatic ring structures, wherein "aryl" refers to those groups that are carbocyclic and "heteroaryl" refers to those groups that are heterocyclic. Examples of aromatic ring structures include phenyl, naphthyl, 1, 2, 3, 4-tetrahydronaphthyl, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, imidazolyl, pyrazinyl, pyridazinyl, 1, 2, 3-triazinyl, 1, 2, 4-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1-H-tetrazol-5-yl, indole, quinolyl, benzofuryl, benzothiophenyl (thioindenyl), and the like. Such groups may optionally be fused ring structures or bridges, e.g. OCH₂-O substitution.

The term "alkoxy" is used to refer to the group-O-alkyl. Illustrative examples include methoxy, ethoxy, propoxy, and the like.

The term "aryloxy" denotes-O-aryl, wherein aryl is as defined above.

The term "cycloalkoxy" denotes-O-cycloalkyl, wherein cycloalkyl is as defined above.

The term "halogen" denotes chlorine, fluorine, bromine or iodine. The term "halo" denotes chloro, fluoro, bromo or iodo.

In general, various moieties or functional groups that are variable in a formula can be optionally substituted with one or more suitable substituents. Typical substituents include halogen (F, Cl, Br or I), lower alkyl, -OH, -NO₂、-CN、-CO₂H. -O-lower alkyl, aryl, -aryl-lower alkyl, -CO₂CH₃、-CONH₂、-OCH₂CONH₂、-NH₂、-SO₂NH₂Haloalkyl (e.g., -CF)₃、-CH₂CF₃) -O-haloalkyl (e.g., -OCF)₃、-OCHF₂) And the like.

The terms "comprising" and "including" are used in an open-ended, non-limiting sense.

It is to be understood that while the compounds of formula I may exhibit tautomerism, the general structures in this specification are only one of the possible tautomeric forms is clearly depicted. It will be understood that the general formulae of the present invention are intended to represent any tautomeric form of the compound shown and are not to be limited to the specific tautomeric form depicted by the general structure.

Certain compounds of the present invention may exist as single stereoisomers (i.e., substantially free of other stereoisomers), racemates and/or enantiomeric and/or diastereomeric mixtures. All such single stereoisomers, racemates and mixtures thereof are within the scope of the present invention. Preferably, the optically active compounds of the present invention are used in optically pure form.

As is generally understood by those skilled in the art, an optically pure compound containing one chiral center is one that consists essentially of one of two possible enantiomers (i.e., is enantiomerically pure), and an optically pure compound containing more than one chiral center is one that is both diastereomerically and enantiomerically pure. Preferably, the compounds of the present invention are used in at least 90% optically pure form, i.e., containing at least 90% of a single isomer (80% enantiomeric excess ("e.e") or diastereomeric excess ("d.e"), more preferably at least 95% (90% e.e. or d.e.), even more preferably at least 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98% e.e. or d.e.).

Additionally, the formulas are intended to include solvates and non-solvates of the identified structures. For example, formula I includes both hydrated and non-hydrated forms of the compounds of the structures shown. Other examples of solvates include structures incorporating isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

In addition to compounds of formulae I, II, III and IV, the present invention also includes pharmaceutically acceptable prodrugs, pharmaceutically active metabolites and pharmaceutically acceptable salts of such compounds.

A "pharmaceutically acceptable prodrug" is a compound that can be converted under physiological conditions or by solvolysis to a particular compound or to a pharmaceutically acceptable salt of such a compound.

"pharmaceutically active metabolite" is used to refer to the pharmacologically active product produced by the metabolism of a particular compound or salt thereof in the body. Metabolites of a compound can be identified using conventional techniques well known in the art and their activity can be determined using assays such as those described herein.

Prodrugs and active metabolites of compounds may be identified using conventional techniques well known in the art. See, for example, Bertolini, g, et al, j.med.chem., 40, 2011-; shan, D, et al, J.pharm.Sci., 86(7), 765-; bagshawe k., Drug dev. res., 34, 220-; bodor, N., Advances in Drug Res., 13, 224-; bundgaard, H., Design of produgs (Elsevier Press 1985); and Larsen, I.K., Design and Application of Prodrugs, Drug Design and development (Krogsgaard-Larsen et al, edited by Harwood Academic Publishers, 1991).

"pharmaceutically acceptable salt" is used to refer to salts that retain the biological effectiveness of the free acids and bases of the particular compound or are not biologically undesirable. The compounds of the present invention may bear sufficiently acidic, sufficiently basic or both functional groups and may therefore react with any number of inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts. Typical pharmaceutically acceptable salts include those prepared by reacting a compound of the invention with an inorganic or organic acid or an inorganic base, such as the following salts: including sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, dihydrogenphosphate, metaphosphate, chloride, bromide, iodide, acetate, propionate, caprylate, acrylate, formate, benzoate, xylenesulfonate, benzoate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, or, Gamma-hydroxybutyrate, glycolate, tartrate, mesylate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.

If the compounds of the invention are bases, the desired pharmaceutically acceptable salts may be prepared by any suitable method known in the art, for example by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid; pyranonic acids, such as glucuronic or galacturonic acids; alpha-hydroxy acids such as citric acid or tartaric acid; amino acids such as aspartic acid or glutamic acid; aromatic acids such as benzoic acid or cinnamic acid; sulfonic acids such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.

If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example by treating the free acid with an inorganic base or with an organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

In the case of solid active agents, those skilled in the art will appreciate that the compounds and salts of the present invention may exist in different crystalline forms or polymorphs and are within the scope of the present invention and specific formulae.

Therapeutically effective amounts of the active agents of the present invention may be used to treat diseases mediated by modulation or modulation of protein kinases. An "effective amount" is intended to mean an amount of active agent that, when administered to a mammal in need of such treatment, is sufficient to effect treatment of a disease mediated by one or more protein kinases, such as tyrosine kinase activity. Thus, for example, a therapeutically effective amount of a compound of formula I, a salt, active metabolite or prodrug thereof, is an amount sufficient to modulate, regulate or inhibit the activity of one or more protein kinases such that the disease condition mediated by such activity is alleviated or alleviated.

The amount of a given active agent that corresponds to this amount will vary depending upon such things as the particular compound, the disease condition and its severity, and the condition of the mammal in need of treatment (e.g., body weight), although it will generally be within the skill of the art. "treatment" is used to indicate that a disease in a mammal, such as a human, that is at least partially affected by the activity of one or more protein kinases, such as tyrosine kinases, is alleviated and includes: preventing the occurrence of a disease condition in a mammal, particularly when the mammal is found to be predisposed to the disease but has not yet been diagnosed as having it; modulating and/or inhibiting such disease conditions; and/or alleviating such disease conditions.

The active agents of the present invention can be prepared using readily available starting materials using the reaction schemes and synthetic schemes described below using techniques available in the art.

In one general synthetic approach, compounds of formula I are prepared according to the following reaction scheme:

6-Nitroindazole (Compound V) is treated with iodine and a base, such as NaOH, in an aqueous/organic mixture preferably containing dioxane. The mixture was acidified and the product isolated by filtration. To the resulting 3-iodo-6-nitroindazole in dichloromethane-50% KOH aqueous solution at 0 ℃, is added a protecting group ("Pg") reagent (where X ═ halogen), preferably trimethylsilylethoxymethyl chloride (SEM-CI) and a phase transfer catalyst, such as tetrabutylammonium bromide (TBABr). After 1-4 hours, the two phases were diluted, the organic phase was separated, dried over sodium sulfate, filtered and concentrated. The crude product is purified by silica gel chromatography to give the compound of formula VI. In the presence of a base, e.g. aqueous sodium carbonate solution, and a suitable catalyst, preferably Pd (PPh)₃)₄With appropriate R in the presence of a suitable organic solvent¹-organometallic reagents, preferably R¹Boronic acid treatment of the compound of formula VI to give the compound of formula VII after an extraction operation and silica gel chromatography. R on the compound of the formula VII can be exchanged¹The substituents or intermediates thereafter throughout this scheme are subjected to oxidative cleavage (e.g., ozonolysis) followed by addition of the resulting aldehyde functionality using Wittig or condensation transformations (typical of examples 42 (a-e)). With reducing agents, preferably SnCl₂Treating the compound of formula VII to obtain a compound of formula VII after conventional water treatment and purificationA compound of VIII. For a series of derivatives, where Y ═ NH or N-lower alkyl, can be in the presence of a base, preferably Cs₂CO₃And treating a compound of formula VIII (and wherein Y ═ N-lower alkyl, followed by an alkylation step) with an aryl or heteroaryl chloride, bromide, iodide or triflate in the presence of a catalyst, preferably Pd-BINAP, to give a compound of formula X. To generate additional Y bonds, sodium nitrite is added to the compound of formula VIII under chilled standard aqueous acidic conditions followed by addition of potassium iodide and moderate heating. Standard workup and purification gives compounds of the formula IX.

Treatment of compounds of formula IX with an organometallic reagent, such as butyl lithium, promotes lithium halide exchange. The intermediate is then passed over other metals and catalysts, preferably zinc chloride and Pd (PPh)₃)₄May mediate a reaction with an R2 electrophile, such as carbonyl or triflate, to give a compound of formula X. On the other hand, in a carbon monoxide atmosphere and in the presence of a catalyst, Pd (PPh) is preferred₃)₄Treating a compound of formula IX with a suitable organometallic reagent, such as an organoboronic acid, in the presence of a suitable organometallic reagent to provide a compound of formula X. On the other hand, as for the derivative of Y ═ NH or S, a base may be present, preferably Cs₂CO₃And K₃PO₄And a catalyst, preferably Pd-BINAP or Pd- (bis-cyclohexyl) diphenylphosphine, with a suitable amine or thiol to give a compound of formula X. The series can then be further derivatized by conventional functional group exchange, such as oxidation, reduction, alkylation, acylation, condensation, and deprotection to give the final compound of formula I.

The compounds of formula I of the present invention may also be prepared according to the general procedure shown in the following scheme:

using iodine and a base in an aqueous/organic mixture, preferably with dioxaneSuch as NaOH treatment of 6-iodoindazole (compound XI). The mixture was acidified and the product XII was isolated by filtration. To the resulting 3, 6-diiodo-indazole in dichloromethane-50% KOH aqueous solution at 0 ℃ is added a protecting group reagent, preferably SEM-CI, and a phase transfer catalyst, such as TBABr. The phases were diluted, the organic phase was separated, dried over sodium sulfate, filtered and concentrated. The crude product is purified by silica gel chromatography to give the compound of formula XIII. With the appropriate R in a suitable organic solvent²-organometallic reagents, preferably R²-ZnCl or boron R²Boron reagent and suitable catalyst, preferably Pd (PPh)₃)₄Treatment of the compound of formula XIII affords, after extraction is complete and chromatography on silica gel, the compound of formula XIV. In the presence of a base such as aqueous sodium carbonate and a suitable catalyst, preferably Pd (PPh)₃)₄With appropriate R in the presence of a suitable organic solvent¹Organometallic reagents (e.g. boron R)²-boron reagent or R²-ZnCl) to give the compound of formula XV after completion of the extraction and chromatography on silica gel. Conventional functional group exchanges such as oxidation, reduction, alkylation, acylation, condensation and deprotection can then be used to further derivatize this class of compounds to give the final compounds of formula I.

In another aspect, the compounds of formula I, wherein R is as shown in the following general scheme, may be prepared²Is substituted or unsubstituted Y-Ar, wherein Y is O or S:

stirring 3-chloro-cyclohex-2-enone (XV), H-R²And anhydrous potassium carbonate in acetone for 15-24 hours, cooled and filtered. Concentrating and subjecting the filtrate to silica gel chromatography to give 3-R²-cyclohex-2-enone (XVI).

Ketones of the general formula XVI can be reacted with a suitable base (M-B), preferably lithium bis (trimethylsilyl) amide, and with R¹-CO-X (wherein X ═ halogen) reaction, after standard acid treatmentAnd purification to give the compound of formula XVII. The product mixed with hydrazine monohydrate in HOAc/EtOH is heated at a suitable temperature for a suitable time, preferably at 60-80 ℃ for 2-4 hours. After cooling, the mixture was poured into saturated sodium bicarbonate solution, extracted with organic solvent, concentrated and purified on silica gel to give the compound of formula XVIII. The compounds of formula XVIII may be oxidized using a variety of known methods to give compounds of formula I.

Other methods for synthesizing the compounds of the invention are as follows:

wherein the conditions of steps a) to i) are as follows:

a)NaNO₂、Br₂HBr, 0 ℃ to 5 ℃; the yield is 48 percent;

b)Pd(OAc)₂pd (o-tolyl)₃Di-isopropyl ethylamine (DIEA), DMF, H₂O, degassing, microwave, 110 ℃ and 1 hour; the yield is 68 percent;

c) iron powder, saturated NH₄Aqueous OH, EtOH, 45 ℃; the yield is 72 percent;

d) methyl 2-bromobenzoate, R-BINAP, Pd₂(dba)₃、Cs₂CO₃Toluene, degassing overnight at 110 ℃; the yield is 74 percent;

e)KOH、MeOH∶THF∶H₂o (3: 1) at 70 ℃ for 2-3 hours; quantifying;

f) protected amines, HATU, NEt₃DMF, room temperature for 2 hours; the yield is 80%;

g) TsOH (12% TsOH in HOAc), EtOH (10% aqueous); yield 44% yield;

h) tributylvinyltin, Pd (PPh)₃)₄2, 6-di-tert-butyl-4-methylphenol, toluene, 10Degassing at 5 ℃; the yield is 31 percent;

i)Pd(OAc)₂pd (o-tolyl)₃DIEA, DMF, degassing at 100 ℃; the yield thereof was found to be about 70%.

Other compounds of formula I may be prepared according to methods analogous to the general methods described above or specific methods described in the examples herein. The affinity of the compounds of the invention for the receptor can be enhanced by providing multiple copies of the ligand in closely related form, preferably using a platform provided by the carrier moiety. It has been demonstrated that providing such multivalent compounds with optimal spacing between the moieties can significantly improve binding to the receptor. See, for example, Lee et al, Biochem, 23, 4255 (1984). Selection of appropriate carrier moieties or linker units can be provided to control multivalence and spacing. Such moieties include molecular carriers comprising a plurality of functional groups that can react with the functional groups attached to the compounds of the present invention. Of course, a variety of carriers may be used, including: proteins such as BSA or HAS; a variety of peptides, including: such as pentapeptides, decapeptides, pentadecapeptides, and the like. The peptide or protein may contain a desired number of amino acid residues having a free amino group in its side chain; however, other functional groups, such as sulfhydryl or hydroxyl groups, may also be used to obtain a stable bond.

Compounds that effectively modulate, modulate or inhibit the activity of protein kinases associated with the receptors VEGF, FGF, CDK complex, TEK, CHK1, LCK, FAK, and phosphorylase kinases, among others, and inhibit angiogenesis and/or cell proliferation are desirable and are a preferred embodiment of the present invention. The invention further relates to methods of modulating or inhibiting protein kinase activity, e.g., in mammalian tissue, by administering the agents of the invention. The activity of the compounds of the invention as modulators of protein kinase activity, such as kinase activity, can be determined by any method available to those skilled in the art, including in vivo and/or in vitro assays. Examples of suitable assays for activity determination include those described in the following references: parast C, et al, BioChemistry, 37, 16788-; jeffrey et al Nature, 376, 313-320 (1995); WIPO International publication No. WO 97/34876; and WIPO International publication No. WO 96/14843. These properties can be assessed, for example, by using one or more of the biological test methods listed in the examples below.

The active agents of the present invention may be formulated into pharmaceutical compositions as described below. The pharmaceutical compositions of the present invention comprise an effective modulating, modulating or inhibiting amount of a compound of formula I, II, III or IV and a pharmaceutically acceptable inert carrier or diluent. In one embodiment of the pharmaceutical composition, the active agent of the invention is provided to an effective level that results in a therapeutic benefit comprising modulation of a protein kinase. By "effective level" is meant a level that minimally modulates the action of the protein kinase. These compositions are prepared in unit dosage forms suitable for administration, e.g., parenteral or oral administration.

The active agent of the present invention may be administered in the form of a usual dosage form prepared by mixing a therapeutically effective amount of the active agent (e.g., a compound of formula I) as an active ingredient with a suitable pharmaceutical carrier or diluent according to a conventional method. These methods may include mixing, granulating and compressing or dissolving the components as necessary to make the desired formulation.

The pharmaceutical carrier used may be solid or liquid. Typical solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Typical liquid carriers are syrup, peanut oil, olive oil, water, and the like. Similarly, the carrier or diluent may include time-delayed or time-release materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, or the like.

Various pharmaceutical dosage forms may be used. Thus, if a solid carrier is used, the formulation may be tableted, filled into hard gelatin capsules as a powder or pellet, or tableted. The amount of solid carrier can vary, but is generally from about 25mg to about 1 g. If a liquid carrier is used, the preparation is in the form of syrup, emulsion, drops, soft gelatin capsule, sterile injectable solution or suspension in ampoules or vials, or non-aqueous liquid suspension.

To obtain a stable water soluble dosage form, the pharmaceutically acceptable salts of the active agents of the present invention are dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3M succinic or citric acid solution. If a soluble salt form is not available, the active agent may be dissolved in a suitable co-solvent or mixture of co-solvents. Examples of suitable co-solvents include, but are not limited to, alcohols, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin, and the like, at concentrations of 0-60% of the total volume. In typical embodiments, the compound of formula I is dissolved in DMSO and diluted with water. The composition may also be in the form of a solution of the salt form of the active ingredient in a suitable aqueous carrier, such as water or isotonic saline or dextrose solution.

It will be appreciated that the actual dosage of active agent employed in the compositions of the invention will vary with the particular complex employed, the particular composition formulated, the mode and site of administration, the host treated and the disease being treated. One skilled in the art can use routine dosimetry tests to determine the optimum dose given a given set of specified conditions based on experimental data for the active agent. For oral administration, typical daily dosages of about 0.001 to about 1000mg/kg body weight, more preferably about 0.001 to about 50mg/kg body weight are generally employed, with the course of treatment being repeated at appropriate intervals. Prodrugs are generally administered at a weight level that is chemically equivalent to the weight level of the intact active agent form.

The compositions of the present invention may be prepared according to methods well known for the preparation of pharmaceutical compositions in general, e.g. using conventional techniques such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical compositions may be formulated in accordance with conventional procedures using one or more physiologically acceptable carriers, which may be selected from excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

Suitable formulations depend on the chosen route of administration. For injection, the active agents of the invention may be formulated as aqueous solutions, preferably in physiologically compatible buffers such as hanks 'solution, ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds are readily formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by the following steps: using a mixture of a solid excipient and an active ingredient (active agent); the resulting mixture is optionally ground and, if desired, the granulate mixture is processed into tablet or pastille cores after addition of suitable auxiliaries. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gums, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added.

The core of the ingot may be suitably coated. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinylpyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings to facilitate identification or to characterize different combinations of active ingredients.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient together with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agent may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be suitable in dosages for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration intranasally or by inhalation, it is convenient to deliver the compounds for use according to the invention in the form of an aerosol spray device or nebuliser from a pressurised pack, in which a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, is used. In the case of a pressurized aerosol, the dosage unit can be measured by installing a valve that delivers a metered amount. Capsules and gelatin cartridges for use in an inhaler or insufflator or the like may be formulated containing the compound in admixture with a suitable powder base, such as lactose or starch.

The compounds may 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, e.g., in ampoules or in multi-dose containers, with an added preservative. These 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.

Pharmaceutical preparations for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. Alternatively, suspensions of the active agents can be prepared as appropriate injectable oil suspensions. Suitable lipophilic solvents or carriers include: fatty oils such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension may also optionally contain suitable stabilizers or agents that increase the solubility of the compounds so that highly concentrated solutions can be prepared.

For administration to the eye, a compound of formula I, II, III or IV may be delivered in a pharmaceutically acceptable ophthalmic vehicle to maintain contact of the compound with the ocular surface for a time sufficient to allow penetration of the compound into the cornea and/or sclera and interior regions of the eye, including: such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary body, lens, choroid/retina and sclera. The pharmaceutically acceptable ophthalmic carrier may be an ointment, vegetable oil or an encapsulating material. The compounds of the invention may be injected directly into the vitreous humor or aqueous humor.

In addition, the compounds may also be administered by well-known accepted methods, such as sub-tenon and/or subconjunctival injection. As is well known in the ophthalmic art, the macula consists primarily of the retinal cone and is the largest area of vision in the retina. The tenon's capsule or tenon's membrane is arranged on the sclera. The conjunctiva 36 covers a small area of the posterior limbal globe (bulbar conjunctiva) and folds up (upper conjunctiva) or down (lower conjunctiva) to cover the inner area of the upper and lower eyelids, respectively. The conjunctiva is arranged above the tenon's capsule.

The sclera and tenon's capsule define the outer surface of the eyeball. For the treatment of ARMD, CNV, retinopathies, retinitis, uveitis, Cystoid Macular Edema (CME), glaucoma, and other diseases or conditions of the posterior segment of the eye, it is preferred to dispose a specific amount of a long acting formulation of an ophthalmically acceptable pharmaceutically active agent directly on the outer surface of the sclera and under the tenon's capsule. Furthermore, for ARMD and CME, it is most preferred that the depot be disposed directly on the outer surface of the sclera, under the tenon's capsule and generally on the spot. In a study using white New Zealand rabbits, a long acting pharmaceutical formulation of the angiostatic steroid 4, 9(11) -pregnadiene-17. alpha., 21-diol-3, 20-dione-21-acetate, available from Steralodids, Inc. of Wilton, NewHampshire, was placed directly on the outer surface of the sclera, under the tenon's capsule and slightly posterior to the equator of a rabbit eye. Such drug depots produce a concentration of vasostabilizing steroid evenly distributed throughout the retina and measured on the day after injection about 10-fold higher than a similar concentration delivered by a depot located under the rabbit subconjunctival eye and on the tenon's capsule. These benefits are highly surprising due to the fact that the Novachin white rabbits have extremely thin Nongchong. It is important to note that the tenon's capsule of the human eye is also extremely thin. 4, 9(11) -pregnadiene-17. alpha., 21-diol-3, 20-dione-21-acetate and the related compound 4, 9(11) -pregnadiene-17. alpha., 21-diol-3, 20-dione are more particularly described in U.S. Pat. Nos. 5,770,592 and 5,679,666, the entire contents of which are incorporated by reference.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile and pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the above formulations, the compounds may be formulated as long acting formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly), by intramuscular injection or by sub-tenon or intravitreal injection as described above.

In a particularly preferred embodiment of the invention, the compounds may be prepared for topical administration in saline (in combination with any preservatives and antimicrobials commonly used in ophthalmic formulations) and administered as eye drops. Anti-angiogenic factors can be prepared in solution or suspension in pure form and administered several times per day. Alternatively, the anti-angiogenic composition prepared as described above may also be administered directly to the cornea.

In a preferred embodiment, the composition is prepared using a mucoadhesive polymer that adheres to the cornea. Thus, for example, the compounds may be formulated using suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. In other embodiments, anti-angiogenic factors or anti-angiogenic compositions can be used as adjuncts to conventional cholesterol therapy.

The pharmaceutical carrier for the hydrophobic compound is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD was a solution of 3% w/v benzyl alcohol, 8% w/v non-polar surfactant polysorbate 80 and 65% w/v polyethylene glycol 300 in absolute ethanol. The VPD cosolvent system (VPD: 5W) contained VPD diluted 1: 1 with 5% aqueous glucose. Such co-solvent systems adequately solubilize hydrophobic compounds and themselves produce low toxicity when administered systemically. Of course, the proportion of the co-solvent system can vary significantly, but without destroying its solubility and toxicity characteristics. Furthermore, the composition of the co-solvent component may vary: for example, other low toxicity non-polar surfactants may be used in place of polysorbate 80; the size of the polyethylene glycol fraction may vary; other biocompatible polymers may be substituted for the polyethylene glycol, such as polyvinylpyrrolidone; and other saccharides or polysaccharides may be substituted for glucose.

On the other hand, other delivery systems for hydrophobic drug compounds may be used. Liposomes and emulsions are examples of vehicles or carriers known to deliver hydrophobic drugs. Certain organic solvents, such as dimethyl sulfoxide, can also be used, but usually at the expense of higher toxicity. In addition, sustained release systems may be used to deliver the compounds, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release substances have been established and are well known to those skilled in the art. Sustained release capsules, depending on their chemical nature, can release the compound for weeks up to 100 days. Other protein stabilization strategies may be used depending on the chemical nature and biological stability of the therapeutic agent.

The pharmaceutical composition may also include a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Certain compounds of the present invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically acceptable compatible salts can be formed with a number of acids including hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts are more soluble in water or other protic solvents than the corresponding free base forms.

The preparation of preferred compounds of the invention is described in detail in the following examples, but those skilled in the art will recognize that the chemistry described is readily adaptable to the preparation of many other protein kinase inhibitors of the invention. For example, atypical compounds of the invention may be successfully synthesized by modifications apparent to those skilled in the art, for example using appropriate protective interfering groups, by varying the use of other suitable reagents well known in the art or by routine modification of reaction conditions. On the other hand, other reactions disclosed herein or known in the art are deemed to have applicability to the preparation of other compounds of the present invention.

Detailed description of the invention and preferred embodiments

Examples

In the following examples, all temperatures are expressed in degrees celsius and all parts and percentages are by weight unless otherwise indicated. Reagents were purchased from commercial suppliers such as Aldrich chemical company or Lancaster Synthesis ltd. Tetrahydrofuran (THF), N-Dimethylformamide (DMF), dichloromethane, toluene and dioxane were purchased from Aldrich in the form of exact sealed bottles and used in a recognized manner. Unless otherwise indicated, all solvents were purified using standard methods readily known to those skilled in the art.

The following reactions are generally carried out under a positive pressure of argon or nitrogen or at ambient temperature (unless otherwise specified) using a drying tube and in anhydrous solvents, and the reaction flask is fitted with a rubber septum for introducing the substrates and reagents via syringe. Drying and/or heat drying glassware. Analytical Thin Layer Chromatography (TLC) was carried out with a glass lining of silica gel 60F 254, Analtech (0.25mm) and eluted with the appropriate solvent ratio (v/v) and, if appropriate, labeled. The reaction was checked by TLC and judged to be terminated by the depletion of starting material.

TLC was developed using anisaldehyde spray reagent or phosphomolybdic acid reagent (Aldrich Chemical 20 wt% in ethanol) and activated using heat. Unless otherwise stated, the work-up is generally carried out by doubling the reaction volume using the reaction solvent or extraction solvent and then washing with the indicated aqueous solution using an extraction volume of 25% by volume. With anhydrous Na₂SO₄The product solution was dried, after which it was filtered and the solvent was evaporated under reduced pressure with a rotary evaporator and the solvent was removed carefully in vacuo. Unless otherwise indicated, flash column chromatography purification using Baker grade flash silica gel (47-61m) and silica gel: crude material of about 20: 1-50: 1 (Still et al, J.Org.Chem., 43, 2923 (1978)). Hydrogenolysis was performed at the pressure or ambient temperature specified in the examples.

Recording with a Bruker instrument operating at 300MHz¹H-NMR spectrum and operation recording at 75MHz¹³C-NMR spectrum. As CDCl₃The solution was subjected to NMR spectroscopy (reported in ppm) using chloroform as a reference standard (7.25ppm and 77.00ppm) or CD₃OD (3.4 and 4.8ppm and 49.3ppm), or, if appropriate, tetramethylsilane (0.00 ppm). Other NMR solvents were used if desired. When peak diversity is reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broad), dd (doublet), dt (doublet triplet). If coupling normality is given, it is reported as Hertz (Hz).

Using a Perkin-Elmer FT-IR spectrometer in neat oil, in KBr tablets or in CDCl₃The Infrared (IR) spectrum is recorded as a solution and, if given, in wave numbers (cm)^-1) And (5) reporting. Mass spectra were obtained using LSIMS or electrospray. All melting points (mp) were not calibrated.

Example 1(a) dimethyl 2- (4-chloro-2-nitro-phenyl) -malonate

To a stirred slurry of NaH (36.0g, 1500 mmol 1) in NMP (1.0L) was added dropwise dimethyl malonate (137.4mL, 1200 mmol). The reaction mixture was cooled as needed to maintain the internal temperature below 30 degrees celsius. After the evolution of gas had ceased, 2, 4-dichloronitrobenzene (192g, 1000mmol) was added to the reaction mixture. The reaction mixture was carefully heated to 65 degrees celsius until the reaction was complete as determined by HPLC. The reaction mixture was cooled to room temperature and then poured onto 500mL of ice mixed with 150mL of concentrated HCl. The pH of the aqueous layer was adjusted to neutral using 1N NaOH. The solids were removed by filtration through a coarse porous filter and rinsed with water (3L). The yellow solid was dried overnight. Yield 261.5g, 91%.

Example 1(b) (4-chloro-2-nitro-phenyl) -acetic acid methyl ester

A solution of 2- (4-chloro-2-nitro-phenyl) -malonic acid dimethyl ester (195g, 679.4mmol) in water (100mL) and NMP (1000mL) was heated to reflux for 3.5 h. The solvent was removed by rotary evaporation to give an oil. The oil was dissolved in EtOAc and then washed with water (5 × 300 mL). The aqueous layer was then extracted with EtOAc (4X 300 mL). The organic layer was washed with water. Combine the organic layers and use MgSO₄And (5) drying. After removal of the solid by filtration, the solvent was evaporated to give the desired product as an orange/brown solid (160.0g, 95%).

Example 1(c) (2-acetylamino-4-chloro-phenyl) -acetic acid methyl ester

To an argon-filled flask were added (4-chloro-2-nitro-phenyl) -acetic acid methyl ester (40g, 175mmol), 10% Pd/C (2.5g), acetic anhydride (64mL, 677mmol), water (9mL) and acetic acid (150 mL). The flask was filled with 30PSI of hydrogen under vacuum and shaken vigorously. After 2 hours, an additional 10% Pd/C (2g) was added and the reaction was complete after a total of 4 hours of reaction time. The 10% Pd/C was removed by filtration and the solvent was removed by rotary evaporation.

Example 1(d) methyl 6-chloro-1H-indazole-3-carboxylate

To a stirred solution of (2-acetylamino-4-chloro-phenyl) -acetic acid methyl ester (32.0g, 133mmol) in acetic acid (200mL) at 90 deg.C was added tert-butyl nitrite (20.5mL.172.3mmol) over 1 hour. The reaction mixture was poured into water (1.4L) and the solid recovered by filtration. The yellow precipitate was dissolved in EtOAc and then washed with saturated NaCl. With MgSO₄The organic layer was dried, filtered and concentrated to a solid. The solid was triturated with hexanes and filtered to give the desired material (21.63g, 77%).

Example 1(e) methyl 6-chloro-1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylate

To a slurry of methyl 6-chloro-1H-indazole-3-carboxylate (8.3g, 39.5mmol) in MeCN (200mL) was added 3, 4-dihydro-2H-pyran (5.4mL, 59.3mmol) and p-toluenesulfonic acid (237mg, 1.25 mmol). After the reaction mixture was stirred for 10 minutes, saturated NaHCO was added₃(1mL) and the solvent was removed by rotary evaporation to a volume of 100 mL. The mixture was diluted with EtOAc and washed with water (50mL) and then saturated NaCl (50 mL). Then using Na₂SO₄The organic layer was dried. After removal of the solids by filtration, the organic layer was concentrated by rotary evaporation to an oil. The product precipitated from the oil with hexane to give the desired product (7.667g, 66% yield).

Example 1(f) methyl 6- (2-methoxycarbonyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylate

To a solution of methyl 6-chloro-1H-indazole-3-carboxylate (2.94g, 10.0mmol) in 1, 2-dimethoxyethane (30mL) was added K₃PO₄(5.32g, 25.0mmol), tris (dibenzylideneacetone) dipalladium (459mg, 0.05mmol), 2- (dicyclohexylphosphino) biphenyl (701mg, 2.0mmol), and methyl anthranilate (2.59mL, 20.0 mmol). The solution was vacuum flushed 3 times with argon and thereafter heated to 18 degrees celsius for 18 hours. The reaction mixture was cooled to room temperature and the solid was filtered off by filtration. After washing the solid with ethyl acetate, the solvent was removed by rotary evaporation. The residual oil was chromatographed (150g silica gel, 10-30% EtOAc/hexanes) to give 1.23g (51%) of the desired product.

Example 1(g)6- (2-methoxycarbonyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylic acid

To a solution of methyl 6- (2-methoxycarbonyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylate (2.05g, 5mmol) in methanol (18mL) and tetrahydrofuran (8mL) was added a solution of sodium hydroxide (0.30g, 7.5mmol) in water (2.7 mL). The reaction mixture was stirred at room temperature for 3 hours and then neutralized to pH1 with 1N HCl. The mixture was diluted with EtOAc (25mL) and water (25 mL). After separation of the layers, with CH₂Cl₂The aqueous layer was washed (3X 25 mL). The combined organic extracts were washed with saturated NaCl (100mL) and then Na₂SO₄And (5) drying. The solid was filtered and the liquid was concentrated to an oil. The product was crystallized from EtOAc and hexanes to give the desired product (1.616g, 82%).

Example 1(H) methyl 2- [ 3-methylcarbamoyl-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoate

To a solution of 6- (2-methoxycarbonyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylic acid (0.50g, 1.27mmol) in DMF was added triethylamine (0.42mL, 3.04mmol), methylamine (1.9mL, 3.81mmol) and HATU (0.578g, 1.52 mmol). The reaction mixture was stirred for 3 hours and then concentrated by rotary evaporation. The crude oil was chromatographed (50g silica gel, 25-50% EtOAc/hexanes) to give the desired product (214mg, 42%).

Example 1(i)2- [ 3-methylcarbamoyl-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid

To 2- [ 3-methylcarbamoyl-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]To a solution of methyl benzoate (0.20g, 0.49mmol) in methanol (1.4mL) and tetrahydrofuran (0.6mL) was added a solution of sodium hydroxide (59mg, 1.47mmol) in water (0.3 mL). The reaction mixture was heated to 60 degrees celsius for 1 hour and then cooled to room temperature. The pH was adjusted to pH 2 with 2N HCl. EtOAc (30mL) and water (30mL) were added and the layers were separated. The aqueous layer was extracted with EtOAc (3X 20mL) and the organic layers were combined. After washing with water (15mL), Na was added₂SO₄The organic layer was dried. The solid was filtered off and the organic layer was evaporated to give a yellow solid (193mg, 100%).

Example 1(j)6- (2-prop-2-ynylcarbamoyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylic acid methylamide

To 2- [ 3-methylcarbamoyl-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]To a solution of benzoic acid (150mg, 0.381mmol) in DMF (3.6mL) was added propargylamine (0.052mL, 0.761mmol), TEA (0.264mL, 1.90mmol) and HATU (217mg, 0.571 mmol). The reaction mixture was stirred for 4 hours and then diluted with EtOAc (30mL) and water (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2X 20 mL). The combined organic layers were washed with saturated NaCl (15mL) and then Na₂SO₄And (5) drying. The solid was removed by filtration and the liquid was concentrated by rotary evaporation to give a yellow oil (164mg, 100%).

Example 1(k)6- (2-prop-2-ynylcarbamoyl-phenylamino) -1H-indazole-3-carboxylic acid methylamide

In 1.5mL of 90: 10: 1CH₂Cl₂To a mixture of TFA: triethylsilane was dissolved 6- (2-prop-2-ynylcarbamoyl-phenylamino) -1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carboxylic acid methylamide (30mg) and heated to reflux for 12 hours. The solution was diluted with toluene (40mL) and concentrated by rotary evaporation to give an oil. The oil was dissolved in DMF (1mL) and filtered using a 0.2-micron syringe filter. The desired compound was isolated using prep-HPLC (12mg, 50%).¹H NMR(CDCl₃-d)δ 9.96(1H，s)，□□□□s). 8.28(1H，d，J＝8.85Hz)，7.47(1H，m)，7.34(1H，m)，7.22(1H，m)，7.15(1H，dd，J1＝8.76Hz，J2＝1.79Hz)，6.99(1H，m)，6.86(1H，t，J＝6.97Hz)，6.31(1H，m)，4.23(2H，dd，J1＝5.18Hz，J2＝2.54Hz)，3.49(3H，s)，2.29(s，1H)。

To C₁₉H₁₇N₅O₂1.0MeOH.0.1TFA, calcd for: c, 62.08; h, 5.44; n, 17.92. Measurement value: c, 61.78; h, 5.45; n, 18.04.

Example 2(a) [ 6-chloro-1- (tetrahydro-pyran-2-yl) -1H-indazol-3-yl ] -methanol

To methyl 6-chloro-1H-indazole-3-carboxylate (2.94g, 10.0mmol) cooled to-78 deg.C in dry CH₂Cl₂DIBAL-H (3.56mL, 20.0mmol) was slowly added to the solution in (50 mL). After the addition was complete, the reaction mixture was warmed to room temperature, where HPLC showed that 10% of the remaining starting material was still present. DIBAL-H (0.35mL) was then added and stirred for 10 min. The reaction mixture was diluted with EtOAc (1000mL) and washed with 1N HCl (2X 100 mL). The system was further treated with 1N naHCO₃Washed (100mL) and then with saturated NaCl (100 mL). With MgSO₄The organic layer was dried, filtered and then concentrated to give a white solid (2.65g, 99.5%).

Example 2(b) 6-chloro-1- (tetrahydro-pyran-2-yl) -1H-indazole-3-carbaldehyde

Reacting [ 6-chloro-1- (tetrahydro-pyran-2-yl) -1H-indazol-3-yl]A solution of methanol (1.75g, 6.58mmol), IBX (2.76g, 9.87mmol) and DMSO (27mL) was stirred overnight. The reaction mixture was diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc (3X 100 mL). The organic layers were combined and washed with saturated NaCl (100 mL). With MgSO₄The organic layer was dried, filtered and then concentrated to a solid. Dissolving the solid in CH₂Cl₂And filtered. The organic layer was evaporated to give the desired product (1.707g, 92%).

Example 2(c)1- (6-chloro-1H-indazol-3-yl) -2- (5-ethyl-pyridin-2-yl) -ethanol

To a stirred solution of 4-ethyl-2-methylpyridine (0.458g, 3.79mmol) in THF (4mL) at-50 deg.C was added butyllithium (1.5mL, 2.5M, 3.79mmol) slowly and stirred for 10 min. To the reaction mixture was slowly added a solution of 6-chloro-1H-indazol-3-aldehyde (0.5g, 1.89mmol) in THF (4 mL). After stirring for 10 min, the reaction was quenched with 1N citric acid (10 mL). The mixture was diluted with EtOAc (50mL), water (20mL) and saturated NaCl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3X 15 mL). The organic layers were combined and washed with saturated NaCl (20 mL). With Na₂SO₄After drying the organic layer, the solids were removed by filtration and the liquid was concentrated by rotary evaporation to an oil. Chromatographic purification (40g silica gel, 60-100% EtOAc/hexanes) afforded the desired product (142mg, 32%) and recovered 6-chloro-1H-indazol-3-aldehyde (348 mg).

Example 2(d) 6-chloro-3- [2- (5-ethyl-pyridin-2-yl) -vinyl ] -1H-indazole

To 1- (6-chloro-1H-indazol-3-yl) -2- (5-ethyl-pyridin-2-yl) -ethanol (232mg, 0.60mmol) in CH₂Cl₂To the stirred solution in (1) was added TEA (0.25mL, 1.81mmol) and methanesulfonyl chloride (0.070mL, 0.90 mmol). The reaction mixture was stirred for 30 minutes and then DBU (2mL) was added. The reaction mixture was refluxed for 18 hours and then quenched with 40mL of 1N citric acid. Separate the layers and use 20mL CH₂Cl₂The aqueous layer was extracted. With Na₂SO₄The combined organic layers were dried, filtered, and concentrated by rotary evaporation (12g silica gel, 50-70% EtOAc/hexanes) to give the desired compound (135mg, 71%).

Example 2(e) methyl 2- {3- [2- (5-ethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzoate

1, 2-Dimethoxymethane (2mL) was added to 6-chloro-3- [2- (5-ethyl-pyridin-2-yl) -vinyl ] -vinyl]-1H-indazole (130mg, 0.354mmol), tris (dibenzylideneacetone) dipalladium (16mg, 0.018mmol), - (dicyclohexylphosphino) biphenyl (25mg, 0.071mmol), K₃PO₄(0.188g, 0.885mmol) and methyl anthranilate (0.092mL, 0.71 mmol). The reaction mixture was flushed under vacuum with argon (4 ×) and then heated to 80 degrees celsius for 19 hours. The reaction mixture was diluted with EtOAc (20mL) and filtered through a pad of silica gel. After washing with EtOAc (50mL), the solvent was removed by rotary evaporation. The crude oil was purified by chromatography (40g silica gel, 30-40% EtOAc/hexanes) to afford the desired product (54mg, 32%).

Example 2(f)2- (3- [2- (5-ethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino) -benzoic acid

To 2- {3- [2- (5-ethyl-pyridin-2-yl) -vinyl]To a solution of methyl (1H-indazol-6-ylamino) -benzoate (50mg, 0.104mmol) in methanol (0.42mL) and THF (0.10mL) was added a solution of sodium hydroxide (12mg, 0.311mmol) in water (0.05 mL). The solution was heated to 60 degrees celsius for 3.5 hours and then saturated NH₄And (4) neutralizing with Cl. The reaction mixture was diluted with water (20mL) and then extracted with EtOAc (2X 20 mL). First with Na₂SO₄The combined extracts were dried and then the solids were removed by filtration. The desired product (48.7mg, 100%) was recovered after removal of the solvent by rotary evaporation.

Example 2(g)2- {3- [2- (5-Ethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N-prop-2-ynyl-benzamide

To 2- {3- [2- (5-ethyl-pyridin-2-yl) -vinyl]To (49mg, 0.105mmol) of (E) -1H-indazol-6-ylamino } -benzoic acid was added 2mL of 90: 10: 1CH₂Cl₂A mixture of TFA and TES. The reaction mixture was stirred under reflux for 1 hour and then diluted with toluene (20 mL). The solvent was removed by rotary evaporation to give a viscous oil. The oil was dissolved in DMF (1mL) and TEA (0.072mL, 0.52mmol), propargylamine (0.014mL, 0.208mmol) and HATU (59mg, 0.156mmol) were added to the solution. The reaction mixture was stirred for 3 hours and then purified by preparative HPLC to give the desired product (29mg, 66%).¹H NMR(CDCl₃-d): δ 9.83(1H, s), 8.63(2H, s), 8.04(2H, m), 7.68(2H, s), 7.47(1H, m), 7.32(1H, d, J ═ 1.51Hz), 7.10(1H, dd, J1 ═ 8.67Hz, J2 ═ 1.88Hz), 6.93(1H, m), 6.07(2H, dd, J1 ═ 5.09Hz, J2 ═ 2.26Hz), 3.15(1H, t, J ═ 2.35Hz), 2.97(2H, s), 2.74(1H, s), 2.29(1H, s), 1.27(3H, t, J ═ 7.44 Hz). To C₂₆H₂₃N₅O·0.3H₂Analytical calculation of O.1.2 TFA: c, 60.51; h, 4.43; n, 12.42. measurement: c, 60.38; h, 4.73; n, 12.44.

Example 2(H) N-cyclopropyl-2- {3- [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

According to the formula 2- {3- [2- (5-ethyl-pyridin-2-yl) -vinyl]-1H-indazol-6-ylamino } -N-prop-2-ynyl-benzamide the title compound was prepared in a similar manner by substituting 2, 4-dimethyl-pyridine for 4-ethyl-2-methyl-pyridine in the step of preparing 1- (6-chloro-1H-indazol-3-yl) -2- (5-ethyl-pyridin-2-yl) -ethanol and cyclopropylamine for propargylamine in the final sequential step.¹HNMR(DMSO-d₆)：δ9.85(1H，s)，8.56(2H，m)，8.20(3H，m)，7.53(5H，m)，7.35(1H，s)，7.2(1H，d，J＝6.5Hz)，7.0(1H，s)，2.83(1H，m)，0.70(2H，m)，0.56(2H，m)。ESIMS(M+H⁺)：410.3。

Example 3(a) N-methoxy-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), O-methyl-hydroxylamine hydrochloride (15mg, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 21.6mg (67%) of the title compound as a yellow solid.¹H NMR(DMSO-d₆)：δ9.23(1H，s)，8.71(1H，d，J＝2.2)，8.05(4H，m)，7.51(5H，m)，7.25(1H，s)，7.10(1H，d，J＝7.7Hz)，6.91(1H，m)，5.98(1H，m)，4.31(1H，d，J＝14.3)，7.20(1H，d，J＝7.3)，4.42(2H，d，J＝3.2)。ESIMS(M+H⁺)：412.1。

Example 3(b) N-allyloxy-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), O-allyl-hydroxylamine hydrochloride (18.3mg, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 25.5mg (74%) of the title compound as a yellow solid.¹H NMR(DMSO-d₆)：δ9.28(1H，s)，8.67(2H，d，J＝3.4)，8.05(4H，m)，7.48(5H，m)，7.23(1H，s)，7.04(1H，d，J＝7.6Hz)，6.91(1H，m)，3.69(3H，s)。ESIMS(M+H⁺)：386.1。

Example 3(c) N-Isopropoxy-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), O-allyl-hydroxylamine hydrochloride (18.7mg, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 17.4mg (50%) of the title compound as a yellow solid.¹H NMR(DMSO-d₆)：δ9.23(1H，s)，8.69(H，d，J＝2.1)，8.03(4H，m)，7.50(5H，m)，7.23(1H，s)，7.04(1H，d，J＝6.7Hz)，6.92(1H，m)，5.98(1H，m)，4.13(1H，m)，1.29(6H，d，J＝8.1)。ESIMS(M+H⁺)：414.1。

Example 3(d) N-cyclopropyl-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), cyclopropylamine (11.6uL, 0.17mmol), triethylamine (58. mu.l, 0.25mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 11.7mg (35%) of the title compound as a yellow solid.¹HNMR(DMSO-d₆)：δ9.81(1H，s)，8.68(1H，d，J＝1.7)，8.51(1H，s)，8.01(4H，m)，7.50(5H，m)，7.24(1H，s)，7.03(1H，d，J＝5.3)，6.89(1H，t，J＝4.2)，2.84(1H，m)，0.72(2H，m)，0.56(2H，m)。ESIMS(M+H⁺)：396.1。

Example 3(f) 1-methyl-1H-pyrrole-2-carboxylic acid N' - (1- {2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -phenyl } -formyl) -hydrazide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), 1-methyl-1H-pyrrole-2-carboxylic acid hydrazide (23.3mg, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 16.1mg (35%) of the title compound as a yellow solid.¹H NMR(DMSO-d₆)：δ10.39(1H，s)，10.00(1H，s)，9.52(1H，s)，8.67(1H，d，J＝2.4)，8.07(4H，m)，7.77(1H，d，J＝5.2)，7.51(4H，m)，7.32(1H，s)，7.09(1H，d，J＝6.3)，6.98(3H，m)，6.13(1H，m)，3.87(3H，s)。ESIMS(M+H⁺)：478.1。

Example 3(g) N-benzyl-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), benzylamine (18.2. mu.l, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 45.2mg (76%) of the title compound as a TFA salt (1.5H)₂O, 2.1TFA, effective MW 711.98).¹H NMR(DMSO-d₆)：δ9.86(1H，s)，9.14(1H，t，J＝5.4)，8.73(1H，d，J＝4.8)，8.29(4H，m)，7.56(1H，d，J＝7.0)，7.74(2H，m)，7.89(2H，m)，7.31(5H，m)，7.16(1H，d，J＝7.8)，6.93(1H，t，J＝7.3)，4.46(2H，d，J＝6.1)。ESIMS(M+H⁺)：446.5。

Example 3(H) N- (2-M ethoxy-benzyl) -2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), o-methoxybenzylamine (21.8uL, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 46mg (81%) of the title compound as a TFA salt (1.5H)₂O, 1.5TFA, effective MW 673.59).¹H NMR(DMSO-d₆)：δ9.83(1H，s)，9.03(1H，t，J＝3.4)，8.70(1H，d，J＝3.7)，8.08(4H，m)，7.82(1H，d，J＝7.4)，7.49(4H，m)，7.21(3H，m)，6.96(4H，m)，4.48(2H，d，J＝6.3)。ESIMS(M+H⁺)：476.1。

Example 3(i) N-furan-2-ylmethyl-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), C-furan-2-yl-methylamine (19 μ L, 0.17mmol), triethylamine (58 μ L, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 45mg (85%) of the title compound as a TFA salt (1.5H)₂O, 1.5TFA, effective MW 633.52).¹H NMR(DMSO-d₆)：δ9.82(1H，s)，9.05(1H，t，J＝2.6)，8.73(1H，d，J＝3.7)，8.13(4H，m)，7.73(1H，d，J＝6.8)，7.57(2H，m)，7.26(1H，s)，7.03(1H，d，J＝7.5)，6.40(1H，m)，6.28(1H，m)，4.48(2H，d，J＝6.5)。ESIMS(M+H+)：436.1。

Example 3(j) N-cyclobutyl-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), cyclobutylamine (18.2uL, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 43.2mg (92%) of the title compound as a TFA salt (1.5H)₂O, 1.1TFA, effective MW 561.92).¹H NMR(DMSO-d₆)：δ9.78(1H，s)，8.72(2H，m)，8.13(4H，m)，7.70(1H，d，J＝7.1)，7.58(2H，m)，7.41(2H，m)，7.27(1H，s)，6.89(1H，t，J＝4.2)，2.84(1H，m)，0.72(2H，m)，0.56(2H，m)。ESIMS(M+H⁺)：396.1。

Example 3(k) N- (2-methyl-allyl) -2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Treatment of 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino with HATU (48mg, 0.13mmol)]A solution of benzoic acid (50.0mg, 0.084mmol), 2-methyl-allylamine (16.4uL, 0.17mmol), triethylamine (58. mu.l, 0.42mmol) in DMF (0.8 mL). The mixture was stirred overnight and then purified by reverse phase HPLC to give 45mg (91%) of the title compound as a TFA salt (1.6H)₂O, 1.3TFA, effective MW 586.53).¹H NMR(DMSO-d₆)：δ9.78(1H，s)，8.72(2H，m)，8.13(4H，m)，7.70(1H，d，J＝7.1)，7.58(2H，m)，7.41(2H，m)，7.27(1H，s)，7.06(1H，d，J＝7.1)，6.91(1H，t，J＝7.5)，4.42(1H，m)，2.22(2H，m)，2.08(2H，m)，1.68(2H，m)。ESIMS(M+H⁺)：410.1。

Example 3(l) 6-Nitro-3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazole

A mixture of 3-iodo-6-nitro-1- (2-trimethylsilyl-ethoxyethyl) -1H-indazole (838mg, 2.0mmol), 2-ethynyl-pyridine (242 μ L, 2.4mmol), and triethylamine (6.0mL) was degassed and purged with argon, then with CuI (8mg, 0.042mmol) and Pd (PPh)₃)₂Cl₂(16mg, 0.023 mmol). The resulting mixture was stirred at room temperature overnight at which point HPLC showed all starting material had been consumed. The mixture was purified by back-extracting the volatiles in high vacuum, then through a pad of silica gel eluted with ethyl acetate. The resulting product was used in the next step without further purification. ESIMS (M + H)⁺)：395.1。

Example 3(m) 3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamine

6-Nitro-3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazole (2mmol), SnCl₂A mixture of (1.37uL, 6.0mmol), water (0.5mL) and di MeOH (10mL) was stirred on an oil bath at 60 deg.C for 30 minutes at which time HPLC showed complete reduction. The resulting mixture was back-extracted with methanol, suspended in EtOAc (50mL) and diluted with 1M NaOH (18 mL). The resulting emulsion was slowly extracted with EtOAc (10X 25 mL). With 1M Na₂SO₄The combined organic layers were extracted with brine, MgSO₄Dried, concentrated and filtered through a pad of silica gel eluted with EtOAc. The yield of the crude product in the two steps is 701mg, and the mass yield is 96%. ES (ES)IMS(M+H⁺)：365.1。

Example 3(n) methyl 2- [ 3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzoate

3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-amine (560mg, 1.54mmol), 2-bromomethylbenzoate (647.5uL, 4.61mmol), biphenyl-2-yl-dicyclohexyl-phosphane (107.8mg, 0.308mmol), Pd, were vacuum flushed with nitrogen₂(dba)₃(70.5mg，0.0768mmol)、K₃PO₄(816mg, 3.844mmol of a mixture with dimethoxyethane (1.7ml), then heating for 24 h on an oil bath at 70 ℃ the black mixture was diluted with dichloromethane and filtered, concentrated and chromatographed (20% -40% ethyl acetate/hexane) to give 260mg of a yellow/orange oil in 35% yield over three steps.

Example 3(o)2- [ 3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzoic acid

Reacting 2- [ 3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino]-methyl benzoate (253mg, 0.517mmol) was added to a solution of NaOH (62mg, 1.55mmol) in THF (1.0mL), MeOH (2.25mL) and water (0.5 mL). The reaction mixture was stirred at room temperature for 1 hour, at which point HPLC showed complete consumption of all starting materials. The reaction mixture was neutralized with 1N HCl, extracted with ethyl acetate, then washed with brine and MgSO₄And (5) drying. After concentration in vacuo, 249mg of a yellow solid were obtained (99% mass yield). The material was used without further purification. ESIMS (M-H)^-)：483.0。

Example 3(p)2- [ 3-pyridin-2-ylethynyl-1H-indazol-6-ylamino ] -benzoic acid

Reacting 2- [ 3-pyridin-2-ylethynyl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino]A solution of benzoic acid (231mg, 0.477), 1M tetrabutylammonium fluoride in THF (3.8mL, 3.816mmol), and a solution of ethylenediamine (127uL, 1.908mmol) were stirred in an oil bath at 80 ℃ for 6 hours. The reaction was quenched with acetic acid (218uL, 3.816mmol), diluted with water and extracted with EtOAc (10X 50 mL). The combined organic layers were washed with brine and MgSO₄And (5) drying. After concentration, the solid formed is taken up in CH₂Cl₂Trituration afforded the product as a yellow powder (124mg, 73%). ESIMS (M-H)^-)：353.0。

Example 3(q) N-prop-2-ynyl-2- [3- ((E) -2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

A solution of 2- (3-pyridin-2-ylethynyl-1H-indazol-6-ylamino) -benzoic acid (41mg, 0.117mmol), propargylamine (24. mu.L, 0.35mmol), triethylamine (81. mu.L, 0.58mmol) in DMF (0.5mL) was treated with HATU (89mg, 0.233 mmol). The mixture was stirred overnight and then purified by reverse phase HPLC to give 27mg (59%) of the title compound as a yellow solid.¹HNMR(DMSO-d₆)：δ9.78(1H，s)，8.99(1H，m)，8.61(1H，d，J＝2.1)，7.88(1H，s)，7.72(3H，m)，7.43(4H，m)，7.29(1H，s)，7.04(1H，d，J＝7.3)，6.91(1H，t，J＝5.2)，4.04(2H，s)，3.04(1H，s)。ESIMS(M+H⁺)：392.1。

Example 4 (a): 2-bromo-4, 6-dimethyl-pyridine

48% HBr solution (aq) (Aldrich, 65mL, 1.2mol, 10eq) was cooled to-5 ℃ and treated with 4, 6-dimethyl-pyridin-2-ylamine (Aldrich, 15.0g, 0.12mol.1.0 eq). The viscous white salt mixture was stirred using a mechanical stirrer while bromine (Aldrich, 19.7mL, 0.38mol, 3.1eq) was added dropwise. Using NaNO within 1 hour₂Aqueous solution of (32mL H)₂O) (Aldrich, 22.1g, 0.32mol, 2.6eq) the resulting red mixture. The temperature was maintained below 5 ℃ during the addition of nitrite and then gradually heated to 20 ℃ over 2 hours. The reaction mixture was adjusted to pH14 with NaOH (aq) and extracted with MTBE. The organic extracts were washed with water, brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (silica gel, 350g) and eluted with 2-7% ethyl acetate-cyclohexane to give an orange oil (11.0g, 48%).¹HNMR(DMSO-d₆，300MHz)δ7.30(1H，s)，7.13(1H，s)，2.39(3H，s)，2.26(3H，s)。¹³C NMR(DMSO-d₆，75MHz)δ159.4，151.3，140.9，125.7，124.0，23.7，20.3。ESI m/z 186/188(M+H)⁺。

Example 4 (b): 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -6-nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole

A suspension of 2-bromo-4, 6-lutidine (2.42g, 13mmol), 3-vinyl-6-nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole (2.37g, 8.67mmol), palladium acetate (0.145g, 0.65mmol), tri-o-tolylphosphine (0.791g, 2.6mmol) and diisopropylethylamine (2.4mL, 13.8mmol) in aqueous DMF (85%, 34.5mL) was degassed by argon bubbling for 5 minutes followed by sonication for 5 minutes, after which it was heated with a microwave instrument (300 Watts, 10% energy) for 40 minutes at 110 ℃. ColdAfter cooling, the mixture was dropped into cold water. The resulting yellow precipitate was collected by filtration. The solid was dissolved in ethyl acetate, dried (sodium sulfate) and concentrated under reduced pressure. The residue was purified on silica gel using a gradient of 0-20% ethyl acetate in a mixture of chloroform and hexane (1: 1) as eluent. The chromatographed product was triturated with MTBE/hexane to give a clear product as a yellow solid. The mother liquor was repurified with silica gel in a similar manner followed by trituration to give a more clear product in 68% yield.¹H NMR(CDCl₃)：δ8.54(1H，s)，8.15(1H，d，J＝9.4Hz)，8.08(1H，dd，J＝9.04，1.9Hz)，7.87(1H，d，J＝9.0，3.0Hz)，4.08-4.01(1H，m)，3.84-3.76(1H，m)，2.56(3H，s)，2.62-2.54(1H，m)，2.34(3H，s)，2.24-2.10(2H，m)，1.88-1.68(3H，m)。

Example 5): 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamine

Reacting 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-6-nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole (4.22g, 11.16mmol), iron powder (2.71g, 48.51mmol) and saturated NH₄A suspension of Cl (25ml) in 25ml ethanol was heated at 45 ℃ for 18 h. The reaction mixture was cooled and filtered through filter paper, washed with methanol. The solvent was removed under reduced pressure and the aqueous layer was extracted with EtOAc (2 ×). The combined organic layers were washed with brine, dried (MgSO)₄) And concentrated under reduced pressure to give 4.02g (quantitative) of a rust-colored solid and used without further purification.

¹H NMR(DMSO-d₆)δ7.79(1H，s)，7.74(1H，d，J＝16.4Hz)，7.35(1H，d，J＝16.4Hz)，7.29(1H，s)，6.96(1H，s)，6.63(2H，m)，5.57(1H，dd，J＝2.4，9.5Hz)，5.44(2H，broad s)，3.88(1H，m)，3.67(1H，m)，2.45(3H，s)，2.37(1H，m)，2.29(3H，s)，1.99(2H，m)，1.73(1H，m)，1.57(2H，m)。

Example 6: 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid methyl ester

Stirring 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamine (870mg, 2.5mmol), 2-bromo-benzoic acid methyl ester (0.44ml, 3.12mmol), R-BINAP (78mg, 0.125mmol), Pd₂(dba)₃A suspension of (29mg, 0.03mmol) and cesium carbonate (1.22g, 3.75mmol) in toluene (6ml) was degassed and heated at 100 ℃ for 18 h. The reaction mixture was cooled and poured into saturated NaHCO₃Neutralized and extracted with EtOAc (2 ×). The combined organic layers were washed with brine, dried (MgSO)₄) And concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using 5-10% EtOA in CH₂Cl₂The gradient of (b) eluted to give 964mg (80%) of a yellow foam.

¹H NMR(DMSO-d₆) δ 9.49(1H, s), 8.13(1H, d, J ═ 8.7Hz), 7.94(1H, dd, J ═ 1.5, 8.0Hz), 7.85(1H, d, J ═ 16.4Hz), 7.58(1H, d, J ═ 1.5Hz), 7.48(1H, d, J ═ 16.4Hz), 7.47(1H, m), 7.37(1H, d, J ═ 7.7Hz), 7.34(1H, s), 7.19(1H, dd, J ═ 1.7, 8.7Hz), 6.99(1H, s), 6.89(1H, t, J ═ 8.1Hz), 5.83(1H, d, J ═ 7.2Hz), 3.88(3H, s), 3.59 (1H, 3.75H, 3H, 3.75(1H, 3 m), 3.75(1H, 3H, 2 m), 3.75(1H, 3H, 3.19, 3.7H, m), 3.7H, 1H, 5.5.5.5, 1H, 5.5.5.5.5, J ═ 7.7, 8.7.7.7.7.7 Hz). To C₂₉H₃₀N₄O₃Analysis of 0.15EtOAc, calculated: c, 71.71; h, 6.34; n, 11.30. Measurement value: c, 71.60; h, 6.14; n, 11.37.

Example 7: 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid

To the stirred 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl group]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]Methyl benzoate (1.98g, 4.11mmol) in THF: MeOH (12ml, 3: 1) to which was added H dissolved₂O (3ml) potassium hydroxide (1.15g, 20.5 mmol). The reaction mixture was heated at 70 ℃ for 2 hours, cooled, concentrated under reduced pressure to about 5ml and diluted with water. The solution was neutralized with 2N HCl and the precipitate was collected by filtration and washed with water to give 2.00g (quantitative) of a pale yellow solid.¹H NMR(DSMO-d₆) δ 13.12(1H, width s), 9.82(1H, s), 8.13(1H, d, J ═ 8.7Hz), 7.95(1H, dd, J ═ 1.5, 8.0Hz), 7.89(1H, d, J ═ 16.4Hz), 7.60(1H, s), 7.50(1H, d, J ═ 16.4Hz), 7.46(1H, d, J ═ 6.9Hz), 7.37(1H, d, J ═ 7.7Hz), 7.20(1H, d, J ═ 8.7Hz), 7.06(1H, s), 6.86(1H, t, J ═ 6.9Hz), 5.85(1H, d, J ═ 7.3Hz), 3.82(2H, m), 2.50(3H, s), 3.59 (1H, 3, 3.76, 3H, m), 3H, 3.76 (1H, m), 3H, m, 3H, 3.76, 3, m, 3H, m, 3, m, 3H, m, 2H, m, and H. To C₂₈H₂₈N₄O₃Analysis of 0.5KOH, calculated: c, 67.72; h, 5.79; n, 11.28. Measurement value: c, 67.65; h, 5.88; n, 11.07.

Example 8: 2- {3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzoic acid p-toluenesulfonate salt

2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]A mixture of-benzoic acid (2mmol) and p-toluenesulfonic acid (10mmol) in aqueous methanol (90%, 20mL) was stirred at 70 ℃ for 18 h. After cooling, the resulting viscous yellow slurry was filtered and the solid was washed with methanol to give 2- {3- [2- (4, 6-dimethyl-pyridin-2-yl)) -vinyl radical]-1H-indazol-6-ylamino } -benzoic acid as tosylate salt in 85% yield as light yellow solid.¹HNMR(DSMO-d₆)δ13.43(1H，s)，9.78(1H，s)，8.24-8.19(2H，m)，8.09(1H，d，J＝9.04Hz)，7.95(1H，dd，J＝7.9，1.1Hz)，7.62-7.55(2H，m)，7.49-7.38(5H，m)，7.20(1H，dd，J＝9.0，1.9Hz)，7.09(2H，d，J＝8.3Hz)，6.86(1H，dt，J＝7.9，1.1Hz)，2.67(3H，s)，2.54(3H，s)，2.27(3H，s)。

Example 9: n- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl ] -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 33(a), step (v) of U.S. patent application Ser. No. US 09/609,335 filed on 30.6.2000, except that 4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynylamine and 2- [3- (2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -was used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]Benzoic acid, the entire content of which is incorporated by reference for all purposes.¹H NMR(DSMO-d₆) δ 9.56(1H, s), 9.01(1H, t, J ═ 5.7Hz), 8.06(1H, d, J ═ 8.7Hz), 7.81(1H, d, J ═ 16.4Hz), 7.66(1H, d, J ═ 7.5Hz), 7.41(4H, m), 7.32(1H, s), 7.09(1H, dd, J ═ 1.8, 8.7Hz), 6.98(1H, s), 6.89(1H, t, J ═ 8.0Hz), 5.79(1H, dd, J ═ 2.4, 9.2Hz), 4.28(2H, s), 4.09(2H, m), 3.86(1H, m), 3.72(1H, m), 2.46(3H, s), 2.42(1H, s), 3.42 (1H, 0, 0.7H, s), 3.74H, 0(1H, m), 3.0H, 0, 5.0, 5.7H, d). To C₃₈H₄₇N₅O₃Si 0.7H₂Analysis of O, calculated: c, 68.89; h, 7.36; n, 10.57. measurement: c, 68.99; h, 7.36; n, 10.21.

Example 10: 2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N- (4-hydroxy-but-2-ynyl) -benzamide

Stirring N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]A solution of-benzamide (737mg, 1.13mmol) and p-toluene-sulfonic acid (8.2ml, 12% solution in HOAc) was heated at 70 deg.C for 2 hours. The reaction mixture was cooled and carefully poured into saturated NaHCO₃And extracted with EtOAc (2 ×). The combined organic layers were washed with brine (2 ×), dried (MgSO)₄) And concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using CH₂Cl₂EtOAc: MeOH (1: 0.1) to give 225mg (44%) of a white solid.¹H NMR(DSMO-d₆) δ 12.91(1H, s), 9.84(s, 1H), 9.01(1H, t, J ═ 5.3Hz), 8.07(1H, d, J ═ 8.7Hz), 7.84(1H, d, J ═ 16.4Hz), 7.70(1H, d, J ═ 7.2Hz), 7.43(3H, m), 7.31(1H, s), 7.26(1H, s), 7.02(1H, dd, J ═ 1.6, 8.7Hz), 6.97(1H, s), 6.89(1H, t, J ═ 6.7Hz), 5.12(1H, t, J ═ 5.8Hz), 4.10(2H, d, J ═ 5.3Hz), 4.07(2H, d, J ═ 5.3Hz), 4.47 (2H, J ═ 2H, 8.47, s), 3H, 3.47 (3H, s). To C₂₇H₂₅N₅O₂1.1H₂Analysis of O, calculated: c, 68.80; h, 5.82; n, 14.86. measurement: c, 68.72; h, 5.81; n, 14.65.

Example 11: 4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynylamine

To an ice-cold stirred solution of the known 4- (tert-butyl-dimethyl-silanyloxy) -but-2-yn-1-ol (3.14g, 15.7mmol) in THF (50ml) was added DBU (2.6ml, 17.4 mmol)) And DPPA (3.8mol, 17.6 mmol). The solution was warmed to room temperature and stirred under an inert atmosphere overnight. The reaction mixture was poured into saturated NaHCO₃And the layers were separated. The aqueous layer was extracted with EtOAc (2 ×) and the combined organic layers were dried (Na)₂SO₄) And concentrated in vacuo. To this crude azide in THF (50ml) was added triphenylphosphine (4.61g, 17.6mmol) followed by H₂O (0.44 ml). The resulting solution was stirred at room temperature overnight, concentrated under reduced pressure and the residue was taken up in 1: 1Et₂Stirring the O/petroleum ether mixture into slurry. The solids were removed and the filtrate was concentrated and purified by flash silica gel chromatography using CH₂Cl₂MeOH (19: 1) gave an amber oil.¹H NMR(CDCl₃)δ4.19(2H，t，J＝1.9Hz)，3.33(2H，t，J＝1.9Hz)，0.79(9H，s)，0.00(6H，s)。

Example 12: 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -N-prop-2-ynyl-benzamide

Prepared in analogy to the procedure described in example 6 above, except that 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and propargylamine.¹H NMR(DSMO-d₆) δ 9.87(1H, s), 9.04(1H, t, J ═ 5.8Hz), 8.08(1H, d, J ═ 8.7Hz), 7.83(1H, d, J ═ 16.4Hz), 7.69(1H, d, J ═ 7.5Hz), 7.44(4H, m), 7.34(1H, s), 7.12(1H, dd, J ═ 1.7, 8.7Hz), 6.99(1H, s), 6.91(1H, t, J ═ 5.8Hz), 5.81(1H, dd, J ═ 2.4, 9.2Hz), 4.07(2H, dd, J ═ 2.5, 5.7Hz), 3.88(1H, m), 3.74(1H, m), 3.12(1H, t, 2.12H, J ═ 2H, 5.7Hz), 3.88(1H, m), 3.74(1H, m), 3.43H, 5.7H, 5H, 5.7H, 1H (1H, 5 m), 3.58H, 5 m), 1H (1H, 5.8H, 5 m), 1H, 5H. To C₃₁H₃₁N₅O₂·1.1H₂Analysis of O.0.3 TBME, calcd for: c, 70.73; h, 6.72; n, 12.69. Measurement value: c, 70.56; h, 6.45; n, 12.49.

Example 13: n- (prop-2-ynyl) -2- {3- [ (E) -2- (2, 4-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7, except that N- (3-cyclopropyl-prop-2-ynyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)δ12.90(1H，s)，9.78(1H，s)，9.01(1H，t，J＝5.3Hz)，8.06(1H，d，J＝8.3Hz)，7.84(1H，d，J＝16.2Hz)，7.68(1H，dd，J＝7.9，1.1Hz)，7.45-7.36(3H，m)，7.30(1H，s)，7.25(1H，d，J＝1.5Hz)，7.01(1H，dd，J＝8.7，1.9Hz)，6.96(1H，s)，6.88(1H，dt，J＝6.8，1.9Hz)，4.04(2H，dd，J＝5.6，2.6Hz)，3.11(1H，t，J＝2.6Hz)，2.46(3H，s)，2.29(3H，s)。

Example 14: 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -N- (2-methyl-allyl) -benzamide

Prepared in analogy to the procedure described in example 6 above, except that 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and 2-methyl-allylamine.¹H NMR(DSMO-d₆)δ9.87(1H，s)，8.82(1H，t，J＝5.8Hz)，8.07(1H，d，J＝8.7Hz)，7.82(1H，d，J＝164Hz), 7.74(1H, d, J ═ 7.3Hz), 7.43(4H, m), 7.33(1H, s), 7.10(1H, d, J ═ 8.7Hz), 6.99(1H, s), 6.92(1H, t, J ═ 7.8Hz), 5.80(1H, dd, J ═ 2.2, 9.2Hz), 4.83(2H, d, J ═ 11.8Hz), 3.83(4H, m), 2.47(3H, s), 2.44(1H, m), 2.31(3H, s), 2.00(2H, m), 1.75(1H, m), 1.73(3H, s), 1.58(2H, m). To C₃₂H₃₅N₅O₂·1.09H₂Analysis of O, calculated: c, 71.00; h, 6.92; n, 12.94. Measurement value: c, 71.40; h, 6.89; n, 12.54.

Example 15: 2- {3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N- (2-methyl-allyl) -benzamide

Prepared in analogy to the procedure described in example 7, except that N- (2-methyl-allyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)δ12.89(1H，s)，9.75(1H，s)，8.79(1H，t，J＝5.6Hz)，8.05(1H，d，J＝8.7Hz)，7.85(1H，d，J＝16.2Hz)，7.74(1H，d，J＝7.9Hz)，7.45-7.33(4H，m)，7.23(1H，d，J＝1.5Hz)，7.00-6.97(2H，m)，6.90(1H，dt，J＝7.9，1.1Hz)，4.81(2H，d，J＝11.3Hz)，3.81(2H，d，J＝5.6Hz)，2.47(3H，s)，2.30(3H，s)，1.71(3H，s)。

Alternative synthetic schemes

Example 16 (a): cyclopropyl-prop-2-yn-1-ol

To a round-bottomed flask containing 70mL of anhydrous THF cooled in an ice bath at-10 deg.C was added 65.6mL of a 1.6M solution of BuLi in hexane (105 mmol). 5-chloro-pent-1-yne (5.13g, 50mmol) was slowly introduced while maintaining the temperature at-10-0 ℃. The mixture was stirred at 0 ℃ under argon for 2 hours. Solid paraformaldehyde (3g, 100mmol) was added. The mixture was slowly warmed to room temperature and stirred under argon overnight. On day 2 water was added and about 50mL of 1N aqueous HCl was added. The mixture was extracted with ethyl acetate and the combined organic layers were washed with brine, Na₂SO₄Dried, filtered and concentrated. The crude product was purified by column, using 20% Et₂Elution of O in hexane afforded 3g of 3-cyclopropyl-prop-2-yn-1-ol as an oil (62% yield).¹H NMR(CDCl₃)δ4.22(dd，2H，J＝6.04，2.01Hz)，1.46(t，1H，J＝6.04Hz)，1.26(m，1H)，0.77(m，2H)，0.70(m，2H)。

Example 16 (b): 3-cyclopropyl-prop-2-ynyl azide

3-cyclopropyl-prop-2-yn-1-ol (3.28g, 34.1mmol) was dissolved in 40mL of toluene, DPPA (11.26g, 40.9mmol) was added followed by DBU (6.24g, 40.9mmol) while maintaining the temperature with a water bath. The mixture was stirred at room temperature for 1 hour and 100mL hexane and 15mL CH were used₂Cl₂And (6) diluting. The mixture was washed 4 times with water and 1 time with brine, Na₂SO₄Dried, filtered and concentrated by rotary evaporation using a cold water bath to remove most of the organic solvent, leaving some toluene (volatile product). The residual oil was used for the next step.¹H NMR(CDCl₃)δ3.85(s，2H)，1.26(m，1H)，0.80(m，2H)，0.72(m，2H)。

Example 16 (c): 3-cyclopropyl-prop-2-ynylamine

3-cyclopropyl-prop-2-ynylazide (ca. 34mmol) was dissolved in 100mL THF, 1mL water was added followed by solid PPh₃(13.37g, 51mmol) while maintaining the temperature with a water bath. The mixture was stirred at room temperature for 1 hour. 150mL of 1N aqueous HCl was added to the mixture. The mixture was washed 3 times with dichloromethane. The aqueous layer was basified to pH10-12 with 5N NaOH. The mixture was extracted with ethyl acetate. The aqueous layer was checked with TLC staining to monitor the progress of the amine extraction into the organic phase. With Na₂SO₄The combined organic layers were dried, filtered and concentrated to give 1.62g of the desired product (volatile product, containing residual EtOAc solvent) (two step yield 50%).¹H NMR(CDCl₃)δ3.37(d，2H，J＝2Hz)，1.22(m，1H)，0.74(m，2H)，0.65(m，2H)。

Example 17: n- (3-cyclopropyl-prop-2-ynyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in analogy to the procedure described in example 6 above, except that 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid E and 3-cyclopropyl-prop-2-ynylamine.¹H NMR(CDCl₃)：δ9.51(1H，s)，7.97(1H，d，J＝8.7Hz)，7.81(1H，d，J＝16.6Hz)，7.51-7.42(3H，m)，7.34-7.29(2H，m)，7.16(1H，s)，7.11(1H，dd，J＝9.0，1.9Hz)，6.85(1H，s)，6.81(1H，dt，J＝7.2，1.1Hz)，6.23(1H，t，J＝7.2Hz)，5.61(1H，dd，J＝9.0，2.6Hz)，4.17(2H，dd，J＝5.3，2.3Hz)，4.07-4.00(1H，m)，3.75-3.66(1H，m)，2.63-2.50(1H，m)，2.54(3H，s)，2.32(3H，s)，2.20-2.02(2H，m)，1.79-1.62(3H，m)，1.29-1.19(1H，m)，0.77-0.67(4H，m)。

Example 18: n- (3-Cycloprop-2-ynyl) -2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7 above, except that 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-N-prop-2-ynyl-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹HNMR(DSMO-d₆)：δ12.90(1H，s)，9.79(1H，s)，8.91(1H，t，J＝5.6Hz)，8.06(1H，d，J＝8.7Hz)，7.83(1H，d，J＝16.2Hz)，7.68(1H，dd，J＝7.9，1.1Hz)，7.49-7.38(3H，m)，7.29(1H，s)，7.25(1H，d，J＝1.9Hz)，6.99(1H，dd，J＝9.0，2.3Hz)，6.96(1H，s)，6.88(1H，dt，J＝7.9，1.5Hz)，4.00(2H，dd，J＝5.6，1.9Hz)，2.46(3H，s)，2.29(3H，s)，1.31-1.23(1H，m)，0.75-0.70(2H，m)，0.57-0.52(2H，m)。

Example 19 (a): 2-butyne-1, 4-diol monoacetate

To a solution of butyne-1, 4-diol (5g, 58mmol) in dry THF at room temperature was added sodium hydride (60% dispersion in oil, 2.32g, 58mmol) dropwise. After 4.3h, acetyl chloride (4.12mL, 58mmol) was added. After stirring at room temperature for 22 hours, the mixture was concentrated under reduced pressure. From tolueneThe residue was twice reduced and thereafter purified on silica gel using ethyl acetate/dichloromethane (1: 3) as eluent to give but-2-yne-1, 4-diol monoacetate as an oil in 49% yield.¹HNMR(DSMO-d₆)δ5.23(1H，bs)，4.70(2H，t，J＝1.8Hz)，4.09(2H，s)，2.03(3H，s)。

Example 19 (b): acetic acid 4-amino-but-2-ynyl ester

Prepared analogously as described in example 8, except that 2-butyne-1, 4-diol monoacetate is used instead of 4- (tert-butyl-dimethyl-silanyloxy) -but-2-yn-1-ol.¹H NMR(DSMO-d₆)δ4.77(2H，s)，4.20(2H，s)，2.04(3H，s)。

Example 20: acetic acid 4- (2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } benzoylamino) -but-2-ynyl ester

Prepared in analogy to the procedure described in example 6 above, except that 2- [3-2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1H-indazol-6-ylamino]-benzoic acid p-toluenesulfonate and acetic acid 4-amino-but-2-ynyl ester.¹H NMR(CD₃CN)：δ11.00(1H，bs)，9.59(1H，s)，7.99(1H，d，J＝8.6Hz)，7.86(1H，d，J＝16.4Hz)，7.58(1H，d，J＝7.8Hz)，7.49-7.37(4H，m)，7.32(1H，s)，7.21(1H，s)，7.06(1H，dd，J＝8.8，1.8Hz)，6.96(1H，s)，6.90(1H，t，J＝7.8Hz)，4.63(2H，s)，4.16(2H，d，J＝5.6Hz)，2.49(3H，s)，2.32(3H，s)，2.01(3H，s)。

Example 21: 2- [3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -nicotinic acid methyl ester

Prepared in analogy to the procedure described in example 3 above, except that 2-bromo-nicotinic acid methyl ester was used instead of 2-bromo-benzoic acid methyl ester.¹H NMR(DSMO-d₆)：δ10.36(1H，s)，8.50(1H，dd，J＝4.7，1.9Hz)，8.36(1H，d，J＝1.4Hz)，8.30(1H，dd，J＝7.8，2.0Hz)，8.08(1H，d，J＝8.8Hz)，7.83(1H，d，J＝16.4Hz)，7.48(1H，d，J＝7.5Hz)，7.44(1H，s)，7.33(1H，s)，6.98-6.93(2H，m)，5.80(1H，d，J＝7.0Hz)，2.93(3H，s)，3.93-3.90(1H，m)，3.80-3.75(1H，m)，2.46(3H，s)，2.30(3H，s)，2.10-1.97(2H，m)，1.89-1.60(3H，m)。

Example 22: 2- [3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -nicotinic acid

Prepared in analogy to the procedure described in example 4, except that 2- [3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-nicotinic acid methyl ester instead of 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid methyl ester.¹H NMR(DSMO-d₆)：δ10.73(1H，s)，8.49(1H，d，J＝1.9Hz)，8.45(1H，s)，8.31(1H，dd，J＝7.7，1.8Hz)，8.16-7.97(3H，m)，7.70(1H，d，J＝16.4Hz)，7.50(1H，d，J＝8.6Hz)，7.37(1H，s)，6.96(1H，dd，J＝7.7，4.8Hz)，5.87(1H，d，J＝8.4Hz)，3.95-3.90(1H，m)，3.79-3.70(1H，m)，2.63(3H，s)，2.47(3H，s)，2.07-1.99(2H，m)，1.81-1.62(3H，m)。

Example 23: 2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N- (4-hydroxy-but-2-ynyl) -nicotinamide

Prepared by analogy with the method described above for example 6 from 2- [3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]Preparation of N- (4-hydroxy-but-2-ynyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] using (E) -nicotinic acid and (E) -4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynylamine]-1-H-indazol-6-ylamino]-nicotinamide and N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2-3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-nicotinamide crude mixture and subsequent conversion to 2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl according to a similar method as described for example 7]-1H-indazol-6-ylamino } -N- (4-hydroxy-but-2-ynyl) -nicotinamide, except that N- (4-hydroxy-but-2-ynyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] amide is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino-1-nicotinamide and N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-mixture of niacinamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹HNMR(DSMO-d₆)：δ12.99(1H，s)，11.21(1H，s)，9.26(1H，t，J＝5.3Hz)，8.50(1H，d，J＝1.9Hz)，8.41(1H，dd，J＝4.9，1.9Hz)，8.16(1H，dd，J＝8.3，1.9Hz)，8.04(1H，d，J＝9.0Hz)，7.85(1H，d，J＝16.6Hz)，7.42(1H，d，J＝16.6Hz)，7.31(1H，s)，7.06(1H，dd，J＝8.7，1.5Hz)，6.96(1H，s)，6.93(1H，dd，J＝7.5，4.9Hz)，5.14(1H，t，J＝5.6Hz)，4.16(2H，d，J＝5.6Hz)，4.08(2H，d，J＝7.2Hz)，2.46(3H，s)，2.30(3H，s)。

Example 24: 2- {3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -nicotinic acid p-toluenesulfonate salt

Prepared in analogy to the procedure described in example 5, except that 2- [3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl 0-1H-indazol-6-ylamino) -nicotinic acid was used instead of 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid.

¹H NMR(DSMO-d₆)：δ13.49(1H，s)，10.80(1H，s)，8.63(1H，d，J＝1.5Hz)，8.49(1H，dd，J＝4.8，1.9Hz)，8.31(1H，dd，J＝7.7，1.9Hz)，8.24-8.19(2H，m)，8.06(1H，d，J＝8.8Hz)，7.60-7.55(2H，m)，7.46(2H，d，J＝8.1Hz)，7.22(1H，dd，J＝8.8，1.7Hz)，7.09(2H，d，J＝7.9Hz)，6.95(1H，dd，J＝7.7，4.7Hz)，2.66(3H，s)，2.54(3H，s)，2.27(3H，s)。

Example 25: n- (3-cyclopropyl-prop-2-ynyl) -2- {3- [ (E) -2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -nicotinamide

Prepared in analogy to the procedure described in example 6 above, except that 2- {3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -vinyl is used]-1H indazol-6-ylamino } -nicotinic acid p-toluenesulfonate and 3-cyclopropyl-prop-2-ynylamine.¹H NMR(DSMO-d₆)：δ13.01(1H，s)，11.20(1H，s)，9.19(1H，bt)，8.51(1H，s)，8.40(1H，d，J＝4.9Hz)，8.15(1H，d，J＝7.5Hz)，8.05(1H，d，J＝8.7Hz)，7.83(1H，d，J＝16.4Hz)，7.42(1H，d，J＝16.4Hz)，7.31(1H，s)，7.05(1H，d，J＝8.3Hz)，6.96(1H，s)，6.92(1H，dd，J＝7.5，4.9Hz)，4.06(2H，d，J＝4.14Hz)，2.46(3H，s)，2.29(3H，s)，1.33-1.28(1H，m)，0.77-0.72(2H，m)，0.60-0.55(2H，m)。

Example 26: 4-methyl-2-vinyl-pyridines

A yellow mixture of 2-bromo-4-methyl-pyridine (Aldrich, 5.2g, 30.5mmol, 1.0eq), 2, 6-di-tert-butyl-4-methyl-phenol (Aldrich, 67mg, 0.3mmol, 1 mol%), tributyl-vinyl-stannane (Aldrich, 26.8mL, 91.5mmol, 3.0eq) and tetrakis (triphenylphosphine) palladium (0) (Strem, 1.8g, 1.5mmol, 5 mol%) in toluene (100mL) was degassed and purged with argon. After heating the mixture to 100 ℃ an amber solution was obtained. After 18 hours the reaction mixture was quenched by addition of 1.0M HCl. The acidic extract was washed with diethyl ether, adjusted to pH9 with sodium bicarbonate and extracted with ethyl acetate. The organic extract was washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by flash (silica gel) chromatography (3.7g brown oil) and eluted with 0-5% ethyl acetate-dichloromethane to give a clear oil (1.9g, 53%).¹H NMR(DSMO-d₆，300MHz)δ8.39(1H，d，J＝4.9Hz)，7.33(1H，s)，7.10(1H，dd，J＝5.0，0.8Hz)，6.77(1H，dd，17.5，10.8Hz)，6.20(1H，dd，J＝17.5，1.7Hz)，5.44(1H，dd，J＝10.8，1.8Hz)，2.31(3H，s)。ESIMS m/z 120(M+H)⁺。

Example 27: 3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -6-nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole

4-methyl-2-vinyl-pyridine (example 23) (1.9g, 15.97mmol), 6-nitro-1- (tetrahydro-pyran-2-yl) -3-vinyl-1H-indazole (4.96g, 13.3mmol), Pd (OAc)₂(149mg，A suspension of 0.66mol), P (o-tolyl) 3 and DIEA (3.5mol, 19.96mmol) in degassed DMF (50ml) was heated at 100 ℃ under argon for 18 h. The reaction mixture was cooled and the solids were removed by filtration, washing with EtOAc. The filtrate was diluted with EtOAc and washed with brine (2 ×), dried (MgSO)₄) And concentrated under reduced pressure. The residue was chromatographed on silica, eluting with hexane: EtOAc (3: 1) to give 3.40g (70%) of a bright yellow solid.

¹H NMR(CDCl₃): δ 8.56(1H, s), 8.50(1H, d, J ═ 5.0Hz), 8.11(2H, m), 7.89(1H, d, J ═ 16.3Hz), 7.61(1H, s), 7.03(1H, d, J ═ 4.3Hz), 5.83(1H, dd, J ═ 2.6, 9.0Hz), 4.06(1H, m), 3.82(1H, m), 2.58(1H, m), 2.39(3H, s), 2.18(2H, m), 1.78(3H, m). To C₂₀H₂₀N₄O₃Analysis, calculated value: c, 65.92; h, 5.53; n, 15.38. Measurement value: c, 65.80; h, 5.52; and N, 15.15.

Example 28: 3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamine

Prepared in analogy to the procedure described in example 2, except using [2- (4-methyl-pyridin-2-yl) -vinyl ]]-6-Nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole (example 24) instead of 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-6-nitro-1- (tetrahydro-pyran-2-yl) -1H-indazole.¹HNMR(DSMO-d₆)：δ8.43(1H，d，J＝4.8Hz)，7.79-7.73(2H，m)，7.50(1H，s)，7.39(1H，d，J＝16.4Hz)，7.09(1H，d，J＝4.8Hz)，6.64-6.62(2H，m)，5.57(1H，dd，J＝9.8，2.5Hz)，5.48(2H，bs)，3.92-3.85(1H，m)，3.72-3.64(1H，m)，2.43-2.34(1H，m)，2.33(3H，s)，2.07-2.00(1H，m)，1.96-1.90(1H，m)，1.79-1.66(1H，m)，1.60-1.53(2H，m)。

Example 29: 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid methyl ester

Prepared in analogy to the procedure described in example 3, except that 3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamine instead of 3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamine.¹H NMR(DSMO-d₆)：δ9.48(1H，s)，8.45(1H，d，J＝4.9Hz)，8.13(1H，d，J＝8.7Hz)，7.93(1H，dd，J＝8.3，1.9Hz)，7.86(1H，d，J＝16.2Hz)，7.58(1H，d，J＝1.9Hz)，7.54-7.44(3H，m)，7.36(1H，d，J＝7.5Hz)，7.18(1H，dd，J＝8.7，1.9Hz)，7.11(1H，d，J＝4.9Hz)，6.87(1H，t，J＝8.3Hz)，5.83(1H，dd，J＝9.4，2.3Hz)，3.87(3H，1H)，3.93-3.84(1H，m)，3.77-3.69(1H，m)，2.46-2.37(1H，m)，2.34(3H，s)，2.10-1.94(2H，m)，1.81-1.53(3H，m)。

Example 30: 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzoic acid

Prepared in analogy to the procedure described in example 4 above, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -vinyl is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid methyl ester instead of 2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-methyl benzoate (example 3).¹H NMR(DSMO-d₆)：δ13.17(1H，broad s)，9.83(1H，s)，8.51(1H，d，J＝5.2Hz)，8.14(1H，d，J＝8.7Hz)，7.95(1H，d，J＝16.4Hz)，7.94(dd，(1H，J＝1.5，8.0Hz)，7.73(1H，s)，7.60(1H，d，J＝1.5Hz)，7.59(1H，s)，7.54(1H，s)，7.46(1H，m)，7.37(1H，d，J＝7.6Hz)，7.23(2H，m)，6.86(1H，t，J＝6.9Hz)，5.87(1H，d，J＝7.6Hz)，3.90(1H，m)，3.76(1H，m)，2.45(1H，m)，2.41(3H，s)，2.03(2H，m)，1.77(1H，m)，1.59(2H，m)。

Example 31: 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -N-prop-2-ynyl-benzamide

Prepared in analogy to the procedure described in example 6 above, except that propargylamine and 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] are used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid.¹H NMR(DSMO-d₆)δ9.87(1H，s)，9.03(1H，t，J＝5.5Hz)，8.46(1H，d，J＝4.9Hz)，8.08(1H，d，J＝8.7Hz)，7.86(1H，d，J＝16.4Hz)，7.69(1H，d，J＝7.3Hz)，7.53(1H，s)，7.44(4H，m)，7.13(2H，m)，6.91(1H，t，J＝7.9Hz)，5.81(1H，dd，J＝2.2，9.6Hz)，4.07(2H，dd，J＝2.5，5.5Hz)，3.89(1H，m)，3.75(1H，m)，3.12(1H，t，J＝2.5Hz)，2.42(1H，m)，2.36(3H，s)，2.00(2H，m)，1.75(1H，m)，1.58(2H，m)。

To C₃₀H₂₉N₅O₂Analysis of 0.25TBME, calcd for: c, 73.07; h, 6.28; and N, 13.64. Measurement value: c, 72.95; h, 6.30; and N, 13.64.

Example 32: 2- {3- [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N-prop-2-ynyl-benzamide

Prepared in analogy to the procedure described in example 7, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -ethylene is usedBase of]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-N-prop-2-ynyl-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹HNMR(DSMO-d₆)δ12.93s).9.79(1H，s)，9.02(1H，t，J＝5.4Hz，8.45(1H，d，J＝4.9Hz)，8.08(1H，d，J＝8.7Hz)，7.88(1H，d，J＝16.4Hz)，7.69(1H，d，J＝7.7Hz)，7.45(4H，m)，7.27(1H，s)，)，7.10(1H，d，J＝4.9Hz)，7.03(1H，d，J＝8.8Hz)，6.90(1H，t，J＝7.9Hz)，4.06(2H，dd，J＝2.4，5.4Hz)，3.12(1H，t，J＝2.4Hz)，2.35(3H，s)。

To C₂₅H₂₁N₅O·0.35CH₂Cl₂Analysis, calculated value: c, 69.64; h, 5.00; and N, 16.02. Measurement value: c, 69.65; h, 5.15; and N, 15.80.

Example 33: n- (2-methyl-allyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in analogy to the procedure described in example 6 above, except that 2-methyl-allylamine and 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] are used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid.¹H NMR(DSMO-d₆) δ 9.86(1H, s), 8.81(1H, t, J ═ 5.5Hz), 8.46(1H, d, J ═ 4.9Hz), 8.07(1H, d, J ═ 8.9Hz), 7.86(1H, d, J ═ 16.4Hz), 7.75(1H, d, J ═ 7.7Hz), 7.54(1H, s), 7.50(1H, d, J ═ 16.4Hz), 7.43(3H, m), 7.11(2H, m), 6.92(1H, t, J ═ 8.1Hz), 5.81(1H, dd, J ═ 2.5, 9.8Hz), 4.83(2H, d, J ═ 11.5Hz), 3.81(4H, m), 2.41(1H, m), 2.35 (1H, 2H, 5H, 1.5H, 5 m), 3.83 (1H, m), 1H, 5.5H, 1H, 1.5H, 5 m), 3.73 (1H, 73H, 5 m), 1H, 5.5H, 1m), 3.58H, 1H, 5H, 1. To C₃₁H₃₃N₅O₂0.80TBME analysis, MeterCalculating the value: c, 72.71; h, 7.43; n, 12.11. Measurement value: c, 72.43; h, 7.57; and N, 12.02.

Example 34: n- (2-methyl-allyl) -2- { [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7 above, except using N- (2-methyl-allyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)δ12.90(1H，s)，9.76(1H，s)，8.80(1H，t，J＝5.5Hz)，8.45(1H，d，J＝5.1Hz)，8.07(1H，d，J＝8.9Hz)，7.88(1H，d，J＝16.4Hz)，7.75(1H，d，J＝7.9Hz)，7.51(1H，s)，7.48(1H，d，J＝16.4Hz)，7.43(2H，m)，7.24(1H，s)，7.10(1H，d，J＝4.9Hz)，7.00(1H，dd，J＝1.9，8.9Hz)，6.91(1H，t，J＝8.1Hz)，4.82(2H，d，J＝11.3Hz)，3.83(2H，d，J＝5.8Hz)，2.35(3H，s)，1.73(3H，s)。

To C₂₆H₂₅N₅O·0.20H₂Analysis of O, calculated: c, 73.11; h, 5.99; and N, 16.40. Measurement value: c, 73.13; h, 6.03; n, 16.13.

Example 35: n- (3-cyclopropyl-prop-2-ynyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described above for example 6,except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and 3-cyclopropyl-prop-2-ynylamine.¹H NMR(DSMO-d₆)：δ9.88(1H，bs)，8.93(1H，bt)，8.46(1H，d，J＝4.9Hz)，8.08(1H，d，J＝8.7Hz)，7.85(1H，d，J＝16.4Hz)，7.69(1H，d，J＝7.6Hz)，7.54-7.40(5H，m)，7.14-7.11(2H，m)，6.90(1H，t，J＝6.1Hz)，5.81(1H，d，J＝7.5Hz)，4.02(2H，d，J＝3.6Hz)，3.95-3.85(1H，m)，3.79-3.72(1H，m)，2.49-2.35(1H，m)，2.35(3H，s)，2.15-2.01(2H，m)，1.87-1.55(3H，m)，1.30-1.25(1H，m)，0.77-0.70(2H，m)，0.59-0.54(2H，m)。

Example 36: n- (3-Cycloprop-2-ynyl) -2- {3- [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7 above, except that N- (3-cycloprop-2-ynyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)：δ12.91(1H，s)，9.79(1H，s)，8.91(1H，t，J＝5.6Hz)，8.44(1H，d，J＝4.9Hz)，8.06(1H，d，J＝8.7Hz)，7.87(1H，d，J＝16.6Hz)，7.67(1H，dd，J＝7.9，1.5Hz)，7.50-7.35(4H，m)，7.24(1H，d，J＝1.9Hz)，7.09(1H，d，J＝4.9Hz)，7.00(1H，dd，J＝8.7，1.5Hz)，6.88(1H，dt，J＝4.1，1.5Hz)，4.00(2H，dd，J＝5.3，1.9Hz)，2.34(3H，s)，1.31-1.23(1H，m)，0.75-0.69(2H，m)，0.56-0.52(2H，m)。

Example 37: 2- {3- [2- (4-methyl-pyridin-2-yl ] -vinyl } -1H-indazol-6-ylamino) -N-pyridin-2-ylmethyl-benzamide

Prepared in analogy to the methods described in example 6 and example 7 above, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and C-pyridin-2-yl-methylamine.¹H NMR(DSMO-d₆，300MHz)δ12.91(1H，s)，9.77(1H，s)，9.19(1H，t，J＝5.8Hz)，8.50(1H，d，J＝4.1Hz)，8.45(1H，d，J＝5.0Hz)，8.06(1H，d，J＝8.8Hz)，7.88(1H，d，J＝16.4Hz)，7.82-7.70(2H，m)，7.51-7.25(7H，m)，7.10(1H，d，J＝4.6Hz)，6.98(1H，dd，J＝8.8，1.8Hz)，6.96-6.91(1H，m)，4.58(2H，d，J＝5.9Hz)，2.35(3H，s)。ESIMS m/z 461(M+H)⁺. To C₂₈H₂₄N₆Analysis of O · 0.3MTBE, calculated: c, 72.71; h, 5.77; and N, 17.25. Measurement value: c, 72.38; h, 5.80; n, 16.88.

Example 38: 2- {3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N-pyridin-4-ylmethyl-benzamide

Prepared in analogy to the methods described in example 6 and example 7 above, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and C-pyridin-4-yl-methylamine.¹H NMR(DSMO-d₆，300MHz)δ12.90(1H，s)，9.73(1H，s)，9.21(1H，t，J＝5.9Hz)，8.49-8.44(3H，m)，8.06(1H，d，J＝8.7Hz)，7.88(1H，d，J＝16.4Hz)，7.81(1H，d，J＝7.5Hz)，7.51-7.40(4H，m)，7.31(2H，d，J＝5.9Hz)，7.24(1H，s)，7.11(1H，d，J＝4.4Hz)，7.00(1H，dd，J＝8.7，1.7Hz)，6.97-6.92(1H，m)，4.50(2H，d，J＝5.9Hz)，2.35(3H，s)。ESIMS m/z 461(M+H)⁺. To C₂₈H₂₄N₆O×0.4H₂Analysis of O × 0.7MTBE, calculated: c, 71.36; h, 6.45; n, 15.85. Measurement value: c, 71.27; h, 6.29; n, 15.53.

Example 39: n- (6-methyl-pyridin-2-ylmethyl) -2- {3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the methods described in example 6 and example 7 above, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and C- (6-methyl-pyridin-2-yl) -methylamine.¹H NMR(DSMO-d₆，300MHz)δ12.92(1H，s)，9.76(1H，s)，9.20(1H，t，J＝5.8Hz)，8.44(1H，d，J＝4.9Hz)，8.05(1H，d，J＝8.6Hz)，7.86(1H，d，J＝16.4Hz)，7.81(1H，d，J＝7.7Hz)，7.59(1H，t，J＝7.7Hz)，7.49-7.37(4H，m)，7.23(1H，s)，7.11-7.08(3H，m)，6.99(1H，dd，J＝8.7，1.6Hz)，6.95-6.90(1H，m)，4.51(2H，d，J＝5.9Hz)，2.42(3H，s)，2.33(3H，s)。ESIMS m/z 475(M+H)⁺. To C₂₉H₂₆N₆Analysis of ox0.4dcm, calculated: c, 68.98; h, 5.29; n, 16.39. Measurement value: c, 68.84; h, 5.42; and N, 16.20.

Example 40: n- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [ (E) -3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described above in example 6, except that 2- {3- [2- (4-methyl-pyridin-2-yl-vinyl) -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino is usedBase of]-benzoic acid and C- (2, 5-dimethyl-2H-pyrazol-3-yl) -methylamine.¹H NMR(DSMO-d₆)δ9.81(1H，s)，9.05(1H，bt)，8.468.7Hz}，7.85(1H，d，J＝16.4Hz)，7.71(1H，d，J＝7.5Hz)，7.54-7.40(5H，m)，7.11-7.09(2H，m)，6.91(1H，t，J＝6.9Hz)，5.94(1H，s)，5.80(1H，d，J＝7.3Hz)，4.45(2H，d，J＝5.5Hz)，3.93-3.85(1H，m)，3.78-3.69(1H，m)，3.73(3H，s)，2.45-2.35(1H，m)，2.35(3H，s)，2.07(3H，s)，2.06-1.95(2H，m)，1.85-1.53(m，3H)。

Example 41: n- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- {3- [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7 above, except that N- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [ (E) -3- [2- (4-methyl-pyridin-2-yl) -vinyl ] is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)δ12.90(1H，s)，9.70(1H，s)，9.03(1H，t，J＝6.0Hz)，8.44(1H，d，J＝4.9Hz)，8.06(1H，d，J＝9.0Hz)，7.87(1H，d，J＝16.2Hz)，7.70(1H，d，J＝7.5Hz)，7.50-7.38(4H，m)，7.22(1H，s)，7.1(1H，d，J＝5.6Hz)，6.99(1H，dd，J＝8.7，1.5Hz)，6.90(1H，dt，J＝7.9，1.9Hz)，5.91(1H，s)，4.43(2H，d，J＝5.6Hz)，3.72(3H，s)，2.34(3H，s)，2.05(3H，s)。

Example 42: 1-methyl-1H-benzimidazole-2-aldoxime

To a stirred solution of 1-methyl-1H-benzimidazole-2-aldehyde (980mg, 6.61mmol) in H₂To a suspension in O (10ml) was added sodium acetate (3.25g, 39.68mmol) and hydroxylamine hydrochloride (1.38g, 19.84mmol) in 10ml H₂Solution in O. The reaction mixture was stirred at room temperature for 2 hours and the viscous precipitate was collected by filtration, washed with water and dried in vacuo to yield 1.02g (94%) of a white solid.¹H NMR(DSMO-d₆) δ 12.06(1H, s), 8.28(1H, s), 7.65(1H, d, J ═ 7.5Hz), 7.60(1H, d, J ═ 6.8Hz), 7.32(1H, t, J ═ 7.2Hz), 7.23(1H, t, J ═ 6.8Hz), 4.00(3H, s). To C₉H₉N₃Analysis of O, calculated: c, 61.70; h, 5.18; and N, 23.99. Measurement value: c, 61.80; h, 5.23; and N, 23.98.

Example 43: c- (1-methyl-1H-benzimidazol-2-yl) -methylamine dihydrochloride

To a Parr pressure bottle was added 1-methyl-1H-benzimidazole-2-aldoxime M (267mg, 1.6mmol), 10% palladium on carbon (75mg), concentrated HCl (2 drops) and EtOH (25 mol). The reaction mixture was brought to 45psi H₂Shaken for 2 hours in the environment, after which the catalyst is removed by filtration. The filtrate was concentrated under reduced pressure and the residue was taken up in Et₂Trituration with O afforded 340mg (90%) of a white solid as the dihydrochloride salt, which was used without further purification.¹H NMR(DSMO-d₆)：δ8.87(2H，broad s)，7.72(2H，m)，7.38(2H，m)，4.50(2H，s)，3.89(3H，s)。

Example 44: n- (1-methyl-1H-benzimidazol-2-ylmethyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in analogy to the procedure described in example 6 above, except using C- (1-methyl-1H-benzoimidazol-2-yl) -methylamine hydrochloride N and 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid.¹H NMR(DSMO-d₆)δ9.82(1H，s)，9.20(1H，t，J＝5.3Hz)，8.46(1H，d，J＝4.9Hz)，8.07(1H，d，J＝8.9Hz)，7.85(1H，d，J＝16.4Hz)，7.74(1H，d，J＝7.3Hz)，7.58(1H，d，J＝7.2Hz)，7.50(6H，m)，7.19(4H，m)，6.92(1H，t，J＝8.1Hz)，5.78(1H，dd，J＝2.5，9.5Hz)，4.79(2H，d，J＝5.5Hz)，3.89(1H，m)，3.83(3H，s)，3.71(1H，m)，2.41(1H，m)，2.35(3H，s)，2.00(2H，m)，1.74(1H，m)，1.57(2H，m)。

To C₃₆H₃₅N₇O₂Analysis of 0.65 hexane, calculated: c, 73.31; h, 6.80; n, 15.00. Measurement value: c, 72.92; h, 6.90; n, 14.71.

Example 45: n- (1-methyl-1H-benzimidazol-2-ylmethyl) -2- {3- [ (E)2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7, except that N- (1-methyl-1H-benzoimidazol-2-ylmethyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide.¹H NMR(DSMO-d₆)δ12.93(1H，s)，9.73(1H，s)，9.19(1H，t，J＝5.3Hz)，8.45(1H，d，J＝4.9Hz)，8.06(1H，d，J＝8.5Hz)，7.88(1H，d，J＝16.4Hz)，7.74(1H，d，J＝7.9Hz)，7.60-7.36(6H，m)，7.29-7.14(3H，m)，7.10(1H，d，J＝4.7Hz)，7.04(1H，dd，J＝1.8，8.9Hz)，6.91(1H，t，J＝7.3Hz)，4.79(2H，d，J＝5.3Hz)，3.83(3H，s)，2.35(3H，s)。

To C₃₁H₂₇N₇O·1.80H₂O·0.40CH₂Cl₂Analysis, calculated value: c, 65.02; h, 5.46; n, 16.91. Measurement value: c, 64.97; h, 5.82; and N, 17.09.

Example 46: 1-methyl-1H-imidazole-2-aldoxime

Prepared in analogy to the procedure described in example 39, except that 1-methyl-1H-imidazole-2-aldehyde was used.¹H NMR(DSMO-d₆)δ11.50(1H，s)，8.05(1H，s)，7.28(1H，s)。

To C₅H₇N₃Analysis of O, calculated: c, 47.99; h, 5.64; n, 33.58. Measurement value: c, 48.22; h, 5.58; n, 33.45.

Example 47: c- (1-methyl-1H-imidazol-2-yl) -methylamine dihydrochloride

Prepared in analogy to the procedure described in example 40, except that 1-methyl-1H-imidazole-2-aldoxime was used instead of 1-methyl-1H-benzimidazole-2-aldoxime.¹H NMR(DSMO-d₆)δ7.45(1H，s)，7.29(1H，s)，4.25(21H，s)，3.79(3H，s)。

Example 48: n- (1-methyl-1H-imidazol-2-ylmethyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in analogy to the procedure described in example 6 above, except using C- (1-methyl-1H-imidazol-2-yl) -methylamine hydrochloride and 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid.¹H NMR(DSMO-d₆) δ 9.83(1H, s 9.03(1H, t, J ═ 5.5Hz), 8.45(1H, d, J ═ 4.7Hz), 8.09(1H, d, J ═ 8.5Hz), 7.85(1H, d, J ═ 16.5Hz), 8.67(1H, d, J ═ 7.3Hz), 7.53-7.39(4H, m), 7.11(3H, m), 6.90(1H, d, J ═ 6.9Hz), 6.86(1H, s), 5.79(1H, d, J ═ 8.9Hz), 5.75(1H, s), 4.54(1H, d, J ═ 5.5Hz), 3.85-3.70(2H, m), 3.66(3H, s), 2.10 (1H, 70H, 1.70H, 1m), 1H, 60(1H, m). To C₃₂H₃₃N₇O₂·0.8CH₂Cl₂Analysis, calculated value: c, 63.99; h, 5.672; n, 15.93. Measurement value: c, 63.95; h, 5.72; and N, 16.01.

Example 49: n- (1-methyl-1H-imidazol-2-ylmethyl) -2- {3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -benzamide

Prepared in analogy to the procedure described in example 7, except that N- (1-methyl-1H-imidazol-2-ylmethyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzamide instead of N- [ 4-tert-butyl-dimethyl-silanyloxy) -but-2-ynyl]-2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid.¹H NMR(DSMO-d₆)δ12.89(1H，s 9.72(1H，s，8.99(1H，t，J＝5.6Hz)，8.44(1H，d，J＝4.9Hz)，8.05(1H，d，J＝8.7Hz)，7.86(1H，d，J＝16.4Hz)，7.66(1H，d，J＝6.7Hz)，7.49-7.36(4H，m)，7.24(1H，m)，6.86(1H，s)，7.09(2H，d，J＝8.1Hz)，7.02(1H，d，J＝8.8Hz)，6.88(1H，s)，4.52(2H，d，J＝5.5Hz)，3.29(3H，s), 2.34(3H, s). To C₂₇H₂₅N₇O·0.35CH₂Cl₂Analysis, calculated value: c, 66.59; h, 5.252; n, 19.88. Measurement value: c, 66.485; h, 5.65; n, 19.56.

Example 50: n- (4-hydroxy-but-2-ynyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide

Prepared in analogy to the procedure described in example 6, except that 2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -vinyl is used]-1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino]-benzoic acid and 4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynylamine.¹H NMR(CDCl₃)δ9.48(H，s)，8.46(1H，d，J＝5.3Hz)，7.92(1H，d，J＝9.0Hz)，7.83(1H，d，J＝16.2Hz)，7.52(1H，d，J＝16.6Hz)，7.46-7.41(2H，m)，7.34-7.31(3H，m)，7.12(1H，dd，J＝8.7，1.9Hz)，6.99(1H，d，J＝4.9Hz)，6.81(1H，t，J＝6.8Hz)，6.40(1H，t，J＝4.9Hz)，5.62(1H，dd，J＝9.4，3.0Hz)，4.28-4.23(4H，m)，4.08-4.01(1H，m)，3.76-3.67(1H，m)，2.63-2.49(1H，m)，2.38(3H，s)，2.22-2.06(2H，m)，1.80-1.60(3H，m).

Example 51: 2- {3- [ (E) -2- (4-methyl-pyridin-2-yl) -vinyl ] -1H-indazol-6-ylamino } -N- (4-hydroxy-but-2-ynyl) -benzamide

Prepared in analogy to the procedure described in example 7, except that a mixture of N- (4-hydroxy-but-2-ynyl) -2- [3- [2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide and N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl) -2- [3- (2- (4-methyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide is used instead of N - [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl) -2- [3- [2- (4, 6-dimethyl-pyridin-2-yl) -vinyl ] -1- (tetrahydro-pyran-2-yl) -1H-indazol-6-ylamino ] -benzamide.

¹H NMR(DSMO-d₆)δ12.92(1H，s)，9.83(1H，s)，9.00(1H，t，J＝5.3Hz)，8.44(1H，d，J＝4.9Hz)，8.06(1H，d，J＝9.0Hz)，7.87(1H，d，J＝16.6Hz)，7.68(1H，d，J＝7.9Hz)，7.50-7.38(4H，m)，7.26(1H，s)，7.09(1H，d，J＝5.3Hz)，7.01(1H，dd，J＝8.7，1.5Hz)，6.88(1H，dt，J＝6.8，1.5Hz)，5.11(1H，t，J＝3.0Hz)，4.10-4.04(4H，m)，2.34(3H，s)。

Example 52: 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzoic acid methyl ester

Prepared in analogy to the methods described in examples 2 and 3 above, except that 6-nitro-3-styryl-1-H-indazole was used instead of 6-iodo-3-styryl-1- (2-trimethylsilyl-ethoxymethyl) -1H-indazole.

This material was taken as a crude mixture of product and methyl 2-amino-benzoate for the next step.

Example 53: 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzoic acid

N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-yne using a method similar to that described in example 11 of U.S. patent application Ser. No. 09/609,335 filed on 30.6.2000Base of]-2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino]-separation of the above compounds as by-products from the reaction mixture of benzamide and TBAF, the entire contents of said documents being incorporated herein by reference for all purposes.¹H NMR(DSMO-d₆)δ13.19(1H，broad s 10.00(1H，s，9.13(1H，s)，8.37(1H，d，J＝8.7Hz)，8.06(1H，d，J＝7.5Hz)，7.75(1H，s)，7.64(2H，t，J＝2.3Hz)，7.54(2H，m)，7.35(1H，dd，J＝1.9，8.7Hz)，6.99(1H，m)，6.33(2H，t，J＝2.3Hz)，5.89(2H，s)，3.68(2H，t，J＝8.1Hz)，0.94(2H，t，J＝8.1Hz)，0.00(9H，s)。

Example 54: n- (3-cyclopropyl-prop-2-ynyl) -2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 6 above, except that 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino is used]-benzoic acid and 3-cyclopropyl-prop-2-ynylamine.¹H NMR(DSMO-d₆) δ 9.93(1H, s 8.99(1H, s), 8.95(1H, d, J ═ 5.6Hz), 8.20(1H, d, J ═ 8.9Hz), 7.68(1H, d, J ═ 8.1Hz), 7.51(4H, m), 7.37(1H, t, J ═ 6.8Hz), 7.14(1H, d, J ═ 9.OHz), 6.91(1H, t, J ═ 7.5Hz), 6.21(2H, t, J ═ 2.3Hz), 5.74(2H, s), 4.00(2H, dd, J0.73(2H, m), 0.55(2H, m) · C, C pair₂₅H₂₂N₆O0.05 Hexane 0.30H₂Analysis of O, calculated: c, 70.31; h, 5.43; n, 19.45. measurement: c, 70.63; h, 5.38; n, 19.18.

Example 55: n- (3-cyclopropyl-prop-2-ynyl) -2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared using a method similar to that described in example 11 of U.S. patent application Ser. No. US 09/609,335 filed on 30.6.2000, except that N- (3-cyclopropyl-prop-2-ynyl) -2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylaminomethyl ] -amide was used]-benzamide instead of N-methyl-N- { 3-styryl-1- [ 2-trimethyl-silyl) -ethoxymethyl]-1H-indazol-6-yl } -benzene-1, 3-diamine, the entire contents of which are incorporated herein by reference for all purposes.¹H NMR(DSMO-d₆) δ 13.29(1H, s), 9.83(1H, s), 8.98(1H, s), 8.95(1H, t, J ═ 5.5Hz), 8.19(1H, d, J ═ 8.9Hz), 7.68(1H, d, J ═ 7.5Hz), 7.52(2H, t, J ═ 2.3Hz), 7.43(2H, m), 7.29(1H, s), 7.07(1H, dd, J ═ 1.9, 8.7Hz), 6.91(1H, t, J ═ 7.4Hz), 6.21(2H, t, J ═ 2.3Hz), 4.01(2H, dd, J ═ 1.7, 5.5Hz), 1.27(1H, m), 0.73(2H, m), 0.55H (m). To C₂₅H₂₂N₆O0.05 Hexane 0.30H₂Analysis of O, calculated: c, 70.31; h, 5.43; n, 19.45. Measurement value: c, 70.63; h, 5.38; n, 19.18.

Example 56: n- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl ] -2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 6 above, except that 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino is used]Benzoic acid and 4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynylamine.¹H NMR(DSMO-d₆)δ10.04(1H，s9.16(1H，t，J＝5.3Hz)，9.10(1H，s)，8.31(1H，d，J＝8.7Hz)，7.78(1H，d，J＝7.9Hz)，7.67(4H，m)，7.49(1H，t，J＝8.5Hz)，7.24(1H，dd，J＝1.7，8.7Hz)，7.03(1H，t，J＝7.4Hz)，6.33(2H，t，J＝2.3Hz)，5.85(2H，s)，4.83(2H，s)，4.19(2H，d，J＝5.5Hz)，3.66(2H，t，J＝7.9Hz)，0.94(2H，m)，0.89(9H，s)，0.13(6H，s)，0.00(9H，s)。

Example 57: n- (4-hydroxy-but-2-ynyl) -2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared using a method similar to that described in example 11 of U.S. patent application Ser. No. US 09/609,335 filed on 30.6.2000, except that N- [4- (tert-butyl-dimethyl-silanyloxy) -but-2-ynyl is used]-2- [3- (pyrrol-1-yliminomethyl) -1- (2-methylsilyl-ethoxymethyl) -1H-indazol-6-ylamino]-benzamide instead of N-methyl-N- { 3-styryl-1- [ 2-trimethyl-silyl) -ethoxymethyl]-1H-indazol-6-yl } -benzene-1, 3-diamine, the entire contents of which are incorporated herein by reference for all purposes.¹H NMR(DSMO-d₆) δ 13.30(1H, s), 9.87(1H, s 9.04(1H, t, J ═ 5.3Hz), 8.99(1H, s), 8.19(1H, d, J ═ 8.5Hz), 7.70(1H, d, J ═ 7.3Hz), 7.46(4H, m), 7.31(1H, s), 7.08(1H, dd, J ═ 1.7, 8.7Hz), 6.91(1H, t, J ═ 7.3Hz), 6.21(2H, t, J ═ 2.1Hz), 5.14(1H, t, J ═ 5.8Hz), 4.10(2H, d, J ═ 5.5Hz), 4.06(2H, d, J ═ 5.8 Hz). To C₂₃H₂₀N₆O₂0.35 Hexane 0.20H₂Analysis of O, calculated: c, 67.45; h, 5.86; n, 18.81. Measurement value: c, 67.70; h, 5.73; n, 18.56.

Example 58: 2, 5-dimethyl-2H-pyrazole-3-carbonitrile

2, 5-dimethyl-2H-pyrazole-3-carbonitrile was prepared from ethyl 1, 3-dimethylpyrazole-5-carboxylate as disclosed for 1-methyl-pyrazole-5-carbonitrile in JCS Perkins TransI (1985)1209-1215 by Castellanos, Maria and Montserrta, Llinas.¹H NMR(CDCl₃)δ6.52(1H，s)，3.96(3H，s)，2.27(3H，s)。

Example 59: c- (2, 5-dimethyl-2H-pyrazol-3-yl) -methylamine

A suspension of 2, 5-dimethyl-2H-pyrazole-3-carbonitrile (654mg, 5.4mmol) and 10% palladium on carbon (200mg) in ethanol (15mL) was stirred at 45psi H₂The mixture was shaken in a Parr hydrogenator for 17 hours. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to give 608mg of oil, which was used without any further purification.¹H NMR(CDCl₃)δ5.91(1H，s)，3.81，3.73(2H，2s)，3.75(3H，s)，2.21(3H，s)。

Example 60: n- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [3- (pyrrol-1-yliminomethyl rl- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 6 above, except that 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylamino is used]-benzoic acid and C- (2, 5-dimethyl-2H-pyrazol-3-yl) -methylamine.¹H NMR(CDCl₃)δ9.56(1H，s)，8.68(1H，s)，8.30(1H，d，J＝8.7Hz)，7.49(1H，d，J＝8.3Hz)，7.43(1H，dd，J＝7.9，1.5Hz)，7.36-7.31(2H，m)，7.23(2H，t，J＝2.6Hz)，7.17(1H，dd，J＝8.7，1.9Hz)，6.83(1H，t，J＝7.2Hz)，6.32(1H，bt)，6.29(2H，t，J＝2.3Hz)，6.01(1H，s)，5.67(2H，s)，4.61(2H，d，J＝5.6Hz)，3.60(3H，s)，3.58(2H，t，J＝8.3Hz)，2.22(3H，s)，0.90(2H，t，J＝8.7Hz)，0.06(9H，s)。

Example 61: n- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino ] -benzamide

Prepared using a method similar to that described in example 11 of U.S. patent application Ser. No. US 09/609,335 filed on 30.6.2000, except that N- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indazol-6-ylaminomethyl) -2]-benzamide instead of N-methyl-N- { 3-styryl-1- [ 2-trimethyl-silyl) -ethoxymethyl]-1H-indazol-6-yl } -benzene-1, 3-diamine, the entire contents of which are incorporated herein by reference for all purposes.¹H NMR(CDCl₃)δ13.27(1H，s)，9.72(1H，s)，9.05(1H，t，J＝5.3Hz)，8.97(1H，s)，8.16(1H，d，J＝8.7Hz)，7.68(1H，dd，J＝8.3，1.9Hz)，7.50(2H，t，J＝2.6Hz)，7.46-7.38(2H，m)，7.25(1H，s)，7.05(1H，dd，J＝8.7，1.9Hz)，6.91(1H，t，J＝6.80Hz)，6.20(2H，t，J＝2.3Hz)，5.91(1H，s)，4.43(2H，d，J＝5.6Hz)，3.71(3H，s)，2.04(3H，s)。

Example 62: 2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino ] -benzoic acid

Prepared using a method analogous to that described in example 11 of U.S. patent application Ser. No. US 09/609,335 filed on 30.6.2000, except that 2- [3- (pyrrol-1-yliminomethyl) -1- (2-trimethylsilyl-ethoxymethyl) -1H-indole is usedAzol-6-ylamino]Benzoic acid instead of N-methyl-N- { 3-styryl-1- [ 2-trimethyl-silyl) -ethoxymethyl]-1H-indazol-6-yl } -benzene-1, 3-diamine, the entire contents of which are incorporated herein by reference for all purposes.¹H NMR(DSMO-d₆)δ13.12(1H，s)，12.70(1H，s)，8.94(1H，s)，8.10(1H，d，J＝8.7Hz)，7.91(1H，dd，J＝1.7，7.7Hz)，7.50(2H，t，J＝2.3Hz)，7.36(1H，d，J＝7.9Hz)，7.27(1H，d，J＝1.5Hz)，7.16(1H，t，J＝7.5Hz)，6.94(1H，dd，J＝1.7，8.7Hz)，6.68(1H，t，J＝7.5Hz)，6.19(2H，t，J＝2.3Hz)。

Example 63: 2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino ] -benzoic acid

Prepared in a similar manner as described in example 6 above, except that 2- [3- (pyrrol-1-yliminomethyl) -1H-indazol-6-ylamino is used]-benzoic acid and propargylamine.¹H NMR(DMSO-d₆) δ 13.30(1H, s), 9.82(1H, s), 9.04(1H, t, J ═ 5.6Hz), 8.98(1H, s), 8.19(1H, d, J ═ 8.6Hz), 7.69(1H, d, J ═ 7.9Hz), 7.45(4H, m), 7.31(1H, s), 7.08(1H, d, J ═ 8.6Hz), 6.91(1H, t, J ═ 7.6Hz), 6.21(2H, s), 4.05(2H, s), 3.13(1H, s). To C₂₂H₁₈N₆O·0.40H₂Analysis of O · 0.05 hexane, calculated: c, 67.97; h, 5.01; n, 21.33. Measurement value: c, 67.91; h, 4.78; n, 21.00.

Example 64: n- (4-hydroxy-but-2-ynyl) -2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 6 above, except that 2- [3- (2-pyridine) is used-2-yl-vinyl) -1H-indazol-6-ylamino]Tetrabutylammonium benzoate and 4-amino-but-2-yn-1-ol.¹HNMR(DMSO-d₆)：δ12.95(1H，s)，9.84(1H，s)，9.02(1H，t，J＝5.6Hz)，8.59(1H，d，J＝4.9Hz)，8.08(1H，d，J＝8.7Hz)，7.90(1H，d，J＝16.2Hz)，7.80(1H，t，J＝7.2Hz)，7.70-7.64(2H，m)，7.51(1H，d，J＝16.2Hz)，7.45-7.36(2H，m)，7.27-7.24(2H，m)，7.02(1H，d，J＝9.0Hz)，6.88(1H，t，J＝7.2Hz)，5.13(1H，t，J＝5.6Hz)，4.10-4.04(4H，m)。

Example 65: n- (2, 5-dimethyl-2H-pyrazol-3-ylmethyl) -2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino ] -benzamide

Prepared in a similar manner as described in example 6 above, except that 2- [3- (2-pyridin-2-yl-vinyl) -1H-indazol-6-ylamino is used]Tetrabutylammonium benzoate and C- (2, 5-dimethyl-2H-pyrazol-3-yl) -methylamine.¹H NMR(DMSO-d₆)δ12.93(1H，s)，9.70(1H，s)，9.04(1H，bt)，8.58(1H，d，J＝4.0Hz)，8.07(1H，d，J＝8.8Hz)，7.88(1H，d，J＝16.4Hz)，7.79(1H，t，J＝8.6Hz)，7.71-7.64(2H，m)，7.50(1H，d，J＝16.4Hz)，7.44-7.39(2H，m)，7.28-7.23(2H，m)，7.00(1H，d，J＝8.8Hz)，6.90(1H，t，J＝8.0Hz)，5.91(1H，s)，4.43(2H，d，J＝5.5Hz)，3.71(3H，s)，2.04(3H，s)。

The activity of the above typical compounds can be tested using the following assay.

Biological test: enzyme assay

The stimulation of cell proliferation by growth factors such as VEFG, FGF and others depends on their induction of autophosphorylation of their respective receptor tyrosine kinases. Thus, the ability of a protein kinase inhibitor to block autophosphorylation can be determined by inhibiting the peptide substrate. To determine the inhibitory activity of the compounds on protein kinases, the following constructs were designed.

VEGF-R2 construct for assay:this construct measures the ability of the test compound to inhibit tyrosine kinase activity. A construct (VEGF-R2A50) that expresses the cytoplasmic domain of human vascular endothelial growth factor receptor 2(VEGF-R2) lacking the 50 central residues of the 68 residues of the kinase insertion domain in a baculovirus/insect cell system. Among the 1356 residues of full-length VEGF-R2, VEGF-R2A50 contained residues 806-939 and 990-1171 and also contained a point mutation within the kinase insertion domain compared to the wild-type VEGF-R2 (E990V). By mixing 3mM ATP and 40mM MgCl at 4 deg.C₂The purified construct was autophosphorylated by incubating the enzyme at a concentration of 4uM for 2 hours in 100mM HEPES pH7.5 containing 5% glycerol and 5mM DTT in the presence of. After autophosphorylation, the construct has been demonstrated to have substantially equivalent catalytic activity to the wild-type autophosphorylation kinase domain construct. See Parast et al, Biochemistry, 37, 16788-.

FGF-R1 constructs were used:the intracellular kinase domain of human FGF-R1 was expressed using a baculovirus vector expression system starting from the endogenous methionine residue at position 456 to the glutamic acid residue at position 766 according to the residue numbering system described by Mohammadi et al, mol.cell.biol, 16, 977-. In addition, the construct also contains the following 3 amino acid substitutions: L457V, C488A, and C584S.

LCK constructs for assay:expressing in insect cells an LCK tyrosine kinase as an N-terminal deletion starting at amino acid residue 223 and ending at a protein at residue 509, wherein at the N-terminus there are two amino acid substitutions: P233M and C224D.

CHK1 construct for assay:the C-terminal His-tagged full-length human CHK1(FL-CHK1) was expressed using a baculovirus/insect cell system. It contains 6 histidine residues (6 × His-tag) at the C-terminus of 476 amino acids human CHK 1. The protein is purified by conventional chromatographic techniques.

Assay with CDK 2/cyclin a construct:CDK2 was purified from insect cells that had been infected with baculovirus expression vectors using published methods (Rosenblatt et al J.MoL biol., 230, 1317-1319 (1993)). Cell cycle regulatory protein A was purified from E.coli cells expressing full-length recombinant cell cycle regulatory protein A and truncated cell cycle regulatory protein A constructs were generated by restriction proteolysis and purified as described above (Jeffrey et al Nature, 376, 313-320 (1995)).

Assay with CDK 4/cyclin a construct:complexes of human CDK4 and cell cycle regulatory protein D3 or complexes of cell cycle regulatory protein D1 and fusion proteins of human CDK4 and glutathione-S-transferase (GST-CDK4) were purified from insect cells that had been co-infected with the corresponding baculovirus expression vectors using conventional biochemical chromatography techniques.

FAK constructs for testingThe catalytic domain of human FAK (FAKcd409) was expressed using a baculovirus vector expression system. The 280 amino acid domain expressed includes methionine at position 409 to glutamic acid residue 689. There is an amino acid substitution (P410T) compared to sequence accession number L13616 as disclosed in Whithey, G.S. et al, DNA Cell Biol, 9, 823-30 (1993). The protein is purified using conventional chromatographic techniques.

TIE-2(TEK) constructs for testing

The N-terminal deleted TIE-2 tyrosine kinase domain is expressed in insect cells as a protein starting at amino acid residue 774 and ending at residue 1124. The construct also carries a R774M mutation that serves as a translation initiating methionine residue.

VEGF-R2 assay

Combined spectrophotometric (FLVK-P) assay

ADP production from ATP with phosphoryl transfer Association with phosphoenolpyruvate (PEP) and with alanineNADH oxidation of ketoacid kinase (PK) and Lactate Dehydrogenase (LDH) systems. The oxidation of NADH was monitored by absorbance decrease at 340nm (. epsilon.340 ═ 6.22 cm) using a Beckman DU650 spectrophotometer as follows^-1mM^-1). The assay conditions for phosphorylated VEGF-R2A50 (indicated as FLVK-P in the following table) were as follows: 1mM PEP; 250 μ M NADH; 50 units LDH/mL; 20 units of PK/mL; 5mM DTT; 5.1mM poly (E)₄Y₁) (ii) a 1mM ATP; and 25mM MgCl₂In 200mM HEPES, pH 7.5. The assay conditions for unphosphorylated VEGF-R2A50 (indicated as FLVK in the following table) were as follows: 1mM PEP; 250uM NADH; 50 units LDH/mL; 20 units of PK/mL; 5mM DTT; 20mM poly (E)₄Y₁) (ii) a 3mM ATP; and 60mM MgCl₂And 2mM MnCl₂In 200mM HEPES, pH 7.5. The assay was initiated using 5-40nM enzyme. Determination of K by measuring enzyme Activity at different test Compound concentrations_IThe value is obtained. Data were analyzed using Enzyme Kinetic and Kaleidagraph software.

ELISA assay

The formation of gastrin phosphate was monitored using biotinylated gastrin peptides (1-17) as substrates. Biotinylated gastrin phosphate was immobilized using streptavidin-coated 96-well microtiter plates followed by detection using anti-phosphotyrosine-antibody conjugated to horseradish peroxidase. Using 2, 2' -azino-bis- [ 3-ethylbenzothiazoline sulfonic acid (6)]Horseradish peroxidase activity was monitored by diammonium salt (ABTS). A typical assay solution contains: 2 μ M biotinylated gastrin peptide; 5mM DTT; 20uM ATP; 26mM MgCl₂(ii) a And 2mM MnCl₂In 200mM HEPES, pH 7.5. The assay was initiated using 0.8nM phosphorylated VEGF-R2A 50. Horseradish peroxidase activity was detected using 10mM ABTS. By addition of acid (H)₂SO₄) The horseradish peroxidase reaction was stopped and absorbance was then read at 405 nm. K determination by measuring enzyme activity in the presence of different concentrations of test compound_IThe value is obtained. Data were analyzed using Enzyme Kinetic and Kaleidagraph software.

FGF-R assay

Spectrophotometric measurements were performed as described above for VEGF-R2, except that the concentrations were varied as follows: FGF-R50 nM, ATP 2mM, and poly (E)₄Y₁)＝15mM。

LCK test

Spectrophotometric measurements were performed as described above for VEGF-R2, except that the concentrations were varied as follows: LCK 60nM, MgCl₂0mM poly (E)₄Y₁)＝20mM。

CHK1 test

Generation of ADP from ATP, which accompanies the transfer of a phosphoryl group to the synthetic substrate peptide synthetic-2 (PLARTLSVAGLPGKK), is correlated with the oxidation of NADH by Pyruvate Kinase (PK) and Lactate Dehydrogenase (LDH) using phosphoenol pyruvate (PEP). The oxidation of NADH was monitored by absorbance decrease at 340nm (. epsilon.40 ═ 6.22 cm) using an HP8452 spectrophotometer^-1mM^-1). A typical reaction solution contains: 4mN PEP; 0.15mM NADH; LDH/mL of 28 units; 16 units of PK/mL; 3mM DTT; 0.125mM Syntide-2; 0.15mM ATP; 25mM MgCl₂pH7.5 in 50mM TRIS; and 400mM NaCl. The assay was initiated using 10nM FL-CHK 1. Determination of K by measuring enzyme Activity at different test Compound concentrations_IThe value is obtained. Data were analyzed using Enzyme Kinetic and Kaleidagraph software.

CDK 2/cyclin A and CDK 4/cyclin D assays

By subjecting a DNA fragment derived from [2 ]³²P]The time-dependent entry of the enzyme-catalyzed fragment of the radioactive phosphate of ATP into the recombinant fragment of the retinoblastoma protein was quantified to determine cyclin-dependent kinase activity. Unless otherwise stated, the assay was carried out in the presence of 10mM HEPES (N- [ 2-hydroxyethyl ]]piperazine-N' - [ 2-ethanesulfonic acid])(pH7.4)、10mM MgCl₂25 μ M Adenosine Triphosphate (ATP), 1mg/mL ovalbumin, 5 μ g/mL leupeptin, 1mM dithiothreitol, 10mM beta-phosphoglycerol, 0.1mM sodium vanadate, 1mM sodium fluoride, sodium chloride, and sodium chloride,2.5mM ethylene glycol-bis (p-aminoethylether) -N, N, N 'N' -tetraacetic acid (EGTA), 2% (v/v) dimethyl sulfoxide and 0.03-0.2. mu. Ci [, [ solution ] ]³²P]In 96-well plates with a total volume of 50. mu.L in the presence of ATP. The substrate (0.3-0.5. mu.g) was purified recombinant retinoblastoma protein fragment (Rb) (residues 386-928 of native retinoblastoma protein; 62.3kDa, containing most of the phosphorylation sites found in the native 106-kDa protein and 6 histidine residues at the tail, which are easy to purify). Reactions were initiated with CDK2(150nM CDK 2/cyclin a complex) or CDK4(50nM CDK 4/cyclin D3 complex), incubated at 30 ℃ and stopped after 20 minutes (min.) by addition of ethylenediaminetetraacetic acid (EDTA) to 250 mM. The phosphorylated substrate was then captured on a nitrocellulose membrane using a 96-well filtration manifold and radioactivity bound was removed by washing with 0.85% phosphoric acid back. Radioactivity was quantified by contacting the dried nitrocellulose membrane with a phosphorus imager. Determination of apparent K by measuring enzyme activity in the presence of different compound concentrations and subtracting background radioactivity measured in the absence of enzyme_IThe value is obtained. Under the usual detection conditions, kinetic parameters (kcat, Km of ATP) were determined for each enzyme by determining the dependence of the initiation rate on ATP concentration. Data were fit to the formula for competitive inhibition using kaleidagraph (synergy software) or to the formula for competitive tight binding inhibition using software KineTic (BioKin, Ltd.). K of known inhibitors assayed against CDK4 and CDK2_IValue and published IC₅₀The values are consistent. The specific activity of CDK4 was the same as if the constructs were complexed with full-length cyclin D3 or truncated cyclin D3; both complexes also produced Ks that were very similar for the selected inhibitors_IThe value is obtained.

FAK test

FAK HTS uses fluorescence polarization assays supplied by LJL Biosystems. The kinase solution contains: 100Hepes pH7.5, 10mM MgCl₂1mM DTT, 1mM ATP, and 1mg/ml polyGlu-Tyr (4: 1). The reaction was initiated by the addition of 5nM FAKcd 409. The reaction was stopped by addition of EDTA, followed by addition of allFluorescently labeled peptides and anti-phosphotyrosine antibodies supplied by LJL Biosystems. The suppression results were read on the analysis (ljl) detector.

TIE-2 metric photometric test

The catalytic generation of ADP by ATP kinase with phosphoryl transfer to the random copolymer poly (GIu4Tyr) is linked to the oxidation of NADH by the activity of Pyruvate Kinase (PK) and Lactate Dehydrogenase (LDH). Conversion of NADH to NAD was monitored by absorbance drop at 340nm using a Beckman DU650 spectrophotometer⁺(ε40＝6.22cm^-1mM^-1). A typical reaction solution contains: 1mM phosphoenolpyruvate, 0.24mM NADH, 40mM MgCl₂5mM DTT, 2.9mg/mL poly (GIu4Tyr), 0.5mM ATP, 15 units/mL PK, 15 units/mL LDH in 100mM HEPES, pH 7.5. The assay was initiated by the addition of 4-12nM phosphorylated Tie-2(aa 775-. Percent inhibition was determined in triplicate at 1uM inhibitor concentration.

TIE-2 DELFIA assay

Phosphotyrosine formation was monitored using biotinylated p34cdc2(aa6-20 ═ KVEKIGEGTYGVVYK) peptide as substrate. Using NeutrAvidin^TMThe biotinylated peptide was immobilized by a coated 96-well microtiter plate followed by detection using an anti-phosphotyrosine-antibody (PY20) conjugated with europium N1 chelate. A typical assay solution contains: mu.M biotinylated p34cdc2 peptide, 150. mu.M ATP, 5mM MgCl₂1mM DTT, 0.01% BSA, 5% glycerol, 2% DMSO, 25mM HEPES pH7.5. The assay was initiated in NeutrAvidin plates using 50nM of the TIE2 intracellular domain. The kinase reaction was stopped using 50mM EDTA. The plates were then washed and europium antibody was added. After incubation, they were washed again and DELFIA was added^TMThe solution is fortified. Plates were read at standard europium time resolution setting (ex 340nm, em 615nm, slow 400 μ sec, window 400 μ sec). Percent inhibition was calculated based on wells in the plate to which DMSO was added instead of compound in DMSO, where background was subtracted from experimental and control based on wells in the plate to which EDTA was added prior to enzyme addition.

HUVEC proliferation assay

This assay measures the ability of a test compound to inhibit growth factor-stimulated human umbilical vein endothelial cell ("HUVEC") proliferation. HUVEC cells (passage 3-4, Clonetics, Corp.) were thawed into EGM2 medium (Clonetics Corp) in T75 flasks. Fresh EGM2 medium was added to the flask after 24 hours. After 4 or 5 days, the cells were re-contacted with medium (F12K medium supplemented with 10% Fetal Bovine Serum (FBS), 60lg/mL Endothelial Cell Growth Supplement (ECGS), and 0.1mg/mL heparin). Exponentially growing HUVEC cells were used for the experiments hereafter. 1 million to 1 million 2 thousand HUVEC cell plates were fixed in 100. mu.l rich medium (above) on 96-well plates. Cells were allowed to attach in this medium for 24 hours. The medium was then removed by aspiration and 105 μ l of starvation medium (F12K + 1% FBS) was added to each well. After 24 hours, 15 μ l of test active or this separate vehicle dissolved in 1% DMSO in starvation medium was added to each treatment well; the final DMSO concentration was 0.1%. After 1 hour, 30 μ l of VEGF in starvation medium (30ng/mL) was added to all wells, but not to wells containing untreated controls; the final VEGF concentration was 6 ng/mL. Cell proliferation was quantified after 72 hours based on the reduction of MTT dye, at which time the cells were exposed to MTT (Promega Corp.) for 4 hours. The dye reduction was stopped by addition of stop solution (Promega Corp.) and the absorbance at 595 λ was determined on a 96-well spectrophotometer plate reader.

By A⁵⁹⁵Curve-fitting calculation of response to different concentrations of test active agent IC₅₀A value; in general, 7 concentrations at 0.5log intervals were used, with triplicate wells for each concentration. To screen compound library plates, one or two concentrations (one well per concentration) were used and% inhibition was calculated by the following formula:

inhibition = (control-test) ÷ (control-starvation), where:

control ═ a when VEGF was present and no active agent tested⁵⁹⁵；

Assay ═ a when VEGF is present and the test active agent is present⁵⁹⁵；

Starvation of A when neither VEGF nor test active is present⁵⁹⁵。

Mouse PK assay

The following experiments were used to analyze the pharmacokinetic properties (e.g., absorption and elimination) of the drug. Test compounds were formulated at 30: 70(PEG 400: acidified H)₂O) solution or suspension in a vehicle or suspension in 0.5% CMC. It was administered orally (p.o.) intraperitoneally (i.p.) at variable doses to two different groups (n-4) of B6 female mice. Blood samples were collected by orbital bleeding at the following time points post-dose: 0 hours (predose), 0.5 hours, 1.0 hour, 2.0 hours and 4.0 hours, and 7.0 hours. Plasma was obtained from each sample by centrifugation at 2500rpm for 5 minutes. Test compounds were extracted from plasma by organic protein precipitation. For each blood draw, 50 μ Ι _ of plasma was mixed with 1.0mL of acetonitrile, vortexed for 2 minutes and then spun at 4000rpm for 15 minutes to precipitate proteins and extract test compounds. The acetonitrile supernatant (extract containing the test compound) was then poured into a new tube and plated on a hot plate (25 ℃) with N₂Evaporation in the vapour stream. To each tube containing the dried test compound extract was added 125. mu.L of mobile phase (60: 40, 0.025M NH)₄H₂PO₄+2.5mL/L TEA: acetonitrile). The test compound was resuspended in the mobile phase by vortexing and more protein was removed by centrifugation at 4000rpm for 5 minutes. Each sample was poured into an HPLC vial for test compound analysis using Hewlett Packard 1100 series HPLC with UV detection. For each sample, 95 □ L was injected into a Phenomenex-Prodigy reversed phase C-18, 150X 3.2mm column and eluted with a 45-50% acetonitrile gradient over 10 minutes. The test compound plasma concentration (μ g/mL) (peak area-concentration μ g/mL) was determined by comparison with a standard curve using known test compound concentrations extracted from plasma samples according to the method described above. Three groups (n-4) were performed in addition to standard and unknownQuality control (0.25. mu.g/mL, 1.5. mu.g/mL, and 7.5. mu.g/mL) was performed to ensure analytical consistency. The standard curve has R2 > 0.99 and the quality control is all within 10% of its expected value. Quantitative test samples were plotted using Kalidagraph software for visual observation and pharmacokinetic parameters were determined using WIN nlin software. Example 1(a) provides the following results: 0.69 (mouse pK, AUC, ip, Rg-h/ml); 0.33 (mouse pK, AUC, po, lg-h/ml).

Phosphorylation of KDR (VEGFR2) in PAE-KDR cell assay

This assay measures the ability of test compounds to inhibit KDR autophosphorylation in Porcine Aortic Endothelial (PAE) -KDR cells. PAE cells overexpressing human KDR were used in this assay. Cells were cultured in Ham's F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 400ug/mL G418. 3 ten thousand cells were seeded into 75 μ L of growth medium in each well of a 96-well plate and allowed to attach for 6 hours at 37 ℃. The cells were then exposed to starvation medium (Ham's F12 medium supplemented with 0.1% FBS) for 16 hours. After the starvation period, 10 μ L of test active in 5% DMSO in starvation medium was added to the test wells and 10 μ L of vehicle (5% DMSO in starvation medium) was added to the control wells. The final DMSO concentration in each well was 0.5%. The plates were incubated at 37. mu.C for 1 hour and then in the presence of 2mM Na₃VO₄VEGF (purchased from R) 500ng/ml was used in the presence&D System) cells were stimulated for 8 minutes. With 1mm of Na in HBSS₃VO₄Cells were washed 1 time and lysed by adding 50 μ Ι _ of lysis buffer per well. 100ul of dilution buffer was then added to each well and the diluted cell lysates transferred to 96-well goat anti-rabbit coated plates (from Pierce) pre-coated with rabbit anti-human anti-flk-1C-20 antibody (from Santa Cruz). The plates were incubated at room temperature for 2 hours and washed 7 times with 1% Tween 20 in PBS. HRP-PY20 (from Santa Cruz) was diluted and added to the plate for 30 min incubation. The plates were then washed again and TMB peroxidase substrate (purchased from Kirkegaard) was added&Perry) for 10-minute incubation. To each well of a 96-well plate, 100. mu.L of 0.09N H was added₂SO₄To terminate the reaction. Phosphorylation status was assessed by reading the data spectrophotometrically at 450 nm. Calculation of IC by Curve fitting Using four parameter analysis₅₀The value is obtained.

PAE-PDGFR beta phosphorylation in PAE-PDGFR beta cell assays

This assay measures the ability of test compounds to inhibit PDGFR β autophosphorylation in Porcine Aortic Endothelial (PAE) -PDGFR β cells. PAE cells overexpressing human PDGFR β were used in this experiment. Cells were cultured in Ham's F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 400ug/mL G418. 2 ten thousand cells were seeded into 50. mu.L of growth medium in each well of a 96-well plate and allowed to attach for 6 hours at 37 ℃. The cells were then exposed to starvation medium (Ham's F12 medium supplemented with 0.1% FBS) for 16 hours. After the starvation period, 10 μ L of test active in 5% DMSO in starvation medium was added to the test wells and 10 μ L of vehicle (5% DMSO in starvation medium) was added to the control wells. The final DMSO concentration in each well was 0.5%. The plates were incubated at 37. mu.C for 1 hour and then in the presence of 2mM Na₃VO₄In the presence of 1ug/mL PDGF-BB (R)&D System) cells were stimulated for 8 minutes. With 1mm of Na in HBSS₃VO₄Cells were washed 1 time and lysed by adding 50 μ Ι _ of lysis buffer per well. 100ul of dilution buffer was then added to each well and the diluted cell lysates were transferred to 96-well goat anti-rabbit coated plates (Pierce) pre-coated with rabbit anti-human PDGFR β antibody (Santa Cruz). The plates were incubated at room temperature for 2 hours and washed 7 times with 1% Tween 20 in PBS. HRP-PY20(Santa Cruz) was diluted and added to the plate for 30 min incubation. The plates were then washed again and TMB peroxidase substrate (Kirkegaard) was added&Perry) for 10-minute incubation. To each well of a 96-well plate, 100. mu.L of 0.09N H was added₂SO₄To terminate the reaction. Phosphorylation status was assessed by reading the data spectrophotometrically at 450 nm. Calculation of IC by Curve fitting Using four parameter analysis₅₀The value is obtained.

Human liver microsome (H)LM) test

Compound metabolism in human liver microsomes was determined by a chemical LC-MS assay method as follows. Human Liver Microsomes (HLM) were first thawed and treated with cold 100mM potassium phosphate (KPO)₄) The buffer was diluted to 5 mg/mL. Adding appropriate amount of KPO at 37 deg.C₄Buffer, NADPH-regenerating solution (containing B-NADP, glucose-6-phosphate dehydrogenase and MgCl)₂) And HLM pre-incubated for 10 minutes in 13 x 100mm glass tubes (3 tubes per test compound-in triplicate). Test compounds (final concentration 5 □ M) were added to each tube to start the reaction and mixed by slow vortexing, followed by incubation at 37 ℃. A250-uL sample was taken from each incubation tube at t 0, 2 hours to isolate a 12X 75mm glass tube containing 1mL of ice-cold acetonitrile and 0.05uM serpentine base. The samples were centrifuged at 4000rpm for 20 minutes to precipitate the proteins and salts (Beckman Allegra 6KR, S/NALK98D06# 634). The supernatant was transferred to a new 12X 75mm glass tube and evaporated by a Speed-Vac vacuum centrifugal evaporator. The sample was redissolved in 200uL of 0.1% formic acid/acetonitrile (90/10) and vortexed vigorously to dissolve. The samples were then transferred to polypropylene microcentrifuge tubes for separation and centrifuged at 14000Xg for 10 minutes (Fisher micro 14, S/N M0017580). For each replicate (#1-3) at each time point (0 and 2 hours), an aliquot of each test compound was combined into a single HPLC vial (6 samples in total) for LC-MS analysis as described below.

The combined compound samples were injected into a LC-MS system consisting of a Hewlett-packard hp1100 diode array HPLC and a MicromassQuattroII triple quadrupole mass spectrometer operating in the anodised SIR mode (a program was designed to scan specifically for the molecular ion of each test compound. the peak of each test compound was integrated at each time point. for each compound, the peak area at each time point (n-3) was averaged and the average peak area at 2 hours was divided by the average peak area at 0 hours to give the percentage of test compound remaining at 2 hours.

The results of testing the compounds using different assays are summarized in the table belowWherein the designation "% @" indicates the percentage of inhibition at that concentration, unless otherwise indicated; ", a^*"values are expressed at 1. mu.M^*Or 50nM^**Ki (nM) or% inhibition at compound concentration. "NT" means that there is no significant inhibition or no test.

TABLE 1

Example #	FLVK Ki％ Inhibition of @50 nM	FLVK-P**	LckP*% inhibition @ 1. mu.M	FGF-P% inhibition @ 1. mu.M	HUVECIC50(nM)	HUVEC + albumin IC50(nM)	% remaining (HLM)	PAEPGFR autophosphorylation IC50(nM)	PAEKDRIC50nMAVG	bFGFHuvecIC50(nM)AVG
											3(a)	98	NT	30	99	12.7	NT	NT	NT	NT	NT
3(b)	98	NT	27	96	57	NT	84@2h	NT	NT	NT
											3(c)	91	NT	9	83	0.43	9.2	46@0.5h	NT	NT	NT
3(d)	89	NT	11	80	0.4	7.5	68@2h	3.5	NT	147
											3(f)3(g)	9595	NTNT	4128	6072	NT1.1	＞100NT	NT72@0.5h	NTNT	NTNT	NTNT
3(h)	96	NT	37	85	1.6	NT	75@0.5h	0.63	NT	NT
											3(i)	88	NT	22	45	0.2	NT	NT	1.9	NT	1000
3(j)	80	NT	17	43	1.7	NT	65@0.5h	4.7	NT	NT
											3(k)	74	NT	19	36	0.8	NT	75@0.5h	5	NT	1000
3(q)	47	NT	7	31	5	NT	82@0.5h	5.2	NT	NT
											2(h)	84	NT	NT	75	1.6	NT	74@0.5h	2.8	NT	70
1(k)	27	NT	NT	12	＞10	NT	NT	NT	NT	NT
											2(g)	83	NT	NT	79	0.71	NT	85@0.5h	10.5	NT	173
64	94	NT	NT	39	0.15	NT	66@0.5h	5.5	NT	1250
											65	3.11nM	NT	NT	NT	3.4	NT	86@0.5h	5.8	NT	NT
61	65	NT	NT	14	6.5	NT	NT	NT	NT	662
											41	45	NT	NT	11	6.4	NT	NT	NT	NT	3775
51	82	NT	NT	52	NT	NT	NT	NT	NT	NT
											15	64	NT	NT	29	1.5	NT	NT	12.3	NT	1613
36	95	0.3nM	NT	69	1.67	NT	NT	NT	1.62	935

TABLE 1 continuation
											Example #	FLVK Ki％ Inhibition of @50 nM	FLVK-P**	LckP*% inhibition @1 □ M	FGF-P% inhibition @1 □ M	HUVECIC50(nM)	HUVEC + albumin IC50(nM)	% remaining (HLM)	PAEPGFR autophosphorylation IC50(nM)	PAEKDRIC50nMAVG	bFGFHuvecIC50(nM)AVG
13	80	NT	NT	63	NT	NT	NT	6	NT	NT
											18	94	NT	NT	59％	NT	NT	NT	NT	NT	1882
20	91	NT	NT	35％	0.084	NT	NT	NT	NT	NT
											37	90	NT	NT	45	NT	NT	NT	NT	0.76	NT
38	75	NT	NT	NT	NT	0.68	NT	2	NT	NT
											39	96	NT	NT	76％	NT	NT	NT	4.7	NT	NT
32	78	NT	NT	70％	0.61	NT	97@0.5h	0.5	NT	NT
											55	97	NT	NT	67％	0.2	NT	NT	3.7	NT	NT
57	91	NT	NT	52％	＜1.8	NT	NT	1.3	NT	NT
											63	85	NT	NT	63％	0.1	NT	NT	2.4	NT	NT
34	72	NT	NT	NT	NT	NT	NT	4.5	NT	NT
											10	76	6.07	NT	38,197nM	0.67	NT	80@0.5h	21	NT	NT
45	28	NT	NT	24	NT	NT	NT	NT	NT	NT
											49	11	NT	NT	36	NT	NT	NT	NT	NT	NT
23	23	NT	NT	56	NT	NT	NT	40	NT	NT
											25	64	NT	NT	13	3	NT	NT	NT	NT	NT

In vivo assay for retinal vessel development in newborn rabbits

Rabbit retinal vascular development occurs from postnatal day 1 to postnatal day 14 (P1-P14). This process is dependent on VEGF activity (j.stone et al j.neurosci., 15, 4738 (1995)). The above situation demonstrates that VEGF also acts as a retinal vascular survival factor during early vascular development (Alon et al nat. med., 1, 1024 (1995)). To assess the ability of a particular compound to inhibit VEGF activity in vivo, the compound is formulated with a suitable carrier, typically 50% polyethylene glycol having an average molecular weight of 400 daltons and 50% 300mM sucrose in deionized water. Generally, 2 microliters (2 μ l) of the drug solution is injected into the middle vitreous of the eye of a rabbit pup on day 8 or 9 after birth. On day 6 after intravitreal injection, animals were sacrificed and the retina was stripped from the remaining ocular tissue. The isolated retinas were then subjected to a histochemical staining protocol (Lutty and McLeod, arch. ophthalmol., 110, 267(1992)) which specifically stains epithelial cells, showing the extent of vascularization in the tissue samples. Each retina was then mounted flat on a slide and examined to determine the extent of vascularization. The effective compounds inhibit further development of the retinal vasculature and induce nearly maximal vascular degeneration within the retina. The amount of vascular regression was used to assess the relative efficacy of the compounds after in vivo administration. Classifying the vascular degeneration into a subjective scale of 1-3+, wherein 1+ is detectably determined about 25% or less than 25% degeneration; 2+ is judged to be about 25-75% degenerated, while 3+ causes nearly complete degeneration of the intraretinal vasculature (about 75% or more than 75%).

To analyze the degeneration more quantitatively, an ADPase-stained, flat fixed retinal image was captured with a digital camera coupled to a dissecting microscope. The retinal images were then input into Image analysis software (Image Pro Plus 4.0, Media Cybernetics, Silver Spring, MD). The software was used to determine the percentage of retinal area containing stained blood vessels. This value for the experimental eye was compared to the value measured in the contralateral eye from the same animal injected with the vehicle. The reduction in the area of blood vessels observed in the eye receiving the compound compared to the eye injected with vehicle was then expressed as a "percent regression" of the sample. The percent regression was averaged for groups of 5-8 animals.

In samples that show nearly total degradation by microscopic observation, a percent degradation value of 65-70% is typically determined. This is due to the presence of pigmented deposits within the retinal folds induced by the vehicle used for drug injection. The image analysis software interprets these pigmented folds as blood vessels. No calibration attempts are made for these folds as they change from eye to eye. Thus, it should be noted that the percent degradation values are reported as results from retention measurements that rank the compounds precisely, underestimating their absolute efficacy.

In vivo testing of retinal vessel development in a neonatal rabbit model of retinopathy of prematurity

A second VEGF-dependent retinal neovascularization model was used to evaluate the activity of the series of compounds. In this model (Penn et al, invest, ophthalmol. vis. sci., 36, 2063, (1995)), rabbit pups (n ═ 16) were placed in a computer-controlled oxygen concentration adjustable room along with their mother rabbits. Animals were exposed to 50% oxygen for 24 hours followed by 10% oxygen for 24 hours. This alternating cycle of hyperoxia and hypooxia was repeated 7 times, after which the animals were taken out of contact with room air (P14). The compounds were administered by intravitreal injection when removed from contact with room air and the animals were sacrificed after 6 days (P20). The retinas were then isolated, fixed by staining and analyzed in a developmental model as described above. Effectiveness was also graded as described for the developmental models.

The above typical compounds were formulated into the pharmaceutical compositions of the following general examples.

Example 1: parenteral composition

To prepare a parenteral pharmaceutical composition suitable for administration by injection, 100mg of a water-soluble salt of a compound of formula I is dissolved in DMSO and then mixed with 10mL of 0.9% sterile saline. The mixture is formulated into unit dosage forms suitable for administration by injection.

Example 2: oral composition

To prepare a pharmaceutical composition for oral delivery, 100mg of a compound of formula I is mixed with 750mg lactose. The mixture is formulated into oral dosage units suitable for oral administration, such as hard gelatin capsules.

Example 3: intraocular composition

To prepare a sustained release pharmaceutical composition for intraocular delivery, a compound of formula I is suspended in a neutral isotonic solution of hyaluronic acid (concentration 1.5%) in phosphate buffer (ph7.4) to form a 1% suspension.

It is to be understood that the above description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. It will be apparent to those skilled in the art that modifications and variations can be made in this embodiment by routine experimentation without departing from the spirit of the invention. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the following claims and their equivalents.

Claims (15)

1. A compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt selected from the group consisting of:

2. a compound represented by the general formula or a pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt thereof:

3. a method of treating age-related macular degeneration in a mammal in need thereof with a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

4. A method of treating choroidal neovascularization in a mammal using a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

5. A method of treating retinopathy in a mammal using a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

6. The method of claim 5, wherein the retinopathy comprises diabetic retinopathy, vitreoretinopathy or retinopathy of prematurity.

7. A method of treating retinitis in a mammal using a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

8. The method of claim 7, wherein the retinitis comprises cytomegalovirus retinitis.

9. A method of treating macular edema in a mammal with a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

10. A method of treating an ocular condition in a mammal using a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

11. A pharmaceutical composition comprising:

(a) a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt of claim 1 or 2; and

(b) a pharmaceutically acceptable carrier, diluent or excipient.

12. A method of treating a mammalian disease mediated by protein kinase activity with a therapeutically effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt as defined in claim 1 or 2.

13. The method of claim 12, wherein the mammalian disease is associated with tumor growth, cell proliferation, or angiogenesis.

14. A method of modulating the activity of a protein kinase receptor comprising contacting said kinase receptor with an effective amount of a compound, pharmaceutically acceptable prodrug, pharmaceutically active metabolite or pharmaceutically acceptable salt as defined in claim 1 or 2.

15. The method of claim 14, wherein the protein kinase receptor is a VEGF receptor.