Substituted 2,10-dihydro-9-oxa-1,2,4A-triazaphenanthren-3-ones and uses thereof

The invention provides a compound of Formula (I)pharmaceutically acceptable salts, pro-drugs, biologically active metabolites, stereoisomers and isomers thereof wherein the variable are defined herein. The compounds of the invention are useful for treating immunological and oncological conditions.

BACKGROUND OF THE INVENTION

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of the PKC, Jak1, Jak2, Jak3, Tyk2, KDR, Flt-3, ROCK, CDK2, CDK4, TANK, Trk, FAK, Abl, Bcr-Abl, cMet, b-RAF, FGFR3, c-kit, PDGF-R, Syk, or Aurora kinases.

Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within cells (see, e.g., Hardie and Hanks,The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif., 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 25-300 amino acid catalytic domain. The kinases may be categorized into families by the substrate that they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids etc.). Sequence motifs have been identified that generally correspond to each of these families (see e.g., Hanks and Hunter, (1995),FASEB J.9:576-596; Knighton et al., (1991),Science253:407-414; Hiles et al., (1992),Cell70:419-429; Kunz et al., (1993),Cell73:585-596; Garcia-Bustos et al., (1994),EMBO J.13:2352-2361).

The protein kinase C family is a group of serine/threonine kinases including at least ten related isoenzymes, including alpha, beta 1, beta 2, gamma, delta, epsilon, eta, lambda, iota, theta and zeta. The isoenzymes have been divided into three groups based on their different expression patterns and co-factor requirements. The classical PKC enzymes (cPKC), including alpha, beta 1, beta 2 and gamma isozymes require diacylglycerol (DAG), phosphatidylserine (PS) and calcium for activation. The novel PKC's (nPKC), including delta, epsilon, theta and eta isozymes, require DAG and PS but are calcium independent. The atypical PKC's (aPKC), including zeta, lambda/iota do not require calcium or DAG.

PKC isoforms have been shown to play key roles in cellular signaling, proliferation, differentiation, migration, survival, and death. In resting cells, PKCs are predominantly localized in the cytosol and are catalytically inactive due to autoinhibition by their pseudosubstrate domain. Upon cell activation, PKC isotype-specific signals trigger translocation from the cytosol to the membrane and induce conformational changes, which displace the pseudosubstrate moiety from the catalytic domain and enable PKC isotypes to phosphorylate specific protein substrates (Biochem. J.370:361-371, 2003). Most isoforms are ubiquitously expressed, except PKCγ and PKCθ. While PKCγ is exclusively found in the brain, high protein levels of PKCθ are seen predominantly in hematopoietic cells and skeletal muscle. PKCα and PKCθ as well as PKCβ and PKCδ are functionally important for T and B cell signaling, respectively (Nat. Immunol.5:785-790, 2004. Curr. Opin. Immunol.16:367-373, 204. Nature.416:860-865, 2002). PKCθ plays an essential role in T cell activation because it is the only isoform that is selectively translocated to the T cell/antigen-presenting cell contact site immediately after cell-cell interaction (Nature.385:83-86, 1997). Furthermore, PKCθ is crucial for IL-2 production, a prerequisite for the proliferation of T cells (Eur. J. Immunol.30:3645-3654, 2000). PKCθ-deficient mice are defective in NF-κB (Cell Mol. Immunol.3:263-270, 2006), NFAT and AP-1 activation (Nature,404 (96776), 402-407, 2000. Journal of Immunology176:6004-6011, 2006) and are resistant to experimental autoimmune encephalomyelitis (J. Immunol.176:2872-2879, 2006), collagen-induced arthritis (Journal of Immunology177 (3), 1886-1893, 2006) and asthma (Journal of Immunology173 (10), 6440-6447, 2004). PKCα in T cells is required for proliferation and IFN-γ production (J. Immunol.176:6004-6011, 2006). B cells require PKCβ for proper antigen receptor function and PKCδ for the induction of tolerance (Nature.416:860-865, 2002). Thus, PKC isoforms in T and B cells are considered attractive therapeutic targets for autoimmune diseases and transplantation (Curr. Opin. Investig. Drugs.7:432-437, 2006).

Further, PKCε and PKCγ have been suggested to play a role in nociception and inflammatory pain (J. Pharm. Exp. Ther. Pain110, 281-289, 2004) and PKCζ has been proposed as an intermediary in the activation of the NF-κB and IL-4/Stat6 pathway (Cell Death Differ.13: 702, 2006). The NF-κB pathway is important for inflammatory and immune diseases, therefore a PKCζ inhibition may serve to reduce the severity of these type of diseases (Allergol. Int.55: 245, 2006. J. Biol. Chem.281: 24124, 2006. Arthritis Rheum.56: 4074, 2007. J. Interferon Cytokine Res.27: 622, 2007).

The novel compounds of this invention inhibit the activity of one or more protein kinases and are, therefore, expected to be useful in the treatment of kinase-mediated diseases.

SUMMARY OF THE INVENTION

In a first embodiment the invention provides a compound of Formula (I):

In a sixth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein:R1is —H, deuterium, halo, —CF3, —(C1-C3)alkyl, or —(C3-C6)cycloalkyl, wherein —(C1-C3)alkyl is optionally substituted with 1-3 groups selected from halo, hydroxyl, or —(C1-C3)alkoxy; and wherein —(C3-C6)cycloalkyl is optionally substituted with 1-3 groups selected from halo, hydroxyl, —(C1-C3)alkyl, or —(C1-C3)alkoxy;R4is —CF3, —CHF2, —CH2F, or —(C1-C6)alkyl optionally substituted with 1-3 groups selected from halo, hydroxyl, or —(C1-C3)alkoxy;Rais independently —H or —(C1-C3)alkyl;Rbis independently —H, phenyl optionally substituted with hydroxyl or —(C1-C3)alkyl, or 4-10 membered heterocyclyl; wherein the 4-10 membered heterocyclyl is optionally substituted with 1-3 groups independently selected from: halo, —(C1-C3)alkyl, -hydroxy(C1-C4)alkyl, -halo(C1-C3)alkyl, —(C1-C4)alkoxy(C1-C3)alkyl, —(C1-C3)alkoxy, -halo(C1-C3)alkoxy, —C(O)(C1-C3)alkyl, —C(O)halo(C1-C3)alkyl, —C(O)hydroxy(C1-C4)alkyl, —C(O)(C1-C3)alkyleneN(CH3)2, —(C1-C3)aralkyl, oxetanyl, —CH2-oxetanyl, tetrahydropyran, or —CH2-tetrahydropyran; and,Rcis independently —CH2-optionally substituted phenyl, optionally substituted phenyl, or 4-10 membered heterocyclyl optionally substituted with one or more (C1-C3)alkyl; wherein each phenyl is independently optionally substituted with —(C1-C3)alkyl.

In a seventh embodiment the invention provides a compound according to any of the foregoing embodiments, having the Formula (II):

a pharmaceutically acceptable salt, an isomer, a stereoisomer, a tautomer, a pro-drug, or a biologically active metabolite thereof.

In an eighth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein R3is represented by structural formula (i):

In a ninth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein the compound is of Formula (III) or (IV):

In a tenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein the compound is of Formula (IV), (V), or (VI):

In an eleventh embodiment the invention provides a compound according to any of the foregoing embodiments 10, wherein R5and R7are each independently —H or —(C1-C3)alkyl; R6is —(C1-C3)alkyl; and R8is —H, —CH3, or —CH2-aryl.

In a twelfth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein R3is represented by structural formula (II):

wherein:m is 1, 2, 3, or 4;Z is absent or —CH2-optionally substituted with —(C1-C3)alkyl; andeach substitutable ring atom of R3is optionally substituted by Rx, and each Rxis independently —H, halo, —CF3, —(C1-C3)alkyl, -hydroxy(C1-C4)alkyl, -halo(C1-C3)alkyl, —(C1-C4)alkoxy(C1-C3)alkyl, —C(O)(C1-C3)alkyl, —C(O)halo(C1-C3)alkyl, —C(O)hydroxy(C1-C4)alkyl, —C(O)—(C1-C3)alkylene-N(CH3)2, —(C1-C3)aralkyl, oxetanyl, —CH2-oxetanyl, tetrahydropyranyl, or —CH2-tetrahydropyranyl; orwhen Z is absent, two Rx, together with the atoms to which they are attached, form a ring fused to the heterocyclic ring to form an optionally substituted bicyclic ring.

In a thirteenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein the compound is of Formula (IV-1), (IV-2), (IV-3), or (IV-4):

In a fourteenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein R2is selected from —H; halo; —CF3; —CH3; ethyl; isopropyl; —CH(CH3)CF3; furanyl; cyclopentyl; cyclopropyl; bicyclo[1.1.1]pentyl, bicyclo[2.1.0]pentyl, benzothiazolyl; optionally substituted cyclohexenyl; optionally substituted naphthyl; optionally substituted tetrahydropyranyl, optionally substituted pyridinyl; or optionally substituted phenyl;wherein the cyclohexenyl is optionally substituted with halo;wherein the naphthyl is optionally substituted with one or more groups selected from halo, —(C1-C3)alkyl, or —(C1-C3)alkoxy; and,wherein the phenyl is optionally substituted with one or more groups independently selected from halo, —CF3, —CH3, —CN, —(C1-C4)alkyl, —(C1-C3)alkoxy, -halo(C1-C3)alkoxy, —(C1-C4)alkoxy(C1-C3)alkyl, -halo(C1-C3)alkyl, —N((C1-C3)alkyl)2, —S(O)CH3, and —S(O)2CH3.

In a fifteenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein R2and R3, together with the carbon atoms to which they are attached, form a 5-7 membered heterocyclyl or a 5-6 membered heteroaryl,wherein each substitutable nitrogen atom in the heterocyclyl and heteroaryl is optionally substituted with: optionally substituted (C1-C4)alkyl, —C(O)— optionally substituted (C1-C3)alkyl, optionally substituted 4-10 membered heterocyclyl, —(C1-C3)alkyl-(4-10 membered) heterocyclyl, or —(C1-C3)aralkyl, or 5-6 membered heteroaryl, andwherein each substitutable carbon atom in the heterocyclyl and heteroaryl is optionally substituted with one or more substituents independently selected from deuterium, halo, —CN, —OH, —NRaRb, —ORc, optionally substituted —(C1-C4)alkyl, —C(O)-optionally substituted (C1-C3)alkyl, —C(O)hydroxy, optionally substituted 4-10 membered heterocyclyl, —(C1-C3)alkyl-(4-10 membered) heterocyclyl, or —(C1-C3)aralkyl.

In a sixteenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein R2and R3, together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl or 5-6 membered heteroaryl,wherein each substitutable nitrogen atom in the heterocyclyl and heteroaryl is optionally substituted with —(C1-C3)alkyl, -hydroxy(C2-C4)alkyl, -halo(C1-C3)alkyl, —(C2-C4)alkoxy(C1-C3)alkyl, —C(O)(C1-C3)alkyl, —C(O)halo(C1-C3)alkyl, —C(O)hydroxy(C1-C4)alkyl, —C(O)(C1-C3)alkylene-N(CH3)2, optionally substituted azetidinyl, oxetanyl, optionally substituted piperidinyl, optionally substituted pyrrolidinyl, —CH2-oxetanyl, -tetrahydropyranyl, —CH2-tetrahydropyranyl, or —(C1-C3)aralkyl; andwherein each substitutable carbon atom in the heterocyclyl and heteroaryl is optionally substituted with one or more substituents independently selected from deuterium, halo, —(C1-C4)alkyl, -hydroxy(C1-C4)alkyl, -halo(C1-C4)alkyl, —(C1-C4)alkoxy(C1-C4)alkyl, —C(O)(C1-C3)alkyl, —C(O)halo(C1-C3)alkyl, —C(O)hydroxy, —C(O)(C1-C3)alkylene-N(CH3)2, oxetanyl, —(C1-C3)alkyl-oxetanyl, -tetrahydropyranyl, —(C1-C3)alkyl-tetrahydropyranyl, or —(C1-C3)aralkyl.

In a seventeenth embodiment the invention provides a compound according to any of the foregoing embodiments, wherein the compound is of Formula (VIII-1) or (VIII-2):

In a twentieth embodiment the invention provides a method of treating a disease or condition, comprising administering a therapeutically effective amount of a compound of any one of the foregoing embodiments.

In a twenty-second embodiment the invention provides a method according to the twentieth or twenty-first embodiment wherein the disease or condition is an autoimmune disease.

In a twenty-fourth embodiment the invention provides a pharmaceutical composition comprising a compound according to any of the foregoing embodiments and a pharmaceutically acceptable carrier or diluent.

DETAILED DESCRIPTION OF THE INVENTION

Protein kinases are a broad and diverse class, of over 500 enzymes, that include oncogenes, growth factors receptors, signal transduction intermediates, apoptosis related kinases and cyclin dependent kinases. They are responsible for the transfer of a phosphate group to specific tyrosine, serine or threonine amino acid residues, and are broadly classified as tyrosine and serine/threonine kinases as a result of their substrate specificity.

The protein kinase C family is a group of serine/threonine kinases that comprises twelve related isoenzymes. Its members are encoded by different genes and are sub-classified according to their requirements for activation. The classical enzymes (cPKC) require diacylglycerol (DAG), phosphatidylserine (PS) and calcium for activation. The novel PKC's (nPKC) require DAG and PS but are calcium independent. The atypical PKC's (aPKC) do not require calcium or DAG.

Upon T cell activation, a supramolecular activation complex (SMAC) forms at the site of contact between the T cell and the antigen presenting cell (APC). PKCtheta is the only PKC isoform found to localize at the SMAC (Monks, C. et al.,Nature,1997, 385, p. 83), placing it in proximity with other signaling enzymes that mediate T cell activation processes.

In another study (Baier-Bitterlich, G. et al.,Mol. Cell. Biol.,1996, 16, p. 842) the role of PKCtheta in the activation of AP-1, a transcription factor important in the activation of the IL-2 gene, was confirmed. In unstimulated T cells, constitutively active PKCtheta stimulated AP-1 activity while in cells with dominant negative PKCtheta, AP-1 activity was not induced upon activation by PMA.

Proliferation of peripheral T cells from PKCtheta knockout mice, in response to T cell receptor (TCR)/CD28 stimulation was greatly diminished compared to T cells from wild type mice. In addition, the amount of IL-2 released from the T cells was also greatly reduced (Sun, Z. et al.,Nature,2000, 404, p. 402). It has also been shown that PKCtheta-deficient mice show impaired pulmonary inflammation and airway hyperresponsiveness (AHR) in a Th2-dependent murine asthma model, with no defects in viral clearance and Th1-dependent cytotoxic T cell function (Berg-Brown, N. N. et al.,J. Exp. Med.,2004, 199, p. 743; Marsland, B. J. et al.,J. Exp. Med.,2004, 200, p. 181). The impaired Th2 cell response results in reduced levels of IL-4 and immunoglobulin E (IgE), contributing to the AHR and inflammatory pathophysiology. Otherwise, the PKCtheta knockout mice seemed normal and fertile.

Regulatory T cells (Treg; CD4+CD25highFoxP3+) have been shown to play an important role in controlling autoimmunity by suppressing inflammatory responses (reviewed in Sakaguchi,Cell,2008, 133 (5), p. 775). A recent study suggests that PKCtheta negatively regulates Treg differentiation and function (Ma et al.,J. Immunol,2012, 188 (11), p. 5337) and demonstrates that inhibiting PKCtheta, using knock out or inhibitor, increases Treg generation in vitro and in vivo. Another report (Zanin-Zhorov et al.,Science,2010, 328, p. 372) shows that PKCtheta inhibition enhances function of Tregs in autoimmune disease. These data suggest that enhancement of Treg differentiation and function through PKCtheta inhibition may be beneficial in controlling autoimmunity.

The studies cited above and others studies confirm the critical role of PKCtheta in T cell activation. Thus an inhibitor of PKCtheta would be of therapeutic benefit in treating immunological disorders and other diseases mediated by the inappropriate activation of T cells.

Many of the kinases, whether a receptor or non-receptor tyrosine kinase or a S/T kinase have been found to be involved in cellular signaling pathways involved in numerous pathogenic conditions, including immunomodulation, inflammation, or proliferative disorders such as cancer.

Many autoimmune diseases and disease associated with chronic inflammation, as well as acute responses, have been linked to excessive or unregulated production or activity of one or more cytokines.

The compounds of the invention are also useful in the treatment of cardiovascular disorders, such as acute myocardial infarction, acute coronary syndrome, chronic heart failure, myocardial infarction, atherosclerosis, viral myocarditis, cardiac allograft rejection, and sepsis-associated cardiac dysfunction. Furthermore, the compounds of the present invention are also useful for the treatment of central nervous system disorders such as meningococcal meningitis, Alzheimer's disease and Parkinson's disease.

Compounds of Formula (I) of the invention can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the compound of the present invention. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent that affects the viscosity of the composition.

It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the compounds of the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other preferred combinations are corticosteroids including prednisolone; the well known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the compounds of this invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which a compound of Formula (I) of the invention can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-21, IL-23, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Compounds of the invention can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; preferred examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7 (U.S. Pat. No. 6,090,382, HUMIRA™), CA2 (REMICADE™), SIMPONI™ (golimumab), CIMZIA™, ACTEMRA™, CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, p75TNFR1gG (ENBREL™) or p55TNFR1gG (Lenercept), and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be effective for the same reason. Other preferred combinations include Interleukin 11. Yet other preferred combinations are the other key players of the autoimmune response which may act parallel to, dependent on or in concert with IL-18 function; especially preferred are IL-12 antagonists including IL-12 antibodies or soluble IL-12 receptors, or IL-12 binding proteins. It has been shown that IL-12 and IL-18 have overlapping but distinct functions and a combination of antagonists to both may be most effective. Yet another preferred combination is non-depleting anti-CD4 inhibitors. Yet other preferred combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors or antagonistic ligands.

Preferred examples of therapeutic agents for multiple sclerosis in which a compound of Formula (I) can be combined to include interferon-β, for example, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspase inhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40 ligand and CD80.

Non-limiting examples of therapeutic agents for HCV with which a compound of Formula (I) can be combined include the following: Interferon-alpha-2α, Interferon-alpha-2β, Interferon-alpha con1, Interferon-alpha-n1, pegylated interferon-alpha-2a, pegylated interferon-alpha-2β, ribavirin, peginterferon alfa-2b+ribavirin, ursodeoxycholic acid, glycyrrhizic acid, thymalfasin, Maxamine, VX-497 and any compounds that are used to treat HCV through intervention with the following targets: HCV polymerase, HCV protease, HCV helicase, and HCV IRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for restenosis with which a compound of Formula (I) can be combined include the following: sirolimus, paclitaxel, everolimus, tacrolimus, ABT-578, and acetaminophen.

Preferred examples of therapeutic agents for SLE (Lupus) with which a compound of Formula (I) can be combined include the following: NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example, celecoxib, rofecoxib, valdecoxib; anti-malarials, for example, hydroxychloroquine; steroids, for example, prednisone, prednisolone, budenoside, dexamethasone; cytotoxics, for example, azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, for example Cellcept®. A compound of Formula (I) may also be combined with agents such as sulfasalazine, 5-aminosalicylic acid, olsalazine, Imuran® and agents which interfere with synthesis, production or action of proinflammatory cytokines such as IL-1, for example, caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra. A compound of Formula (I) may also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors; or molecules that target T cell activation molecules, for example, CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family antibodies. A compound of Formula (I) can be combined with IL-11 or anti-cytokine antibodies, for example, fonotolizumab (anti-IFNg antibody), or anti-receptor receptor antibodies, for example, anti-IL-6 receptor antibody and antibodies to B-cell surface molecules. A compound of Formula (I) may also be used with LJP 394 (abetimus), agents that deplete or inactivate B-cells, for example, Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, for example, anti-TNF antibodies, D2E7 (U.S. Pat. No. 6,090,382; HUMIRA™), CA2 (REMICADE™), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL™) and p55TNFRIgG (LENERCEPT™).

In this invention, the following definitions are applicable:

A “therapeutically effective amount” is an amount of a compound of Formula (I) or a combination of two or more such compounds, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition. A therapeutically effective amount can also be an amount which is prophylactically effective. The amount which is therapeutically effective will depend upon the patient's size and gender, the condition to be treated, the severity of the condition and the result sought. For a given patient, a therapeutically effective amount can be determined by methods known to those of skill in the art.

“Pharmaceutically acceptable salts” refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid or organic acids such as sulfonic acid, carboxylic acid, organic phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, salicylic acid, lactic acid, tartaric acid (e.g. (+) or (−)-tartaric acid or mixtures thereof), amino acids (e.g. (+) or (−)-amino acids or mixtures thereof), and the like. These salts can be prepared by methods known to those skilled in the art.

Certain compounds of Formula (I) which have acidic substituents may exist as salts with pharmaceutically acceptable bases. The present invention includes such salts. Examples of such salts include sodium salts, potassium salts, lysine salts and arginine salts. These salts may be prepared by methods known to those skilled in the art.

Certain compounds of Formula (I) and their salts may exist in more than one crystal form and the present invention includes each crystal form and mixtures thereof.

Certain compounds of Formula (I) and their salts may also exist in the form of solvates, for example hydrates, and the present invention includes each solvate and mixtures thereof.

Certain compounds of Formula (I) may contain one or more chiral centers, and exist in different optically active forms. When compounds of Formula (I) contain one chiral center, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as racemic mixtures. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may be separated, for example, by crystallization; formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

When a compound of Formula (I) contains more than one chiral center, it may exist in diastereoisomeric forms. The diastereoisomeric compounds may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers may be separated as described above. The present invention includes each diastereoisomer of compounds of Formula (I), and mixtures thereof.

Certain compounds of Formula (I) may exist in different tautomeric forms or as different geometric isomers, and the present invention includes each tautomer and/or geometric isomer of compounds of Formula (I) and mixtures thereof.

Certain compounds of Formula (I) may exist in different stable conformational forms which may be separable. Torsional asymmetry due to restricted rotation about an asymmetric single bond, for example because of steric hindrance or ring strain, may permit separation of different conformers. The present invention includes each conformational isomer of compounds of Formula (I) and mixtures thereof.

Certain compounds of Formula (I) may exist in zwitterionic form and the present invention includes each zwitterionic form of compounds of Formula (I) and mixtures thereof.

As used herein the term “pro-drug” refers to an agent which is converted into the parent drug in vivo by some physiological chemical process (e.g., a pro-drug on being brought to the physiological pH is converted to the desired drug form). Pro-drugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The pro-drug may also have improved solubility in pharmacological compositions over the parent drug. An example, without limitation, of a pro-drug would be a compound of the present invention wherein it is administered as an ester (the “pro-drug”) to facilitate transmittal across a cell membrane where water solubility is not beneficial, but then it is metabolically hydrolyzed to the carboxylic acid once inside the cell where water solubility is beneficial.

Pro-drugs have many useful properties. For example, a pro-drug may be more water soluble than the ultimate drug, thereby facilitating intravenous administration of the drug. A pro-drug may also have a higher level of oral bioavailability than the ultimate drug. After administration, the pro-drug is enzymatically or chemically cleaved to deliver the ultimate drug in the blood or tissue.

Exemplary pro-drugs upon cleavage release the corresponding free acid, and such hydrolyzable ester-forming residues of the compounds of this invention include but are not limited to carboxylic acid substituents wherein the free hydrogen is replaced by (C1-C4)alkyl, (C1-C12)alkanoyloxymethyl, (C4-C9)1-(alkanoyloxy)ethyl, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N—(C1-C2)alkylamino(C2-C3)alkyl (such as β-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)-alkylcarbamoyl-(C1-C2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl.

Other exemplary pro-drugs release an alcohol of Formula (I) wherein the free hydrogen of the hydroxyl substituent is replaced by (C1-C6)alkanoyloxymethyl, 1-((C1-C6)alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C12)alkoxycarbonyloxymethyl, N—(C1-C6)alkoxycarbonylamino-methyl, succinoyl, (C1-C6)alkanoyl, α-amino(C1-C4)alkanoyl, arylacetyl and α-aminoacyl, or α-aminoacyl-α-aminoacyl wherein said α-aminoacyl moieties are independently any of the naturally occurring L-amino acids found in proteins, P(O)(OH)2, —P(O)(O(C1-C6)alkyl)2or glycosyl (the radical resulting from detachment of the hydroxyl of the hemiacetal of a carbohydrate).

Other exemplary pro-drugs release an amine of Formula (I) wherein the free hydrogen of the amine group is replaced by —C(O)alkyl, —C(O)O-alkyl, N-phosphonoxyalkyl, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl, wherein the alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl can be optionally substituted with, for example, halogen and hydroxyl.

As used herein, “hydrate” is a solvate wherein the solvent molecule is water.

As used herein, the term “bridged (C5-C12) cycloalkyl group” means a saturated or unsaturated, bicyclic or polycyclic bridged hydrocarbon group having two or three C3-C10cycloalkyl rings. Non bridged cycloalkyls are excluded. For purposes of exemplification, which should not be construed as limiting the scope of this invention, bridged cyclic hydrocarbon may include bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl, bicyclo[4.3.1]decyl, bicyclo[3.3.1]nonyl, bornyl, bornenyl, norbornyl, norbornenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, tricyclobutyl, and adamantyl.

As used herein the term “bridged (C2-C10) heterocyclyl” means bicyclic or polycyclic bridged hydrocarbon groups containing one or more heteroatoms such as nitrogen, oxygen and sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, bridged (C2-C10) heterocyclyl may include azanorbornyl, quinuclidinyl, isoquinuclidinyl, tropanyl, azabicyclo[3.2.1]octanyl, azabicyclo[2.2.1]heptanyl, 2-azabicyclo[3.2.1]octanyl, azabicyclo[3.2.1]octanyl, azabicyclo[3.2.2]nonanyl, azabicyclo[3.3.0]nonanyl, and azabicyclo[3.3.1]nonanyl.

As used herein, “spirocyclic (C2-C10) heterocyclyl” means bicyclic or polycyclic hydrocarbon group having two or three (C3-C10) rings at least one of which contains a heteroatom such as nitrogen, oxygen or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, spirocyclic (C2-C10) heterocyclyl may include diazaspiro[3.5]nonane and diazaspiro[4.5]decane.

As used herein, “spirocyclic (C5-C11) carbocyclyl” means a saturated or unsaturated, bicyclic or polycyclic hydrocarbon group having two or three (C3-C10) cycloalkyl rings. For purposes of exemplification, which should not be construed as limiting the scope of this invention, spirocyclic (C5-C11) carbocyclyl includes spiro[5.5]undecane, spiro[4.5]decane and spiro[4.4]nonane.

The term “heterocyclic”, “heterocyclyl” or “heterocyclylene”, as used herein, include non-aromatic ring systems, including, but not limited to, monocyclic, bicyclic, and tricyclic rings, which can be completely saturated or which can contain one or more units of unsaturation. (for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system) and have 5 to 12 atoms including at least one heteroatom, such as nitrogen, oxygen, or sulfur. For purposes of exemplification, which should not be construed as limiting the scope of this invention, the following are examples of heterocyclic rings: azepinyl, azetidinyl, indolinyl, isoindolinyl, morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, quinucludinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydroindolyl, thiomorpholinyl and tropanyl.

As used herein, “alkyl” and “alkylene” include straight chained or branched hydrocarbons which are completely saturated. For purposes of exemplification, which should not be construed as limiting the scope of this invention, examples of alkyls are methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and isomers thereof.

As used herein, “alkenyl”, “alkenylene”, “alkynylene” and “alkynyl” mean hydrocarbon moieties containing two to eight carbons and include straight chained or branched hydrocarbons which contain one or more units of unsaturation, one or more double bonds for alkenyl and one or more triple bonds for alkynyl. For purposes of exemplification, which should not be construed as limiting the scope of this invention, examples of alkenyl are ethenyl, propenyl and butenyl, and examples of alkynyl are ethynyl, propynyl and butynyl.

As used herein, “aryl” or “arylene” groups include aromatic carbocyclic ring systems (e.g. phenyl) and fused polycyclic aromatic ring systems. For purposes of exemplification, which should not be construed as limiting the scope of this invention, aryl groups include naphthyl, biphenyl and 1,2,3,4-tetrahydronaphthyl.

As used herein, “cycloalkyl” or “cycloalkylene” means C3-C12monocyclic or multicyclic (e.g., bicyclic, tricyclic, etc.) hydrocarbons that are completely saturated or have one or more unsaturated bonds but do not amount to an aromatic group. For purposes of exemplification, which should not be construed as limiting the scope of this invention, examples of a cycloalkyl group are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl.

As used herein, many moieties or substituents are termed as being either “substituted” or “optionally substituted”. When a moiety is modified by one of these terms, unless otherwise noted, it denotes that any portion of the moiety that is known to one skilled in the art as being available for substitution can be substituted, which includes one or more substituents, where if more than one substituent then each substituent is independently selected. Such means for substitution are well-known in the art and/or taught by the instant disclosure. For purposes of exemplification, which should not be construed as limiting the scope of this invention, some examples of groups that are substituents are: deuterium, optionally substituted (C1-C8)alkyl groups, optionally substituted (C2-C8)alkenyl groups, (C2-C8)alkynyl groups, optionally substituted (C3-C10)cycloalkyl groups, halogen (F, Cl, Br or I), halogenated (C1-C8)alkyl groups (for example but not limited to —CF3), —O—(C1-C8)alkyl groups, —OH, —S—(C1-C8)alkyl groups, —SH, —NH(C1-C8)alkyl groups, —N((C1-C8)alkyl)2groups, —NH2, —NH—(C1-C6)alkyl-optionally substituted heterocycle, —NH-heterocycle, —C(O)NH2, —C(O)NH(C1-C8)alkyl groups, —C(O)N((C1-C8)alkyl)2, —NHC(O)H, —NHC(O)(C1-C8)alkyl groups, —NHC(O)(C3-C8)cycloalkyl groups, —N((C1-C8)alkyl)C(O)H, —N((C1-C8)alkyl)C(O)(C1-C8)alkyl groups, —NHC(O)NH2, —NHC(O)NH(C1-C8)alkyl groups, —N((C1-C8)alkyl)C(O)NH2groups, —NHC(O)N((C1-C8)alkyl)2groups, —N((C1-C8)alkyl)C(O)N((C1-C8)alkyl)2groups, —N((C1-C8)alkyl)C(O)NH((C1-C8)alkyl), —C(O)H, —C(O)(C1-C8)alkyl groups, —CN, —NO2, —S(O)(C1-C8)alkyl groups, —S(O)2(C1-C8)alkyl groups, —S(O)2N((C1-C8)alkyl)2groups, —S(O)2NH(C1-C8)alkyl groups, —S(O)2NH(C3-C8)cycloalkyl groups, —S(O)2NH2groups, —NHS(O)2(C1-C8)alkyl groups, —N((C1-C8)alkyl)S(O)2(C1-C8)alkyl groups, —(C1-C8)alkyl-O—(C1-C8)alkyl groups, —O—(C1-C8)alkyl-O—(C1-C8)alkyl groups, —C(O)OH, —C(O)O(C1-C8)alkyl groups, —NHOH, —NHO(C1-C8)alkyl groups, —O-halogenated (C1-C8)alkyl groups (for example but not limited to —OCF3), —S(O)2-halogenated (C1-C8)alkyl groups (for example but not limited to —S(O)2CF3), —S-halogenated (C1-C8)alkyl groups (for example but not limited to —SCF3), —(C1-C6)alkyl-optionally substituted heterocycle (for example but not limited to azetidine, piperidine, piperazine, pyrrolidine, tetrahydrofuran, pyran or morpholine), —(C1-C6)alkyl-heteroaryl (for example but not limited to tetrazole, imidazole, furan, pyrazine or pyrazole), -optionally substituted phenyl, —NHC(O)O—(C1-C6)alkyl groups, —N((C1-C6)alkyl)C(O)O—(C1-C6)alkyl groups, —C(═NH)—(C1-C6)alkyl groups, —C(═NOH)—(C1-C6)alkyl groups, or —C(═N—O— (C1-C6)alkyl)-(C1-C6)alkyl groups.

One or more compounds of this invention can be administered to a human patient by themselves or in pharmaceutical compositions where they are mixed with biologically suitable carriers or excipient(s) at doses to treat or ameliorate a disease or condition as described herein. Mixtures of these compounds can also be administered to the patient as a simple mixture or in suitable formulated pharmaceutical compositions. A therapeutically effective dose refers to that amount of the compound or compounds sufficient to result in the prevention or attenuation of a disease or condition as described herein. Techniques for formulation and administration of the compounds of the instant application may be found in references well known to one of ordinary skill in the art, such as “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

Alternatively, one may administer the compound in a local rather than a systemic manner, for example, via injection of the compound directly into an edematous site, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Many of the compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art.

For any compound used in a method of the present invention, the therapeutically effective dose can be estimated initially from cellular assays. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the IC50as determined in cellular assays (i.e., the concentration of the test compound which achieves a half-maximal inhibition of a given protein kinase activity). In some cases it is appropriate to determine the IC50in the presence of 3 to 5% serum albumin since such a determination approximates the binding effects of plasma protein on the compound. Such information can be used to more accurately determine useful doses in humans. Further, the most preferred compounds for systemic administration effectively inhibit protein kinase signaling in intact cells at levels that are safely achievable in plasma.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) and the ED50(effective dose for 50% maximal response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between MTD and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1). In the treatment of crises, the administration of an acute bolus or an infusion approaching the MTD may be required to obtain a rapid response.

Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g. the concentration necessary to achieve 50-90% inhibition of protein kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90% until the desired amelioration of symptoms is achieved. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.

In some formulations it may be beneficial to use the compounds of the present invention in the form of particles of very small size, for example as obtained by fluid energy milling.

The use of compounds of the present invention in the manufacture of pharmaceutical compositions is illustrated by the following description. In this description the term “active compound” denotes any compound of the invention but particularly any compound which is the final product of one of the following Examples.

In the preparation of capsules, 10 parts by weight of active compound and 240 parts by weight of lactose can be de-aggregated and blended. The mixture can be filled into hard gelatin capsules, each capsule containing a unit dose or part of a unit dose of active compound.

Tablets can be prepared, for example, from the following ingredients.

The active compound, the lactose and some of the starch can be de-aggregated, blended and the resulting mixture can be granulated with a solution of the polyvinylpyrrolidone in ethanol. The dry granulate can be blended with the magnesium stearate and the rest of the starch. The mixture is then compressed in a tabletting machine to give tablets each containing a unit dose or a part of a unit dose of active compound.

Tablets can be prepared by the method described in (b) above. The tablets can be enteric coated in a conventional manner using a solution of 20% cellulose acetate phthalate and 3% diethyl phthalate in EtOH:DCM (1:1).

In the preparation of suppositories, for example, 100 parts by weight of active compound can be incorporated in 1300 parts by weight of triglyceride suppository base and the mixture formed into suppositories each containing a therapeutically effective amount of active ingredient.

In the compositions of the present invention the active compound may, if desired, be associated with other compatible pharmacologically active ingredients. For example, the compounds of this invention can be administered in combination with another therapeutic agent that is known to treat a disease or condition described herein. For example, with one or more additional pharmaceutical agents that inhibit or prevent the production of VEGF or angiopoietins, attenuate intracellular responses to VEGF or angiopoietins, block intracellular signal transduction, inhibit vascular hyperpermeability, reduce inflammation, or inhibit or prevent the formation of edema or neovascularization. The compounds of the invention can be administered prior to, subsequent to or simultaneously with the additional pharmaceutical agent, whichever course of administration is appropriate. The additional pharmaceutical agents include, but are not limited to, anti-edemic steroids, NSAIDS, ras inhibitors, anti-TNF agents, anti-IL1 agents, antihistamines, PAF-antagonists, COX-1 inhibitors, COX-2 inhibitors, NO synthase inhibitors, Akt/PTB inhibitors, IGF-1R inhibitors, PI3 kinase inhibitors, calcineurin inhibitors and immunosuppressants. The compounds of the invention and the additional pharmaceutical agents act either additively or synergistically. Thus, the administration of such a combination of substances that inhibit angiogenesis, vascular hyperpermeability and/or inhibit the formation of edema can provide greater relief from the deletrious effects of a hyperproliferative disorder, angiogenesis, vascular hyperpermeability or edema than the administration of either substance alone. In the treatment of malignant disorders combinations with antiproliferative or cytotoxic chemotherapies or radiation are included in the scope of the present invention.

The present invention also comprises the use of a compound of Formula (I) as a medicament.

Abbreviations

Following a sixty minute incubation at room temperature, the reaction was quenched with 5 μL of 0.5 M EDTA pH 7.5. Detection buffer is added to each well (50 mM Hepes pH 7.0, 0.4 M KF, 0.01% Tween (BioRad), 0.1% BSA, 0.9 ng/well batch 2 Phospho-(Ser) 14-3-3 Binding Motif 4E2 Monoclonal Antibody-K (Cell Signaling #9606, labeled by Perkin Elmer W1024), and 0.055 μg/well CR130-100 (Perkin Elmer). Plates are incubated for 10 min at rt and read on the RubyStar HTRF micro-plate analyzer, measuring fluorescence counts at 665 nm and 620 nm (20 counts/sec).

Background counts from a minus enzyme control is subtracted from all data. Data (after background correction) are converted to percent activity by dividing by the signal obtained from PKC without drug sample. IC50values are determined by fitting the percent activity vs. inhibitor concentration data set to percent activity=1/1+[I]/IC50by non-linear least means squares curve fitting.

Human T-Blasts are generated using PHA following standard protocols. T-Blasts are freeze aliquoted and vials thawed as required. To set up the assay, thaw vial and wash cells using standard growth medium (RPMI 1640 medium with 2 mM L-glutamine, 10 mM HEPES, 100 U/mL Pen/Strep, and 10% FBS) Count and resuspend cells at 2×106/mL in growth medium. Seed 2×105/100 μL cell suspension per well in flat bottomed 96 well plate. Compound dilutions are prepared from 100% DMSO stocks. A total of 8 serial dilutions are made (1/3 in 100% DMSO). Compounds are diluted 1/50 in growth medium to prepare 4× stocks of each tested concentration. 50 μL from each 4× stock concentration is added to the 100 μL cell suspension and incubated for 30 minutes prior stimulus addition. A 4× stimulus stock is prepared using anti-CD3+anti-CD28 antibodies. Antibody mix is added to the cell/compounds suspension in 50 μL at 5 μg/mL final concentration. Compounds are tested in duplicates. Plates are transferred to 37° C./CO2incubator for 24 hours. After incubation 130 μL of supernatant is collected for IL-2 determination while MTT toxicity analysis is performed on remaining cells/medium.

Human T-Blasts are generated using PHA following standard protocols. T-Blasts are freeze aliquoted and vials thawed as required. To set up the assay, vial is thawed and cells washed using standard growth medium (RPMI 1640 medium with 2 mM L-glutamine, 10 mM HEPES, 100 U/mL Pen/Strep, and 10% FBS). Maintain cells in culture with growth medium plus 50 μ/mL rhIL-2 for 48 hours prior stimulation. Count and resuspend cells at 6×106/mL in growth medium. Add 1.5×106/250 μL cell suspension to 1.5 mL Eppendorf tubes. Compounds dilutions are prepared from 100% DMSO stocks. A total of 6 serial dilutions are made (1/3 in 100% DMSO) Compounds are diluted 1/4 in growth medium to prepare 25× stocks of each tested concentration. 10 μL from each 25× stock concentration is added to the 250 μL cell suspension and incubated for 30 minutes prior stimulus addition. A 50× stimulus stock is prepared using anti-CD3+anti-CD28 antibodies. Antibody mix is added to the cell/compounds suspension in 5 μL at 5 μg/mL final concentration. Tubes are transferred to 37° C./CO2incubator for 60 min. After incubation, cells are collected by quick centrifugation in table top Eppendorf centrifuge (12,000 rpm/20 sec.) and washed quickly using 750 μL of ice cold PBS containing 1 mM sodium orthovanadate. Cells are collected by centrifugation (12,000 rpm/20 sec.) and lysed using 50 μL of buffer A. Incubate lysates on ice for 30 min and clear lysates by centrifugation in table top Eppendorf centrifuge (12,000 rpm/10 min./4° C.) Cleared lysates are transferred to new Eppendorf tubes and 50 μL of 2× reducing sample buffer added. Samples are incubated at 90° C. for 5 min before being loaded into gels and separated by SDS-PAGE. Western blot analysis is performed after transferring the gels onto PVDF membranes. A specific monoclonal antibody recognizing a phosphorylated S32/S36 form of IκB-α is used.

In Vivo

Concanavalin A (Con A)-Induced Cytokine Production in Lewis Rats

The test compound is formulated in an inert vehicle (for example but not limited to 0.5% hydroxypropylmethyl cellulose/0.02% Tween 80 in water) at the desired concentration to achieve doses in the range of 0.01-100 mg/kg. Eight-week-old male Lewis rats (200 g) (Charles River Laboratories) are dosed with the compound orally, at time zero (0 min). After about 60 min the rats are injected intravenously (i.v.) with 10 mg/kg Concanavalin A dissolved in PBS About 2 h later, the rats are cardiac bled and their plasma is analyzed for levels of IL-2 (ELISA kit) and IFN-γ (ELISA kit).

Start with a volume of cold CFA 4× the volume of the collagen solution, because ¾ of the CFA oil will be discarded. Keep the CFA on ice. Centrifuge CFA @ 1,000 rpm for 10 minutes at 4° C., remove ¾ of the oil (7.5 mL) and discard it, then resuspend the remaining CFA.

To Prepare Emulsion: Load two Luer-Lok glass syringes. Using a long hypodermic needle, load one glass syringe with 4×CFA or 1×IFA, then load a second glass syringe with an equal amount of collagen. Wear goggles for this step in case some of the liquid sprays out. Connect the 2 syringes with a metal Luer-Lok connector and inject the two solutions back and forth to mix them. Keep the apparatus on ice, alternating with forcing the liquid back and forth between the syringes 8-10 times every few minutes until emulsified. To test for emulsification, remove one syringe and let one drop of the liquid fall into a beaker of water. The drop should hold together in the water without spreading over the surface. Keep the collagen/adjuvant on ice after emulsification and while injecting into the animals.

The mice were dosed according to the study design with vehicle, compound or dexamethasone at 1 mg/kg from day 0-17. Seven days after immunization, mice were monitored for arthritis. Rear paws were evaluated for paw-edema using Dyer spring calipers on days 7, 10, 13, 15, and 17. Mice began to show signs of paw swelling between day 7 and 10. At the termination of the experiment, a full 12 hour exposure AUC was performed on the final day.

Adjuvant Induced Arthritis (AIA) in a Lewis Rat

Female Lewis rats, (8 weeks of age, 170 g in weight from Charles River Laboratories) are immunized intradermally (i.d.) in the right hind-footpad with 100 μL of a suspension of mineral oil and containing 200 μgM. tuberculosis(H37RA). The inflammation appears in the contra-lateral (left) hind paw seven days after the initial immunization. Seven days post immunization, the compound is formulated in an inert vehicle (for example but not limited to 0.5% hydroxypropylmethyl cellulose/0.02% Tween 80 in water) and dosed orally once or twice a day for at least 10 days. Baseline paw volume is taken on day 0 using a water displacement pleythsmograph (Vgo Basile North America Inc. PA 19473, Model #7140). Rats are lightly anesthetized with an inhalant anesthetic (isoflurane) and the contra-lateral (left) hind paw is dipped into the plethysmograph and the paw volume is recorded. The rats are scored every other day up to day 17 after immunization. On day 17 after immunization, all rats are exsanguinated by cardiac puncture under isoflurane anesthesia, and the left hind paw is collected to assess the impact on bone erosion using micro-CT or histology. For assessing bone erosion by micro CT, paw samples are scanned (SCANCO Medical, Southeastern, PA, Model # μCT 40) at a voxel size of 18 μm, a threshold of 400, sigma-gauss 0.8, support-gauss 1.0. Bone volume and density is determined for a 360 μm (200 slice) vertical section encompassing the tarsal section of the paw. The 360 μm section is analyzed from the base of the metatarsals to the top of the tibia, with the lower reference point fixed at the tibiotalar junction. In addition, the changes in bone are assessed by histology. Essentially, the left hind paws are collected from various groups and are fixed and decalcified in Cal-Rite following which the paws are embedded in paraffin blocks. 5 μm sections are cut, routinely de-paraffinized and stained by standard hematoxylin and eosin procedure. The sections are then evaluated for inflammation, bone and cartilage erosion using a subjective scoring system with a scale of 0-5. Drug exposure is determined in the plasma using LC/MS.

The teachings of all references, including journal articles, patents and published patent applications, are incorporated herein by reference in their entirety.

The following examples are for illustrative purposes and are not to be construed as limiting the scope of the present invention.

General Synthetic Schemes

Compounds of the invention may be prepared using the synthetic transformations illustrated in Schemes I-VI. Starting materials are commercially available or may be prepared by the procedures described herein, by literature procedures, or by procedures that would be well known to one skilled in the art of organic chemistry. All times and temperatures are approximate. All drying reagents are anhydrous. Unless stated, all aqueous solutions are saturated. If desired, chiral separation of compounds may be accomplished by methods known to one skilled in the art such as chiral SFC or chiral preparative HPLC.

Methods for preparing 3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one and 3,5-dihydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]triazin-2(1H)-one compounds of the invention are illustrated in Schemes I-V. In Scheme I, step a, commercially available 2-amino-4-nitrophenols are reacted with chloroacetyl chloride using conditions described in Preparation #2, Step A, or by methods known to one skilled in the art (for example,Organic Preparations and Procedures International1982, 14 (3), 195-197) to give benzooxazinones 3. Alternatively, 4-chloro-5-nitroanilines are cyclized with methyl 2-mercaptoacetate as described in Example #97, Step A to give benzothiazinones 4. In Scheme I, step c, benzooxazinones 3 or benzothiazinones 4 can be alkylated using conditions such as those described in Preparation #2, Step B, or Example #97, Step B, or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). Conversion of lactams 5 and 6 to thiolactams 7 or 8, respectively, may be accomplished using conditions such as those described in Preparation #2, Step C, or Example #97, Step C, or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). The materials may be cyclized to dihydrobenzooxazinotriazinones 9 and dihydrobenzothiazinotriazinones 10 using conditions such as those described in Preparation #2, Step D, or Example #97, Step D or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). In Scheme I, step g, the materials 9 and 10 may be reduced to the anilines 12 and 13, respectively using methods such as those described in Preparation #2, Step E, or Example #97, Step E, or by a variety of methods known to one skilled in the art (for example, Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2ndedition”, 1999, Wiley-VCH). Alternatively, reduction of compounds 7 (Scheme I, step f) followed by cyclization (Scheme I, step h) to give dihydrobenzooxazinotriazinones 9 may be accomplished using methods known to one skilled in the art (for example, Example #56, Steps E and F). Anilines 12 or 13 may undergo further functionalization using reactions known to one skilled in the art (for example, Larock, R. C. referenced above). For example, formation of secondary anilines 14 and 15 may be accomplished using standard conditions such as those described in Example #1, Step A, or Example #97, Step F, or Example #79, Step A, or by methods known to one skilled in the art (for example, Larock, R. C. referenced above). Additional functionalization of secondary anilines 14 to form compounds 16, if desired, can be performed using conditions such as those described in Example #55, Step B, or using reactions known to one skilled in the art (for example, Larock, R. C. referenced above). If R2or R3contain a protecting group, deprotection of dihydrobenzooxazinotriazinone and dihydrobenzothiazinotriazinone compounds 14, 15, or 16 can be performed using conditions such as those described in Greene, T. W. and Wuts. P. G. M “Protective Groups in Organic Synthesis, 3rdEdition, 1999, Wiley-Interscience”. For example, a protecting group such as a t-butyloxycarbonyl (Boc) group can be removed from a protected amine to yield the unprotected amine (for example, Example #55, Step C) and the deprotected compound 14, 15, or 16 may then be reacted further as described above. A protecting group such as a benzylhydryl group can be removed from a protected amine to yield the unprotected amine (for example, Example #80, Step H) and the deprotected compound 14, 15, or 16 may then be reacted further as described above. If desired, chiral separation of compounds 5, 6, 9, 10, 12, 13, 14, 15, or 16 may be done using methods known to one skilled in the art such as chiral SFC or chiral preparative HPLC (for example, Example #1, Step C).

In Scheme II, step a, nitration of commercially available 4-bromophenols 17 provides nitrophenols 18 using methods known to one skilled in the art (for example, Example #53, Step A). Reduction to aminophenols 19 may be accomplished using methods such as those described in Example #53, Step B, or by a variety of methods known to one skilled in the art (for example, Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2ndedition”, 1999, Wiley-VCH). In Scheme II, step c, cyclization of aminophenols 19 with chloroacetyl chloride and subsequent alkylation is performed using conditions described in Example #53, Step C, or using reactions known to one skilled in the art (for example, Larock, R. C. referenced above, andIndian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). The material may be cyclized to dihydrobenzooxazinotriazinones 21 using conditions such as those described in Example #53, Step D, or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). Dihydrobenzooxazinotrizinones 21 may be 2-(trimethylsilyl)ethoxymethyl (SEM) protected using conditions such as those described in Example #53, Step E, or by methods known to one skilled in the art (for example, Greene, T. W. and Wuts. P. G. M “Protective Groups in Organic Synthesis, 3rdEdition, 1999, Wiley-Interscience”.) In Scheme II, step f, an amine is introduced by reaction with dihydrobenzooxazinotriazinones 22 under Buchwald-Hartwig amination conditions (for example, Example #53, Step F, orAdvanced Synthesis&Catalysis2004, 346, 1599-1626) to give dihydrobenzooxazinotriazinones 23. Deprotection of dihydrobenzooxazinotriazinones 23 may be accomplished using conditions such as those described in Example #53, Step G, or by methods known to one skilled in the art (for example, the books from Larock, R. C. or Greene, T. W. and Wuts, P. G. M. referenced above). If R2and/or R3contain a protecting group, deprotection of compound 24 can be performed using conditions such as those described in Example #53, Step H, or Greene, T. W. and Wuts, P. G. M. described above. For example, a protecting group such as a t-butoxycarbonyl group can be removed with acid using conditions such as those described in Example #53, Step H or by methods known to one skilled in the art (for example, the books from Larock, R. C. or Greene, T. W. and Wuts, P. G. M. referenced above). If desired, chiral separation of compounds 20-24 may be accomplished using methods known to one skilled in the art such as chiral SFC or chiral preparative HPLC (for example, Example #53, Step G). Alternatively R1can be introduced at a later stage, for example after step f or step g, utilizing the methodology outlined in Scheme IV, steps a and b, or steps a, c and d.

In Scheme III, step a, dihydrobenzooxazinones 25 can be alkylated using conditions such as those described in Example #65, Step A, or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). The formation of esters 26 by oxidative rearrangement is accomplished using conditions such as those described in Example #65, Step B or by methods known to one skilled in the art (for example, Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2ndedition”, 1999, Wiley-VCH). Deprotection of compounds 27 to yield phenols 28 can be performed using conditions such as those described in Example #65, Step C, or Greene, T. W. and Wuts. P. G. M “Protective Groups in Organic Synthesis, 3rdEdition, 1999, Wiley-Interscience”. The formation of ethers 29 can be achieved by reaction of phenols 28 with appropriately substituted alkyl mesylates by methods known to one skilled in the art (for example, Example #65, Step D, or Larock, R. C. referenced above). In Scheme III, step e, conversion to thiolactams 30 may be achieved using methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183, or Example #65, Step E). Thiolactams 30 may be cyclized to dihydrobenzooxazinotriazinones 31 using conditions such as those described in Example #65, Step F or by methods known to one skilled in the art (for example,Indian Journal of Pharmaceutical Sciences1991, 53 (4), 180-183). Alternatively, formation of the boronic esters 32 from dihydrobenzooxazinotriazinones 22 (Scheme III, step g) can be performed using conditions such as those described in Example #107, Step A, or using reactions known to one skilled in the art (for example,Journal of Organic Chemistry1995 60, 7508-7510). Formation of phenols 33 can be accomplished by oxidative cleavage using methods known to one skilled in the art (for example, Example #107, Step B, or Larock, R. C. referenced above). The formation of ethers 34 can be achieved by methods known to one skilled in the art (for example, Example #107, Step C, or Larock, R. C. referenced above). Deprotection of dihydrobenzooxazinotriazinones 34 may be accomplished using methods known to one skilled in the art (for example, the books from Larock, R. C. or Greene, T. W. and Wuts, P. G. M. referenced above, or Example #107, Step D). If R2contains a protecting group, deprotection of compounds 29, 30, 31 or 34 can be performed using conditions such as those described in Example #80, Step L, or Example #107, Step D, or Greene, T. W. and Wuts, P. G. M. described above. For example, a protecting group such as a t-butoxycarbonyl group can be removed with acid using conditions such as those described in Example #80, Step L, or by methods known to one skilled in the art (for example, the books from Larock, R. C. or Greene, T. W. and Wuts, P. G. M. referenced above). A protecting group such as a benzylhydryl group can be removed from a protected amine to yield the unprotected amine (for example, Example #107, Step D). The deprotected compounds 29, 30, 31, or 34 may then be reacted further as described above. If desired, chiral separation of compounds 22, 31 or 34 may be done using methods known to one skilled in the art such as chiral SFC or chiral preparative HPLC (for example, Example #96, Step A).

In Scheme IV, step a, bromination of dihydrobenzooxazinones 35 can be accomplished using conditions such as those described in Example #1, Step B, or by methods known to one skilled in the art (for example, Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2ndedition”, 1999, Wiley-VCH). Further functionalization of dihydrobenzooxazinones 36 can be performed to yield substituted dihydrobenzooxazinones 37 using reactions known to one skilled in the art (for example, Example #1, Step C or Larock, R. C. referenced above). Alternatively, substituted dihydrobenzooxazinones 39 may be generated by introducing a substituted alkenyl group using conditions such as those described in Example #112, Step A, followed by reduction using reactions known to one skilled in the art (for example, Example #112, Step B, or Larock, R. C. referenced above). If R1contains a protecting group, deprotection of compounds 37, 38 or 39 can be performed using conditions such as those described in Example #112, Step C, or Greene, T. W. and Wuts. P. G. M “Protective Groups in Organic Synthesis, 3rdEdition, 1999, Wiley-Interscience”. For example, a protecting group such as a t-butoxycarbonyl group can be removed with acid using conditions such as those described in Example #112, Step C, or by methods known to one skilled in the art (for example, the books from Larock, R. C. or Greene, T. W. and Wuts, P. G. M. referenced above). If desired, chiral separation of compounds 35-39 may be done using methods known to one skilled in the art such as chiral SFC or chiral preparative HPLC (for example, Example #1, Step C).

In Scheme V, step a, an alkenyl group is introduced by reaction of bromodihydrooxazinotriazinones 21 (Scheme II, step d) under Suzuki or Heck cross-coupling conditions (for example, Example #71, Step A, or Example #76, Step A, or de Meijere, A. and Diederich, F. “Metal-Catalyzed Cross-Coupling Reactions, 2ndedition”, 2004, Wiley-VCH). Reduction of compounds 41 to yield dihydrooxazinotriazinones 42 can be performed using conditions such as those described in Example #71, Step B, or by methods known to one skilled in the art (for example, Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2ndedition”, 1999, Wiley-VCH). Alternatively, the Suzuki coupling can be performed from the boronic ester dihydrooxazinotriazinones 40 to provide compounds 41 using conditions such as those described in Example #86, Step B, or de Meijere, A. and Deiderich, F. as referenced above.

Methods for preparing 5,6-dihydro-1H-[1,2,4]triazino[4,3-a]quinolin-2(3H)-one compounds of the invention are illustrated in Scheme VI. In Scheme VI, step a, protection of aminodihydroquinolinones 43 (prepared by methods described inJournal of Medicinal Chemistry2005, 48 (1), 71-90) to yield dimethylpyrrolodihydroquinolinones 44 can be performed using conditions known to one skilled in the art (for example, Example #108, Step C, or Greene, T. W. and Wuts. P. G. M. “Protective Groups in Organic Synthesis, 3rdEdition, 1999, Wiley-Interscience”). In Scheme VI, step b, dihydroquinolinones 44 can be alkylated using conditions such as those described in Example #108, Step D, or by methods known to one skilled in the art (for example, Larock, R. C. referenced above). Conversion to thiolactams 46 is accomplished using conditions such as those described in Example #108, Step E. Dihydrotriazinoquinolinones 47 can be prepared by reaction of thiolactams 46 with hydrazine (for example, Example #108, Step F). Deprotection of compounds 47 to yield aminodihydrotriazinoquinolinones 48 can be performed using conditions such as those described in Example #108, Step G, or Greene, T. W. and Wuts, P. G. M. referenced above. Aminodihydrotriazinoquinolinones 48 may undergo further functionalization using reactions known to one skilled in the art (for example, Larock, R. C. referenced above). For example, formation of secondary amines 49 may be accomplished using standard conditions such as those described in Example #108, Step H, or by methods known to one skilled in the art (for example, the books from Larock, R. C. referenced above). If R1contains a protecting group, deprotection of compounds 49 can be performed using conditions such as those described in Greene, T. W. and Wuts. P. G. M. referenced above. For example, a protecting group such as a t-butoxycarbonyl group can be removed from a protected amine to yield the unprotected amine (for example, Example #108, Step I) and the deprotected compound may then be reacted further as described above. Alternatively R1can be introduced at a later stage, for example after step a, step b, or step c, utilizing the methodology outlined in Scheme IV, steps a and b, or steps a, c and d.

Analytical data is included within the procedures below, in the illustrations of the general procedures, or in the tables of examples. Unless otherwise stated, all1H NMR data were collected on a Bruker Avance III 400 MHz FT-NMR spectrometer with 5 mm BBO(F) probe or a Varian 400 MHz, 1H/19F/31P/13C 5 mm PFG 4Nuc probe instrument and chemical shifts are quoted in parts per million (ppm). LC/MS was performed on instrument with Shimadzu pump LC-20AB, PDA SPD-M20, MS MS-2010EV. LC/MS and HPLC data is referenced to LC/MS and HPLC conditions using the method number provided in Table 1.

Chiral purification is performed using Varian 218 LC pumps, a Varian CVM 500 with switching valves and heaters for automatic solvent, column and temperature control and a Varian 701 Fraction collector. Detection methods include a Varian 210 variable wavelength detector, an in-line polarimeter (PDR-chiral advanced laser polarimeter, model ALP2002) used to measure qualitative optical rotation (+/−) and an evaporative light scattering detector (ELSD) (a PS-ELS 2100 (Polymer Laboratories)) using a 100:1 split flow. ELSD settings are as follows: evaporator: 46° C., nebulizer: 24° C. and gas flow: 1.1 SLM.

Preparations of intermediate and final compounds obtained via the General Procedures can be optionally degassed using one or more of the Degassing Methods described below. The reaction mixtures may be degassed by a single or multiple applications of any technique or combination of techniques known to one skilled in the art. Some examples that are not limiting include bubbling a continuous stream of an inert gas (e.g. nitrogen, argon, etc.) through a mixture of reagents and a solvent suitable for the transformation (e.g. THF, 1,4-dioxane, EtOAc, DCM, toluene, MeOH, EtOH, DMF, MeCN, water, etc.); freeze-thawing of a mixture of reagents in a solvent (e.g. THF, 1,4-dioxane, EtOAc, DCM, toluene, MeOH, EtOH, DMF, MeCN, water, etc.) where the resulting solution is cooled below its freezing point and evacuated under reduced pressure, then allowed to warm above the freezing point and purged with an atmosphere of inert gas (e.g. nitrogen, argon, etc.); evacuation under reduced pressure of a mixture of reagents with or without a suitable solvent for the transformation (e.g. THF, 1,4-dioxane, EtOAc, DCM, toluene, MeOH, EtOH, DMF, MeCN, water, etc.) followed by purging of the mixture with an inert gas (e.g. nitrogen, argon, etc.); evacuation under reduced pressure of a mixture of reagents in a suitable solvent for the transformation (e.g. THF, 1,4-dioxane, EtOAc, DCM, toluene, MeOH, EtOH, DMF, MeCN, water, etc.) with the aid of mechanical agitation (e.g. stirring, shaking, sonication, etc.) followed by purging of the mixture with an inert gas (e.g. nitrogen, argon, etc.). Some descriptions of these techniques can be found in the following references, Gordon, A. J. and Ford, R. A. “The Chemist's Companion”, 1972; Palleros, D. R. “Experimental Organic Chemistry”, 2000; Harwood, L. M., Moody, C. J. and Percy, J. M. “Experimental Organic Chemistry Standard and Microscale, 2ndEdition”, 1999; Landgrebe, J. A. “Theory and Practice in the Organic Laboratory, 4thEdition”, 1993; Leonard, J., Lygo, B. and Procter, G. “Advanced Practical Organic Chemistry, 2ndEdition”, 1998; Meyers, A. G.; Dragovich, P. S.Organic Syntheses,1995, 72, 104; Hajos, Z. G., Parrish, D. R.Organic Syntheses,1985, 63, 26.

PREPARATIONS AND EXAMPLES

All starting materials are commercially available from Sigma-Aldrich (including Fluka and Discovery CPR) unless otherwise noted after the chemical name. Reagent/reactant names given are as named on the commercial bottle or as generated by IUPAC conventions, CambridgeSoft® ChemDraw Ultra 9.0.7, CambridgeSoft® Chemistry E-Notebook 9.0.127, or AutoNom 2000. None of the specific conditions and reagents noted herein is to be construed as limiting the scope of the invention and are provided for illustrative purposes only. All reactions were run under a nitrogen atmosphere.

To a 0° C. mixture of 4-bromo-3-(trifluoromethyl)phenol (Apollo, 10.8 g, 44.8 mmol) in AcOH (40 mL) was added concentrated sulfuric acid (1.5 mL) followed by fuming nitric acid (5.2 g) and the mixture was stirred for 30 min. Additional concentrated sulfuric acid (9 mL) was added and the temperature was allowed to rise to ambient temperature and stirred for 3 h at rt. The mixture was poured into ice water (500 mL) and the aqueous solution was extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4and filtered. The filtrate was evaporated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 10-20% EtOAc in petroleum ether) to give 4-bromo-2-nitro-5-(trifluoromethyl)phenol (4.7 g, 36%) as yellow oil.1H NMR (DMSO-d6, 400 MHz): δ 12.0 (brs, 1H), 8.31 (s, 1H), 7.54 (s, 1H).

To a mixture of zinc powder (16.23 g, 248 mmol) and ammonium chloride (13.28 g, 248 mmol) in MeOH (80 mL) was added 4-bromo-2-nitro-5-(trifluoromethyl)phenol (7.1 g, 24.8 mmol) and the reaction mixture was stirred overnight at ambient temperature. The reaction mixture was filtered and the filtrate was evaporated in vacuo. The residue was partitioned between EtOAc (100 mL) and water (300 mL). The organic phase was separated and washed with brine (30 mL), dried over Na2SO4, filtered and concentrated in vacuo to give 2-amino-4-bromo-5-(trifluoromethyl)phenol as a black oil (5.9 g, 93%), which was used in the next step without further purification.1H NMR (DMSO-d6, 400 MHz): δ 9.89 (brs 1H), 6.98 (s, 1H), 6.92 (s, 1H), 5.53 (brs, 2H).

To a solution of 6-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (Preparation #2, Step A, 5 g, 25.8 mmol) in THF (80 mL) at 0° C. was added methylmagnesium bromide solution (3N in THF, 25.8 mL, 77.4 mmol, 3 equiv) dropwise. The reaction mixture was stirred for 15 min at 0° C. then allowed to warm to ambient temperature and stirred for 1 h. The reaction mixture was cooled to 0° C. and additional methylmagnesium bromide solution (3N in THF, 25.8 mL, 77.4 mmol) was added dropwise. The reaction mixture was allowed to warm to ambient temperature and stirred for an additional 1 h. The reaction mixture was poured into a 0° C. solution of KMnO4(0.270 g) in acetone (4 mL) and water (4 mL) and stirred for 15 min. The reaction mixture was allowed to warm to rt over 15 min. The resulting mixture was filtered and washed with EtOAc (25 mL). The aqueous solution was separated and the organic phase was concentrated in vacuo to give the crude product (0.870 g, crude) as a 1:1 mixture of 6-nitro-4H-benzo[1,4]oxazin-3-one and 7-methyl-6-nitro-4H-benzo[1,4]oxazin-3-one which was used in the next step directly.1H NMR (CDCl3, 400 MHz): δ 8.51 (brs, 1H), 7.56 (s, 1H), 6.84 (s, 1H), 4.65 (s, 2H), 2.52 (s, 3H).

To 7-methyl-6-nitro-4H-benzo[1,4]oxazin-3-one (50% purity; 0.870 g, 1.44 mmol) and K2CO3(1.15 g, 8.36 mmol) in acetone (20 mL) was added 2-bromo-propionic acid ethyl ester (1.5 g, 8.36 mmol) and the mixture was heated at 70° C. for 5 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo and the residue was dissolved in DCM (15 mL). The solution was washed with water (15 mL), dried over anhydrous Na2SO4, filtered and the filtrate was evaporated in vacuo. The residue was purified by chromatography on silica gel (eluting with 20% EtOAc in petroleum ether) to give a mixture of the title compound as a yellow solid (total 0.5 g crude, 48% desired product as indicated by HPLC at 254 or 220 nm), HPLC (Column: Ultimate XB-C18, 3 um, 50×3.0 mm; Mobile phase: MeCN (0.02% TFA) in water (0.04% TFA), from 30% to 90% within 7 min; Flow rate: 1.5 mL/min; Wavelength: 220 nm) Rt=3.42 min.), which was used directly in the next step.

To a solution of 2-(7-methyl-6-nitro-3-thioxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester (0.130 g, 0.40 mmol) in EtOH (8 mL) was added hydrazine hydrate (98%, 1.4 mmol, 0.070 g) and the reaction mixture was heated at 120° C. for 4 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo to give 4,7-dimethyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.085 g, 75%) as yellow solid, which was used directly in the next step. TLC (eluting with 25% EtOAc in petroleum ether) Rf=0.4.

To a solution of 4,7-dimethyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.085 g, 0.31 mmol) and ammonium chloride (0.164 g, 3.1 mmol) in MeOH (10 mL) and THF (10 mL) was added zinc powder (0.200 g, 3.1 mmol) and the resulting mixture was heated at 70° C. for 4 h. The reaction mixture was cooled to ambient temperature, filtered and the filtrate was washed with MeOH (15 mL). The filtrate was concentrated in vacuo and the residue was dissolved in water (20 mL). The aqueous solution was extracted with EtOAc (3×15 mL) and the combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated to give 6-amino-4,7-dimethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.060 g, 0.24 mmol, 79%) as a solid, which was used directly in the next step. TLC (eluting with 25% EtOAc in petroleum ether) Rf=0.2.

A solution of 6-amino-4,7-dimethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one ((Example #54, Step E, 0.060 g, 0.24 mmol) and 3-oxo-azetidine-1-carboxylic acid tert-butyl ester (0.083 g, 0.41 mmol) in MeOH (4.5 mL) and acetic acid (0.5 mL) was stirred at ambient temperature for 1 h. Sodium cyanoborohydride (0.031 g, 0.41 mmol) was added and the resulting dark solution was stirred at ambient temperature for 14 h. The solvent was removed in vacuo to give the crude product, which was purified by preparative TLC (eluting with 33% EtOAc in petroleum ether) to give 3-(4,7-dimethyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.042 g, 44%) as an off-white solid. LC/MS (Table 1, Method 2) Rt=1.065 min.; MS m/z: 424 [M+23]+.

A solution of 3-(4,7-dimethyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.16 g, 0.40 mmol) and paraformaldehyde (neat, 0.024 g, 0.80 mmol) in MeOH (10 mL) and HOAc (1 mL) was heated at 70° C. for 14 h. The mixture was cooled to ambient temperature and sodium cyanoborohydride (0.050 g, 0.80 mmol) was added. The resulting dark solution was stirred at 70° C. for 3 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (eluting with 33-60% EtOAc in petroleum ether) to give 3-[(4,7-dimethyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-methyl-amino]-azetidine-1-carboxylic acid tert-butyl ester as yellow solid (0.081 g, 49%). LC/MS (Table 1, Method 2) Rt=1.168 min.; MS m/z: 416 [M+H]+.

A suspension of 7-fluoro-6-nitro-4H-benzo[1,4]oxazin-3-one (3.50 g, 16.50 mmol) and K2CO3(3.42 g, 24.75 mmol) in acetone (100 mL) was stirred at rt for 15 min. A solution of ethyl 2-bromopropanoate (5.97 g, 33.0 mmol) in acetone (60 mL) was added dropwise and the reaction mixture was heated at reflux for 5 h. The reaction mixture was cooled to ambient temperature and concentrated in vacuo. The residue was dissolved in DCM (50 mL) and washed with water (50 mL). The organic phase was separated and dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 0-30% EtOAc in petroleum ether) to give 2-(7-fluoro-6-nitro-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester as a white solid (3.29 g, 64%).1H NMR (DMSO-d6, 400 MHz): δ 7.89 (d, J=7.2 Hz, 1H), 7.39 (d, J=11.6 Hz, 1H), 5.28 (q, J=7.2 Hz 1H), 4.86 (s, 2H), 4.02-4.16 (m, 2H), 1.48 (d, J=7.2 Hz, 3H), 1.12 (t, J=7.2 Hz, 3H).

A solution of 6-amino-7-fluoro-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.100 g, 0.400 mmol) and tert-butyl 3-oxoazetidine-1-carboxylate (0.171 g, 0.999 mmol) in MeOH (10 mL) and HOAc (1 mL) was stirred at rt for 1 h. Sodium cyanoborohydride (0.176 g, 2.80 mmol) was added and the reaction mixture was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo and the residue was purified by column on silica gel (eluting with 0-10% MeOH in DCM to give 3-(7-fluoro-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.115 g, 71%) as a crude gum, which was used directly in the next step. LC/MS (Table 1, Method 3) Rt=1.374 min.; MS m/z: 428 [M+23]+.

To a suspension of 3-(7-fluoro-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.110 g, 0.271 mmol) in EtOAc (10 mL) was added an EtOAc solution saturated with hydrogen chloride (3 mL). The reaction mixture was stirred at rt for 3 h then concentrated in vacuo and the residue was purified by preparative HPLC (Table 3, Method 1)) to give 6-(azetidin-3-ylamino)-7-fluoro-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloric acid as a white solid (0.026 g, 31%). LC/MS (Table 1, Method 5) Rt=1.853 min.; MS m/z: 306 [M+H]+.

A suspension of 3-(7-isopropenyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (1.15 g, 2.69 mmol) and 10% Pd/C (0.2 g) in MeOH (150 mL) was stirred under H2at 55 PSI overnight. The reaction mixture was filtered through a pad of Celite® and the filtrate was evaporated in vacuo. The residue was purified by preparative HPLC (Table 3, Method 2) to give 3-(7-isopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester as a white solid (0.86 g, 74%). LC/MS (Table 1, Method 2) Rt=1.186 min.; MS m/z: 452.1 [M+23]+.

To a suspension of urea-hydrogen peroxide (5.93 g, 63 mmol) and NaHCO3(5.93 g, 63 mmol) in DCM (60 mL) at 0° C. was added 2-(6-acetyl-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester (6.14 g, 21 mmol). The reaction mixture was stirred for 10 min and TFAA (5.84 mL, 42 mmol) was added dropwise. The reaction mixture was stirred at 0° C. for 1 h and then 3 days at ambient temperature. The reaction mixture was diluted with DCM (60 mL), quenched with saturated Na2S2O3solution (30 mL) and washed with water (30 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give 2-(6-acetoxy-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester, which was used in the next step directly without purification. TLC (eluting with 25% EtOAc/heptane) Rf=0.4.

To a solution of 3-[4-(1-ethoxycarbonyl-ethyl)-3-thioxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-yloxy]-azetidine-1-carboxylic acid tert-butyl ester (0.530 g, 1.22 mmol) in EtOH (10 mL) was added hydrazine hydrate (0.486 g, 9.72 mmol) and the mixture was heated at reflux for 2 h. The reaction mixture was cooled to ambient temperature, concentrated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 20% EtOAc in petroleum ether) to give 3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.234 g, 52%) as a white solid. LC/MS (Table 1, Method 3) Rt=1.332 min.; MS m/z: 389 [M+H]+.

To a solution of 3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.234 g, 0.603 mmol) in DCM (4 mL) and MeOH (2 mL) was added tetrabutylammonium tribromide (0.291 g, 0.603 mmol) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (eluting with 30% EtOAc in petroleum ether) to give 3-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.267 g, 95%). LC/MS (Table 1, Method 2) Rt=1.217 min.; MS m/z: 467/469 [M+H]+.

To a solution of 3-(7-cyclopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.029 g, 0.068 mmol) in DCM (2 mL) was added TFA (0.35 mL) dropwise and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 6-(azetidin-3-yloxy)-7-cyclopropyl-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a white solid (0.020 g, 90%). LC/MS (Table 1, Method 5) Rt=2.056 min.; MS m/z: 329 [M+H]+.

A suspension of 3-(7-isopropenyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.030 g, 0.070 mmol) and 10% Pd/C (10 mg) in MeOH (10 mL) was stirred under H2(50 psi) at ambient temperature overnight. The reaction mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by preparative TLC (eluting with 30% EtOAc in petroleum ether) to give 3-(7-isopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester as a white solid (0.016 g, 53%). LC/MS (Table 1, Method 2) Rt=1.275 min.; MS m/z: 431 [M+H]+.

To a solution of 3-(7-isopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.016 g, 0.067 mmol) in DCM (2 mL) was added TFA (0.35 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 6-(azetidin-3-yloxy)-7-isopropyl-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a pale solid (0.015 g, 51%). LC/MS (Table 1, Method 5) Rt=2.160 min.; MS m/z: 331 [M+H]+.

A suspension of 2-[6-(1-benzhydryl-3-methyl-azetidin-3-yloxy)-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl]-propionic acid ethyl ester (0.300 g, 0.070 mmol) and Pd(OH)2/C (10%, 0.170 g) in EtOH (15 mL) was stirred under H2(50 psi) at ambient temperature overnight. The reaction mixture was filtered and the filtrate was evaporated in vacuo to give 2-[6-(3-methyl-azetidin-3-yloxy)-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl]-propionic acid ethyl ester (0.250 g, crude) as an oil, which was used in the next step directly. LC/MS (Table 1, Method 2) Rt=0.806 min.; MS m/z: 335 [M+H]+.

To a solution of 3-[4-(1-ethoxycarbonyl-ethyl)-3-thioxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-yloxy]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.095 g, 0.211 mmol) in EtOH (5 mL) was added hydrazine hydrate (0.105 g, 2.109 mmol) and the mixture was heated at reflux for 2 h. The reaction mixture was cooled to ambient temperature, concentrated in vacuo and the residue was purified by preparative TLC (eluting with 30% EtOAc in petroleum ether) to give 3-methyl-3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.064 g, 75%) as a white solid. LC/MS (Table 1, Method 2) Rt=1.143 min.; MS m/z: 403 [M+H]+.

To a solution of 3-methyl-3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (0.064 g, 0.159 mmol) in DCM (2 mL) was added TFA (0.5 mL) dropwise and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 4-methyl-6-(3-methyl-azetidin-3-yloxy)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as white solid (0.045 g, 68%). LC/MS (Table 1, Method 5) Rt=1.691 min; MS m/z: 303 [M+H]+.

A solution of 6-(1-benzhydryl-3-methyl-azetidin-3-ylamino)-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.020 g, 0.034 mmol) in DCM (0.5 mL) and TFA (0.25 mL) was stirred at ambient temperature for 1 h. The solvent was removed in vacuo. The residue was dissolved in THF (0.5 mL) and aqueous ammonium hydroxide solution (25%, 0.25 mL) was added. The mixture was stirred for additional 2 h at ambient temperature. The mixture was diluted with water (10 mL) and extracted with EtOAc (3×10 mL). The combined organic phase was washed with brine (10 mL), dried over Na2SO4and filtered. The filtrate was evaporated in vacuo to give crude 6-(1-benzhydryl-3-methyl-azetidin-3-ylamino)-4-methyl-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one as a yellow solid (0.015 g, 84%), which was used in next step directly. LC/MS (Table 1, Method 2) Rt=0.997 min.; MS m/z: 536 [M+H]+.

To a solution of 4-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-piperidine-1-carboxylic acid tert-butyl ester (Example #71, Step B, 0.50 g, 1.249 mmol) in DCM (5 mL) and MeOH (5 mL) was added tetrabutylammonium tribromide (0.662 g, 1.373 mmol) in portions and the mixture was stirred for 30 min at ambient temperature. The reaction was quenched by addition of saturated aqueous Na2S2O3solution (5 mL) and the pH was adjusted to 7 with saturated aqueous NaHCO3solution. The mixture was extracted with DCM (3×20 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 20% EtOAc in petroleum ether) to give 4-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.34 g, 57%). LC/MS (Table 1, Method 2) Rt=1.289 min.; MS m/z: 479/481 [M+H]+.

To a solution of 3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethylene)-azetidine-1-carboxylic acid tert-butyl ester (0.1 g, 0.16 mmol) in MeOH (10 mL) was added Pd/C (10%, 0.02 g) and the mixture was stirred under H2(50 psi) for 14 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo and the residue was purified by preparative TLC (eluting with 50% EtOAc in petroleum ether) to give 3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethyl)-azetidine-1-carboxylic acid tert-butyl ester (0.075 mg, 69%) as a pale yellow powder. LC/MS (Table 1, Method 2) Rt=1.217 min; MS m/z: 409 [M+23]+.

To a mixture of 1-(2-fluoro-4-hydroxy-phenyl)-ethanone (5 g, 32.5 mmol) in concentrated sulfuric acid (50 mL) at −5° C. was added KNO3(3.29 g, 32.6 mmol) in portions and the mixture was stirred for 3 h at −5° C. The mixture was poured into ice water (120 mL) and the aqueous solution was extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4and filtered. The filtrate was evaporated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 2-10% EtOAc in petroleum ether) to give 1-(2-fluoro-4-hydroxy-5-nitro-phenyl)-ethanone as a yellow solid (4.59 g, 71%).1H-NMR (CDCl3, 400 MHz): δ 10.95 (s 1H), 8.79 (d, J=7.2 Hz, 1H), 6.92 (d, J=11.2 Hz, 1H), 2.63 (d, J=5.2 Hz, 3H).

To a solution of 1-(2-fluoro-4-hydroxy-5-nitro-phenyl)-ethanone (3 g, 15.7 mmol) in EtOH (30 mL) and THF (6 mL) was added Pd/C (10%, 1.5 g, 1.4 mmol) and the reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was filtered and the filtrate was evaporated in vacuo to give 1-(5-amino-2-fluoro-4-hydroxy-phenyl)-ethanone as a black solid (2.5 g, crude), which was used in the next step directly without further purification.1H-NMR (DMSO-d6, 400 MHz): δ 7.01 (d, J=8.0 Hz, 1H), 6.48 (d, J=12.4 Hz, 1H), 2.39 (d, J=4.8 Hz, 3H).

A suspension of 2-[7-fluoro-6-(1-benzhydryl-3-methyl-azetidin-3-yloxy)-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl]-propionic acid ethyl ester (0.280 g, 0.540 mmol) and Pd(OH)2/C (10%, 0.140 g, 0.1 mmol) in EtOH (15 mL) was stirred under H2(50 psi) at ambient temperature overnight. The reaction mixture was filtered and the filtrate was evaporated in vacuo to give 2-[7-fluoro-6-(3-methyl-azetidin-3-yloxy)-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl]-propionic acid ethyl ester (0.275 g, crude) as an oil, which was used in the next step directly. LC/MS (Table 1, Method 3) Rt=1.026 min.; MS m/z: 353 [M+H]+.

To a solution of 3-[4-(1-ethoxycarbonyl-ethyl)-7-fluoro-3-thioxo-3,4-dihydro-2H-benzo[1,4]oxazin-6-yloxy]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.080 g, 0.171 mmol) in EtOH (5 mL) was added hydrazine hydrate (0.085 g, 1.707 mmol) and the mixture was heated to reflux for 2 h. The reaction mixture was cooled to ambient temperature and concentrated in vacuo. The residue was purified by preparative TLC (eluting with 30% EtOAc in petroleum ether) to give 3-(7-fluoro-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.033 g, 46%) as a white solid. LC/MS (Table 1, Method 2) Rt=1.144 min.; MS m/z: 421 [M+H]+.

To a solution of 3-(7-fluoro-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.033 g, 0.078 mmol) in DCM (2 mL) was added TFA (0.5 mL) dropwise and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 7-fluoro 4-methyl-6-(3-methyl-azetidin-3-yloxy)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a white solid (0.017 g, 67%). LC/MS (Table 1, Method 5) Rt=1.806 min; MS m/z: 321 [M+H]+.

To a solution of 3-methyl-3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino]-azetidine-1-carboxylic acid tert-butyl ester (0.085 g, 0.142 mmol) in THF (5 mL) was added a solution of TBAF in THF (1M, 0.709 mL, 0.709 mmol). The reaction mixture was heated to reflux overnight. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. The residue was purified by preparative HPLC (Table 3, Method 9) to give 3-methyl-3-(4-methyl-3-oxo-7-trifluoromethyl-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester as a pale yellow solid (0.010 g, 15%). LC/MS (Table 1, Method 5) Rt=1.207 min; MS m/z: 492 [M+23]+.

A solution of 3-methyl-3-(4-methyl-3-oxo-7-trifluoromethyl-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.010 g, 0.021 mmol) in DCM (1 mL) and TFA (0.25 mL) was stirred for 1 h at ambient temperature. The solvent was removed in vacuo and the residue was purified by preparative HPLC (Table 3, Method 10) to give 4-methyl-6-(3-methyl-azetidin-3-ylamino)-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a white solid (0.0045 g, 44%). LC/MS (Table 1, Method 4) Rt=1.441 min; MS m/z: 370 [M+H]+.

To a solution of 3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-azetidine-1-carboxylic acid tert-butyl ester (Example #69, Step E, 0.400 g, 0.994 mmol) in DCM (4 mL) and MeOH (2 mL) was added tetra-N-butylammonium tribromide (0.483 g, 0.994 mmol) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (eluting with 25% EtOAc in petroleum ether) to give 3-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.422 g, 88%). LC/MS (Table 1, Method 2) Rt=1.198 min.; MS m/z: 481/483 [M+H]+

A suspension of 3-(7-isopropenyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.054 g, 0.122 mmol) and Pd/C (10%, 0.015 g) in MeOH (10 mL) was stirred under H2(50 psi) at ambient temperature overnight. The reaction mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by preparative TLC (eluting with 30% EtOAc in petroleum ether) to give 3-(7-isopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester as a white solid (0.049 g, 95%). LC/MS (Table 1, Method 2) Rt=1.327 min.; MS m/z: 467 [M+23]+.

To a solution of 3-(7-isopropyl-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.049 g, 0.110 mmol) in DCM (2 mL) was added TFA (0.35 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo. The residue was purified by preparative HPLC (Table 3, Method 7) to give 7-isopropyl-4-methyl-6-(3-methyl-azetidin-3-yloxy)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a pale solid (0.042 g, 82%). LC/MS (Table 1, Method 5) Rt=2.233 min.; MS m/z: 345 [M+H]+

To a solution of 3-(4,7-dimethyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yloxy)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.024 g, 0.058 mmol) in DCM (2 mL) was added TFA (0.35 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo. The residue was purified by preparative HPLC (Table 3, Method 6) to give 4,7-dimethyl-6-(3-methyl-azetidin-3-yloxy)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as a pale solid (0.008 g, 44%). LC/MS (Table 1, Method 5) Rt=1.913 min.; MS m/z: 317 [M+H]+

A mixture of 6-amino-7-fluoro-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Example #56, Step F, 0.100 g, 0.400 mmol), methanesulfonic acid 1-benzhydryl-3-methyl-azetidin-3-yl ester (Preparation #3, Step B, 0.159 g, 0.480 mmol) and K2CO3(0.166 g, 1.199 mmol) in DMF (2 mL) was stirred at 80° C. overnight. The reaction mixture was cooled to ambient temperature and poured into water (8 mL). The aqueous mixture was extracted with EtOAc (3×10 mL) and the combined organic phase was washed with brine (3×10 mL), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 5-10% EtOAc in petroleum ether) to give 6-(1-benzhydryl-3-methyl-azetidin-3-ylamino)-7-fluoro-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one as a white solid (0.055 g, 28%). LC/MS (Table 1, Method 2) Rt=1.199 min.; MS m/z: 486 [M+H]+.

To a solution of 9-((1-benzhydryl-3-methylazetidin-3-yl)amino)-8-fluoro-1-methyl-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one (0.055 g, 0.113 mmol) in EtOH (4 mL) and THF (1 mL) was added Pd(OH)2/C (10%, 0.100 g, 0.07 mmol) under an atmosphere of argon and the mixture was stirred at ambient temperature for 18 h under an atmosphere of H2(50 psi). The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC (Table 3, Method 5) to give 8-fluoro-1-methyl-9-((3-methylazetidin-3-yl)amino)-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]-triazin-2(1H)-one trifluoroacetic acid as a pale solid (0.015 g, 31%). LC/MS (Table 1, Method 5) Rt=1.472 min; MS m/z: 320 [M+H]+.

To a solution of 3-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (Example 1, Step B, 0.053 g, 0.114 mmol) in DCM (2 mL) was added TFA (0.5 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 6-(azetidin-3-ylamino)-7-bromo-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (0.040 g, 76%) as a white solid. LC/MS (Table 1, Method 5) Rt=1.865 min.; MS m/z: 366 [M+H]+.

To a solution of 2-benzylpiperidin-4-one (BETAPHARMA, 0.868 g, 3 mmol) in anhydrous THF (10 mL) was added LiHMDS (1M solution in THF, 6 mL) dropwise at −78° C. After addition, the mixture was allowed to warm to −30° C. and stirred for 20 min. The mixture was cooled to −78° C. again and 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (2.144 g, 6 mmol) was added. The mixture was allowed to warm to ambient temperature and stirred for 2 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel (eluting with 2% EtOAc in petroleum ether) to give tert-butyl 6-benzyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate as an oil (0.72 g, crude), which was used in the next step without further purification. TLC (eluting with 10% EtOAc/PE) Rf=0.5.

To a solution of tert-butyl 6-benzyl-4-(1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)-5,6-dihydropyridine-1(2H)-carboxylate (0.147 g, 0.3 mmol) in MeOH (30 mL) was added Pd/C (10%, 0.096 g, 0.1 mmol) and the mixture was stirred under an atmosphere of H2(50 psi) for 12 h. After filtration, the filtrate was concentrated in vacuo to give tert-butyl 2-benzyl-4-(1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.147 g), which was used in the next step without further purification. LC/MS (Table 1, Method 2) Rt=2.453 min; MS m/z: 491 [M+H]+.

To a solution of tert-butyl 2-benzyl-4-(1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.147 g, 0.3 mmol) in DCM (2.5 mL) was added TFA (0.5 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and EtOAc (2 mL) was added to the residue. The precipitate was collected by filtration and washed with EtOAc (1 mL) and petroleum ether (2 mL) to give 9-(2-benzylpiperidin-4-yl)-1-methyl-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one trifluoroacetic acid (0.030 g, 20% yield for three steps) as a solid. LC/MS (Table 1, Method 4) Rt=1.651 min; MS m/z: 391 [M+H]+.

A mixture of paraformaldehyde (0.016 g, 0.053 mmol) in methanol (10 mL) and AcOH (1 mL) was heated to reflux overnight. The reaction was cooled to ambient temperature and 4-methyl-6-(3-methyl-azetidin-3-ylamino)-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (Example #79, Step D, 0.020 g, 0.053 mmol) was added into the solution and the mixture was stirred for 2 h. Sodium cyanoborohydride (0.013 g, 0.212 mmol) was added and the mixture was stirred overnight at rt. The solvent was removed in vacuo and the residue was purified by prep-HPLC (Table 3, Method 8) to give 6-[(1,3-dimethyl-azetidin-3-yl)-methyl-amino]-4-methyl-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (0.015 g, 0.036 mmol, 68%). LC/MS (Table 1, Method 4) Rt=1.689 min.; MS m/z: 406 [M+H]+.

6-(Azetidin-3-ylamino)-4-methyl-2,10-dihydro-9-thia-1,2,4a-triaza-phenanthren-3-one hydrochloric acid salt

A mixture of 6-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one (0.512 g, 2.436 mmol), ethyl 2-bromopropanoate (0.573 g, 3.17 mmol) and K2CO3(1.01 g, 7.31 mmol) in acetone (40 mL) was heated at reflux for 16 h. After cooling to ambient temperature, the mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 9% EtOAc in petroleum ether) to give ethyl 2-(6-nitro-3-oxo-2H-benzo[b][1,4]thiazin-4(3H)-yl)propanoate (0.376 g, 50%). TLC (eluting with 20% EtOAc/PE) Rf=0.6.

A mixture of ethyl 2-(6-nitro-3-oxo-2H-benzo[b][1,4]thiazin-4(3H)-yl)propanoate (0.189 g, 0.61 mmol) and Lawesson's reagent (0.493 g, 1.22 mmol) in toluene (40 mL) was heated at reflux for 16 h. After cooling to ambient temperature, the mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 8% EtOAc in petroleum ether) to give ethyl 2-(6-nitro-3-thioxo-2H-benzo[b][1,4]thiazin-4(3H)-yl)propanoate (0.109 g, 55%). TLC (eluting with 25% EtOAc in petroleum ether) Rf=0.3.

To a solution of ethyl 2-(6-nitro-3-thioxo-2H-benzo[b][1,4]thiazin-4(3H)-yl)propanoate (0.475 g, 1.455 mmol) in EtOH (30 mL) was added hydrazine hydrate (0.364 g, 7.28 mmol) and the mixture was then heated at reflux for 16 h. After cooling to ambient temperature gradually, the precipitate was collected by filtration to give 1-methyl-9-nitro-3,5-dihydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]-triazin-2(1H)-one (0.312 g, 77%), which was used in the next step directly without further purification. TLC (eluting with 33% EtOAc in petroleum ether) Rf=0.3.

To a solution of 1-methyl-9-nitro-3,5-dihydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]triazin-2(1H)-one (0.308 g, 1.11 mmol) in MeOH (25 mL) and THF (25 mL) was added powdered zinc (0.724 g, 11.1 mmol) and NH4Cl (0.592 g, 11.1 mmol) and the mixture was heated at reflux for 16 h. After cooling to ambient temperature, the mixture was filtered to give crude 9-amino-1-methyl-3,5-dihydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]triazin-2(1H)-one (100%), which was used in the next step directly without further purification. TLC (eluting with 50% EtOAc/PE) Rf=0.3.

A mixture of 9-amino-1-methyl-3,5-dihydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]triazin-2(1H)-one (0.700 g, 2.82 mmol) and tert-butyl 3-oxoazetidine-1-carboxylate (0.145 g, 8.46 mmol) in MeOH/AcOH (50 mL, v:v=10:1) was stirred for 1 h at ambient temperature. NaBH3CN (0.007 g, 0.121 mmol) was added and the mixture was heated at 60° C. for 2 h. The reaction mixture was cooled to ambient temperature, the solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (eluting with 17% EtOAc in petroleum ether) to give 3-((1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]thiazino[3,4-c][1,2,4]triazin-9-yl)amino)azetidine-1-carboxylate (0.369 g, 32%). TLC (eluting with 50% EtOAc/PE) Rf=0.5.

To a solution of 3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethylene]-azetidine-1-carboxylic acid tert-butyl ester (0.072 g, 0.127 mmol) in CH3OH (10 mL) was added Pd(OH)2/C (0.02 g) and the mixture was stirred under an atmosphere of H2(50 psi) for 14 hr. The reaction mixture was filtered and the filtrate was concentrated to give 3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethyl]-azetidine-1-carboxylic acid tert-butyl ester (0.052 g, 70%) as a pale yellow powder. LC/MS (Table 1, Method 2) Rt=1.502 min.; MS m/z: 607 [M+23]+.

To a solution of 3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethyl]-azetidine-1-carboxylic acid tert-butyl ester (0.052 g, 0.08 mmol) in THF (1 mL) was added TBAF (1M in THF, 1 mL) and the solution was heated to 80° C. for 14 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. The residue was partioned between EtOAc (5 mL) and water (5 mL). The aqueous phase was extracted with EtOAc (3×10 mL). The combined organic portion was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (eluting with 50% EtOAc in petroleum ether) to give 3-(4-methyl-3-oxo-7-trifluoromethyl-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylmethyl)-azetidine-1-carboxylic acid tert-butyl ester (0.019 g, 44%). LC/MS (Table 1, Method 2) Rt=1.230 min.; MS m/z: 477 [M+23]+.

To a solution of 3-methyl-3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino]-azetidine-1-carboxylic acid tert-butyl ester (4 g, 6.67 mmol) in THF (10 mL) was added a solution of TBAF (1M in THF, 80 mL, 80 mmol) The reaction mixture was heated at 80° C. for 15 h. The reaction mixture was cooled to ambient temperature and solvent was removed in vacuo. The residue was purified by preparative HPLC (Table 3, Method 17) and further separated by chiral SFC (Table 2, Method 10) to give (R)-tert-butyl 3-methyl-3-((1-methyl-2-oxo-8-(trifluoromethyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)amino)azetidine-1-carboxylate (Enantiomer 1, SFC (Table 1, Method 16) Rt: 6.686 min, 0.600 g, 19%) as a white solid, LC/MS (Table 1, Method 2) Rt=1.192 min.; MS m/z: 492 [M+23]+.

To a solution of tert-butyl 3-methyl-3-(methyl(1-methyl-2-oxo-8-(trifluoromethyl)-3-((2-(tri methylsilyl)ethoxy)methyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)amino)azetidine-1-carboxylate (0.586 g, 0.360 mmol) in DCM (4 mL) was added TFA (0.8 mL) and the mixture was stirred at ambient temperature for 1.5 h. The solvent was removed in vacuo and dioxane (1 mL) and ammonium hydroxide (25%, 1 mL) were added and the reaction mixture was stirred for 1.5 h at ambient temperature. The solvent was removed in vacuo and the residue was purified by HPLC (Table 3, Method 19) and further separated by chiral SFC (Table 2, Method 12) to give (R)-1-methyl-9-(methyl(3-methylazetidin-3-yl)amino)-8-(trifluoromethyl)-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one trifluoroacetic acid (SFC (Table 1, Method 17) Rt: 6.57 min, 0.033 g, 7%)) as a pale solid. LC/MS (Table 1, Method 5) Rt=2.319 min.; MS m/z: 384 [M+H]+.

To a mixture of (E)-tert-butyl 3-((8-(2-ethoxyvinyl)-1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)amino)azetidine-1-carboxylate (0.020 g, 0.044 mmol) in THF (5 mL) was added aqueous HCl (1N, 1 mL) and the reaction mixture was stirred at rt overnight. The solvent was removed in vacuo to give the residue, which was purified by prep-HPLC (Table 3, Method 14) to give 10-(azetidin-3-yl)-1-methyl-5,10-dihydro-1H-[1,2,4]-triazino[4′,3′:4,5]oxazino[2,3-f]indol-2(3H)-one trifluoroacetic acid (0.0052 g, 38%) as a pale yellow powder. LC/MS (Table 1, Method 5) Rt=1.748 min.; MS m/z: 312 [M+H]+.

To a solution of 6-(1-benzhydryl-3-methyl-azetidin-3-ylamino)-4-methyl-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Example #79, Step C, 0.10 g, 0.18 mmol) in THF (20 mL), MeOH (20 mL) and aqueous HCl (12N, 0.3 mL) was added Pd(OH)2/C (10%, 0.02 g, 0.014 mmol) and the solution was heated at 50° C. under an atmosphere of H2(55 psi) for 4 h. The reaction mixture was cooled to ambient temperature and filtered. The filtrate was evaporated in vacuo to give 4-methyl-6-(3-methyl-azetidin-3-ylamino)-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.095 g, 57% pure, 82%), which was used in the next step directly without further purification. LC/MS (Table 1, Method 2) Rt=0.890 min; MS m/z: 378 [M+H]+.

A solution of 4-methyl-6-(3-methyl-azetidin-3-ylamino)-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.095 g, 57% purity, 0.15 mmol) and paraformaldehyde (0.015 g, 0.45 mmol) in MeOH (10 mL) and AcOH (1 mL) was heated at 80° C. for 2 h. NaBH3CN (0.019 g, 0.30 mmol) was added and the reaction mixture was stirred at 80° C. for 0.5 h. The reaction mixture was cooled to ambient temperature. The solvent was removed in vacuo and the residue was washed with aqueous saturated Na2CO3solution (2 mL). The aqueous phase was extracted with EtOAc (4×20 mL). The combined organic layer was dried, filtered and concentrated in vacuo to give crude 6-(1,3-dimethyl-azetidin-3-ylamino)-4-methyl-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.060 g, 78% over two steps), which was used in the next step directly. LC/MS (Table 1, Method 2) Rt=0.924 min; MS m/z: 392 [M+H]+. A solution of 6-(1,3-dimethyl-azetidin-3-ylamino)-4-methyl-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.060 g, 0.15 mmol) in ammonium hydroxide (25%, 5 mL) and dioxane (5 mL) was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and the residue was purified by preparative HPLC (Table 3, Method 16) to give 6-(1,3-dimethyl-azetidin-3-ylamino)-4-methyl-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (0.022 g, 27%). LC/MS (Table 1, Method 4) Rt=1.722 min; MS m/z: 392 [M+H]+.

A mixture of 7-fluoro-4-methyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.30 g, 1.07 mmol), K2CO3(0.296 g, 2.14 mmol) and phenol (0.151 g, 1.61 mmol) in acetone (10 mL) was stirred at ambient temperature for 48 h and then heated to reflux for 3 h. After cooling to ambient temperature, the solvent was removed in vacuo and the residue was partitioned between EtOAc (30 mL) and water (10 mL). The organic portion was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by chromatography on silical gel (eluting with 15% EtOAc in petroleum ether) to give 4-methyl-6-nitro-7-phenoxy-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.090 g, 24%) as an orange solid. LC/MS (Table 1, Method 2) Rt=1.117 min; MS m/z: 355 [M+H]+

A mixture of 4-methyl-6-nitro-7-phenoxy-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.090 g, 0.25 mmol) and Raney Ni (0.030 g) in MeOH (5 mL) was stirred overnight under an atmosphere of H2(30 psi) at ambient temperature. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give 6-amino-4-methyl-7-phenoxy-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one as a colorless oil (0.090 g, crude), which was used in next step without further purification. LC/MS (Table 1, Method 2) Rt=0.938 min; MS m/z: 325 [M+H]+

A mixture of 6-bromo-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Example #53, Step E, 0.610 g, 1.234 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.627 g, 2.468 mmol), Pd(dppf)Cl2(0.180 g, 0.405 mmol) and KOAc (0.303 g, 3.08 mmol) in dioxane (20 mL) was stirred at 90° C. overnight. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (eluting with 5-10% EtOAc in petroleum ether) to give 4-methyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.500 g, crude) as a white solid containing 20% of the de-Br byproduct and was used in the next step directly. LC/MS (Table 1, Method 2) Rt=1.499 min.; MS m/z: 564 [M+23]+.

A mixture of 6-hydroxy-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.055 g, 0.127 mmol), 1-benzhydryl-3-methylazetidin-3-ylmethanesulfonate (Preparation #3, Step B, 0.084 g, 0.255 mmol) and Cs2CO3(0.083 g, 0.255 mmol) in DMF (2 mL) was stirred at 80° C. overnight. The reaction mixture was cooled to ambient temperature and poured into water (150 mL). The aqueous mixture was extracted with EtOAc (3×20 mL) and the combined organic phase was washed with brine (3×20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 5-10% EtOAc in petroleum ether) to give 6-(1-benzhydryl-3-methyl-azetidin-3-yloxy)-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.031 g, 36%) as an oil. LC/MS (Table 1, Method 2) Rt=1.024 min.; MS m/z: 667 [M+H]+.

A suspension of 6-(1-benzhydryl-3-methyl-azetidin-3-yloxy)-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.031 g, 0.046 mmol) and Pd(OH)2/C (10%, 0.030 g) in EtOH (10 mL) was stirred at 50° C. under an atmosphere of H2(50 psi) overnight. The reaction mixture was cooled to ambient temperature and filtered. The filtrate was evaporated in vacuo to give 4-methyl-6-(3-methyl-azetidin-3-yloxy)-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.021 g crude) as an oil, which was used directly without purification. To a solution of 4-methyl-6-(3-methyl-azetidin-3-yloxy)-7-trifluoromethyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.021 g, 0.042 mmol) in DCM (3 mL) was added TFA (1 mL). The reaction was stirred at ambient temperature for 1.5 h then the solvent was removed in vacuo and the residue was diluted with dioxane (1 mL). Aqueous ammonium hydroxide (25%, 1 mL) was added and the reaction mixture was stirred at ambient temperature for 1.5 h. The solvent was removed in vacuo and the residue was purified by preparative HPLC (Table 3, Method 15) to give 4-methyl-6-(3-methyl-azetidin-3-yloxy)-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (0.0039 g, 25%). LC/MS (Table 1, Method 5) Rt=2.088 min; MS m/z: 371 [M+H]+.

A suspension of Pd(OH)2/C (10%, 3.0 g) and (E)-ethyl 3-(2,4-dinitrophenyl)acrylate (12.0 g, 45.1 mmol) in MeOH (80 mL) was stirred at 25° C. under an atmosphere of H2(45 psi) for 15 h. The mixture was filtered and the filtrate was concentrated in vacuo to give 3-(2,4-diamino-phenyl)-propionic acid ethyl ester (8.6 g, 98%), which was used directly in the next step without further purification. A solution of ethyl 3-(2,4-diaminophenyl)propanoate (8.6 g, 41.3 mmol) in EtOH (50 mL) was stirred at 85-100° C. for 48 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. EtOAc (10 mL) was added to the residue and the precipitate was collected by filtration to give 7-amino-3,4-dihydro-1H-quinolin-2-one (5.8 g, 87%).1H NMR (DMSO-d6, 400 MHz): δ 9.83 (brs, 1H), 6.76 (d, J=7.6 Hz, 1H), 6.12 (m, 2H), 4.95 (brs, 2H), 2.65 (t, J=7.2 Hz, 2H), 2.36 (t, J=7.2 Hz, 2H).

To a solution of HCl in EtOAc (4M, 5 mL, 20 mmol) was added slowly tert-butyl-3-(1-methyl-2-oxo-2,3,5,6-tetrahydro-1H-[1,2,4]triazino[4,3-a]quinolin-9-ylamino)azetidine-1-carboxylate (0.022 g, 0.057 mmol) and the reaction mixture was stirred at ambient temperature for 2 h. The solution was concentrated in vacuo to give 9-(azetidin-3-ylamino)-1-methyl-5,6-dihydro-1H-[1,2,4]triazino[4,3-a]quinolin-2(3H)-one hydrochloric acid (0.015 g, 95%). LC/MS (Table 1, Method 5) Rt=2.252 min; MS m/z: 286 [M+H]+.

To a solution of (R)-tert-butyl 2-benzyl-4-oxopiperidine-1-carboxylate (enantiomer 2, SFC (Table 1, Method 19) Rt=3.17 min, 0.579 g, 2 mmol) in anhydrous THF (15 mL) was added LiHMDS solution (1M, 3 mL, 3 mmol) dropwise at −78° C. The mixture was allowed to warm to −30° C. and stirred for 20 min then cooled to −78° C. 1,1,1-Trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (1.072 g, 3 mmol) was added. The mixture was allowed to warm to rt and stirred for 2 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel (eluting with 2% EtOAc in petroleum ether) to give (R)-tert-butyl 6-benzyl-4-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydropyridine-1(2H)-carboxylate as oil (0.9 g, 99%). TLC (eluting with 10% EtOAc in petroleum ether) Rf=0.4.

To a solution of (R)-tert-butyl 6-benzyl-4-((R)-1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)-5,6-dihydropyridine-1(2H)-carboxylate (0.330 g, 0.675 mmol) in MeOH (20 mL) was added Pd/C (10%, 0.033 g, 0.028 mmol) and the mixture was stirred under an atmosphere of H2(50 psi) at 50° C. for 12 h. The reaction mixture was cooled to ambient temperature and filtered. The filtrate was concentrated in vacuo to give crude (2S)-tert-butyl 2-benzyl-4-((R)-1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.320 g). LC/MS (Table 1, Method 4) Rt=2.813 min; MS m/z: 513 [M+23]+.

To a solution of (2S,4R)-tert-butyl 2-benzyl-4-((R)-1-methyl-2-oxo-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.190 g, 0.387 mmol) in DCM (3 mL) was added TFA (1 mL) and the mixture was stirred at ambient temperature for 1 h. The solvent was removed in vacuo and EtOAc (2 mL) was added to the residue. The precipitate was collected by filtration and washed with EtOAc (1 mL) and petroleum ether (2 mL) to give (R)-9-((2S,4R)-2-benzylpiperidin-4-yl)-1-methyl-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one trifluoroacetic acid (Example #109-2, Enantiomer 2, SFC (Table 1, Method 21) Rt=4.521 min, 0.190 g, 97%) as a solid. LC/MS (Table 1, Method 4) Rt=1.617 min; MS m/z: 391 [M+H]+.

A mixture of crude {1-[4-methyl-3-oxo-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl]-pyrrolidin-2-ylmethyl}-carbamic acid tert-butyl ester (0.117 mol) in DCM (2 mL) and TFA (1 mL) was stirred for 1 h at ambient temperature. The solvent was removed in vacuo and the residue was re-dissolved in MeOH (2 mL) and aqueous ammonium hydroxide (25%, 0.5 mL). The mixture was stirred for 2 h then purified by preparative HPLC (Table 3, Method 20) to give 6-(2-aminomethyl-pyrrolidin-1-yl)-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid as white solid (0.0068 g, 13% for 2 steps). LC/MS (Table 1, Method 4) Rt=1.111 min; MS m/z: 316 [M+H]+

To a −15° C. solution of 2-((tert-butoxycarbonyl)amino)propanoic acid (2.5 g, 13.21 mmol) in THF (66 mL) was added Et3N (1.337 g, 13.21 mmol) and ethyl carbonochloridate (1.434 g, 13.21 mmol). The reaction mixture was stirred for 15 min then allowed to warm to 0° C. A solution of diazomethane (prepared according to the standard procedure (Organic Syntheses, Coll. Vol. 5, p. 351 (1973); Vol. 41, p. 16 (1961)) in Et2O (150 mL) was added, until the rich yellow color persisted. The reaction mixture was allowed to warm to rt and stirred for 3 h. Excess CH2N2was destroyed by the addition of a small amount of aqueous AcOH (20%, 3 mL). The reaction mixture was extracted with saturated NaHCO3(20 mL), and the organic portion was separated and washed with saturated aqueous NH4Cl (20 mL) and brine (20 mL). The organic portion was separated, dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 5-20% EtOAc in petroleum ether) to give (3-diazo-1-methyl-2-oxo-propyl)-carbamic acid tert-butyl ester (2.8 g, 99%) as a brown solid.1H NMR (DMSO-d6, 400 MHz): δ 7.27 (d, J=7.2, 1H), 6.02 (s, 1H), 4.01 (m, 1H), 1.39 (s, 9H), 1.20 (m, 3H).

(3-Diazo-1-methyl-2-oxo-propyl)-carbamic acid tert-butyl ester (0.250 g, 1.17 mmol) was dissolved in anhydrous DCM (6 mL) and Et3N (1.18 mg, 0.012 mmol) was added. The reaction mixture was cooled to 0° C. and rhodium (II) acetate (0.010 g, 0.023 mmol) was added. The reaction mixture was stirred at 0° C. for 1 h then concentrated in vacuo to give 2-methyl-3-oxo-azetidine-1-carboxylic acid tert-butyl ester, which was used in the next step without further purification. TLC (eluting with 20% EtOAc/heptane) Rf=0.3.

To a solution of 2-methyl-3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.016 g, 0.040 mmol) in DCM (2 mL) was added TFA (0.4 mL) and the mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo to give 4-methyl-6-(2-methyl-azetidin-3-ylamino)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one trifluoroacetic acid (0.012 g, 100%) LC/MS (Table 1, Method 5) Rt=1.582 min.; MS m/z: 302 [M+H]+.

To a solution of 4-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-piperidine-1-carboxylic acid tert-butyl ester (Example #71, Step B, 0.50 g, 1.249 mmol) in DCM (5 mL) and MeOH (5 mL) was added tetrabutylammonium tribromide (0.662 g, 1.373 mmol) portionwise and the reaction mixture was stirred for 30 min at ambient temperature. Saturated aqueous Na2S2O3solution (5 mL) was added and the pH was adjusted to 7 by the addition of saturated aqueous NaHCO3solution. The mixture was extracted with DCM (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 20% EtOAc in petroleum ether) to give 4-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.34 g, 57%). LC/MS (Table 1, Method 2) Rt=1.289 min.; MS m/z: 479/481 [M+H]+.

To a solution of tert-butyl 4-(1-methyl-2-oxo-8-(trifluoromethyl)-3-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)-5,6-dihydropyridine-1(2H)-carboxylate (0.07 g, 0.117 mmol) in MeOH (15 mL) was added Pd/C (10%, 0.010 g) and the reaction mixture was stirred under an atmosphere of H2(1 atm) for 20 h. The reaction mixture was filtered, washing with methanol (3×5 mL). The filtrate was concentrated in vacuo to give tert-butyl 4-(1-methyl-2-oxo-8-(trifluoromethyl)-3-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.07 g, 100%), which was used directly in the next step.

To a solution of tert-butyl 4-(1-methyl-2-oxo-8-(trifluoromethyl)-3-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.07 g, 0.117 mmol) in THF (2 mL) was added a solution of TBAF in THF (1M, 0.58 mL, 0.585 mmol) and the reaction mixture was heated at reflux for 24 h. The reaction mixture was cooled to rt and the solvent was removed in vacuo. The residue was purified by preparative HPLC (Table 3, Method 23) to give tert-butyl 4-(1-methyl-2-oxo-8-(trifluoromethyl)-1,2,3,5-tetrahydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-9-yl)piperidine-1-carboxylate (0.04 g, 73%). LC/MS (Table 1, Method 3) Rt=1.665 min; MS m/z: 469 [M+H]+.

Step C 1-Methyl-9-(piperidin-4-yl)-8-(trifluoromethyl)-3,5-dihydrobenzo[5,6][1,4]oxazino[3,4-c][1,2,4]triazin-2(1H)-one trifluoroacetic acid

A solution of 7-methoxy-4-methyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (prepared from 2-amino-5-methoxyphenol using the similar procedure detailed in Example #56, Step A-D and Example #106, Step A, 1 g, 3.42 mmol) and lithium chloride (0.44 g, 10.27 mmol) in DMF (10 mL) was heated at 150° C. for 3 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (eluting with 50% EtOAc in petroleum ether) to give 7-hydroxy-4-methyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.65 g, 68%) as a red solid. TLC (eluting with 50% EtOAc in petroleum ether) Rf=0.5.

To a solution of 7-benzyloxy-4-methyl-6-nitro-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.14 g, 0.38 mmol) in MeOH (10 mL) was added Raney-Ni (0.03 g). Then the mixture was stirred under an atmosphere of H2(1 atm) at rt for 2 h. The reaction mixture was filtered and washed with 10% MeOH/DCM (3×10 mL). The filtrate was concentrated in vacuo to give 6-amino-7-benzyloxy-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.065 g, 54%), which was used in the next step directly without further purification. TLC (eluting with 50% EtOAc in petroleum ether) Rf=0.3.

A solution of 6-amino-7-benzyloxy-4-methyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.065 g, 0.19 mmol) and tert-butyl 3-oxoazetidine-1-carboxylate (0.061 g, 0.36 mmol) in MeOH (10 mL) and AcOH (1 mL) was heated at 70° C. for 14 h. The reaction mixture was cooled to ambient temperature and sodium cyanoborohydride (0.022 g, 0.36 mmol) was added. The reaction mixture was heated at 70° C. for 1 hr then cooled to ambient temperature. The solvent was removed in vacuo and the residue was purified by preparative TLC (eluting with 50% EtOAc in petroleum ether) to give 3-(7-benzyloxy-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-azetidine-1-carboxylic acid tert-butyl ester (0.028 g, 32%). LC/MS (Table 1, Method 2) Rt=1.205 min; MS m/z: 516 [M+23]+

TABLE 15The following analogs were prepared from (R)-4-methyl-7-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Preparation#10, Step F) using the procedure detailed in Example #130, Steps A-B & Step D.m/zExampleRtminESI+Structure#Halogen(method)(M + H)+1311.792 (Table 1, Method 4)412132(See Preparation #12, Step B for intermediate synthesis)2.454 (Table 1, Method 5)446

Using a similar procedure as detailed in Example #133, Steps E and G followed by Example #134, Step A, and Preparation #14, Step A, (R)-6-[(R)-1-(1,3-dimethyl-azetidin-3-yl)-ethyl]-7-[2-(1-hydroxy-1-methyl-ethyl)-phenyl]-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one was prepared from 3-[(S)-1-((R)-7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-ethyl]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (Example #133, Step D) and (2-acetylphenyl)boronic acid. LC/MS (Table 1, Method 4) Rt=1.542 min.; MS m/z: 463 [M+H]+.

TABLE 17The following analogs were prepared from (R)-7-(2-fluoro-phenyl)-4-methyl-6-[(R)-1-(3-methyl-azetidin-3-yl)-ethyl]-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-onehydrochloric acid (Example #146, Step B) using the similar procedure detailed in Example#134, Step A. In cases where a TBS group was present, the TBS group was also removedunder the reaction conditions.m/zExampleRtminESI+Structure#Reagent(method)(M + H)+1411.869 (Table 1, Method 4)4531421.973 (Table 1, Method 4)5071441.785 (Table 1, Method 4)4791451.817 (Table 1, Method 4)467

To a solution of HCl (4M in EtOAc, 160 mL) was added 3-[(R)-7-((E)-2-ethoxy-vinyl)-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (16 g, 33.9 mmol) at 0° C. and the reaction mixture was stirred for 30 min. The reaction mixture was concentrated in vacuo to give crude (R)-1-methyl-10-(3-methyl-azetidin-3-yl)-3,5-dihydro-10H-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-2-one hydrochloride (10 g, 91%), which was used in the next step without further purification. LC/MS (Table 1, Method 25) Rt=0.627 min; MS m/z: 326 [M+H]+

TABLE 19The following analog was prepared from 6-amino-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Preparation #2, Step E) and tert-butyl 3-oxopiperidine-1-carboxylate using the similar procedure detailed in Example #151, Steps A-D without thechiral separation in Step Am/zExampleRtminESI+Structure#Reagent(method)(M + H)+1521.3 (Table 1, Method 4)340

A mixture of 3-((R)-8-iodo-1-methyl-2-oxo-1,2,3,5-tetrahydro-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-10-yl)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.3 g, 0.544 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (0.838 g, 5.44 mmol), K2CO3(0.15 g, 1.088 mmol) and Pd(dppf)Cl2.CH2Cl2(0.044 g, 0.054 mmol) in dioxane (6 mL) and water (1 mL) was heated at 68° C. for 14 h. The reaction mixture was cooled to ambient temperature and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (eluting with 10%˜100% EtOAc in petroleum ether) to give 3-methyl-3-((R)-1-methyl-2-oxo-8-vinyl-1,2,3,5-tetrahydro-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-10-yl)-azetidine-1-carboxylic acid tert-butyl ester (0.22 g, 90%) as yellow gum, which was used directly in the next step. LC/MS (Table 1, Method 25) Rt=0.892 min.; MS m/z: 452 [M+H]+.

TABLE 20The following analog was prepared from 3-((R)-8-iodo-1-methyl-2-oxo-1,2,3,5-tetrahydro-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-10-yl)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (Example #156, Step A) and methylboronicacid using the procedure detailed in Example #156, Steps B & D.m/zExampleRtminESI+Structure#Reagent(method)(M + H)+1571.502 (Table 1, Method 4)340

To a solution of 1-methyl-10-(3-methyl-azetidin-3-yl)-3,5,9,10-tetrahydro-8H-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-2-one 2,2,2-trifluoroacetate (Example #158, 0.65 g, crude, 1.985 mmol) in DCM (15 mL) was added TEA (0.603 g, 5.96 mmol), DMAP (0.024 g, 0.199 mmol) and BOC2O (0.867 g, 3.97 mmol). The resultant mixture was stirred at ambient temperature for 3 h. Water (2×10 mL) was added and the reaction mixture was extracted with EtOAc (2×40 mL). The combined organic phase was washed with brine (20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was dried in vacuo and the residue was purified by column chromatography on silica gel (eluting with 10%-50% EtOAc in petroleum ether) to give racemic 3-methyl-3-(1-methyl-2-oxo-1,2,3,5,8,9-hexahydro-6-oxa-3,4,10,11b-tetraaza-cyclopenta[b]phenanthren-10-yl)-azetidine-1-carboxylic acid tert-butyl ester (0.43 g, 1.006 mmol). LC/MS (Table 1, Method 25) Rt=0.823 min; MS m/z: 428 [M+1]+.

The above compound was separated by Chiral SFC (Table 2, Method 25) to give two isomers.

(R)-3-(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-3-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester was separated by SFC (Table 2, Method 25) to give two isomers.

A solution of 3-[7-(2-fluoro-phenyl)-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino]-3-methyl-azetidine-1-carboxylic acid tert-butyl estercarboxylate (0.27 g, 0.545 mmol) in TFA (1 mL) and DCM (4 mL) was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and the residue was dissolved in MeOH (10 mL). Then, aqueous solution of ammonium (5 mL, 25%) was added and the mixture was concentrated in vacuo to give the crude 7-(2-fluoro-phenyl)-4-methyl-6-(3-methyl-azetidin-3-ylamino)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (0.4 g), which was used in the next step directly. LC/MS (Table 1, Method 4) Rt=1.570 min.; MS m/z: 396 [M+H]+.

To a solution of 3-{[(R)-7-(2-fluoro-phenyl)-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl]-methyl-amino}-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.250 g, 0.491 mmol) in EtOAc (2 mL) was added HCl (4M in EtOAc, 3 mL). The reaction mixture was stirred at ambient temperature for 30 min and then concentrated in vacuo to give (R)-7-(2-fluoro-phenyl)-4-methyl-6-[methyl-(3-methyl-azetidin-3-yl)-amino]-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride acid which was used directly in the next step without further purification. LC/MS (Table 1, Method 25), Rt=0.722 min., MS m/z: 410 [M+H]+

TABLE 25The following analogs were prepared from (R)-7-(2-fluoro-phenyl)-4-methyl-6-[methyl-(3-methyl-azetidin-3-yl)-amino]-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride acid (Example #183, Step B) using the procedure detailedin Example #138. In cases where a TBS group was present, the TBS group was alsoremoved under the reaction conditions.m/zExampleRtminESI+Structure#Reagent(method)(M + H)+1911.816 (Table 1, Method 4)4541922.504 (Table 1, Method 4)4681932.065 (Table 1, Method 4)5061941.595 (Table 1, Method 4)466

TABLE 25bThe following analogs were prepared from (R)-7-(2-hydroxymethyl-phenyl)-4-methyl-6-[methyl-(3-methyl-azetidin-3-yl)-amino]-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride acid (prepared from 3-[((R)-7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-methyl-amino]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester) and(2-(hydroxymethyl)phenyl)boronic acid using the similar procedure detailed inExample #183, Steps A-B) using the procedure detailed in Example #138, Step A.In cases where a TBS group was present, the TBS group was also removedunder the reaction conditions.m/zExampleRtminESI+Structure#Reagent(method)(M + H)+1951.515 (Table 1, Method 4)466

TABLE 25cThe following analog was prepared from 4-methyl-6-[methyl-(3-methyl-azetidin-3-yl)-amino]-7-phenyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-onehydrochloride acid (prepared from 3-[(7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-methyl-amino]-3-methyl-azetidine-1-carboxylic acid tert-butyl ester) and phenylboronic acid using the similar procedure detailed in Example #183,Steps A-B) using the procedure detailed in Example #138, Step A.m/zExampleRtminESI+Structure#Reagent(method)(M + H)+1961.72 (Table 1, Method 4)448

A mixture of 3-methyl-3-[4-methyl-3-oxo-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester (prepared using a similar procedure as detailed in Example #162, Step C from 3-amino-3-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester, 1.1 g, 2.016 mmol) in a solution of TBAF in THF (1 M, 20 mL, 20 mmoL) was stirred at 80° C. for 16 h. The reaction mixture was cooled to ambient temperature and diluted with water (25 mL). The aqueous solution was extracted with EtOAc (3×25 mL) and the combined organic phase was washed with brine (10 mL), dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 10% EtOAc in petroleum) to give 3-methyl-3-(4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-ylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester (0.45 g, 54%). LC/MS (Table 1, Method 2) Rt=0.890 min; MS m/z: 438 [M+Na]+.

Chiral SFC (Table 2, Method 24) separation gave the following four isomers.

TABLE 26The following analogs were prepared from (3R,4R)-4-((R)-7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-3-methyl-piperidine-1-carboxylic acid tert-butylester (Example #199, Step D) using the procedure detailed in Example #76, Steps A-C.m/zExampleRtminESI+Structure#Boronate(method)(M + H)+2021.577 (Table 1, Method 4)357

TABLE 28aThe following analog was prepared from (R)-7-(2-fluoro-phenyl)-4-methyl-6-((3R,4R)-3-methyl-piperidin-4-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride acid (Example #199, Step F) using the procedure detailed in Example #138.The TBS group was also removed under the reaction conditions.m/zExampleRtminESI+Structure#PiperidineAldehyde(method)(M + H)+2062.035 (Table 1, Method 44)453

TABLE 28The following analog was prepared from (R)-7-(2-fluoro-phenyl)-4-methyl-6-((3S,4S)-3-methyl-piperidin-4-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride acid (Example #203, Step D) using the procedure detailed in Example #138.The TBS group was also removed under the reaction conditions.m/zExampleRtminESI+Structure#PiperidineAldehyde(method)(M + H)+2071.683 (Table 1, Method 4)453

TABLE 29aThe following analog was prepared from 4-((R)-7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-piperidine-1-carboxylic acid tert-butyl ester (Preparation #8,Step B) using the procedure detailed in Example #199, Steps E-F and Example #76, Step B.m/zExampleRtminESI+Structure#Boronate(method)(M + H)+2081.585 (Table 1, Method 4)343

Step C

TABLE 30The following analog was prepared from (R)-4-methyl-7-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (Preparation#10, Step F), using the procedure detailed in Example #212, Steps A-E.m/zExampleRtminESI+Structure#Halogen(method)(M + H)+2132.415 (Table 1, Method 4)413

A solution of (3R,4R)-4-((R)-7-bromo-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl)-3-methyl-piperidine-1-carboxylic acid tert-butyl ester (Example #199, Step D, 3 g, 6.08 mmol) in HCl (4M in EtOAc, 10 mL) was stirred at ambient temperature for 1 h. The organic solvent was removed in vacuo to give crude (R)-7-bromo-4-methyl-6-((3R,4R)-3-methyl-piperidin-4-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride (2.2 g, 82%) as a white solid, which was used directly in the next step. LC/MS (Table 1, Method 25) Rt=0.720 min.; MS m/z: 393 [M+H]+& 395 [M+H+2]+.

A solution of (3R,4R)-4-[(R)-7-(2,6-difluoro-phenyl)-4-methyl-3-oxo-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl]-3-methyl-piperidine-1-carboxylic acid tert-butyl ester (0.005 g, 0.01 mmol) in HCl (4M in EtOAc, 1 mL) was stirred at ambient temperature for 1 h. The solvent was removed in vacuo to give crude (R)-7-(2,6-difluoro-phenyl)-4-methyl-6-((3R,4R)-3-methyl-piperidin-4-yl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one hydrochloride (0.005 g, 100%), which was used directly in the next step without purification. LC/MS (Table 1, Method 25) Rt=0.717 min.; MS m/z: 427 [M+H]+.

To a solution of (2S,5R)-2,5-dimethyl-4-[4-methyl-3-oxo-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl]-piperazine-1-carboxylic acid tert-butyl ester (1.39 g, 2.483 mmol) in DCM (10 mL) and MeOH (10 mL) was added a solution of n-Bu4NBr3(1.197 g, 2.483 mmol) in DCM (10 mL) drop-wise and the reaction mixture was stirred at ambient temperature for 1 h. Aqueous saturated Na2S2O3solution (10 mL) was added followed by addition of aqueous NaHCO3solution to adjust the pH to 8. The reaction mixture was extracted with EtOAc (3×30 mL) and the combined organic phase was washed with brine (3×30 mL) and dried over anhydrous Na2SO4. The mixture was concentrated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 0-20% EtOAc in petroleum ether) to give (2S,5R)-4-[7-bromo-4-methyl-3-oxo-2-(2-trimethylsilanyl-ethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-phenanthren-6-yl]-2,5-dimethyl-piperazine-1-carboxylic acid tert-butyl ester (0.422 g, 27%) as a solid. LC/MS (Table 1, Method 25) Rt=1.159 min.; MS m/z: 638 [M+H]+& 640 [M+H+2]+.

TABLE 33bThe following analog was prepared from 6-bromo-4-methyl-2-(2-trimethylsilanyl-ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one(Preparation #1, Step E) using the similar procedure detailed in Example #246, Steps A-G.m/zExamplePiperazineRtminESI+Structure#(Step A)(method)(M + H)+2471.777 (Table 1, Method 4)438

To a solution of 6-amino-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (5.0 g, 21.5 mmol) in MeOH (20 mL) and DCM (40 mL) was added tetrabutylammonium tribromide (10.9 g, 22.6 mmol) portionwise and the resulting reaction mixture was stirred for 0.5 h at 25° C. The reaction was quenched by addition of saturated aqueous sodium thiosulphate (10 mL). The mixture was neutralized by the addition of saturated aqueous NaHCO3, diluted with water (100 mL) and extracted with DCM (3×50 mL). The combined organic layer was concentrated, washed with brine (3×30 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product, which was purified by column chromatography on silica gel (eluting with 5% MeOH in DCM) to give 6-amino-7-bromo-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (3.0 g, 46%) as a brown solid.1H NMR (DMSO-d6, 400 MHz): δ 10.78 (s, 1H), 7.02 (s, 1H), 6.68 (s, 1H), 4.98 (brs, 2H), 4.50 (m, 3H), 1.31 (d, J=6.8 Hz, 3H).

To a solution of methanesulfonic acid 1-benzhydryl-3-methyl-azetidin-3-yl ester (12 g, 36.3 mmol) in DCM (100 mL) was added a saturated solution of ammonia in MeOH (200 mL) and the mixture was stirred for 3 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (eluting with 0-100% EtOAc in petroleum ether) to give 1-benzhydryl-3-methyl-azetidin-3-ylamine (5.31 g, 58%), which was used directly in step F. Alternatively, 1-benzhydryl-3-methyl-azetidin-3-ylamine can be prepared via steps D and E. TLC (eluting with 20% EtOAc/heptane) Rf=0.2.

A mixture of 1-benzhydryl-3-chloro-3-methyl-azetidine (2 g, 7.39 mmol) and NaN3(0.8 g, 12.31 mmol) was dissolved in DMF (30 mL) and the mixture was stirred at 100° C. overnight. The mixture was cooled to ambient temperature and partitioned between water (150 mL) and DCM (50 mL). The organic phase was washed with brine (3×30 mL), dried over anhydrous Na2SO4and concentrated in vacuo to give 3-azido-1-benzhydryl-3-methyl-azetidine as a yellow oil (2.1 g, 100%), which was used directly in the next step. LC/MS (Table 1, Method 2) Rt=0.915 min; MS m/z: 279 [M+H]+

A solution of triphenylphosphine (3.97 g, 15.14 mmol) and 3-azido-1-benzhydryl-3-methyl-azetidine (2.1 g, 7.57 mmol) in THF (20 mL) and water (2 mL) was stirred for 48 h. The solvent was removed in vacuo and a solution of hydrochloric acid (1M, 100 mL) was added to the residue. The aqueous solution was washed with DCM (50 mL) and the aqueous solution was basified to pH 8 with a solution of sodium hydroxide in water (1M). Then, the aqueous solution was extracted with EtOAc (3×50 mL). The combined organic phase was concentrated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 10-100% EtOAc in petroleum ether) to give crude 1-benzhydryl-3-methyl-azetidin-3-ylamine (1.6 g, 6.37 mmol, 84%).1H NMR (CDCl3, 400 MHz): δ7.40-7.33 (m, 4H), 7.21-7.25 (m, 4H), 7.12-7.18 (m, 2H), 4.22-4.30 (m, 1H), 3.10-3.20 (m, 2H), 2.80-2.92 (m, 2H), 1.41-1.47 (m, 3H). The two NH2protons were not observed in CDCl3.

To a solution of 3-(methoxy-methyl-carbamoyl)-azetidine-1-carboxylic acid tert-butyl ester (6.0 g, 24.56 mmol) in THF (15 mL) at −78° C. was added a solution of methymagnesium bromide (3M in toluene, 12.3 mL, 36.9 mmol). The reaction mixture was stirred at −78° C. for 1 h. The reaction mixture was warmed to ambient temperature gradually and stirred for 1 h. The reaction was quenched by the addition of aqueous KHSO4solution (20 mL). The aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 10% EtOAc in petroleum ether) to give 3-acetyl-azetidine-1-carboxylic acid tert-butyl ester (3.2 g, 65.4%).1H NMR (DMSO-d6, 400 MHz): δ 3.88 (m, 4H), 3.53 (m, 1H), 2.08 (s, 3H), 1.34 (s, 9H).

To a −78° C. solution of 3-acetyl-azetidine-1-carboxylic acid tert-butyl ester (0.1 g, 0.5 mmol) in THF (10 mL) was added a solution of LiHMDS (1M, 1.0 mL, 1.0 mmol). The reaction mixture was warmed to ambient temperature gradually and stirred for 4 h. The reaction mixture was cooled to −78° C. and a solution of 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (0.36 g, 1.0 mmol) in THF (5 mL) was added. The reaction mixture was warmed to ambient temperature gradually and stirred overnight. The reaction was quenched by the addition of aqueous NH4Cl solution (20 mL). The aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 10% EtOAc in petroleum ether) to give 3-(1-trifluoromethanesulfonyloxy-vinyl)-azetidine-1-carboxylic acid tert-butyl ester (0.092 g, 55.3%).1H NMR (DMSO-d6, 400 MHz): 5.56 (d, J=4.8 Hz, 1H), 5.36 (d, J=4.8 Hz, 1H), 4.05 (m, 2H), 3.79 (m, 2H), 3.56 (m, 1H), 1.35 (s, 9H).

To a mixture of 1-(2-fluoro-4-hydroxy-phenyl)-ethanone (Alfa, 5 g, 32.5 mmol) in concentrated sulfuric acid (50 mL) at −5° C. was added KNO3(3.29 g, 32.6 mmol) and the reaction mixture was stirred for 3 h. The reaction mixture was poured into ice water (500 mL) carefully and the aqueous solution was extracted with EtOAc (3×150 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4and filtered. The filtrate was evaporated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 2-10% EtOAc in petroleum ether) to give 1-(2-fluoro-4-hydroxy-5-nitro-phenyl)-ethanone as yellow solid (4.59 g, 71%).1H NMR (CDCl3, 400 MHz): δ 10.95 (s, 1H), 8.79 (d, J=7.2 Hz, 1H), 6.92 (d, J=11.2 Hz, 1H), 2.63 (d, J=5.2 Hz, 3H).

To a solution of 1-(2-fluoro-4-hydroxy-5-nitro-phenyl)-ethanone (3 g, 15.7 mmol) in EtOH (30 mL) and THF (6 mL) was added 10% Pd/C (1.5 g, 1.4 mmol) and the reaction mixture was stirred at ambient temperature under an atmosphere of H2for 2 h. The reaction mixture was filtered and the filtrate was evaporated in vacuo to give 1-(5-amino-2-fluoro-4-hydroxy-phenyl)-ethanone as black solid (2.5 g, 94%), which was used in the next step directly without further purification.1H NMR (DMSO-d6, 400 MHz): δ 7.01 (d, J=8.0 Hz, 1H), 6.48 (d, J=12.4 Hz, 1H), 2.39 (d, J=4.8 Hz, 3H).

To a mixture of 2-amino-5-methyl-phenol (Alfa, 15.0 g, 122 mmol), K2CO3(50.5 g, 365 mmol) and Bu4NBr (3.94 g, 12.18 mmol) in acetonitrile (400 mL) at 0° C. was added 2-chloroacetyl chloride (15.1 g, 10.0 mL, 134 mmol) drop-wise. After addition, the mixture was heated at reflux for 2.5 h. The reaction mixture was cooled to ambient temperature and evaporated in vacuo. Water was added (500 mL) and the aqueous solution was extracted with EtOAc (3×150 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 25% EtOAc in petroleum ether) to give 7-methyl-4H-benzo[1,4]oxazin-3-one as a brown solid (14.1 g, 71%).1H NMR (DMSO-d6, 400 MHz): δ 10.58 (s, 1H), 6.76 (m, 3H), 4.52 (s, 2H), 2.21 (s, 3H).

To a mixture of 7-methyl-4H-benzo[1,4]oxazin-3-one (14.1 g, 86 mmol) and Cs2CO3(70.4 g, 216 mmol) in DMF (500 mL) was added ethyl 2-bromopropanoate (31.3 g, 173 mmol) at ambient temperature and the reaction mixture was heated to 70° C. for 3 h. The reaction mixture was cooled to ambient temperature and the DMF was removed in vacuo. Water (500 mL) was added to the residue and the aqueous solution was extracted with EtOAc (3×150 mL). The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 10% EtOAc in petroleum ether) to give 2-(7-methyl-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester as brown solid (22.2 g, 98%). TLC (eluting with 20% EtOAc/heptane) Rf=0.3.

To a solution of 2-(7-methyl-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester (21.0 g, 80 mmol) in DCM (250 mL) was added 1-bromopyrrolidine-2,5-dione (15.6 g, 88 mmol) and the reaction mixture was stirred at ambient temperature for 3 h. The reaction mixture was quenched by addition of a saturated solution of thiosulfate in water (50 mL). The organic phase was separated and the aqueous portion was extracted with DCM (3×20 mL). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 20% EtOAc in petroleum ether) to give -(6-bromo-7-methyl-3-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-propionic acid ethyl ester as solid (23.1 g, 85%). LC/MS (Table 1, Method 3) Rt=1.259 min.; MS m/z: 342/344 [M+H]+.

To a solution of 4-bromo-5-fluoro-2-nitrophenol (40.0 g, 169 mmol) in MeOH (250 mL) and THF (250 mL) was added zinc powder (91.0 g, 1700 mmol) and ammonium chloride (111.0 g, 1700 mmol) and the reaction mixture was stirred at ambient temperature overnight. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was washed with cold water to give 2-amino-4-bromo-5-fluorophenol (38.0 g, crude) which was used in the next step directly without further purification.1H NMR (DMSO-d6, 400 MHz): δ 9.82 (br s, 1H), δ 6.76 (d, J=7.2 Hz, 1H), 6.60 (d, J=10.0 Hz, 1H), 4.65 (br s, 2H).

To a solution of (S)-ethyl lactate (80 g, 677 mmol) in DCM (800 mL) was added trifluoromethansulfonicanhydride (210 g, 745 mmol) at 0° C. drop-wise. The solution was stirred for 15 min at 0° C. and then pyridine (58.9 g, 745 mmol) was added drop-wise. The suspension was stirred for another 15 min at 0° C. followed by the removal of the solvent in vacuo. To the residue was added water (800 mL) and the aqueous solution was extracted with petroleum ether (3×800 mL). The combined organic layer was washed with brine (2×1500 mL), dried over Na2SO4, filtered and concentrated in vacuo to give (S)-2-trifluoromethanesulfonyloxy-propionic acid ethyl ester (150 g, 89%) as a brown liquid, which was used in the next step directly without further purification.1H NMR (400 MHz, CDCl3) δ 5.14-5.28 (m, 1H) 4.21-4.35 (m, 1H) 1.63-1.73 (m, 3H) 1.25-1.37 (m, 3H)

To a mixture of 3-methyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester (0.4 g, 1.876 mmol) in THF (5 mL) was added KHMDS (1M in THF, 2.81 mL, 2.81 mmol) dropwise at −78° C. and the reaction mixture was stirred for 30 min then allowed to warm to ambient temperature and stirred for 3 h. The reaction mixture was cooled at −78° C. and a solution of N-(5-chloropyridin-2-yl)-1,1,1-trifluoro-N-((trifluoromethyl)sulfonyl)methanesulfonamide (0.884 g, 2.251 mmol) in THF (5 mL) was added dropwise. The reaction mixture was allowed to warm to rt and stirred overnight. Saturated aqueous NH4Cl (20 mL) was added and the reaction mixture was extracted with EtOAc (3×10 mL). The combined organic layer was washed with brine (20 mL), dried over Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluting with 1-15% EtOAc in petroleum ether) to give 3-methyl-4-trifluoromethanesulfonyloxy-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester (0.31 g, 47%) as a colorless oil.1H NMR (CDCl3, 400 MHz): δ 5.73 (brs, 1H), 4.24-3.89 (m, 2H), 3.76-3.31 (m, 2H), 2.63 (brs, 1H), 1.48 (s, 9H), 1.16 (d, J=7.0 Hz, 3H)

To a solution of methylmagnesium bromide (263 mL, 790 mmol) in anhydrous THF (800 mL) was added a solution of 1-benzhydrylazetidin-3-one (75 g, 316 mmol) in THF (300 mL) rapidly with vigorous stirring at 0° C. The reaction mixture was stirred for 10 min, then allowed to warm to 25° C. and stirred for 2 h. The reaction mixture was quenched by the addition of saturated aqueous ammonium chloride (1 L), diluted with water (1 L) and extracted with EtOAc (3×1 L). The combined organic phase was washed with brine (0.5 L), dried over anhydrous Na2SO4and filtered. The filtrate was evaporated in vacuo and the residue was purified by column chromatography on silica gel (eluting with 10-20% EtOAc in petroleum ether) to give 1-benzhydryl-3-methylazetidin-3-ol (65 g, 81%) as a white solid.1H NMR (CDCl3, 400 MHz): δ 7.42 (m, 4H), 7.31 (m, 4H), 7.22 (m, 2H), 4.36 (s, 1H), 3.20 (m, 2H), 2.99 (m, 2H), 1.53 (s, 3H).

A mixture of (R)-4-methyl-6-(3-methyl-azetidin-3-ylamino)-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (9 g, 21.21 mmol) and paraformaldehyde (3.82 g, 127 mmol) in MeOH/AcOH (300 mL/30 mL) was stirred at ambient temperature for 0.5 h. Sodium cyanotrihydroborate (4.0 g, 63.6 mmol) was added and the reaction mixture was stirred at ambient temperature for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography on basified silica gel (eluting with 10% MeOH in DCM) to give (R)-6-[(1,3-dimethyl-azetidin-3-yl)-methyl-amino]-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (6 g, 86%). LC/MS (Table 1, Method 3) Rt=0.967 min.; MS m/z: 330 [M+H]+.

To a mixture of (R)-6-[(1,3-dimethyl-azetidin-3-yl)-methyl-amino]-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (3 g, 9.11 mmol) in DCM (40 mL) and MeOH (20 mL) was added tetra-n-butylammonium tribromide (6.59 g, 13.66 mmol) in portions and the reaction mixture was stirred at 20° C. for 16 h. Saturated aqueous Na2S2O3(10 mL) was added and saturated aqueous sodium bicarbonate (5 mL) was added to neutralize the mixture. The organic phase was separated, dried over anhydrous Na2SO4and concentrated in vacuo. The residue was purified by column chromatography on basified silica gel (eluting with 10% MeOH in DCM) to give (R)-7-bromo-6-[(1,3-dimethyl-azetidin-3-yl)-methyl-amino]-4-methyl-2,10-dihydro-9-oxa-1,2,4a-triaza-phenanthren-3-one (3 g, 81%) as a brown solid. LC/MS (Table 1, Method 3), Rt=1.060 min.; MS m/z: 410 [M+H+2]+& 408 [M+H]+. SFC (Table 1, Method 49), Rt=5.174 min.

To a solution of 3-acetyl-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (prepared from 1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid using the similar procedure detailed in Preparation #4, Steps A-B, 0.10 g, 0.47 mmol) and triethylamine (0.5 mL, 3.8 mmol) in EtOH (2 mL) was added aqueous hydrazine hydrate (85%, 0.117 g, 2.34 mmol) and the solution was heated at reflux for 4 h. The reaction mixture was cooled to ambient temperature and poured into water. The mixture was extracted with DCM (3×15 mL) and the combined organic phase was washed with brine (10 mL) and concentrated in vacuo to give 3-(1-hydrazono-ethyl)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.076 g, 71%), which was used directly in the next step without further purification. TLC: Rf=0.15 (eluting with 50% EtOAc in petroleum ether).

To a solution of 3-(1-hydrazono-ethyl)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.076 g, 0.33 mmol) and triethylamine (0.169 g, 1.67 mmol) in THF (3 mL) was added dropwise a solution of iodine (0.127 g, 0.50 mmol) in THF (1 mL). The reaction mixture was stirred for 1 h at ambient temperature then concentrated in vacuo. The residue was dissolved in DCM (10 mL) and washed with aqueous Na2S2O3(5%, 2 mL). The organic phase was separated and concentrated in vacuo. The residue was purified by silica gel chromatography (eluting with 20% EtOAc in petroleum ether) to give 3-(1-iodo-vinyl)-3-methyl-azetidine-1-carboxylic acid tert-butyl ester (0.060 g, 55%), which was unstable and used without further purification. TLC: Rf=0.30 (eluting with 10% EtOAc in petroleum ether).