NOVEL INHIBITORS OF MAP4K1 TECHNICAL FIELD

The present patent application is directed to novel inhibitors of the mitogen-activated protein kinase kinase kinase kinase 1, also known as MAP4K1 or HPK1 (hematopoietic progenitor kinase 1). BACKGROUND OF THE INVENTION

Protein kinases represent a large family of proteins which play a variety of crucial roles in the regulation of a wide range of cellular processes. Such kinases include Akt, Axl, Aurora A, Aurora B, DYRK2, EPHAa2, FGFR3, FLT-3, VEGFr3, IGFLr, IKK2, JNK3, VEGFr2, MEK1, MET, P70s6K, Plk1, RSK1, Src, TrkA, Zap70, cKit, bRaf, EGFR, Jak2, PI3K, NPM-Alk, c-Abl, BTK, FAK, PDGFR, TAK1, LimK, Flt1, PDK1, Erk and RON. Inhibition of various protein kinases, especially selective inhibition, has become an important strategy in treating many diseases and disorders.

MAP4K1 is a serine/threonine kinase of the Ste20 family. MAP4K enzymes (MAP kinase kinases) are generally involved at the highest level of a largely linear kinase activation pathway. A MAP4K will phosphorylate and activate a particular substrate which is a MAP3K (a MAP kinase kinase). A MAP3K in turn phosphorylates and activates a MAP2K (a MAP kinase kinase). A MAP2K in turn phosphorylates and activates a MAPK (MAP kinase). The MAP kinase is the final effector of the pathway and it in turn phosphorylates a substrate to control key cellular processes such as cell proliferation, cell differentiation, gene expression, transcription regulation, and apoptosis. The substrate of MAPK is generally a nuclear protein, such as nuclear factor kappa-B (NF-кB). Activation of the MAPK by its phosphorylation by an MAP2K results in translocation of this final enzyme in the cascade into the nucleus.

MAP4K1, also known as HPK1, is primarily expressed in the immune system’s Tcells and B cells, which are critical in regulation of the immune system. Overstimulation of T cell and B cell activation pathways can result in auto-immune diseases, while understimulation of these pathways can result in immune dysfunction, susceptibility to viral and bacterial infection and increased susceptibility to cancer. MAP4K1 is activated by its interaction with activated T cell receptors (TCRs) and B cell receptors (BCRs), so MAP4K1 activation serves to convey the cellular activation signal from the surface of a T or B cell to the effector proteins in the nucleus. There is also evidence that MAP4K1 can be activated via the TGF-β receptor, the erythropoietin receptor and the FAS protein (which is involved in apoptosis  

signaling). MAP4K1 activation ultimately results in activation of several identified nuclear effector proteins, including those involved in the NF-к1, AP-1, ERK2, and Fos signaling pathways.

MAP4K1 is considered a negative regulator of T cell receptor (TCR) activation signals, and it is one of the effector molecules that mediates immunosuppression of T cell responses upon exposure to prostaglandin E2 (PGE2). Studies have shown that MAPK1 activity dampens the strength of the T cell receptor signal transduction cascade, and thus, targeted genetic disruption of MAP4K1 results in strengthened TCR activation signals.

One particularly important pathway that MAP4K1 appears to be involved with is the JNK pathway. MAP4K1 regulates the MAP3K’s MEKK1, TAK1 and MLK3. These in turn regulate the MAP2K’s MKK4 and MKK7. These in turn regulate the MAPK JNK. JNK then regulates important transcription factors and other proteins, including p53, SMAD4, NFAT-2, NFAT-4, ELK1, ATF2, HSF1, c-Jun, and JunD. JNK has been implicated in apoptosis, neurodegeneration, cell differentiation and proliferation, inflammatory conditions and cytokine production.

The JNK signal transduction pathway is activated in response to environmental stress and by the engagement of several classes of cell surface receptors, including cytokine receptors, serpentine receptors and receptor tyrosine kinases. In mammalian cells, the JNK pathway has been implicated in biological processes such as oncogenic transformation and mediating adaptive responses to environmental stress. JNK has also been associated with modulating immune responses, including maturation and differentiation of immune cells, as well as effecting programmed cell death in cells identified for destruction by the immune system. Among several neurological disorders, JNK signaling is particularly implicated in ischemic stroke and Parkinson's disease, but also in other diseases as mentioned further below.

It is noteworthy that the MAPK p38alpha was shown to inhibit cell proliferation by antagonizing the JNK-c-Jun-pathway. p38alpha appears to be active in suppression of proliferation in both normal cells and cancer cells, and this strongly suggests the involvement of JNK in hyperproliferative diseases (see, e.g., Hui et al., Nature Genetics, Vol. 39, No. 6, June 2007). JNK signaling has also been implicated in diseases such as excitotoxicity of hippocampal neurons, liver ischemia, reperfusion, neurodegenerative diseases, hearing loss, deafness, neural tube birth defects, cancer, chronic inflammatory diseases, obesity, diabetes, in particular, insulin-resistant diabetes, and it has been proposed that selective JNK inhibitors

 

are needed for treatment of various diseases with a high degree of specificity and lack of toxicity.

Because MAP4K1 is an upstream regulator of JNK, effective inhibitors of MAP4K1 would be useful in treating the same diseases which have been suggested or implicated for JNK inhibitors, especially where such disease or dysfunction is manifested in hematopoietic cells such as T cells and B cells.

Targeted disruption of MAP4K1 (HPK1) alleles has been shown to confer T cells with an elevated Th1 cytokine production in response to TCR engagement. Burakoff et al., Immunologic Research, 54(1): 262-265 (2012). HPK1−/− T cells were found to proliferate more rapidly than the haplotype-matched wild-type counterpart and were resistant to prostaglandin E2 (PGE2)-mediated suppression. Most strikingly, mice that received adoptive transfer of HPK1−/− T cells became resistant to lung tumor growth. Also, the loss of HPK1 from dendritic cells (DCs) endowed them with superior antigen presentation ability, enabling HPK1−/− DCs to elicit a more potent anti-tumor immune response when used as cancer vaccine. It was considered probable that blocking the MAP4K1 kinase activity with a small molecule inhibitor may activate the superior antitumor activity of both cell types, resulting in a synergistic amplification of anti-tumor potential. Given that MAP4K1 is not expressed in any major organs, it is less likely that a selective inhibitor of MAP4K1 would cause any serious side effects.

The relationship between MAP4K1 and PGE2 is particularly noteworthy because PGE2 is the predominant eicosanoid product released by cancer cells, including lung, colon and breast cancer cells. Tumor-produced PGE2 is known to contribute significantly to tumor- mediated immune suppression.

Zhang et al., J. Autoimmunity, 37:180-189 (2011), described diminished HPK1 expression in CD4 T cells of lupus patients due to the selective loss of JMJD3 histone demethylase binding to the HPK1 locus. This suggests that HPK1 is one of the key molecules involved in the maintenance of peripheral tolerance. Peripheral tolerance is one of the major obstacles to the development of effective anti-tumor immunity.

Several small molecule inhibitors of MAP4K1 have been reported, but they do not inhibit MAP4K1 selectively, or even preferentially. Such inhibitors include staurosporine, bosutinib, sunitinib, lestaurtinib, crizotinib, foretinib, dovitinib and KW-2449. Staurosporine, for example, broadly inhibits a wide range of protein kinases across both the serine/threonine and tyrosine kinase families. Bosutinib is primarily an inhibitor of the tyrosine kinase BCR- Abl, with additional activity against the Src family tyrosine kinases. Sunitinib is a broad   inhibitor of tyrosine kinases. Lestaurtinib is primarily an inhibitor of the FLT, JAK and TRK family tyrosine kinases. Crizotinib is primarily an inhibitor of the c-met and ALK tyrosine kinases. Foretinib was under study as an inhibitor of the c-Met and VEGFR tyrosine kinases. Dovitinib is primarily an inhibitor of the FGFR receptor tyrosine kinase. KW-2449 is an experimental inhibitor primarily of the FLT3 tyrosine kinase.

Sunitinib inhibits MAP4K1 at nanomolar concentrations, but it is a broad-spectrum receptor tyrosine kinase inhibitor. Treating T-cells with sunitinib results in enhanced cytokine product similar to that observed with HPK1−/− T cells, which suggests that in T cells a selective MAP4K1 inhibitor could produce the same enhanced immune response phenotype.

Currently, there is a largely unmet need for an effective way of treating disease and disorders associated disrupted protein kinase signaling. Autoimmune diseases, inflammatory diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, are all diseases and disorder which can be affected by dysfunctional protein kinase signaling. Improved therapeutic compounds, compositions and methods for the treatment for these disease and disorders are urgently required. MAP4K1 inhibition is an especially attractive target for cancer immunotherapy.

The major challenge currently faced in the field is the lack of MAP4K1 specific inhibitors. The present disclosure provides novel, highly effective small-molecule inhibitors of MAP4K1.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a compound of formula (I)

stereoisomer, diastereoisomer, enantiomer or a pharmaceutically acceptable salt thereof,

wherein

A is selected from CH and N;

D is selected from CR ¹ R ² and CO;

E is selected from (CR ³ R ⁴ )m, NR ¹ and CO;   F is selected from O, CH2, CHOH and CO;

each occurrence of R ⁵ is selected from hydrogen, halogen, cyano, hydroxyl and C _{1- 8} alkyl;

R ⁷ is selected from hydrogen and C1-8alkyl;

each occurrence of R ⁶ is selected from halogen, cyano, hydroxyl, C _1-8 alkyl, haloC _{1- 8} alkyl, hydroxyC _1-8 alkyl, C _1-8 alkoxy, C _1-8 alkoxyC _1-8 alkyl, C _3-6 cycloalkyl and C _3- 6cycloalkylC1-8alkyl;

R ¹ , R ² , R ³ and R ⁴ which may be same or different, are each independently selected from hydrogen, amine, C _1-8 alkyl, C _3-6 cycloalkyl, haloC _1-8 alkyl, hydroxyC _1-8 alkyl, C _3- 6cycloalkylC1-8alkyl, C1-8alkoxy, 3-15 membered heterocyclyl, C1-8alkyl3-15 membered heterocyclyl and CR ^a R ^b NR ^a R ^b ;

R ^a and R ^b , which may be the same or different, are each independently selected from hydrogen and C _1-8 alkyl;

Z is selected from O, NH and S;

L is selected from and

x and y represents point of attachment;

Ring Q is selected from

each occurrence of R ⁸ is selected from halogen, cyano, cyanoC1-8alkyl, cyanohaloC1- 8alkyl, cyanoC _3-6 cycloalkyl, C _1-8 alkyl, haloC _1-8 alkyl, hydroxyC _1-8 alkyl, hydroxyC _1-8 haloalkyl, and -SO ₂ R ¹ ;

  each occurrence of R ⁹ is selected from halogen, cyano, hydroxyl, C1-8alkyl, haloC1-

8alkyl, hydroxyC _1-8 alkyl, C _1-8 alkoxy,

 

R ¹⁰ is selected from halogen, hydroxyl, cyano, C1-8alkyl, haloC1-8alkyl, C3-6cycloalkyl and C _6-14 aryl; wherein C _6-14 aryl is optionally substituted with one or more substituents selected from halogen, hydroxyl, cyano, amide or C _1-8 alkyl;

‘m’ is 1 or 2;

‘n’ is 0, 1 or 2;

‘p’ is 0 or 1;

‘q’ is 0 or 1; and

‘t’ is 1 or 2.

The compounds of formula (I) may involve one or more embodiments. It is to be understood that the embodiments below are illustrative of the present invention and are not intended to limit the claims to the specific embodiments exemplified. It is also to be understood that the embodiments defined herein may be used independently or in conjunction with any definition, any other embodiment defined herein. Thus the invention contemplates all possible combinations and permutations of the various independently described embodiments. For example, the invention provides compounds of formula (I) as defined above wherein A is N (according to an embodiment defined below); F is O (according to another embodiment defined below); R ⁷ is hydrogen (according to yet another embodiment defined below).

According to one embodiment specifically provided are compounds of formula (I), in which A is CH.

According to another embodiment specifically provided are compounds of formula (I), in which A is N.

According to yet another embodiment specifically provided are compounds of formula (I), in which D is CR ¹ R ² or CO.   According to yet another embodiment specifically provided are compounds of formula (I), in which R ¹ is hydrogen or C _1-8 alkyl (e.g. methyl) and R ² is hydrogen, C _1-8 alkyl (e.g. methyl, ethyl or isopropyl), hydroxyC _1-8 alkyl (e.g. hydroxyl methyl), C _1-8 alkoxyC _1- 8alkyl (e.g. methoxymethyl), 3-15 membered heterocyclylC1-8alkyl (e.g. or CR ^a R ^b NR ^a R ^b . In this embodiment, R ^a and R ^b are independently hydrogen or methyl. In another embodiment, R ^a and R ^b are hydrogen. In yet another embodiment, R ^a and R ^b are methyl.

According to yet another embodiment specifically provided are compounds of formula (I), in which D is CH2, CH-CH3, CH-CH2-CH3, C(CH3)2, CH-CH(CH3)2, CH-

CH2OH, CH-CH2-O-CH3, CH-CH2-N(CH3)2 or CO.

According to yet another embodiment specifically provided are compounds of formula (I), in which E is (CR ³ R ⁴ ) _m , NR ¹ or CO.

According to yet another embodiment specifically provided are compounds of formula (I), in which R ³ is hydrogen or C _1-8 alkyl (e.g. methyl) and R ⁴ is hydrogen or C _1-8 alkyl (e.g. methyl) and‘m’ is 1 or 2.

According to yet another embodiment specifically provided are compounds of formula (I), in which E is CH2, CH-CH3, C(CH3)2, (CH2)2, N-CH3 or CO.

According to yet another embodiment specifically provided are compounds of formula (I), in which F is O, CH2, CHOH or CO.

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁵ is hydrogen, halogen (e.g. chloro) or cyano.

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁵ is hydrogen, chloro or cyano.

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁷ is hydrogen or C _1-8 alkyl (e.g. methyl).

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁷ is hydrogen or methyl.

According to yet another embodiment specifically provided are compounds of formula (I), in which Z is O.   According to yet another embodiment specifically provided are compounds of formula (I), in which Z is NH.

According to yet another embodiment specifically provided are compounds of formula (I), in which Z is S.

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁶ is halogen (e.g. chloro or fluoro), C _1-8 alkyl (e.g. methyl), haloC _1- 8alkyl (e.g. trifluoromethyl) or C1-8alkoxy (e.g. methoxy).

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁶ is chloro, fluoro, methyl, trifluoromethyl or methoxy.

According to yet another embodiment specifically provided are compounds of formula (I), in which‘n’ is 0, 1 or 2.

According to yet another embodiment specifically provided are compounds of

formula (I), in which L is x and y represents point of attachment.

According to yet another embodiment specifically provided are compounds of

formula (I), in which L is x and y represents point of attachment.

According to yet another embodiment specifically provided are compounds of

formula (I), in which L is x and y represents point of attachment.

According to yet another embodiment specifically provided are compounds of

formula (I), in which L is ; x and y represents point of attachment.

According to yet another embodiment specifically provided are compounds of formula (I), in which Ring Q is

 

According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is

According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is .

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁸ is halogen (e.g. chloro or bromo), cyano, cyanoC1-8alkyl (e.g. cyanomethyl or cyanoisopropyl), cyanohaloC1-8alkyl (e.g. cyanodifluoromethyl), cyanoC3- 6cycloalkyl (e.g. cyanocyclopropane), C _1-8 alkyl (e.g. methyl), haloC _1-8 alkyl (e.g. trifluoromethyl or difluoromethyl), hydroxyC1-8haloalkyl (e.g. hydroxyl difluoromethyl) or - SO2R ¹ . In this embodiment, R ¹ is C1-8alkyl (e.g. methyl, ethyl or amine).

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁸ is chloro, bromo, cyano, cyanomethyl, cyanoisopropyl, cyanodifluoromethyl, cyanocyclopropane, methyl, trifluoromethyl, difluoromethyl, hydroxyl difluoromethyl, -SO ₂ Me, -SO ₂ Et or -SO ₂ NH ₂ .

According to yet another embodiment specifically provided are compounds of formula (I), in which R ⁹ is halogen (e.g. fluoro or bromo), cyano, hydroxyl, C1-8alkyl (e.g. methyl), haloC _1-8 alkyl (e.g. trifluoromethyl), C _1-8 alkoxy (e.g. methoxy),

 

 

According to yet another embodiment specifically provided are compounds of formula I in which R ⁹ is fluoro bromo c ano hdroxl methl trifluoromethl methox

 

 

According to yet another embodiment specifically provided are compounds of formula (I), in which Ring Q is

 

 

  According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is In this embodiment, R ¹⁰ is C _1-8 alkyl (e.g. methyl) or haloC _1-8 alkyl (e.g. trifluoromethyl). In yet another embodiment,‘t’ is 2.

According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is

According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is

According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is

According to yet another embodiment specifically provided are compounds of formula (I), in which R ¹⁰ is C1-8alkyl (e.g. methyl) or C6-14aryl. In this embodiment, C6-14aryl is optionally substituted with one or more substituents selected from cyano, or amide.

  According to yet another embodiment specifically provided are compounds of

formula (I), in which Ring Q is or C

According to yet another embodiment specifically provided are compounds of formula (I), in which‘p’ is 0 or 1.

According to yet another embodiment specifically provided are compounds of formula (I), in which‘q’ is 0 or 1.

According to yet another embodiment, specifically provided are compounds of formula (I), in which

A is CH or N;

D is CH ₂ , CH-CH ₃ , CH-CH ₂ -CH ₃ , C(CH ₃ ) ₂ , CH-CH(CH ₃ ) ₂ , CH-CH ₂ OH, CH-CH ₂ -O-

CH , , CH-CH2-N(CH3)2, CO or cycloalkyl;

E is CH2, CH-CH3, C(CH3)2, (CH2)2, N-CH3 or CO;

F is O, CH ₂ , CHOH or CO;

R ⁵ is hydrogen, chloro or cyano;

R ⁷ is hydrogen or methyl;

Z is O, NH or S;

R ⁶ is chloro, fluoro, methyl, trifluoromethyl or methoxy;

L is  

Ring Q is

 

 

 

‘m’ is 1 or 2; and

‘n’ is 0, 1 or 2.

According to an embodiment, specifically provided are compounds of formula (I) with an IC50 value of less than 500 nM, preferably less than 100 nM, more preferably less than 50 nM, with respect to MAP4K1 inhibition.

Compounds of the present invention include the compounds in Examples 1-261. It should be understood that the formulas (I) structurally encompasses all geometrical isomers, stereoisomers, enantiomers and diastereomers, N-oxides, and pharmaceutically acceptable salts that may be contemplated from the chemical structure of the genera described herein.

The present application also provides a pharmaceutical composition that includes at least one compound described herein and at least one pharmaceutically acceptable excipient (such as a pharmaceutically acceptable carrier or diluent). Preferably, the pharmaceutical composition comprises a therapeutically effective amount of at least one compound described  

herein. The compounds described herein may be associated with a pharmaceutically acceptable excipient (such as a carrier or a diluent) or be diluted by a carrier, or enclosed within a carrier which can be in the form of a tablet, capsule, sachet, paper or other container.

Dosages employed in practicing the present invention will of course vary depending, e.g. on the particular disease or condition to be treated, the particular compound used, the mode of administration, and the therapy desired. The compound may be administered by any suitable route, including orally, parenterally, transdermally, or by inhalation. In general, satisfactory results, e.g. for the treatment of diseases as hereinbefore set forth are indicated to be obtained on oral administration at dosages of the order from about 0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicated daily dosage for oral administration will accordingly be in the range of from about 0.75 to 300 mg, conveniently administered once, or in divided doses 2 to 4 times, daily or in sustained release form. Unit dosage forms for oral administration thus for example may comprise from about 0.2 to 75 or 150 mg or 300 mg, e.g. from about 0.2 or 2.0 to 10, 25, 50, 75, 100, 150, 200 or 300 mg of the compound disclosed herein, together with a pharmaceutically acceptable diluent or carrier therefor.

Pharmaceutical compositions comprising Compounds of the Invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets, capsules, solutions, suspensions and the like. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms“halogen” or“halo” means fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo).

The term“alkyl” refers to a hydrocarbon chain radical that includes solely carbon and hydrogen atoms in the backbone, containing no unsaturation, having from one to eight carbon atoms (i.e. C _1-8 alkyl), and which is attached to the rest of the molecule by a single bond, such as, but not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, and 1,1-dimethylethyl (t-butyl). The term“C1-6alkyl” refers to an alkyl chain having 1 to 6 carbon atoms. The term“C _1-4 alkyl” refers to an alkyl chain having 1 to 4 carbon atoms. Unless set forth or recited to the contrary, all alkyl groups described or claimed herein may be straight chain or branched.

The term“alkoxy” denotes an alkyl group attached via an oxygen linkage to the rest of the molecule (i.e. C1-8 alkoxy). Representative examples of such groups are -OCH3 and -

 

OC2H5. Unless set forth or recited to the contrary, all alkoxy groups described or claimed herein may be straight chain or branched.

The term“alkoxyalkyl” or“alkyloxyalkyl” refers to an alkoxy or alkyloxy group as defined above directly bonded to an alkyl group as defined above (i.e. C1-8alkoxyC1-8alkyl or C _1-8 alkyloxyC _1-8 alkyl). Example of such alkoxyalkyl moiety includes, but are not limited to, - CH ₂ OCH ₃ (methoxymethyl) and -CH ₂ OC ₂ H ₅ (ethoxymethyl). Unless set forth or recited to the contrary, all alkoxyalkyl groups described herein may be straight chain or branched.

The term“haloalkyl” refers to at least one halo group (selected from F, Cl, Br or I), linked to an alkyl group as defined above (i.e. haloC _1-8 alkyl). Examples of such haloalkyl moiety include, but are not limited to, trifluoromethyl, difluoromethyl and fluoromethyl groups. The term“haloC1-4alkyl” refers to at least one halo group linked an alkyl chain having 1 to 4 carbon atoms. Unless set forth or recited to the contrary, all haloalkyl groups described herein may be straight chain or branched.

The term“haloalkoxy” refers to an alkoxy group substituted with one or more halogen atoms (i.e. haloC _1-8 alkoxy). Examples of“haloalkoxy” include but are not limited to fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, pentachloroethoxy, chloromethoxy, dichlorormethoxy, trichloromethoxy and 1-bromoethoxy. Unless set forth or recited to the contrary, all haloalkoxy groups described herein may be straight chain or branched.

The term“hydroxyC1-8alkyl” refers to a C1-8alkyl group as defined above wherein one to three hydrogen atoms on different carbon atoms is/are replaced by hydroxyl groups (i.e. hydroxyC _1-4 alkyl). Examples of hydroxyC _1-4 alkyl moieties include, but are not limited to

The term“cyanoalkyl” refers to a alkyl group as defined above directly bonded to cyano group (i.e. cyanoC _1-8 alkyl). Examples of such cyanoC _1-8 alkyl moiety include, but are not limited to, cyanomethyl, cyanoethyl and cyanoisopropyl. Unless set forth or recited to the contrary, all cyanoalkyl groups described herein may be straight chain or branched.

The term“cyanohaloalkyl” refers to cyanoalkyl group substituted with one or more halogen atoms (i.e.cyanohaloC _1-8 alkyl). Example of cyanohaloalkyl include but are not limited to cyanodifluoromethyl. Unless set forth or recited to the contrary, all cyanohaloalkyl groups described herein may be straight chain or branched.

The term“cycloalkyl” denotes a non-aromatic mono or multicyclic ring system of 3 to about 12 carbon atoms, (i.e.C3-12cycloalkyl). Examples of monocyclic cycloalkyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of  

multicyclic cycloalkyl groups include, but are not limited to, perhydronapthyl, adamantyl and norbornyl groups, bridged cyclic groups or spirobicyclic groups, e.g., spiro(4,4)non-2-yl. The term“C _3-6 cycloalkyl” refers to the cyclic ring having 3 to 6 carbon atoms. Examples of“C _3- 6cycloalkyl” include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term“cycloalkylalkyl” refers to a cyclic ring-containing radical having 3 to about 6 carbon atoms directly attached to an alkyl group (i.e. C3-6cycloalkylC1-8alkyl). The cycloalkylalkyl group may be attached to the main structure at any carbon atom in the alkyl group that results in the creation of a stable structure. Non-limiting examples of such groups include cyclopropylmethyl, cyclobutylethyl, and cyclopentylethyl.

The term“cyanocycloalkyl” refers to a cyclic ring-containing radical having 3 to about 6 carbon atoms directly attached to cyano group (i.e.“cyanoC _3-6 cycloalkyl). Non- limiting example of such groups include cyanocyclopropane.

The term“aryl” refers to an aromatic radical having 6 to 14 carbon atoms (i.e. C6- ₁₄ aryl), including monocyclic, bicyclic and tricyclic aromatic systems, such as phenyl, naphthyl, tetrahydronapthyl, indanyl, and biphenyl.

The term“heterocyclic ring” or“heterocyclyl” unless otherwise specified refers to substituted or unsubstituted non-aromatic 3 to 15 membered ring radical (i.e. 3 to 15 membered heterocyclyl) which consists of carbon atoms and from one to five hetero atoms selected from nitrogen, phosphorus, oxygen and sulfur. The heterocyclic ring radical may be a mono-, bi- or tricyclic ring system, which may include fused, bridged or spiro ring systems, and the nitrogen, phosphorus, carbon, oxygen or sulfur atoms in the heterocyclic ring radical may be optionally oxidized to various oxidation states. In addition, the nitrogen atom may be optionally quaternized; also, unless otherwise constrained by the definition the heterocyclic ring or heterocyclyl may optionally contain one or more olefinic bond(s). Examples of such heterocyclic ring radicals include, but are not limited to azepinyl, azetidinyl, benzodioxolyl, benzodioxanyl, chromanyl, dioxolanyl, dioxaphospholanyl, decahydroisoquinolyl, indanyl, indolinyl, isoindolinyl, isochromanyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxazolinyl, oxazolidinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2- oxoazepinyl, octahydroindolyl, octahydroisoindolyl, perhydroazepinyl, piperazinyl, 4- piperidonyl, pyrrolidinyl, piperidinyl, phenothiazinyl, phenoxazinyl, quinuclidinyl, tetrahydroisquinolyl, tetrahydrofuryl or tetrahydrofuranyl, tetrahydropyranyl, thiazolinyl, thiazolidinyl, thiamorpholinyl, thiamorpholinyl sulfoxide and thiamorpholinyl sulfone. The

 

heterocyclic ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.

The term“heterocyclylalkyl” refers to a heterocyclic ring radical directly bonded to an alkyl group (i.e. 3 to 15 membered heterocyclylC1-8alkyl). The 20 heterocyclylalkyl radical may be attached to the main structure at any carbon atom in the alkyl group that results in the creation of a stable structure.

The term“heteroaryl” unless otherwise specified refers to 5 to 14 membered aromatic heterocyclic ring radical with one or more heteroatom(s) independently selected from N, O or S (i.e. 5 to 14 membered heteroaryl). The heteroaryl may be a mono-, bi- or tricyclic ring system. The heteroaryl ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure. Examples of such heteroaryl ring radicals include, but are not limited to oxazolyl, isoxazolyl, imidazolyl, furyl, indolyl, isoindolyl, pyrrolyl, triazolyl, triazinyl, tetrazoyl, thienyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, benzofuranyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzothienyl, benzopyranyl, carbazolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, naphthyridinyl, pteridinyl, purinyl, quinoxalinyl, quinolyl, isoquinolyl, thiadiazolyl, indolizinyl, acridinyl, phenazinyl and phthalazinyl.

The term “pharmaceutically acceptable salt” includes salts prepared from pharmaceutically acceptable bases or acids including inorganic or organic bases and inorganic or organic acids. Examples of such salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate. Examples of salts derived from inorganic bases include, but are not limited to, aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, and zinc.

The term“treating” or“treatment” of a state, disorder or condition includes: (a) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or  

condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (b) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof; or (c) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

The term“subject” includes mammals (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non-domestic animals (such as wildlife).

A“therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a state, disorder or condition, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

The compounds of formula (I) may contain asymmetric or chiral centers, and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of formula (I) as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolysing) the individual diastereomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of chiral HPLC column. The chiral centres of the present invention can have the S or R configuration as defined by the IUPAC 1974.

The terms "salt" or "solvate", and the like, is intended to equally apply to the salt, solvate and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers or racemates of the inventive compounds. PHARMACEUTICAL COMPOSITIONS

The compounds of the invention are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared using procedures well  

known in the pharmaceutical art and comprise at least one compound of the invention. The pharmaceutical compositions described herein comprise one or more compounds described herein and one or more pharmaceutically acceptable excipients. Typically, the pharmaceutically acceptable excipients are approved by regulatory authorities or are generally regarded as safe for human or animal use. The pharmaceutically acceptable excipients include, but are not limited to, carriers, diluents, glidants and lubricants, preservatives, buffering agents, chelating agents, polymers, gelling agents, viscosifying agents, solvents and the like.

Examples of suitable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid, lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, fatty acid esters, and polyoxyethylene.

The pharmaceutical compositions described herein may also include one or more pharmaceutically acceptable auxiliary agents, wetting agents, suspending agents, preserving agents, buffers, sweetening agents, flavouring agents, colorants or any combination of the foregoing.

The pharmaceutical compositions may be in conventional forms, for example, capsules, tablets, solutions, suspensions, injectables or products for topical application. Further, the pharmaceutical composition of the present invention may be formulated so as to provide desired release profile.

Administration of the compounds of the invention, in pure form or in an appropriate pharmaceutical composition, can be carried out using any of the accepted routes of administration of such compounds or pharmaceutical compositions. The route of administration may be any route which effectively transports the active compound of the patent application to the appropriate or desired site of action. Suitable routes of administration include, but are not limited to, oral, nasal, buccal, dermal, intradermal, transdermal, parenteral, rectal, subcutaneous, intravenous, intraurethral, intramuscular, and topical.

Solid oral formulations include, but are not limited to, tablets, capsules (soft or hard gelatin), dragees (containing the active ingredient in powder or pellet form), troches and lozenges.

Liquid formulations include, but are not limited to, syrups, emulsions, and sterile injectable liquids, such as suspensions or solutions.  

Topical dosage forms of the compounds include, but are not limited to, ointments, pastes, creams, lotions, powders, solutions, eye or ear drops, impregnated dressings, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration.

Suitable doses of the compounds for use in treating the diseases and disorders described herein can be determined by those skilled in the relevant art. Therapeutic doses are generally identified through a dose ranging study in humans based on preliminary evidence derived from the animal studies. Doses must be sufficient to result in a desired therapeutic benefit without causing unwanted side effects. Mode of administration, dosage forms, and suitable pharmaceutical excipients can also be well used and adjusted by those skilled in the art.

METHODS OF TREATMENT

The compounds of Formula I, as described herein are highly effective inhibitors of the MAP4K1 kinase, producing inhibition at nanomolar concentrations. MAP4K1 inhibitors according to the invention are therefore useful for treatment and prophylaxis of diseases associated with protein kinase signaling dysfunction. Accordingly, without being bound by any theory, it is believed that inhibition of MAP4K1 could, for example, reverse or prevent the cellular dysfunction associated with perturbations of the JNK signaling pathway, especially in T and B cells. Therefore, administration of a MAP4K1 inhibitor as described herein could provide a potential means to regulate MAPK signal transduction pathways, especially the JNK pathway, and by extension provide a treatment for a variety of diseases and disorders including autoimmune, neurodegenerative, neurological, inflammatory, hyperproliferative, and cardiovascular diseases and disorders.

In addition, without being bound by theory, selective MAP4K1 inhibition, as provided by the Compounds of the Invention, may provide a novel means of cancer treatment. Traditional signal transduction strategies relate to interference with the pathways that promote tumor cell proliferation or metastasis. The present invention provides instead a means of enhancing the activity and effectiveness of the body’s T cells, for example, to overcome the immunosuppressive strategies used by many cancers. The U.S. Food and Drug Administration (FDA) has recently approved some monoclonal antibody-based treatments that achieve the same result by interfering with T-cell surface receptors which promote inhibition of TCR activity (e.g., anti-CTLA-4 and anti-PD-1 antibodies, marketed as Ipilimumab and Pembrolizumab, respectively). The success of the treatments demonstrate proof of the concept that cancer can be effectively treated by interfering with pathways which  

inhibit TCR signaling, Targeting these pathways using a small molecule inhibitor of MAP4K1 should produce improved results using more patient-friendly administration techniques.

Therefore, in the third aspect, the invention provides a method for the treatment or prophylaxis of a disease or disorder which may be ameliorated by modulating (e.g., inhibiting) MAP4K1-dependent signaling pathways, including the JNK pathway, e.g., autoimmune, neurodegenerative, neurological, inflammatory, hyperproliferative, and cardiovascular diseases and disorders, comprising administering to a patient in need thereof an effective amount of the compound of Formula I as described herein, in free or pharmaceutically acceptable salt form.

In particular embodiments, administration of the compound of Formula I results in enhanced T cell receptor (TCR) signaling, such as resulting in an enhanced T cell-mediated immune response (e.g., increased T cell cytokine production).

In other particular embodiments, administration of the compound of Formula I results in increased T cell resistance to PGE2-mediated T cell suppression.

The disease or disorder may be selected from the group consisting of: neurodegenerative diseases, such as Parkinson's disease or Alzheimer's disease; stroke and associated memory loss; autoimmune diseases such as arthritis; allergies and asthma; diabetes, especially insulin-resistant diabetes; other conditions characterized by inflammation, including chronic inflammatory diseases; liver ischemia; reperfusion injury; hearing loss or deafness; neural tube birth defects; obesity; hyperproliferative disorders including malignancies, such as leukemias, e.g. chronic myelogenous leukemia (CML); oxidative damage to organs such as the liver and kidney; heart diseases; and transplant rejections. In certain embodiments, the disease or disorder to be treated may also relate to impaired MAP4K1-dependent signaling. Impaired MAP4K1 signaling can lead to reduced immune cell, e.g. T and B cell, function which can permit or enhance the escape of nascent cancer cells from immune surveillance. Restoration of T and B cell function via treatment with a MAP4K1-inhibitor can therefore promote the clearance of carcinogenic and pre- carcinogenic cells from the body. Thus, in a particular embodiment, the invention provides a method for the treatment or prevention of hyperproliferative diseases, such as cancer, including melanomas, thyroid cancers, adenocarcinoma, breast cancer, central nervous system cancers such as glioblastomas, astrocytomas and ependymomas, colorectal cancer, squamous cell carcinomas, small and non-small cell lung cancers, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, sarcoma and skin cancers. In  

particular embodiments, owing to the unique role of immune cell dysfunction in hematologic cancers, the invention provides a method of treatment or prevention of hematologic cancers such as leukemias, acute myelogenous leukemia (AML), myelodysplastic syndromes, chronic myelogenous leukemia (CML), Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, megakaryoblastic leukemia, and multiple myeloma.

The MAP4K1 inhibitor compounds described herein for the treatment or prophylaxis of disease or disorder according to the foregoing methods may be used as a sole therapeutic agent or may be used in combination with one or more other therapeutic agents useful for the treatment of said diseases or disorders. Such other agents include inhibitors of other protein kinases in the JNK pathway, including, for example, inhibitors of JNK (e.g., JNK1 or JNK2), MKK4, MKK7, p38, MEKK (e.g., MEKK1, MEKK2, MEKK5), and GCK,

Therefore, in a particular embodiment, the MAP4K1 inhibitor of the invention may be administered in combination with inhibitors of JNK (e.g., JNK1 or JNK2), MKK4, MKK7, p38, MEKK (e.g., MEKK1, MEKK2, MEKK5), and GCK.

In another aspect, the invention provides the following:

(i) the compound of Formula I as described herein, in free or pharmaceutically acceptable salt form, for use in any of the methods or in the treatment or prophylaxis of any disease or disorder as set forth herein, (ii) a combination as described hereinbefore, comprising a MAP4K1 inhibitor of the invention, e.g., the compound of Formula I as described herein, in free or pharmaceutically acceptable salt form and a second therapeutic agent useful for the treatment or prophylaxis of any disease or disorder set forth herein;

(iii) use of the compound of Formula I in free or pharmaceutically acceptable salt form, or the combination described herein, (in the manufacture of a medicament) for the treatment or prophylaxis of any disease or condition as set forth herein,

(iv) the compound of Formula I in free or pharmaceutically acceptable salt form, the combination described herein or the pharmaceutical composition of the invention as hereinbefore described for use in the treatment or prophylaxis of any disease or condition as set forth herein. GENERAL METHODS OF PREPARATION

  The compounds, described herein, including those of general formula (I), intermediates and specific examples are prepared through the synthetic methods as depicted in Schemes 1 to 14. Furthermore, in the following schemes, where specific acids, bases, reagents, coupling reagents, solvents, etc. are mentioned, it is understood that other suitable acids, bases, reagents, coupling reagents, solvents etc. may be used and are included within the scope of the present invention. The modifications to reaction conditions, for example, temperature, duration of the reaction or combinations thereof, are envisioned as part of the present invention. The compounds obtained using the general reaction sequences may be of insufficient purity. These compounds can be purified using any of the methods for purification of organic compounds known to persons skilled in the art, for example, crystallization or silica gel or alumina column chromatography using different solvents in suitable ratios. All possible geometrical isomers and stereoisomers are envisioned within the scope of this invention.

General schemes

A general approach for the preparation of compounds of the formulae (Ia) (wherein R ⁵ , R ⁶ , R ⁷ , A, E, F, D, Z, Q and n are as defined in the general description) is depicted in synthetic scheme 1.

Synthetic Scheme 1

The substitution reaction of compound of formula (1) (wherein R’ = Me or Et) with halogen bearing compound of formula (2) in the presence of a suitable reagent and solvent yields the compound of formula (3). The reaction may be carried out in the presence of   suitable base such as cesium carbonate, potassium carbonate, sodium carbonate, cesium fluoride etc., and the solvent can be selected from DMF, DMSO, acetonitrile, 1,4-dioxane or a mixture thereof. The reaction can also be performed by Buchwald reaction using a suitable base such as tripotassium phosphate, sodium or potassium tert-butoxide, cesium carbonate, etc., in the presence of palladium acetate as catalyst and a suitable hindered ligand (eg. XPhos, di-tBuXPhos, JohnPhos, DavePhos, Sphos, etc.) in an appropriate solvent such as toluene, 1,4-doxane, water or a mixture thereof. (Ref: Angew. Chem. Int. Ed.2006, 45, 4321) The coupling reaction of the ester of formula (3) with amine of formula (4) in the presence of a suitable reagent and solvent affords the compound of general formula (Ia). The suitable base used in the reaction may be potassium tert-butoxide or trimethyl aluminium solution. The coupling reaction may be carried out in a suitable solvent or mixture thereof. The suitable solvent may be selected from dichloromethane, THF, toluene, or a combination thereof. Alternatively, the coupling reaction of compound of formula (1) with the amine of formula (4) gives amide of formula (5) which on substitution reaction with compound of formula (2) furnishes the compound of general formula (I). A general approach for the preparation of compounds of the formulae (IIa) (wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , A, F, Z, Q and n are as defined in the general description) (and P = protecting group, like Boc or PMB) is depicted in synthetic scheme 2. Synthetic Scheme 2

The substitution reaction of compound of formula (1) (wherein R’ = Me or Et) with halogen bearing compound of formula (6) in the presence of a suitable reagent and solvent yields the compound of formula (7). The reaction may be carried out in the presence of

  suitable base such as cesium carbonate, potassium carbonate, sodium carbonate, cesium fluoride etc., and the solvent can be selected from DMF, DMSO, acetonitrile, 1,4-dioxane or a mixture thereof. The reaction can also be performed by Buchwald reaction using a suitable base such as tripotassium phosphate, sodium or potassium tert-butoxide, cesium carbonate, etc., in the presence of palladium acetate as catalyst and a suitable hindered ligand (eg. XPhos, di-tBuXPhos, JohnPhos, DavePhos, Sphos, etc.) in an appropriate solvent such as toluene, 1,4-doxane, water or a mixture thereof. The coupling reaction of the ester of formula (7) with amine of formula (4) in the presence of a suitable reagent and solvent directly affords the deprotected final compound of general formula (IIa). The suitable base used in the reaction may be potassium tert-butoxide or trimethyl aluminium solution. The coupling reaction may be carried out in a suitable solvent or mixture thereof. The suitable solvent may be selected from dichloromethane, THF, toluene, or a combination thereof. Alternatively, the coupling reaction of compound of formula (1) with the amine of formula (4) gives amide of formula (5) which on substitution reaction with compound of formula (6) followed by deprotection furnishes the compound of general formula (IIa). The reaction conditions for the alternative sequence may remain the same as described in scheme 1. Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc. A general approach for the preparation of compounds of the formulae (IIb) (wherein R ⁵ , R ⁶ , R ⁷ , A, E, F, D and n are as defined in the general description) is depicted in synthetic scheme 3.

n h i h m

The benzoic acid derivative of formula (8) on coupling reaction with ethyl 2-(3- aminophenyl)-2,2-difluoroacetate (9) yields the amide derivative of formula (10). The reaction may be carried out via acid chloride formation using oxalyl chloride or thionyl

  chloride followed by coupling with the amine in the presence of suitable base and solvent. The suitable base for the reaction may be triethylamine, N,N-diisopropylethylamine, pyridine or DMAP and solvent may be selected from THF, chloroform, dichloromethane or 1,4- dioxane. The reductive amination and deprotection of compound of formula (10) using ammonia solution in methanol at elevated temperature (above 50 °C) affords the compound of formula (11). Substitution of compound of formula (11) with halogen derivative (2) yields the compound of formula (12). The reaction may be carried out in the presence of suitable base such as cesium carbonate, potassium carbonate, sodium carbonate, cesium fluoride etc.; and the solvent can be selected from DMF, DMSO, acetonitrile, 1,4-dioxane or a mixture thereof. The amide group of compound of formula (12) converts to the nitrile group by reaction with Burgess Reagent to give compound of general formula (IIb). The reaction may be carried out in a suitable solvent such as dichloromethane. A general approach for the preparation of compounds of the formula (IIIa) and (IIIb) (wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , A, F, Z, Q and n are as defined in the general description) (and wherein R’ = Me or Et and P = protecting group, like Cbz, Boc or PMB) is depicted in synthetic scheme 4.

Synthetic Scheme 4

 

The amino phenol compound of formula (13) on reaction with compound of formula (6) yields the compound of formula (14). The reaction may be carried out in the presence of suitable base such as cesium carbonate, potassium carbonate, sodium carbonate, cesium fluoride etc.; and the solvent can be selected from DMF, DMSO, toluene, acetonitrile, 1,4- dioxane or a mixture thereof. The amine compound of formula (14) on step wise reaction with triphosgene and amine of formula (4) in THF; followed by deprotection yields the urea derivative of general formula (IIIa). Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc. Alternatively, Compound of general formula (IIIa) may be synthesized by the reaction of a carbamate derivative of amine (4’) with compound (14) in the presence of a suitable base and solvent. The suitable base for the reaction may be triethylamine, DIPEA, etc. and the suitable solvent may be DMSO. Compound of general formula (IIIa) can also be synthesized by an alternative sequence of reaction starting from Z- protected analogue (13’) of compound (13). The compound of formula (13’) on step wise reaction with triphosgene and amine of formula (4) in THF; followed by Z-deprotection yields the urea derivative of formula (14’). Compound (14’) on reaction with compound of formula (6) yields the compound general formula (IIIa). The reaction conditions may remain same as discussed above.

Alternatively, the amine of formula (14) on reaction with acid compound of formula (4’) yields the amide compound of formula (15). The coupling reaction may be carried out in the presence of suitable coupling agent such as HATU, EDCI.HCl with or without HOBt, T3P or DCC. The reaction may be carried out in suitable solvent selected from THF, dichloromethane, dichloroethane, chloroform, 1,4-dioxane or a mixture thereof. The compound of formula (16) on deprotection yields the compound of general formula (IIIb). Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc. A general approach for the preparation of compounds of the formula (IIc) (wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , A, F, Q and n are as defined in the general description) (and wherein R’ = Me or Et; P = protecting group, like Boc or PMB) is depicted in synthetic scheme 5. Synthetic Scheme 5

 

The coupling reaction of the nitro ester of formula (16) with amine of formula (4) in the presence of a suitable reagent and solvent affords the compound of general formula (17). The suitable base used in the reaction may be potassium tert-butoxide or trimethyl aluminium solution. The coupling reaction may be carried out in a suitable solvent or mixture thereof. The suitable solvent may be selected from dichloromethane, THF, toluene, or a combination thereof. Nitro reduction compound of formula (17) yields the amine of formula (18). The reaction may be carried out using iron powder in the presence of acetic acid or ammonium chloride in appropriate solvent such as methanol, ethanol, THF, water or a mixture thereof. The substitution reaction of compound of formula (18) with halogen bearing compound of formula (6) in the presence of a suitable reagent and solvent yields the compound of formula (19). The reaction may be performed using Buchwald coupling method in the presence of suitable base, catalyst, ligand and solvent. The reaction may be performed using base such as sodium or potassium tert-butoxide, cesium or potassium carbonate, etc. palladium acetate can be used as a catalyst along with a suitable ligand (eg. XPhos, t-BuXPhos, JohnPhos) and appropriate solvent can be selected from 1,4-dioxane, toluene, water or a mixture thereof. The compound of formula (19) on deprotection yields the compound of general formula (IIc). Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc. A general approach for the preparation of compounds of the formula (IId) and (IIe), (IIf) and (IIg) (wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , A, Q and n are as defined in the general description) (and P = protecting group, like Boc or PMB) is depicted in synthetic scheme 6. Synthetic Scheme 6

 

The compound of formula (5a) with phenolic hydroxyl group on substitution with halogen bearing compound of formula (20) yields the ether of formula (21). The reaction may be carried out in the presence of suitable base and solvent. Suitable base may be potassium carbonate, cesium carbonate, cesium fluoride etc. and the suitable solvent may be DMF, DMSO, 1,4-dioxane, etc. The compound of formula (21) on N-deprotection affords the compound of general formula (IId). Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc.

Alternatively compound of formula (21) on reaction with sodium borohydride in a suitable solvent yields the gives the hydroxyl derivative of formula (22). The suitable solvent for the reaction may be THF, methanol or a mixture thereof. The compound (22) undergoes further reduction in the presence of trimethylsilane and trifluoroacetic acid followed by N- deprotection to furnish the compound of general formula (IIf). Deprotection reaction may be carried out using hydrochloric acid or trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc.

The compound of general formula (IId) on reduction using sodium borohydride yields the compound of general formula (IIe). The suitable solvent for the reaction may be THF, methanol or a mixture thereof. In another embodiment the compound of formula (5a) on reaction with formyl derivative of formula (23) in the presence of base and solvent affords the compound of formula (24). Suitable base may be potassium carbonate, cesium carbonate, cesium fluoride etc. and the suitable solvent may be DMF, DMSO, 1,4-dioxane, etc.

The reaction of compound of formula (24) with methylamine solution in the presence of catalytic amount of acetic acid followed by N-deprotection yields the compound of general formula (IIg). Deprotection reaction may be carried out using hydrochloric acid or

 

trifluoroacetic acid in suitable solvent such as methanol, ethanol, ethyl acetate, 1,4-dioxane, dichloroethane, etc. A general approach for the preparation of compounds of the formula (2a) (wherein R ¹ , R ² , R ³ and R ⁴ are as defined in the general description) (and P = protecting group, like Boc or PMB) is depicted in synthetic scheme 7.

Synthetic Scheme 7

4,6-Dichloro-5-methoxypyrimidine (25) on reaction with appropriately substituted ethanolamine derivative of formula (26) in the presence of suitable base and solvent yields the compound of formula (27). The suitable base for the reaction may be potassium carbonate and solvent may be DMF or 1,4-dioxane. In attempt to cyclize the compound of formula (27) using boron tribromide in a suitable solvent such as THF, affords either of the compound of formula (28) or (29) or a mixture thereof in varied ratio, which on reaction with a suitable protecting agent in the presence of a suitable base and solvent furnishes the compound of formula (2a). The N-protecting agent can be tert-butyl dicarbonate (Boc anhydride) or 4- methoxybenzylchloride (PMB-Cl). The suitable base for the reaction may be triethylamine, DIPEA, DMAP, or a mixture thereof and solvent can be selected from THF, dichloromethane, 1,4-dioxane, DMF or a mixture thereof. A general approach for the preparation of compounds of the formula (2b) (wherein R ³ and R ⁴ are as defined in the general description) is depicted in synthetic scheme 8.

Synthetic Scheme 8

The demethylation reaction of 4,6-dichloro-5-methoxypyrimidine (25) with aluminium chloride in a suitable solvent such as dichloroethane yields 4,6-dichloropyrimidin-

  5-ol (30) which on reaction with appropriately substituted glycolate ester of formula (31) under Mitsunobu condition affords the ethyloxy-ester of formula (32). The Mitsunobu reaction may be carried out in the presence of triphenylphosphine, diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) in THF as a solvent. The compound of formula (32) on reaction with 4-methoxybenzylamine (PMBA) in the presence of a suitable base and solvent at elevated temperature (more than 100 °C) affords the cyclized compound of formula (2b). The suitable base for the reaction may be triethylamine, DIPEA, DMAP, or a mixture thereof and solvent can be selected from DMF, 1,4-dioxane, DMSO or a mixture thereof. A general approach for the preparation of compounds of the formula (2d) (wherein R ¹ and R ² , are as defined in the general description) (and P = protecting group, like Boc or PMB) is depicted in synthetic scheme 9.

Synthetic Scheme 9

4,6-Dichloro-5-methoxypyrimidine (25) on reaction with appropriately substituted ethanolamine derivative of formula (26a) in the presence of suitable base and solvent yields the compound of formula (27a). The suitable base for the reaction may be potassium carbonate and solvent may be DMF or 1,4-dioxane. In attempt to cyclize the compound of formula (27a) using boron tribromide in a suitable solvent such as THF, affords the compound of formula (33) which on N-protection with a suitable protecting agent in the presence of suitable base and solvent furnishes the compound of formula (34). The N- protecting agent can be tert-butyl dicarbonate (Boc anhydride) or 4-methoxybenzylchloride (PMB-Cl). The suitable base for the reaction may be triethylamine, DIPEA, DMAP, or a mixture thereof and solvent can be selected from THF, dichloromethane, 1,4-dioxane, DMF or a mixture thereof. The compound of formula (34) on self-Mitsunobu reaction yields the cyclized compound of formula (2d).

 

A general approach for the preparation of compounds of the formula (2e) and (2f) (wherein P = protecting group, like Boc or PMB) is depicted in synthetic scheme 10.

Synthetic Scheme 10

Bromination of 2-aminopyridin-3-ol (35) using bromine in absolute ethanol yields the hydrobromide salt of 2-amino-4-bromopyridin-3-ol (36). The compound (36) on reaction with dibromoethane (37) in the presence of suitable base and solvent at elevated temperature (> 50 °C) yields 8-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (38). The suitable base for the reaction may be potassium or cesium carbonate and solvent can be selected from acetonitrile, dichloromethane, 1,4-dioxane, THF or a mixture thereof. Compound (38) on N- protection with a suitable protecting agent in the presence of suitable base and solvent furnishes the compound of formula (2e). The N-protecting agent can be tert-butyl dicarbonate (Boc anhydride) or 4-methoxybenzylchloride (PMB-Cl). The suitable base for the reaction may be LiHMDS, triethylamine, DIPEA, DMAP, or a mixture thereof. The suitable solvent can be selected from THF, dichloromethane, 1,4-dioxane, DMF or a mixture thereof. Alternatively, 2-amino-4-bromopyridin-3-ol hydrobromide (36) undergoes cyclization with chloroacetyl chloride (39) at elevated temperature (> 50 °C) in the presence of suitable base solvent to yield 8-bromo-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (40). The suitable base for the reaction may be sodium bicarbonate and solvent may be 2-butanone, water or a mixture thereof. Compound (40) on N-protection with a suitable protecting agent in the presence of suitable base and solvent furnishes the compound of formula (2f). The N-protecting agent can be tert-butyl dicarbonate (Boc anhydride) or 4-methoxybenzylchloride (PMB-Cl). The suitable base for the reaction may be cesium carbonate, and solvent can be selected from THF, 1,4-dioxane, DMF or a mixture thereof. A general approach for the preparation of compounds of the formula (2g) is depicted in synthetic scheme 11.  

Synthetic Scheme 11

4,6-Dichloropyrimidine-5-carbaldehyde (41) on reaction with vinylmagnesium chloride (42) in THF affords 1-(4,6-dichloropyrimidin-5-yl)prop-2-en-1-ol (43) which on oxidation yields 1-(4,6-dichloropyrimidin-5-yl)prop-2-en-1-one (44). The oxidation reaction may be carried out using Des -Martin periodinane in dichloromethane. The compound of formula (44) on reaction with 4-methoxybenzylamine in the presence of a suitable base (optional) and solvent at elevated temperature (> 45 °C) affords the cyclized compound of formula (2g). The suitable base for the reaction may be triethylamine, DIPEA, DMAP, or a mixture thereof and solvent can be selected from DMF, 1,4-dioxane, DMSO or a mixture thereof.

A general approach for the preparation of compounds of the formula (1a) (wherein R ⁶ and n are as defined in the general description) and X is halogen is depicted in synthetic scheme 12.

Synthetic Scheme 12

The compound of formula (45) on reaction with benzyl alcohol (46) in the presence of suitable base and solvent yields the compound of formula (47). The suitable base for the reaction may be potassium tert-butoxide and solvent may be DMSO. The compound of formula (47) undergoes esterification using sulfuric acid in methanol under reflux conditions to give the compound of formula (48) which on palladium (palladium on carbon 5-10%, 50% wet) catalyzed hydrogenation affords the compound of formula (1a). The hydrogenation reaction may be performed in a suitable solvent such as ethanol, methanol, ethyl acetate, or a combination thereof. Alternatively, the benzoic acid of formula (49) on esterification reaction using sulfuric acid in methanol affords the methyl ester of formula (1a).   A general approach for the preparation of compounds of the formula (1a) (wherein R ⁶ and n are as defined in the general description) and X is halogen is depicted in synthetic scheme 13.

Synthetic Scheme 13

The amino benzoic acid derivative of formula (50) on diazotization reaction using sodium nitrite and hydrochloric acid followed by reaction with potassium ethyl xanthate (51) in the presence of a suitable base such as sodium bicarbonate yields the thiophenol-benzoic acid of formula (52). The suitable solvent for the reaction is water. The benzoic acid of formula (52) on esterification reaction using sulfuric acid in methanol affords the methyl ester of formula (1b). A general approach for the preparation of compounds of the formula (4a) (wherein R ⁸ and R ⁹ are as defined in the general description) is depicted in synthetic scheme 14.

Synthetic Scheme 14

The compound of formula (53) (wherein‘hal’ = halogen) on reaction with compound of formula (54) affords the compound of formula (55). The reaction may be done in the presence of suitable base and solvent. The suitable base may be sodium, potassium or cesium carbonate, sodium or potassium tert-butoxide, sodium hydride, cesium fluoride, etc. The solvent may be selected from THF, DMF, toluene, DMSO, chloroform, dichloromethane, acetonitrile, dichloroethane, 1,4-dioxane or a mixture thereof. Nitro reduction of compound (55) yields the compound of formula (4a). The reaction may be carried out using iron powder in the presence of acetic acid or ammonium chloride in appropriate solvent such as methanol, ethanol, THF, water or a mixture thereof. Nitro reduction can also be done by palladium (palladium on carbon 5-10%, 50% wet) catalyzed hydrogenation. The hydrogenation reaction may be performed in a suitable solvent such as ethanol, methanol, ethyl acetate, or a combination thereof. Intermediates  

Methods for the Synthesis of Intermediate A

Intermediate A1

tert-Butyl 4-chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate

Step 1: 2-((6-Chloro-5-methoxypyrimidin-4-yl)amino)ethanol

To a stirred solution of 4,6-dichloro-5-methoxypyrimidine (10 g, 55.8 mmol) in 1,4-dioxane (100 mL) were added ethanolamine (3.42 mL, 56.9 mmol) and potassium carbonate (9.26 g, 67.0 mmol) and the mixture was refluxed at 125 °C for 8 h. The mixture was cooled to RT and partitioned between ethyl acetate and water. The organic layer was separated and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure to yield 10.5 g of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.44 (t, J = 5.6 Hz, 2H), 3.52 (t, J = 6.8 Hz, 2H), 3.72 (s, 3H), 4.80-4.85 (br s, 1H), 7.55 (s, 1H), 8.04 (s, 1H); ESI-MS (m/z) 204 (M+H) ⁺ .

Step 2: 4-Chloro-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine

A mixture of 2-((6-chloro-5-methoxypyrimidin-4-yl)amino)ethanol (step 1 intermediate) (6.0 g, 29.4 mmol) and boron tribromide in dichloromethane (1.0M, 100 mL) was refluxed for 3-4 h. The mixture was concentrated and the residue was diluted with water. The solution was neutralized with saturated sodium bicarbonate solution and the product was extracted in ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield 4.0 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 4.02 (t, J = 10.4 Hz, 2H), 4.66 (t, J = 10.0 Hz, 2H), 8.46 (s, 1H), 10.31 (br s, 1H).

Step 3: tert-Butyl 4-chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate

To a stirred solution of 4-chloro-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine (step 2 intermediate) (4.0 g, 23.3 mmol) in dichloromethane (40 mL) were added di-tert-butyl dicarbonate (Boc anhydride) (7.6 g, 34.9 mmol) at 0 °C followed by triethylamine (9.7 mL) and the mixture was stirred at 0 °C for 3 h and then 1 h at RT. DMAP (1.4 g, 11.6 mmol) was  

added in small portions to the reaction mixture at 0 °C and stirred for 1 h at RT. The solvent was evaporated under reduced pressure and the residue was diluted with ice-water mixture. The aqueous mixture was neutralized using sodium bicarbonate solution and the product was extracted in dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue thus obtained was purified by silica gel column chromatography to yield 1.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.49 (s, 9H), 3.92 (t, J = 4.8 Hz, 2H), 4.12 (t, J = 4.4 Hz, 2H), 8.31 (s, 1H). Intermediate A2

4-Chloro-8-(4-methoxybenzyl)-6H-pyrimido[5,4-b][1,4]oxazi n-7(8H)-one

Step 1: 4,6-Dichloropyrimidin-5-ol

A suspension of 4,6-dichloro-5-methoxypyrimidine (2.0 g, 11.2 mmol) and aluminum chloride (2.0 g, 15.0 mmol) in DCE (10 mL) was heated to reflux for 3-4 h. The mixture was concentrated under reduced pressure, cooled to 0 °C and quenched with ice-water. The mixture was diluted with 1M HCl (10 mL). The precipitated solid was filtered and dried well to yield 1.0 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.39 (s, 1H), 11.67 (br s, 1H).

Step 2: Ethyl 2-((4,6-dichloropyrimidin-5-yl)oxy)acetate

To a mixture of 4,6-dichloropyrimidin-5-ol (step 1 intermediate) (4.0 g, 24.2 mmol), ethyl glycolate (3.02 g, 29.1 mmol) and triphenylphosphine (12.7 g, 48.5 mmol) in THF (40 mL) was slowly added diethyl azodicarboxylate (DEAD) (9.8 g, 48.5 mmol) at RT. The mixture was stirred overnight at RT. The mixture was diluted with diethyl ether and filtered off the precipitated solid. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to yield 450 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.34 (t, J = 7.2 Hz, 3H), 4.31 (q, J = 7.2 Hz, 2H), 4.80 (s, 2H), 8.51 (s, 1H).

  Step 3: 4-Chloro-8-(4-methoxybenzyl)-6H-pyrimido[5,4-b][1,4]oxazin-7 (8H)-one To a solution of ethyl 2-((4,6-dichloropyrimidin-5-yl)oxy)acetate (step 2 intermediate) (1.0 g, 3.98 mmol) in DMF (10 mL) were added 4-methoxybenzylamine (818 mg, 5.97 mmol) followed by DIPEA (513 g, 3.98 mmol) at 0 °C. The mixture was stirred overnight at RT and the 1 h at 130 °C. The mixture was cooled and quenched with water. The aqueous mixture was extracted twice with dichloromethane. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue was purified by silica gel column chromatography to yield 660 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.71 (s, 3H), 5.05 (s, 2H), 5.12 (s, 2H), 6.86 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 8.44 (s, 1H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 1. Table 1: Chemical name, structure and analytical data of Intermediates A15-A17

Intermediate A3

(S)-tert-Butyl 4-chloro-6-methyl-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carbo xylate

 

Step 1: (S)-1-((6-Chloro-5-methoxypyrimidin-4-yl)amino)propan-2-ol

To a stirred solution of 4,6-dichloro-5-methoxypyrimidine (5.0 g, 27.9 mmol) in 1,4-dioxane (50 mL) were added potassium carbonate (4.6 g, 33.5 mmol) followed by (S)-(+)-1-amino-2- propanol (2.3 g, 30.7 mmol) at RT. The resultant mixture was stirred at 125 °C for 8 h. The mixture was cooled to RT and diluted with ethyl acetate and water. The layers were separated and the organic layer was washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated to yield 4.85 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.05 (d, J = 6.4 Hz, 3H), 3.28-3.34 (m, 2H), 3.73 (s, 3H), 3.78-3.87 (m, 1H), 4.77 (d, J = 4.8 Hz, 1H), 7.46 (t, J = 5.6 Hz, 1H), 8.04 (s, 1H); ESI-MS (m/z) 218 (M+H) ⁺ .

Step 2: (S)-4-((2-Bromopropyl)amino)-6-chloropyrimidin-5-ol

A mixture of (S)-1-((6-chloro-5-methoxypyrimidin-4-yl)amino)propan-2-ol (step 1 intermediate) (4.8 g, 22.1 mmol) and boron tribromide (1M in dichloromethane, 25 mL) was heated at 80 °C for 18 h. The solvent was removed under reduced pressure and the residue was quenched with ice-cooled water. The aqueous solution was neutralized using sodium bicarbonate and extracted twice with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated to yield 3.9 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.63 (d, J = 6.4 Hz, 3H), 3.58-3.64 (m, 1H), 3.72-3.79 (m, 1H), 4.39-4.47 (m, 1H), 7.48 (t, J = 5.6 Hz, 1H), 7.86 (s, 1H), 9.99 (br s, 1H).

Step 3: (S)-tert-Butyl 4-chloro-6-methyl-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carbo xylate The titled compound was prepared by the reaction of (S)-4-((2-bromopropyl)amino)-6- chloropyrimidin-5-ol (step 2 intermediate) (500 mg, 2.69 mmol) with di-tert-butyl dicarbonate (881 mg, 4.84 mmol) in the presence of DMAP (296 mg, 2.48 mmol) in dichloromethane (10 mL) as per the procedure described in step 3 of Intermediate A1 to yield   154 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.50 (s, 9H), 1.63 (d, J = 6.8 Hz, 3H), 3.57-3.63 (m, 1H), 3.75-3.81 (m, 1H), 4.39 (q, J = 6.8 Hz, 1H), 8.27-8.33 (m, 1H); ESI- MS (m/z) 285 (M) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 2. Table 2: Chemical name, structure and analytical data of Intermediate A10

Intermediate A4

tert-Butyl 4-chloro-7,7-dimethyl-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-c arboxylate

Step 1: 2-((6-Chloro-5-methoxypyrimidin-4-yl)amino)-2-methylpropan-1 -ol

To a stirred solution of 4,6-dichloro-5-methoxypyrimidine (5.0 g, 27.9 mmol) in 1,4-dioxane (50 mL) were added potassium carbonate (4.6 g, 33.5 mmol) followed by 2-amino-2- methylpropanol (2.71 mL, 28.4 mmol) at RT. The resultant mixture was stirred at 125 °C for 8 h. The mixture was cooled to RT and diluted with ethyl acetate and water. The layers were separated and the organic layer was washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated to yield 3.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.37 (s, 6H), 3.50 (d, J = 5.6 Hz, 2H), 3.74 (s, 3H), 5.10 (t, J = 6.0 Hz, 1H), 6.30 (s, 1H), 8.06 (s, 1H).

Step 2: 4-Chloro-6-((1-hydroxy-2-methylpropan-2-yl)amino)pyrimidin-5 -ol

 

To a solution of 2-((6-chloro-5-methoxypyrimidin-4-yl)amino)-2-methylpropan-1 -ol (step 1 intermediate) (3.5 g, 15.1 mmol) in dichloromethane (35 mL) was added boron tribromide (1M in THF, 75.8 mL) at 0 °C and the mixture was stirred at RT for 16 h. The reaction mixture was quenched with methanol at 0 °C and concentrated under reduced pressure. The residue was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated to yield 2.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.36 (s, 6H), 3.48 (s, 2H), 5.13-5.15 (br s, 1H), 6.11 (s, 1H), 7.88 (s, 1H), 9.97 (s, 1H).

Step 3: tert-Butyl (6-chloro-5-hydroxypyrimidin-4-yl)(1-hydroxy-2-methylpropan- 2- yl)carbamate

To a stirred solution of 4-chloro-6-((1-hydroxy-2-methylpropan-2-yl)amino)pyrimidin-5 -ol (step 2 intermediate) (2.5 g, 11.4 mmol) in dichloromethane (25 mL) were added di-tert-butyl dicarbonate (2.5 g, 11.4 mmol) at 0 °C followed by triethylamine (2.4 mL, 17.1 mmol) and the mixture was stirred at RT for 15 h. The solvent was evaporated under reduced pressure and the residue thus obtained was purified by silica gel column chromatography to yield 1.9 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.36 (br s, 6H), 1.50 (br s, 9H), 3.50 (d, J = 6.0 Hz, 2H), 5.06 (t, J = 10.0 Hz, 1H), 6.55 (s, 1H), 8.20 (s, 1H).

Step 4: tert-Butyl 4-chloro-7,7-dimethyl-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-c arboxylate To a stirred solution of tert-butyl (6-chloro-5-hydroxypyrimidin-4-yl)(1-hydroxy-2- methylpropan-2-yl)carbamate (step 3 intermediate) (1.9 g, 5.97 mmol) in anhydrous THF (20 mL) were added triphenylphosphine (1.88 g, 7.18 mmol) and diisopropyl azodicarboxylate (DIAD) (1.39 mL, 7.17 mmol) at 0 °C under nitrogen atmosphere. The mixture was stirred for 1 h at 0 °C. The solvent was evaporated under reduced pressure and the residue thus obtained was purified by silica gel column chromatography to yield 350 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.36 (s, 6H), 1.51 (s, 9H), 4.13 (s, 2H), 8.19 (s, 1H); ESI-MS (m/z) 300 (M+H) ⁺ .

  The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 3.

Table 3: Chemical name, structure and analytical data of Intermediates A5-A6 and A11-A14

 

Intermediate A7

tert-Butyl 8-bromo-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate

Step 1: 2-Amino-4-bromopyridin-3-ol hydrobromide

To a solution of 2-aminopyridin-3-ol (45 g, 409 mmol) in absolute ethanol (225 mL) at 0-10 °C was drop wise added bromine (64 mL, 818 mmol) and the mixture was stirred for 3 days at RT. The solvent was removed under reduced pressure at low temperature and the residue was cooled to 0 °C. Ethyl acetate was added to the residue and stirred for 1 h. The solid was filtered and dried well to yield 48 g of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 5.94 (br s, 2H), 6.65 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 5.6 Hz, 1H).

Step 2: 8-Bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine

To a solution of 2-amino-4-bromopyridin-3-ol hydrobromide (step 1 intermediate) (15 g, 58.6 mmol) in acetonitrile (150 mL) were added cesium carbonate (57.1 g, 175 mmol) followed by 1,2-dibromoethane (16.4 g, 87.7 mmol) and the mixture was refluxed for 48 h. The mixture was filtered and the filtrates was concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 3.0 g of the titled compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.33-3.43 (m, 2H), 4.13-4.20 (m, 2H), 6.71 (d, J = 5.2 Hz, 1H), 7.02 (s, 1H), 7.40 (d, J = 5.2 Hz, 1H); ESI-MS (m/z) 215 (M+H) ⁺ .

Step 3: tert-Butyl 8-bromo-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate

To a solution of 8-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (step 2 intermediate) (2.1 g, 9.76 mmol) in anhydrous THF (20 mL) were dropwise added lithium bis(trimethylsilyl)amide (LiHMDS) (1M, 11.6 mL, 11.7 mmol) followed by di-tert-butyl dicarbonate (3.3 mL, 14.6 mmol) at 0 °C and the mixture was stirred at the same temperature for 1 h. The reaction was quenched with saturated ammonium chloride solution and extracted

  with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 2.4 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.46 (s, 9H), 3.86 (d, J = 4.4 Hz, 2H), 4.35 (d, J = 4.4 Hz, 2H), 7.38 (d, J = 5.2 Hz, 1H), 7.81 (d, J = 5.2 Hz, 1H); ESI-MS (m/z) 316 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 4.

Table 4: Chemical name, structure and analytical data of Intermediates A26

Intermediate A8

8-Bromo-4-(4-methoxybenzyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H )-one

Step 1: 8-Bromo-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one

To a solution of 2-amino-4-bromopyridin-3-ol hydrobromide (step 1-Intermediate A7) (12 g, 63.4 mmol) in a mixture of 2-butanone and water (1:1, 120 mL) was added aqueous solution of sodium bicarbonate (16 g, 190 mmol) at 0 °C and the mixture was stirred for 10 min. Chloroacetyl chloride (7.16 g, 63.4 mmol) was added to the mixture and stirred for 3-4 h at 0 °C. Then the mixture was heated to 80 °C and stirred for 10 h. The mixture was cooled to RT and extracted twice with ethyl acetate. The combined organic layers were washed with water, brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue obtained was purified by silica gel column chromatography to yield 2.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 4.77 (s, 2H), 7.26 (d, J = 5.2 Hz, 1H), 7.75 (d, J = 5.2 Hz, 1H), 11.44 (s, 1H); ESI-MS (m/z) 229 (M+H) ⁺ .  

Step 2: 8-Bromo-4-(4-methoxybenzyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H )-one To a solution of 8-bromo-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (step 1 intermediate) (250 mg, 1.09 mmol) in DMF (5.0 mL) were added cesium carbonate (709 mg, 2.18 mmol) followed by 4-methoxybenzyl chloride (256 mg, 1.63 mmol) and the mixture was stirred for 3 h at RT. The mixture was quenched with water and the product was extracted twice in ethyl acetate. The combined organic layers were washed with water, brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the residue obtained was purified by silica gel column chromatography to yield 270 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.78 (s, 3H), 4.80 (s, 2H), 5.30 (s, 2H), 6.83 (d, J = 5.2 Hz, 2H), 7.16 (d, J = 5.2 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 5.2 Hz, 1H).

Intermediate A9

4-Chloro-8-(4-methoxybenzyl)-7,8-dihydropyrido[2,3-d]pyri midin-5(6H)-one

Step 1: 1-(4,6-Dichloropyrimidin-5-yl)prop-2-en-1-ol

To a stirred solution of 4,6-dichloropyrimidine-5-carbaldehyde (10 g, 56.5 mmol) in anhydrous THF (100 mL) was slowly added vinylmagnesium chloride (1M, 67.6 mL, 67.8 mmol) at -20 °C and the mixture was stirred for 3 h at RT. The reaction mixture was quenched with aqueous ammonium chloride solution and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated to yield 5.0 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 5.36-5.39 (m, 1H), 5.43 (s, 1H), 5.90-5.92 (br s, 1H), 6.15-6.23 (m, 1H), 8.73 (s, 1H).

Step 2: 1-(4,6-Dichloropyrimidin-5-yl)prop-2-en-1-one

To a stirred solution of 1-(4,6-dichloropyrimidin-5-yl)prop-2-en-1-ol (step 1 intermediate) (100 mg, 0.49 mmol) in dichloromethane (5.0 mL) was added Dess–Martin periodinane (415 mg, 0.97 mmol) at 0 °C and the mixture was stirred overnight at RT. The mixture was filtered

 

through celite and the filtrate was washed with water. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography to yield 20 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 6.10 (d, J = 17.6 Hz, 1H), 6.33 (d, J = 10.8 Hz, 1H), 6.62-6.69 (m, 1H), 8.89 (s, 1H).

Step 3: 4-Chloro-8-(4-methoxybenzyl)-7,8-dihydropyrido[2,3-d]pyrimid in-5(6H)-one To a solution of 1-(4,6-dichloropyrimidin-5-yl)prop-2-en-1-one (step 2 intermediate) (1.1 g, 5.40 mmol) in DMF (28 mL) was added 4-methoxybenzylamine (1.1 g, 8.12 mmol) at RT. The mixture was stirred overnight at 50 °C. The mixture was cooled and quenched with water. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue was purified by silica gel column chromatography to yield 900 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.29 (t, J = 6.0 Hz, 2H), 3.78-3.82 (m, 5H), 4.66 (d, J = 5.6 Hz, 2H), 6.89 (dd, J1 = 2.0 Hz, J2 = 6.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 8.38 (s, 1H).

Intermediate A18

(R)-tert-Butyl 4-chloro-7-(morpholinomethyl)-6H-pyrimido[5,4-b][1,4]oxazine -8(7H)- carboxylate

Step 1: (R)-Methyl 2-((tert-butoxycarbon l amino -3- meth lsulfonyl)oxy)propanoate

To a stirred solution (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate (2.0 g, 9.13 mmol) in dichloromethane (20 mL) were added DIPEA (3.9 mL, 22.8 mmol) followed by methanesulfonyl chloride (Mesyl chloride) (850 µL, 10.9 mmol) at 0 °C and the mixture was stirred at 0 °C for 1 h. The mixture was diluted with ethyl acetate and water. The organic layer was separated and washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure to yield 2.95 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.41 (s, 9H), 3.20 (s, 3H), 3.67 (s, 3H), 4.35-4.45 (m, 3H), 7.53 (d, J = 8.0 Hz, 1H).

Step 2: (R)-Methyl 2-((tert-butoxycarbonyl)amino)-3-morpholinopropanoate  

To a stirred solution of (R)-methyl 2-((tert-butoxycarbonyl)amino)-3- ((methylsulfonyl)oxy)propanoate (step 1 intermediate) (2.9 g, 9.76 mmol) in dichloromethane (20 mL) were added morpholine (1.7 mL, 19.5 mmol) followed by N,N- diisopropylethylamine (DIPEA) (3.3 mL, 19.5 mmol) at 0 °C. The mixture stirred at RT for 18 h. The reaction mixture was concentrated under reduced pressure and the residue obtained was purified by column chromatography to yield 1.46 g of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.96 (t, J = 7.2 Hz, 3H), 2.28-2.51 (m, 10H), 3.72 (s, 2H), 8.09 (d, J = 8.4 Hz, 1H), 8.40 (s, 1H), 8.51 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H).

Step 3: (R)-tert-Butyl (1-hydroxy-3-morpholinopropan-2-yl)carbamate

To a solution of (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-morpholinopropanoate (step 2 intermediate) (1.4 g, 4.86 mmol) in THF (15 mL) was added DIBAL solution (1M in toluene, 19.4 mL, 19.4 mmol) at -78 °C and the mixture was stirred at the same temperature for 2 h. The reaction was quenched with brine and stirred for 1 h. The solution was filtered and washed with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 1.2 g of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9H), 2.20-2.38 (m, 6H), 3.52-3.60 (m, 6H), 4.60 (t, J = 5.6 Hz, 1H), 8.41 (d, J = 8.8 Hz, 1H), 8.32 (s, 1H); ESI-MS (m/z) 260 (M+H) ⁺ .

Step 4: (R)-tert-Butyl (1-((4,6-dichloropyrimidin-5-yl)oxy)-3-morpholinopropan-2- yl)carbamate

The titled compound was prepared by the reaction of (R)-tert-butyl (1-hydroxy-3- morpholinopropan-2-yl)carbamate (step 3 intermediate) (1.2 g, 4.61 mmol) with 4,6- dichloropyrimidin-5-ol (step 1 of Intermediate A2) (912 mg, 5.51 mmol) in the presence of DIAD (1.4 g, 6.92 mmol) and triphenylphosphine (1.8 g, 6.92 mmol) in THF (10 mL) as per  

the procedure described in step 2 of Intermediate A2 to yield 2.96 g of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.18 (d, J = 6.0 Hz, 9H), 2.50-2.51 (m, 4H), 3.50-3.55 (m, 4H), 3.90-4.0 (m, 1H), 4.09-4.13 (m, 1H), 4.21-4.23 (m, 1H), 4.74-4.80 (m, 2H), 8.32 (s, 1H); ESI-MS (m/z) 406 (M+H) ⁺ .

Step 5: (R)-1-((4,6-Dichloropyrimidin-5-yl)oxy)-3-morpholinopropan-2 -amine hydrochloride

To a solution of (R)-tert-butyl (1-((4,6-dichloropyrimidin-5-yl)oxy)-3-morpholinopropan-2- yl)carbamate (step 4 intermediate) (2.9 g, 7.13 mmol) in ethyl acetate (10 mL) was added hydrochloric acid in 1,4-dioxane (4M, 40 mL) and the mixture was stirred at RT for 3 h. The mixture was concentrated under reduced pressure to give 536 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.28-3.66 (m, 6H), 3.90-4.05 (m, 4H), 4.44-4.48 (m, 3H), 8.76 (s, 1H), 9.01 (br s, 3H).

Step 6: (R)-4-Chloro-7-(morpholinomethyl)-7,8-dihydro-6H-pyrimido[5, 4-b][1,4]oxazine

To a solution of (R)-1-((4,6-dichloropyrimidin-5-yl)oxy)-3-morpholinopropan-2 -amine hydrochloride (step 5 intermediate) (520 mg, 1.51 mmol) in DMF (5.0 mL) was added DIPEA (2.6 mL, 15.1 mmol) and the mixture was stirred overnight at RT. The mixture was quenched with water and extracted twice with ethyl acetate. The combined organic extracts were washed with water, brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue obtained was purified by column chromatography to yield 271 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.38-2.46 (m, 4H), 2.49-2.51 (m, 2H), 3.57 (t, J = 4.8 Hz, 4H), 3.77-3.82 (m, 1H), 4.15-4.16 (s, 2H), 7.89 (s, 1H), 8.09 (s, 1H); ESI-MS (m/z) 271 (M+H) ⁺ .

Step 7: (R)-tert-Butyl 4-chloro-7-(morpholinomethyl)-6H-pyrimido[5,4-b][1,4]oxazine - 8(7H)-carboxylate

The titled compound was prepared by the reaction of (R)-4-chloro-7-(morpholinomethyl)-7,8- dihydro-6H-pyrimido[5,4-b][1,4]oxazine (step 6 intermediate) (260 mg, 0.96 mmol) with di-

  tert-butyl dicarbonate (230 mg, 1.05 mmol) in the presence of DMAP (106 mg, 0.86 mmol) in dichloromethane (20 mL) as per the procedure described in step 3 of Intermediate A1 to yield 231 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.50 (s, 9H), 3.51-2.29 (m, 6H), 3.52 (d, J = 4.4 Hz, 4H), 4.03 (q, J = 7.2 Hz, 1H), 4.21 (dd, J ₁ = 2.4 Hz, J ₂ = 11.2 Hz, 1H), 8.27-8.33 (m, 1H); ESI-MS (m/z) 285 (M) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 5.

Table 5: Chemical name, structure and analytical data of Intermediate A23-A25

Intermediate A19

(S)-tert-Butyl 7-(((tert-butoxycarbonyl)oxy)methyl)-4-chloro-6H-pyrimido[5, 4- b][1,4]oxazine-8(7H)-carboxylate

Step 1: N-(6-Chloro-5-hydroxypyrimidin-4-yl)acetamide

 

To a solution of 4-amino-6-chloropyrimidin-5-ol (1.5 g, 10.3 mmol) in dichloromethane (15 mL) was added acetyl chloride (0.8 mL, 10.3 mmol) followed by dropwise addition of triethylamine (4.2 mL, 30.9 mmol) at 0 °C and the mixture was stirred at RT for 3 h. The mixture was diluted with ethyl acetate and water. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic extracts were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 790 mg of the desired compound. ESI-MS (m/z) 188 (M+H) ⁺ .

Step 2: (S)-1-(4-Chloro-7-(hydroxymethyl)-6H-pyrimido[5,4-b][1,4]oxa zin-8(7H)- yl)ethanone

To a suspension of potassium carbonate (1.47 g, 10.7 mmol) in acetonitrile (20 mL) were added N-(6-chloro-5-hydroxypyrimidin-4-yl)acetamide (step 1 intermediate) (500 mg, 2.67 mmol) followed by (2R)-(-)-glycidyl tosylate (669 mg, 2.93 mmol) and the mixture was refluxed for 24 h. The mixture was cooled to RT and diluted with ethyl acetate and water. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic extracts were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 270 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.12 (s, 3H), 3.96-4.16 (m, 1H), 4.16-4.30 (m, 4H), 6.23 (s, 1H), 8.06 (s, 1H).

Step 3: (S)-(4-Chloro-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-7-yl )methanol

A solution of (S)-1-(4-chloro-7-(hydroxymethyl)-6H-pyrimido[5,4-b][1,4]oxa zin-8(7H)- yl)ethanone (step 2 intermediate) (150 mg, 0.62 mmol) in hydrochloric acid in 1,4-dioxane (3.0 mL) was stirred at RT for 3 h. The mixture was concentrated under reduced pressure and the residue was diluted with water. The aqueous mixture was basified using saturated sodium  

bicarbonate solution till pH 8-9 at -20 °C. The mixture was extracted twice with ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 170 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.34-3.41 (m, 1H), 3.47-3.57 (m, 2H), 4.11-4.32 (m, 2H), 5.09 (t, J = 5.6 Hz, 1H), 7.88 (s, 1H), 8.21-8.22 (br s, 1H).

Step 4: (S)-tert-Butyl 7-(((tert-butoxycarbonyl)oxy)methyl)-4-chloro-6H-pyrimido[5, 4- b][1,4]oxazine-8(7H)-carboxylate

The titled compound was prepared by the reaction of (S)-(4-chloro-7,8-dihydro-6H- pyrimido[5,4-b][1,4]oxazin-7-yl)methanol (step 3 intermediate) (170 mg, 0.84 mmol) with di-tert-butyl dicarbonate (476 mg, 2.18 mmol) in the presence of DMAP (246 mg, 2.01 mmol) in dichloromethane (10 mL) as per the procedure described in step 3 of Intermediate A1 to yield 100 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 9H), 1.50 (s, 9H), 4.02-4.13 (m, 1H), 4.15-4.35 (m, 1H), 4.31 (dd, J1 = 2.8 Hz, J2 = 11.2 Hz, 1H), 4.55- 4.79 (m, 1H), 4.80-4.81 (m, 1H), 8.33 (s, 1H).

Intermediate A20

tert-Butyl 9-bromo-3,4-dihydropyrido[3,2-b][1,4]oxazepine-5(2H)-carboxy late

Step 1: 4-Bromo-3-(3-chloropropoxy)p ridin-2-amine

The titled compound was prepared by the reaction of 2-amino-4-bromopyridin-3-ol hydrobromide (step 1 of Intermediate A7) (1.0 g, 3.90 mmol) and 1-bromo-3-chloropropane (925 mg, 5.80 mmol) in the presence of cesium carbonate (3.8 g, 11.7 mmol) in acetonitrile (10 mL) as per the procedure described in step 2 of Intermediate A7 to yield 400 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.19-2.25 (m, 2H), 3.95 (t, J = 6.0 Hz, 2H), 4.39 (t, J = 5.6 Hz, 2H), 6.17 (s, 2H), 6.74 (d, J = 5.6 Hz, 1H), 7.55 (d, J = 5.6 Hz, 1H).

Step 2: 9-Bromo-2,3,4,5-tetrahydropyrido[3,2-b][1,4]oxazepine

 

To a stirred solution of 4-bromo-3-(3-chloropropoxy)pyridin-2-amine (step 1 intermediate) (400 mg, 1.20 mmol) in DMF (5.0 mL) was added sodium hydride (60% w/w, 96 mg, 2.40 mmol) at RT. The mixture stirred at 80 °C for 1 h. The reaction mixture was quenched with water and diluted with ethyl acetate. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated sodium bicarbonate solution followed by brine. The organic layer was dried over anhydrous sodium, filtered and concentrated under reduced pressure to yield 250 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.94-1.98 (m, 2H), 3.25-3.29 (m, 2H) 4.15 (t, J = 6.0 Hz, 2H), 6.39 (br s, 1H), 6.86 (d, J = 5.6 Hz, 1H), 7.52 (d, J = 5.2 Hz, 1H),

Step 3: tert-Butyl 9-bromo-3,4-dihydropyrido[3,2-b][1,4]oxazepine-5(2H)-carboxy late The titled compound was prepared by the reaction of 9-bromo-2,3,4,5-tetrahydropyrido[3,2- b][1,4]oxazepine (step 2 intermediate) (2.0 g, 8.77 mmol) and di-tert-butyl dicarbonate (2.84 g, 13.0 mmol) in the presence of lithium bis(trimethylsilyl)amide (LiHMDS) (1M, 11 mL, 10.6 mmol) in anhydrous THF (20 mL) as per the procedure described in step 3 of Intermediate A7 to yield 2.1 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.35 (s, 9H), 1.94-1.97 (m, 2H), 3.60-3.63 ( br s, 2H), 4.12-4.14 (br s, 2H), 7.63 (d, J = 5.2 Hz, 1H), 8.02 (d, J = 5.2 Hz, 1H). Intermediate A21

tert-Butyl 8-bromo-7-nitro-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxyla te

Step 1: 8-Bromo-7-nitro-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine

To a solution of 8-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (step 2 of Intermediate A7) (802 mg, 3.73 mmol) in trifluoroacetic acid (5.0 mL) was added potassium nitrate (415 mg, 4.10 mmol) at 0 °C. The mixture was gradually warmed to RT and stirred overnight at RT. The mixture was quenched with ice-water and neutralized with aqueous sodium hydroxide solution at 0 °C. The precipitated solid was filtered, washed with water and dried. The crude solid was triturated with diethyl ether and dried well to yield 732 mg of the desired

  compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.52 (t, J = 4.0 Hz, 2H), 4.26 (t, J = 4.0 Hz, 2H), 8.46 (s, 1H), 8.60 (s, 1H).

Step 2: tert-Butyl 8-bromo-7-nitro-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxyla te

The titled compound was prepared by the reaction of 8-bromo-7-nitro-3,4-dihydro-2H- pyrido[3,2-b][1,4]oxazine (step 1 intermediate) (875 mg, 3.36 mmol) with di-tert-butyl dicarbonate (1.15 mL, 5.04 mmol) in the presence of triethylamine (0.94 mL, 6.72 mmol) and DMAP (41 mg, 0.34 mmol) in dichloromethane (20 mL) as per the procedure described in step 3 of Intermediate A4 to yield 1.02 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.49 (s, 9H), 3.94 (t, J = 4.4 Hz, 2H), 4.46 (t, J = 4.4 Hz, 2H), 8.58 (s, 1H).

Methods for the Synthesis of Intermediate B

Intermediate B1

Preparation of methyl 4-chloro-3-hydroxybenzoate

To a stirred solution of 4-chloro-3-hydroxybenzoic acid (690 mg, 4.00 mmol) in methanol (8.0 mL) was added sulfuric acid (10 µL, 0.20 mmol) and the mixture was heated at 75 °C for 14 h. The mixture was concentrated, residue was cooled to 0 °C and neutralized using aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated to yield 902 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ^3.91 (s, 3H), 5.66 (d, J = 4.4 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.56 (dd, J = 8.3, 2.0 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H); ESI-MS (m/z) 187 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 6.

Table 6: Chemical name, structure and analytical data of Intermediates B2-B12

 

Intermediate B13  

Methyl 3-hydroxy-4-(trifluoromethyl)benzoate

Step 1: Methyl 3-(benzyloxy)-4-(trifluoromethyl)benzoate

A mixture of 3-fluoro-4-(trifluoromethyl)benzoic acid (1.0 g, 4.80 mmol) and benzyl alcohol (3.67 mL, 36.4 mmol) and potassium tert-butoxide (1.58 g, 14.2 mmol) in DMSO was stirred at RT for 2 days. The mixture was diluted with ethyl acetate and water. The aqueous layer was separated and acidified with conc. HCl and extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated. The residue was dissolved in methanol (5.0 mL). Sulfuric acid (0.5 mL) was added to the mixture and refluxed for 3 h. The mixture was concentrated, residue was cooled to 0 °C and neutralized using aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated to yield 510 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.97 (s, 3H), 5.27 (s, 2H), 7.35-7.50 (m, 5H), 7.69-7.70 (m, 2H), 7.74 (s, 1H).

Step 2: Methyl 3-hydroxy-4-(trifluoromethyl)benzoate

A solution of methyl 3-(benzyloxy)-4-(trifluoromethyl)benzoate (step 1 intermediate) (500 mg, 2.27 mmol) in methanol (5.0 mL) with catalytic amount of 10% palladium on carbon (50% wet) was hydrogenated at RT for 16 h. The mixture was filtered through celite and the celite bed was rinsed with methanol. The combined filtrate and washings were concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 400 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.87 (s, 3H), 7.47 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 11.12 (br s, 1H).

Intermediate B14

Methyl 3-mercapto-4-methylbenzoate

Step 1: 3-Mercapto-4-methylbenzoic acid  

To a stirred suspension of 3-amino-4-methylbenzoic acid (2.0 g, 13.22 mmol) in conc. hydrochloric acid (8.0 mL) and water (20 mL) at 0 °C was added a solution of sodium nitrite (958 mg, 13.88 mmol) in water (20 mL) over a period of 20 min. The diazotization mixture was added lot wise to a stirred mixture of potassium ethyl xanthate (2.5 g, 15.8 mmol) and 2M aqueous sodium carbonate solution (22 mL, 43.6 mmol) over a period of 20 min. The mixture was heated for 1 h at 45 °C. The mixture was acidified with conc. hydrochloric acid and the product was extracted twice in ethyl acetate. The combined organic extracts were washed with water and dried over anhydrous sodium sulfate. The residue obtained after removal of solvents was refluxed overnight with potassium hydroxide (2.9 g, 52.9 mmol) in a mixture of ethanol and water (1:1, 20 mL). The reaction mixture was cooled to RT and acidified with conc. hydrochloric acid and extracted thrice with ethyl acetate. The combined organic extracts were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to yield 760 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 2.23 (s, 3H), 5.64 (s, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.60 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 7.98 (s, 1H), 12.90 (s, 1H); ESI-MS (m/z) 167 (M-H)-.

Step 2: Methyl 3-mercapto-4-methylbenzoate

To a stirred solution of 3-mercapto-4-methylbenzoic acid (760 g, 4.52 mmol) in methanol (10 mL) was added sulfuric acid (250 µL) and the mixture was refluxed for 2 h. The mixture was concentrated, residue was cooled to 0 °C and neutralized using aqueous sodium bicarbonate solution. The mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated to yield 502 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.28 (s, 3H), 3.83 (s, 3H), 5.75 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.62 (dd, J ₁ = 1.6 Hz, J ₂ = 7.6 Hz, 1H), 8.01 (s, 1H).

Methods for the Synthesis of Intermediate C Intermediate C1

4-((4-Ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)anil ine

Step 1: 1-(Bromomethyl)-4-nitro-2-(trifluoromethyl)benzene  

To a solution of 2-methyl-5-nitrobenzotrifluoride (10 g, 48.5 mmol) and AIBN (800 mg, 4.85 mmol) in ethylene dichloride (150 mL) was added N-bromosuccinimide (8.6 g, 48.5 mmol) at RT and the reaction mixture was refluxed for 18 h. The mixture was concentrated and the residue was diluted with water and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 6.21 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 4.88 (s, 2H), 8.04 (d, J = 8.4 Hz, 1H), 8.43 (s, 1H), 8.54 (dd, J ₁ = 2.4 Hz, J ₂ = 8.4 Hz, 1H).

Step 2: 1-Ethyl-4-(4-nitro-2-(trifluoromethyl)benzyl)piperazine

To a stirred solution of 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (step 1 intermediate) (4.0 g, 14.1 mmol) in dichloromethane (40 mL) were added N-ethylpiperazine (1.88 mL, 14.7 mmol) followed by N,N-diisopropylethylamine (DIPEA) (3.27 mL, 19.1 mmol) at RT. The mixture stirred at RT for 16 h. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution followed by brine. The organic layer was dried over anhydrous sodium, filtered and concentrated under reduced pressure. The residue obtained was purified by column chromatography to yield 3.5 g of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 0.96 (t, J = 7.2 Hz, 3H), 2.28-2.51 (m, 10H), 3.72 (s, 2H), 8.09 (d, J = 8.4 Hz, 1H), 8.40 (s, 1H), 8.51 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H).

Step 3: 4-((4-Ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline

A solution of 1-ethyl-4-(4-nitro-2-(trifluoromethyl)benzyl)piperazine (step 2 intermediate) (800 mg, 2.52 mmol) in methanol (20 mL) with catalytic amount of 10% palladium on carbon (50% wet) was hydrogenated at RT for 16 h. The mixture was filtered through celite and the celite bed was rinsed with methanol. The combined filtrate and washings were concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 600 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.97 (t, J = 7.2 Hz, 3H), 2.26-2.51 (m, 10H), 3.38 (s, 2H), 5.44 (s, 2H), 6.74 (dd, J ₁ = 2.4 Hz, J ₂ = 8.4 Hz, 1H), 6.85 (s, 1H), 7.29 (t, J = 8.4 Hz, 1H).

  The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 7.

Table 7: Chemical name, structure and analytical data of Intermediates C2-C15

 

 

Intermediate C16

4-(2-(Dimethylamino)ethoxy)-3-(trifluoromethyl)aniline

Step 1: N,N-Dimethyl-2-(4-nitro-2-(trifluoromethyl)phenoxy)ethanamin e

To a stirred solution of N,N-dimethylethanolamine (2.9 mL, 48.6 mmol) in DMF (10 mL) was added sodium hydride (60% w/w, 421 mg, 10.5 mmol) at 0 °C and stirred for 30 min.1- Fluoro-4-nitro-2-(trifluoromethyl)benzene (2.0 g, 9.56 mmol) was added to the mixture and stirred at RT for 2 h. The reaction mixture was quenched with water extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine, dried over anhydrous sodium, filtered and concentrated under reduced pressure. The residue obtained was purified by column chromatography to yield 2.3 g of the desired product. The compound was as such taken forward to the next step without characterization.

Step 2: 4-(2-(Dimethylamino)ethoxy)-3-(trifluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation reaction of N,N-dimethyl- 2-(4-nitro-2-(trifluoromethyl)phenoxy)ethanamine (2.2 g, 7.91 mmol) in methanol (15 mL) as per the procedure described in step 3 of Intermediate C1 to yield 1.8 g of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.20 (s, 6H), 2.58 (t, J = 6.0 Hz, 2H), 4.0 (t, J = 6.0 Hz, 2H), 5.03 (s, 2H), 6.77 (dd, J ₁ = 2.8 Hz, J ₁ = 8.4 Hz, 1H), 6.81-6.82 (br s, 1H), 6.97 (d, J = 8.8 Hz, 1H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 8.

Table 8: Chemical name, structure and analytical data of Intermediates C17 and C67

 

Intermediate C18

3-(4-Isopropylpiperazin-1-yl)-5-(trifluoromethyl)aniline

To a stirred solution of 3-bromo-5-(trifluoromethyl)aniline (1.5 g, 6.25 mmol) in DMF (10 mL) were added 1-isoprpylpiperazine (3.6 mL, 24.4 mmol), cesium carbonate (4.06 g, 12.5 mmol), copper (I) iodide (595 mg, 3.12 mmol) and 8-hydroxyquinoline (272 mg, 1.87 mmol) at RT. The mixture was heated in a sealed tube at 120 °C for 16 h. The reaction mixture was cooled to RT and diluted with ethyl acetate. The solution was washed with water followed by brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by column chromatography to yield 660 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.00 (d, J = 6.4 Hz, 6H), 2.50-2.55 (m, 4H), 2.62-2.69 (m, 1H), 3.07 (t, J = 4.8 Hz, 4H), 5.33-5.35 (br s, 2H), 6.28 (s, 1H), 6.32 (s, 2H); ESI-MS (m/z) 288 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 9.

Table 9: Chemical name, structure and analytical data of Intermediates C29-C23

 

Intermediate C24

4-(4-Methylpiperazin-1-yl)-3-(trifluoromethyl)aniline

Step 1: 1-Methyl-4-(4-nitro-2-(trifluoromethyl)phenyl)piperazine   A mixture of 2-fluoro-5-nitrobenzotrifluride (5.0 g, 23.9 mmol) and 1-methylpiperazine (7.18 g, 71.7 mmol) in DMSO (20 mL) was heated at 100 °C for 5 h. The mixture was cooled to RT and diluted with ethyl acetate and water. The organic layer was separated; and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water followed by brine and concentrated under reduced pressure to give 5.2 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.23 (s, 3H), 2.45-2.51 (m, 4H), 3.13 (t, J = 4.8 Hz, 4H), 7.54 (d, J = 8.8 Hz, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.40 (d, J = 1.6 Hz, 1H). Step 2: 4-(4-Methylpiperazin-1-yl)-3-(trifluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation reaction of 1-methyl-4-(4- nitro-2-(trifluoromethyl)phenyl)piperazine (step 1 intermediate) (5.0 g, 17.3 mmol) in methanol (200 mL) as per the procedure described in step 3 of Intermediate C1 to yield 4.12 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.19 (s, 3H), 2.49-2.51 (m, 4H), 2.71 (t, J = 4.4 Hz, 4H), 5.34 (s, 2H), 6.76 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.4 Hz, 1H); ESI-MS (m/z) 260 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 10.

Table 10: Chemical name, structure and analytical data of Intermediates C25-C26

Intermediate C27

tert-Butyl 4-(4-amino-2-(trifluoromethyl)phenyl)piperazine-1-carboxylat e

Step 1: tert-Butyl 4-(4-nitro-2-(trifluoromethyl)phenyl)piperazine-1-carboxylat e  

To a stirred solution of 2-fluoro-5-nitrobenzotrifluride (5.0 g, 23.9 mmol) in acetonitrile (50 mL) were added potassium carbonate (6.6 g, 47.8 mmol) and tert-butyl piperazine-1- carboxylate (4.9 g, 26.3 mmol) at RT and the mixture refluxed 24 h. The mixture was cooled to RT and diluted with ethyl acetate and water. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water followed by brine and concentrated under reduced pressure to give 5.23 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.43 (s, 9H), 3.06 (t, J = 5.2 Hz, 4H), 3.47 (t, J = 4.4 Hz, 4H), 7.61 (d, J = 8.8 Hz, 1H), 8.39-8.45 (m, 2H).

Step 2: tert-Butyl 4-(4-amino-2-(trifluoromethyl)phenyl)piperazine-1-carboxylat e

The titled compound was prepared by the catalytic hydrogenation reaction of tert-butyl 4-(4- nitro-2-(trifluoromethyl)phenyl)piperazine-1-carboxylate (step 1 intermediate) (3.0 g, 8.00 mmol) in methanol (200 mL) as per the procedure described in step 3 of Intermediate C1 to yield 2.3 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.41 (s, 9H), 2.66 (t, J = 4.8 Hz, 4H), 3.34 (s, 4H), 5.38 (m, 2H), 6.75 (dd J ₁ = 2.4 Hz, J ₂ = 8.4 Hz, 1H), 6.81 (s, 1H), 7.23 (d, J = 8.4 Hz, 1H); ESI-MS (m/z) 346 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 11.

Table 11: Chemical name, structure and analytical data of Intermediates C99

Intermediate C28

4-((4-Ethylpiperazin-1-yl)methyl) nilin

Step 1: 1-Ethyl-4-(4-nitrobenzyl)pip r zin  

The titled compound was prepared by the reaction of 1-(bromomethyl)-4-nitrobenzene (1.8 g, 8.32 mmol) with N-ethylpiperazine (1.16 mL, 9.16 mmol) in the presence of N,N- diisopropylethylamine (DIPEA) (2.15 mL, 12.5 mmol) in dichloromethane (10 mL) as per the procedure described in step 2 of Intermediate C1 to yield 1.23 g of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.98 (t, J = 7.2 Hz, 3H), 2.34-2.40 (m, 10H), 3.59 (s, 2H), 7.58 (d, J = 8.4 Hz, 2H), 8.19 (d, J = 8.4 Hz, 2H).

Step 2: 4-((4-Ethylpiperazin-1-yl)methyl)aniline

To a stirred solution of 1-ethyl-4-(4-nitrobenzyl)piperazine (step 1 intermediate) (200 mg, 0.88 mmol) in a mixture of methanol and water (1:1, 20 mL) were added ammonium chloride (215 mg, 4.08 mmol) followed by iron powder (224 mg. 4.028 mmol) in small portions at 100 °C. The mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was diluted with a mixture of ethyl acetate and water. The organic layer was separated and washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue obtained was purified by column chromatography to yield 3.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 0.95 (t, J = 7.2 Hz, 3H), 2.24-2.29 (m, 10H), 3.23 (s, 2H), 4.93 (br s, 2H), 6.48 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 12.

Table 12: Chemical name, structure and analytical data of Intermediates C29-C32 and C77- C78

 

Intermediate C33

N ¹ -(4-Amino-2-(trifluoromethyl)benzyl)-N ¹ ,N ² ,N ² -trimethylethane-1,2-diamine

Step 1: tert-Butyl (4-methyl-3-(trifluoromethyl)phenyl)carbamate

To a solution of 4-methyl-3-(trifluoromethyl)aniline (2.5 g, 14.2 mmol) in a mixture of 1,4- dioxane (20 mL) and water (20 mL) were added sodium carbonate (2.3 g, 21.3 mmol) and di-tert-butyl dicarbonate (3.72 g, 17.0 mmol) and the mixture was stirred at RT for 16 h. The reaction mixture was diluted with water and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed

 

with water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated to yield 3.85 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.48 (s, 9H), 2.35 (s, 3H), 7.31 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.87 (s, 1H), 9.59 (s, 1H).

Step 2: tert-Butyl (4-(bromomethyl)-3-(trifluoromethyl)phenyl)carbamate

To a solution of tert-butyl (4-methyl-3-(trifluoromethyl)phenyl)carbamate (step 1 intermediate) (3.8 g, 13.8 mmol) in carbon tetrachloride (30 mL) were added N- bromosuccinimide (4.13 g, 27.6 mmol) and AIBN (226 mg, 1.38 mmol) at RT and the reaction mixture was refluxed for 16 h. The mixture was concentrated and the residue was diluted with water and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 3.05 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.49 (s, 9H), 4.62 (s, 2H), 6.68 (s, 1H), 7.49- 7.57 (m, 2H), 7.71 (s, 1H).

Step 3: tert-Butyl (4-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-3- (trifluoromethyl)phenyl)carbamate

To a solution of tert-butyl (4-(bromomethyl)-3-(trifluoromethyl)phenyl)carbamate (step 2 intermediate) (200 mg, 0.56 mmol) in THF (10 mL) were added N,N,N’- trimethylethylenediamine (176 µL, 1.35 mmol) and triethylamine (236 µL, 1.69 mmol) at RT and the reaction mixture was stirred at RT for 2 h. The mixture was diluted with water and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 315 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.48 (s, 9H), 2.12 (s, 9H), 2.35 (q, J = 4.4 Hz, 2H), 2.41-2.44 (m, 2H), 3.53 (s, 2H), 7.62 (s, 2H), 7.87 (s, 1H), 9.66 (s, 1H); ESI-MS (m/z) 376 (M+H) ⁺ . Step 4: N ¹ -(4-Amino-2-(trifluoromethyl)benzyl)-N ¹ ,N ² ,N ² -trimethylethane-1,2-diamine

 

To a solution of tert-butyl (4-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)-3- (trifluoromethyl)phenyl)carbamate (step 3 intermediate) (300 mg, 0.80 mmol) in dichloromethane (5.0 mL) was added trifluoroacetic acid (2.0 mL) and the mixture was stirred at RT for 3 h. The reaction mixture was diluted with ethyl acetate and the organic solution was washed with saturated sodium bicarbonate solution followed by water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated to yield 102 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.11 (s, 9H), 2.34-2.39 (m, 4H), 3.33-3.40 (br s, 2H), 5.42 (s, 2H), 6.75 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 6.85 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H). Intermediate C34

N ¹ -(2-(Dimethylamino)ethyl)-N ¹ -methyl-5-(trifluoromethyl)benzene-1,3-diamine

Step 1: Di-tert-butyl (3-bromo-5-(trifluoromethyl)phenyl)imidodicarbonate

The titled compound was prepared by the reaction of 3-bromo-5-trifluoromethylaniline (5.0 g, 20.8 mmol) with di-tert-butyl dicarbonate (11.3 g, 52.1 mmol) in the presence of DMAP (254 mg, 2.08 mmol) in dichloromethane (20 mL) as per the procedure described in step 3 of Intermediate A1 to yield 3.8 g of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.41 (s, 18H), 7.77 (d, J = 6.0 Hz, 1H), 7.93-7.96 (m, 1H), 8.05 (s, 1H).

Step 2: Di-tert-butyl (3-((2-(dimethylamino)ethyl](methyl)amino)-5- (trifluoromethyl)phenyl)imidodicarbonate

To a solution of di-tert-butyl (3-bromo-5-(trifluoromethyl)phenyl)imidodicarbonate (step 1 intermediate) (2.2 g, 4.89 mmol) in 1,4-dioxane (20 mL) were added N,N,N’- trimethylethylenediamine (636 µL, 4.89 mmol), sodium tert-butoxide (1.40 g, 14.7 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ) (447 mg, 0.49 mmol) and (2- biphenyl)di-tert-butylphosphinetriethylamine (JohnPhos) (37 mg, 1.47mmol) at RT and the reaction mixture was stirred at 45 °C for 4 h. The mixture was cooled to RT and filtered   through celite. The filtrate was concentrated and the residue was dissolved in ethyl acetate. The organic solution was washed with 0.1 N HCl followed by water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 2.01 g of the desired compound. The compound was as such carried forward to the next step without characterization.

Step 3: N ¹ -(2-(Dimethylamino)ethyl)-N ¹ -methyl-5-(trifluoromethyl)benzene-1,3-diamine The titled compound was prepared by the reaction of di-tert-butyl (3-((2- (dimethylamino)ethyl](methyl)amino)-5-(trifluoromethyl)pheny l)imidodicarbonate (step 2 intermediate) (100 mg, 0.22 mmol) with trifluoroacetic acid (0.5 mL) in dichloromethane (3.0 mL) as per the procedure described in step 4 of Intermediate C33 to yield 50 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.17 (s, 6H), 2.34 (t, J = 7.2 Hz, 2H), 3.3-3.37 (m, 5H), 5.29 (s, 2H), 6.07 (s, 1H), 6.10 (s, 1H), 6.14 (s, 1H); ESI-MS (m/z) 262 (M+H) ⁺ . The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 13.

Table 13: Chemical name, structure and analytical data of Intermediate C35

Intermediate C36

1-(3-Aminophenyl)cyclopropanecarboni ril

Step 1: 1-(3-Nitrophenyl)cyclopropanec r ni ril

To a stirred solution of 2-(3-nitrophenyl)acetonitrile (500 mg, 3.08 mmol) in DMSO (10 mL) were added 1,2-dibromoethane (266 µL, 3.08 mmol) and sodium hydride (60% w/w, 123 mg, 3.08 mmol) at RT. The mixture stirred at RT for 16 h. The reaction mixture was quenched

 

with aqueous sodium sulfate solution, diluted with ethyl acetate and the organic layer was washed with saturated sodium bicarbonate solution followed by brine. The organic layer was dried over anhydrous sodium, filtered and concentrated under reduced pressure. The residue obtained was purified by column chromatography to yield 635 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.69 (t, J = 2.8 Hz, 2H), 1.87 (t, J = 2.8 Hz, 2H), 7.70 (t, J = 8.0 Hz, 1H), 7.78-7.80 (m, 1H), 8.17-8.19 (m, 2H).

Step 2: 1-(3-Aminophenyl)cyclopropanecarbonitrile

To a stirred solution of 1-(3-nitrophenyl)cyclopropanecarbonitrile (step 1 intermediate) (630 mg, 3.35 mmol) in a mixture of ethyl acetate and water (1:1, 20 mL) were added ammonium chloride (1.79 g, 33.4 mmol) followed by iron powder (747 mg.13.4 mmol) in small portions at 100 °C. The mixture was stirred at 100 °C for 2 h. The mixture was concentrated and the residue was diluted with a mixture of ethyl acetate and water. The organic layer was separated and washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue obtained was purified by column chromatography to yield 350 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.36 (t, J = 4.8 Hz, 2H), 1.66 (t, J = 4.8 Hz, 2H), 5.22 (br s, 2H), 6.36-6.38 (m, 1H), 6.46- 6.48 (m, 1H), 6.57 (s, 1H), 6.99 (t, J = 4.0 Hz, 1H).

Intermediate C37

(S)-3-(3-Fluoropyrrolidin-1-yl)-5-(trifluoromethyl)anilin e

Step 1: (S)-3-Fluoro-1-(3-nitro-5-(trifluoromethyl)phenyl)pyrrolidin e

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (1.2 g, 4.44 mmol) with (S)-3-fluoropyrrolidine (593 mg, 6.66 mmol) in the presence of cesium carbonate (4.34 g, 13.3 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (81 mg, 0.09 mmol) and (±)-2,2′- bis(diphenylphosphino)-1,1′-binaphthalene (rac-BINAP) (83 mg, 0.13 mmol) in 1,4-dioxane (20 mL) as per the procedure described in step 2 of Intermediate C34 to yield 109 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.16-2.22 (m, 1H), 2.27-2.33 (m, 1H),

  3.47-3.58 (m, 1H), 3.60-3.67 (m, 2H), 3.72 (s, 1H), 5.43-5.57 (m, 1H), 7.20 (s, 1H), 7.52 (s, 1H), 7.62 (s, 1H); ESI-MS (m/z) 279 (M+H) ⁺ .

Step 2: (S)-3-(3-Fluoropyrrolidin-1-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation of (S)-3-fluoro-1-(3-nitro- 5-(trifluoromethyl)phenyl)pyrrolidine (step 1 intermediate) (100 mg, 0.36 mmol) in the presence of palladium on carbon (10% w/w, 50% wet) as per the procedure described in step 3 of Intermediate C1 to yield 55 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.10-2.26 (m, 2H), 3.27-3.57 (m, 4H), 5.33-5.36 (br s, 2H), 5.49 (br s, 1H), 5.98 (s, 2H), 6.18 (s, 1H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 14.

Table 14: Chemical name, structure and analytical data of Intermediates C38-C39

Intermediate C40

tert-Butyl 4-(3-amino-5-(trifluorometh l hen l i eridine-1-carboxylate

Step 1: tert-Butyl 4-(3-nitro-5-(trifluoromethyl)phenyl)-5,6-dihydropyridine-1( 2H)- carboxylate

 

To a solution of 1-bromo-3-nitro-5-(trifluoromethyl)benzene (1.0 g, 3.70 mmol) in 1,4- dioxane (35 mL) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6- dihydropyridine-1(2H)-carboxylate (1.1 g, 3.70 mmol), tetrakis(triphenylphosphine)palladium(0) (213 mg, 0.18 mmol) and saturated aqueous sodium bicarbonate solution (15 mL) and the mixture was heated at 120 °C for 6 h. The mixture was cooled to RT and diluted with ethyl acetate. The organic layer was washed with water followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 891 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.52 (s, 9H), 2.60 (br s, 2H), 3.71 (t, J = 5.6 Hz, 2H), 4.18 (br s, 2H), 6.32 (br s, 1H), 7.94 (s, 1H), 8.39 (s, 1H), 8.42 (s, 1H).

Step 2: tert-Butyl 4-(3-amino-5-(trifluoromethyl)phenyl)piperidine-1-carboxylat e

The titled compound was prepared by the catalytic hydrogenation of tert-butyl 4-(3-nitro-5- (trifluoromethyl)phenyl)piperidine-1-carboxylate (step 1 intermediate) (880 mg, 2.35 mmol) in the presence of palladium on carbon (10% w/w, 50% wet) (catalytic) as per the procedure described in step 3 of Intermediate C1 to yield 728 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.50 (s, 9H), 1.55-1.66 (m, 2H), 1.82 (d, J = 12.4 Hz, 2H), 2.58-2.66 (m, 1H), 2.80 (t, J = 12.0 Hz, 2H), 4.26 (br s, 2H), 6.69 (s, 1H), 6.78 (s, 1H), 6.86 (s, 1H). Intermediate C41

3-(1-Methylpiperidin-4-yl)-5-(trifluorometh l aniline

To a stirred suspension of lithium aluminium hydride (275 mg, 7.25 mmol) in THF (20 mL) was slowly added a solution of tert-butyl 4-(3-amino-5-(trifluoromethyl)phenyl)piperidine-1- carboxylate (Intermediate C40) (500 mg, 1.45 mmol) in THF (20 mL) at RT and the mixture was heated at 90 °C for 18 h. The mixture was cooled to RT and quenched with saturated aqueous sodium sulfate solution. The mixture was filtered and the filtrate was diluted with ethyl acetate and water. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered

 

and concentrated. The residue obtained was purified by silica gel column chromatography to yield 181 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.56-1.59 (m, 2H), 1.62-1.71 (m, 2H), 1.89-1.90 (m, 2H), 2.17 (s, 3H), 2.3-2.51 (m, 1H), 2.83 (d, J = 7.6 Hz, 2H), 5.48 (s, 2H), 6.29 (s, 1H), 6.73 (s, 2H).

Intermediate C42

4-(3-Amino-5-(trifluoromethyl)phenyl)thiomorpholine 1,1-dioxide

Step 1: 4-(3-Nitro-5-(trifluoromethyl)phenyl)thiomorpholine

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (2.0 g, 7.40 mmol) with thiomorpholine (1.1 g, 11.1 mmol) in the presence of cesium carbonate (7.2 g, 22.2 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ) (135 mg, 0.15 mmol) and (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (rac-BINAP) (138 mg, 0.22 mmol) in 1,4-dioxane (30 mL) as per the procedure described in step 2 of Intermediate C34 to yield 1.05 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.66-2.69 (m, 4H), 3.79-3.81 (m, 4H), 7.60 (s, 1H), 7.69 (s, 1H), 7.86 (s, 1H). Step 2: 4-(3-Nitro-5-(trifluoromethyl)phenyl)thiomorpholine 1,1-dioxide

To a solution of 4-(3-nitro-5-(trifluoromethyl)phenyl)thiomorpholine (step 1 intermediate) (1.0 g, 3.42 mmol) in dichloromethane (20 mL) was added mCPBA (1.7 g, 10.3 mmol) at RT and the mixture was stirred at RT for 2 h. The mixture was diluted with saturated sodium bicarbonate solution and the layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue was purified by silica gel column chromatography to yield 523 mg of the desired product. ¹ H NMR (400 MHz,

 

DMSO-d6) δ 3.19 (t, J = 5.2 Hz, 4H), 4.01 19 (t, J = 5.2 Hz, 4H), 7.75 (s, 1H), 7.97 (s, 1H), 8.0 (s, 1H).

Step 3: 4-(3-Amino-5-(trifluoromethyl)phenyl)thiomorpholine 1,1-dioxide

The titled compound was prepared by the catalytic hydrogenation of 4-(3-nitro-5- (trifluoromethyl)phenyl)thiomorpholine 1,1-dioxide (step 2 intermediate) (510 mg, 1.57 mmol) in the presence of palladium on carbon (10% w/w, 50% wet) (catalytic) as per the procedure described in step 3 of Intermediate C1 to yield 346 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.12 (s, 4H), 3.72 (s, 4H), 5.43 (s, 2H), 6.35 (s, 1H), 6.42 (d, J = 6.8 Hz, 2H).

Intermediate C43

4-Amino-N-(2-(dimethylamino)ethyl)-2-(trifluoromethyl)ben zamide

Step 1: N-(2-(Dimethylamino)ethyl)-4-nitro-2-(trifluoromethyl)benzam ide

To a stirred solution of 4-nitro-2-(trifluoromethyl)benzoic acid (500 mg, 2.12 mmol) in dichloromethane (20 mL) were added triethylamine (612 µL, 4.24 mmol), N ¹ ,N ¹ - dimethylethane-1,2-diamine hydrochloride (633 µL, 5.0 mmol) followed by HATU (967 mg, 2.54 mmol) at RT. The mixture was stirred at RT for 18 h before quenching it with water. The layers were separate and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure to yield 311 mg of the titled compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.23 (s, 6H), 2.45-2.50 (m, 2H), 3.33- 3.38 (m, 2H), 7.80 (d, J = 8.4 Hz, 1H), 8.49 (s, 1H), 8.56 (dd, J ₁ = 2.0 Hz, J ₂ = 8.4 Hz, 1H), 8.76 (t, J = 5.2 Hz, 1H); ESI-MS (m/z) 306 (M+H) ⁺ .

Step 2: 4-Amino-N-(2-(dimethylamino)ethyl)-2-(trifluoromethyl)benzam ide

The titled compound was prepared by the catalytic hydrogenation of N-(2- (dimethylamino)ethyl)-4-nitro-2-(trifluoromethyl)benzamide (step 1 intermediate) (300 mg, 0.98 mmol) in the presence of palladium on carbon (10% w/w, 50% wet) in methanol (10 mL) as per the procedure described in step 3 of Intermediate C1 to yield 151 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.16 (s, 6H), 2.33 (t, J = 7.2 Hz, 2H), 3.18-3.26

 

(m, 2H), 5.79 (s, 2H), 6.73 (dd, J1 = 2.0 Hz, J2 = 10 Hz, 1H), 6.87 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 8.0 (t, J = 5.6 Hz, 1H); ESI-MS (m/z) 276 (M+H) ⁺ .

Intermediate C44

(4-Amino-2-(trifluoromethyl)phenyl)(4-ethylpiperazin-1-yl )methanone

Step 1: (4-Ethylpiperazin-1-yl)(4-nitro-2-(trifluoromethyl)phenyl)me thanone

To a stirred solution of 4-nitro-2-(trifluoromethyl)benzoic acid (2.0 g, 8.51 mmol) in dichloromethane (20 mL) were added triethylamine (8.5 mL, 59.5 mmol), N-ethylpiperazine (1.24 mL, 9.35 mmol) followed by T3P (11.1 mL, 18.7 mmol) at RT. The mixture was stirred at RT for 16 h before quenching it with water. The layers were separate and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography to yield 2.2 g of the titled compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.99 (d, J = 6.8 Hz, 3H), 2.19-2.50 (m, 6H), 3.03-3.16 (m, 2H), 3.58-3.72 (m, 2H), 7.82 (d, J = 8.4 Hz, 1H), 8.53- 8.58 (m, 2H).

Step 2: (4-Amino-2-(trifluoromethyl)phenyl)(4-ethylpiperazin-1-yl)me thanone

The titled compound was prepared by the catalytic hydrogenation of (4-ethylpiperazin-1- yl)(4-nitro-2-(trifluoromethyl)phenyl)methanone (step 1 intermediate) (2.0 g, 6.04 mmol) in the presence of 10% palladium on carbon (50% wet) in methanol (50 mL) as per the procedure described in step 3 of Intermediate C1 to yield 1.9 g of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.98 (t, J = 7.2 Hz, 3H), 2.19-2.50 (m, 6H), 3.09-3.18 (m, 2H), 3.57- 3.58 (m, 2H), 5.78 (s, 2H), 6.78 (dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 1H), 6.88 (s, 1H), 7.01 (d, J = 8.0 Hz, 1H).

Intermediate C45

Ethyl 2-(3-aminophenyl)-2,2-difluoro Step 1: Ethyl 2,2-difluoro-2-(3-nitrophenyl)acetate

 

To a stirred suspension of 1-iodo-3-nitrobenzene (5.0 g, 20.1 mmol) and copper powder (5.0 g, 80.3 mmol) in DMSO (20 mL) was added ethyl 2-bromo-2,2-difluoroacetate (5.1 mL, 40.2 mmol) at RT. The mixture was stirred overnight at 60 ºC in a sealed tube. The reaction mixture was cooled to RT and quenched with aqueous ammonium chloride solution. The aqueous mixture was poured into water and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material obtained was purified by silica gel column chromatography to yield 3.2 g of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.23 (t, J = 7.2 Hz, 3H), 4.26 (q, J = 7.2 Hz, 2H), 7.89 (t, J = 8.4 Hz, 1H), 8.09 (dd, J ₁ = 0.8 Hz, J ₂ = 8.0 Hz, 1H), 8.33 (t, J = 2.0 Hz, 1H), 8.47 (dd, J1 = 0.8 Hz, J2 = 6.8 Hz, 1H); ESI-MS (m/z) 245 (M+H) ⁺ .

Step 2: Ethyl 2-(3-aminophenyl)-2,2-difluoroacetate

The titled compound was prepared by the reaction of ethyl 2,2-difluoro-2-(3- nitrophenyl)acetate (step 1 intermediate) (600 mg, 2.44 mmol) with iron powder (567 mg, 10.2 mmol) and ammonium chloride (1.30 g, 24.4 mmol) in a mixture of ethyl acetate and water (7:2, 9.0 mL) as per the procedure described in step 2 of Intermediate C28 to yield 350 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.23 (t, J = 7.2 Hz, 3H), 4.20 (q, J = 7.2 Hz, 2H), 5.53 (br s, 2H), 6.65 (d, J = 7.6 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H); ESI-MS (m/z) 216 (M+H) ⁺ .

Intermediate C46

3-(4,4-Difluoropiperidin-1-yl)-5-(trifluoromethyl)aniline

Step 1: 4,4-Difluoro-1-(3-nitro-5-(trifluoromethyl)phenyl)piperidine

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (250 mg, 0.93 mmol) with 4,4-difluoropiperidine (354 mg, 2.77

  mmol) in the presence of sodium tert-butoxide (140 mg, 1.45 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ) (44 mg, 0.05 mmol) and 1,1- bis(diphenylphosphinoferrocene (54 mg, 0.10 mmol) in 1,4-dioxane (3.0 mL) as per the procedure described in step 2 of Intermediate C34 to yield 150 mg of the desired compound. 1H NMR (400 MHz, DMSO-d ₆ ) δ 2.062.09 (m, 4H), 3.60 (t, J = 5.60 Hz, 4H), 7.72 (s, 1H), 7.67 (s, 1H), 7.97 (s, 1H).

Step 2: 3-(4,4-Difluoropiperidin-1-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation of 4,4-difluoro-1-(3-nitro- 5-(trifluoromethyl)phenyl)piperidine (step 1 intermediate) (220 mg, 0.71 mmol) in the presence of palladium on carbon (10% w/w, 50% wet) in methanol (5.0 mL) as per the procedure described in step 3 of Intermediate C1 to yield 70 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.97-2.07 (m, 4H), 3.28-3.33 (m, 4H), 5.39 (s, 2H), 6.32 (s, 1H), 6.40 (s, 2H); ESI-MS (m/z) 281 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 15.

Table 15: Chemical name, structure and analytical data of Intermediates C47-C49 and C63

 

Intermediate C50

N-(3-Amino-5-(trifluoromethyl)phenyl)acrylamide

To a stirred solution of 5-(trifluoromethyl)benzene-1,3-diamine (200 mg, 1.13 mmol) in dichloromethane (9.0 mL) were added triethylamine (164 µL, 1.13 mmol) followed by acryloyl chloride (31 µL, 0.34 mmol) at 0 °C. The mixture was stirred overnight at RT. The mixture was quenched with water and extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue was purified by silica gel column chromatography to yield 60 mg of the desired product. ESI-MS (m/z) 231 (M+H) ⁺ .

Intermediate C51

6-Methyl-5-(trifluoromethyl)pyridin-3-amine

Step 1: Diethyl 2-(5-nitro-3-(trifluoromethyl)pyridin-2-yl)malonate

To a stirred solution of diethylmalonate (5.0 g, 23.5 mmol) in THF (25 mL) was added potassium tert-butoxide (1M in THF, 70.5 mmol) at -10 °C and the mixture was stirred 10 min at the same temperature followed by 30 min at RT. A solution of 2-chloro-5-nitro-3- (trifluoromethyl)pyridine (5.0 g, 23.52 mmol) in THF (25 mL) was added slowly to the mixture at 0 °C and the resultant mixture was stirred for 4-5 h at RT. The mixture was quenched with aqueous ammonium chloride solution and extracted twice with ethyl acetate.   § The combined organic layers were washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated to yield 5.1 g of the desired product. The crude amine was as such carried forward to the next step.

Step 2: 2-Methyl-5-nitro-3-(trifluorometh l ridine

A solution of diethyl 2-(5-nitro-3-(trifluoromethyl)pyridin-2-yl)malonate (step 1 intermediate) (5.0 g, 14.3 mmol) in 50% sulfuric acid (50 mL) was heated at 85-90 °C for 2-3 h. The mixture was cooled to 0 °C and basified with 3N NaOH solution. The aqueous solution was extracted twice with diethyl ether. The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated to yield 3.2 g of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.79 (s, 3H), 8.73 (d, J = 2.4 Hz, 1H), 9.50 (d, J = 2.4 Hz, 1H).

Step 3: 6-Methyl-5-(trifluoromethyl)pyridin-3-amine

The titled compound was prepared by the reaction of 2-methyl-5-nitro-3- (trifluoromethyl)pyridine (250 mg, 1.21 mmol) with iron powder (679 mg, 12.1 mmol) and ammonium chloride (519 mg, 9.70 mmol) in a mixture of ethanol and water (6:1, 3.5 mL) as per the procedure described in step 2 of Intermediate C28 to yield 170 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.41 (s, 3H), 5.57 (br s, 2H), 7.19 (d, J = 2.8 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H).

Intermediate C52

tert-Butyl 4-(3-amino-5-(trifluoromethyl)phenyl)-1,4-diazepane-1-carbox ylate

Step 1: tert-Butyl 4-(3-nitro-5-(trifluoromethyl)phenyl)-1,4-diazepane-1-carbox ylate

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (100 mg, 0.37 mmol) with tert-butyl 1,4-diazepane-1-carboxylate

  (222 mg, 1.11 mmol) in the presence of sodium tert-butoxide (53 mg, 0.55 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ) (20 mg, 0.02 mmol) and 1,1- bis(diphenylphosphinoferrocene (24 mg, 0.04 mmol) in 1,4-dioxane (1.0 mL) as per the procedure described in step 2 of Intermediate C34 to yield 85 mg of the desired compound. 1H NMR (400 MHz, DMSO-d ₆ ) δ 1.42 (s, 9H), 1.630 (br s, 2H), 1.99-2.02 (m, 2H), 3.30-3.40 (m, 2H), 3.67-3.69 (m, 4H), 7.14 (s, 1H), 7.66 (s, 1H), 7.74 (s, 1H).

Step 2: tert-Butyl 4-(3-amino-5-(trifluoromethyl)phenyl)-1,4-diazepane-1-carbox ylate The titled compound was prepared by the reaction of tert-butyl 4-(3-nitro-5- (trifluoromethyl)phenyl)-1,4-diazepane-1-carboxylate (step 1 intermediate )(80 mg, 0.21 mmol) with iron powder (114 mg, 2.05 mmol) and ammonium chloride (88 mg, 1.64 mmol) in ethanol (2.0 mL) as per the procedure described in step 2 of Intermediate C28 to 35 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.34 (s, 9H), 1.71-1.99 (m, 2H), 3.16-3.32 (m, 2H), 3.45-3.56 (m, 4H), 5.23-5.24 (m, 4H), 6.12-6.15 (m, 3H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 16.

Table 16: Chemical name, structure and analytical data of Intermediates C53-C56 and C69- C70

 

nterme ate

3-(4-Methyl-1,4-diazepan-1-yl)-5-(trifluoromethyl)aniline

Step 1: 1-(3-Nitro-5-(trifluoromethyl)phenyl)-1,4-diazepane

 

A solution of tert-butyl 4-(3-nitro-5-(trifluoromethyl)phenyl)-1,4-diazepane-1-carbox ylate (step 1-Intermediate C52) (600 mg, 1.54 mmol) in hydrochloric acid in 1,4-dioxane (5.0 mL) was stirred at RT for 3 h. The mixture was concentrated under reduced pressure and the residue was diluted with water. The aqueous mixture was basified using saturated sodium bicarbonate solution till pH 8-9 at -20 °C. The mixture was extracted twice with dichloromethane. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 250 mg of the desired product. The crude amine was as such carried forward to the next step.

Step 2: 1-Methyl-4-(3-nitro-5-(trifluoromethyl)phenyl)-1,4-diazepane

To a stirred solution of 1-(3-nitro-5-(trifluoromethyl)phenyl)-1,4-diazepane (step 1 intermediate) (250 mg, 0.87 mmol) in acetonitrile (35 mL) were added methyl iodide (126 mg, 0.89 mmol) and potassium carbonate (125 mg, 0.91 mmol) at 0 °C and the mixture was stirred overnight at RT. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. The solution was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 160 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.90-1.92 (m, 2H), 2.26 (s, 3H), 2.26-2.51 (m, 2H), 2.64 (t, J = 4.8 Hz, 2H), 3.55 (t, J = 6.0 Hz, 2H), 3.65 (t, J = 4.8 Hz, 2H),7.32 (s, 1H), 7.56 (s, 1H), 7.63 (t, J = 2.0 Hz, 1H); ESI-MS (m/z) 304 (M+H) ⁺ .

Step 3: 3-(4-Methyl-1,4-diazepan-1-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the reaction of 1-methyl-4-(3-nitro-5- (trifluoromethyl)phenyl)-1,4-diazepane (step 2 intermediate ) (180 mg, 0.59 mmol) with iron powder (331 mg, 5.92 mmol) and ammonium chloride (253 mg, 4.73 mmol) in ethanol (3.0 mL) as per the procedure described in step 2 of Intermediate C28 to 140 mg of the compound. The crude amine was as such carried forward to the next step due to poor solubility and instability in solution form.

  The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 17.

Table 17: Chemical name, structure and analytical data of Intermediate C58

Intermediate C59

3-(4-(Oxetan-3-yl)piperazin-1-yl)-5-(trifluoromethyl)aniline

Step 1: 1-(3-Nitro-5-(trifluoromethyl)phenyl)-4-(oxetan-3-yl)piperaz ine

A mixture of zinc chloride (277 mg, 2.03 mmol) and molecular sieves (200 mg) was dried under vacuum followed with addition of 1-(3-nitro-5-(trifluoromethyl)phenyl)piperazine hydrochloride (100 mg, 0.40 mmol) and oxetan-3-one (150 mg, 2.08 mmol) in methanol (1.0 mL). The resultant mixture was stirred at RT for 2 h. The mixture was cooled to 0 °C and was added sodium cyanoborohydride (126 mg, 2.08 mmol) in small portions. The mixture was stirred for 2 h at RT. The mixture was filtered through celite and the filtrate was diluted with sodium bicarbonate solution. The mixture was again passed through celite and the filtrate was diluted with ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 90 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 2.42-2.51 (m, 4H), 3.43-3.47 (m, 4H), 4.34-4.37 (m, 1H), 4.48 (t, J = 6.0 Hz, 2H), 4.56-4.68 (m, 2H), 7.64 (s, 1H), 7.75 (s, 1H), 7.90 (t, J = 2.0 Hz, 1H).  

Step 2: 3-(4-(Oxetan-3-yl)piperazin-1-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the reaction of 1-(3-nitro-5-(trifluoromethyl)phenyl)-4- (oxetan-3-yl)piperazine (step 1 intermediate ) (100 mg, 0.30 mmol) with iron powder (179 mg, 3.19 mmol) and ammonium chloride (138 mg, 2.55 mmol) in ethanol (4.0 mL) as per the procedure described in step 2 of Intermediate C28 to 70 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.388 (t, J = 4.8 Hz, 4H), 3.12 (t, J = 4.8 Hz, 4H), 3.42-3.45 (m, 1H), 4.47 (t, J = 5.6 Hz, 2H), 4.56 (t, J = 6.4 Hz, 2H), 5.37 (s, 2H), 6.30 (s, 1H), 6.34 (s, 2H). Intermediate C60

(R)-1-(3-Amino-5-(trifluoromethyl)phen l -N N-dimeth l yrrolidin-3-amine

To a stirred solution of 3-bromo-5-(trifluoromethyl)aniline (200 mg, 0.83 mmol) and (R)- N,N-dimethylpyrrolidin-3-amine (200 mg, 1.75 mmol) in DMF (2.0 mL) were added copper iodide (83 mg, 0.44 mmol) followed by 8-hydroxyquinoline (40 mg, 0.26 mmol) at RT in a sealed tube. The mixture was stirred overnight at 120 °C. The mixture was cooled to RT and diluted with water. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 80 mg of the desired product. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 1.73-1.83 (m, 1H), 2.12-2.17 (m, 6H), 2.74-2.78 (m, 1H), 2.98 (t, J = 8.4 Hz, 1H), 3.16- 3.22 (m, 1H), 3.33-3.41 (m, 4H), 5.28 (s, 2H), 5.95 (s, 1H), 6.14 (s, 1H); ESI-MS (m/z) 274 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 18.

Table 18: Chemical name, structure and analytical data of Intermediate C61

  Intermediate C62

tert-Butyl 3-amino-5-(trifluoromethyl)benzyl(isopropyl)carbamate

Step 1: N-(3-Nitro-5-(trifluoromethyl)benzyl)propan-2-amine

A solution of 2-propanamine (408 mg, 7.04 mmol) in dichloromethane (5.0 mL) was cooled to 0 °C and to that was slowly added a solution of 1-(bromomethyl)-3-nitro-5- (trifluoromethyl)benzene (500 mg, 1.76 mmol) in dichloromethane (5.0 mL). The mixture was warmed to RT and stirred for 18 h. The mixture was poured into water and the layers were separated. The aqueous layer was extracted with dichloromethane. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to yield 170 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.13 (d, J = 6.0 Hz, 6H), 2.96 (br s, 1H), 4.11 (br s, 2H), 8.32 (s, 1H), 8.43 (s, 1H), 8.64 (s, 1H).

Step 2: tert-Butyl isopropyl(3-nitro-5-(trifluoromethyl)benzyl)carbamate

The titled compound was prepared by the reaction of N-(3-nitro-5- (trifluoromethyl)benzyl)propan-2-amine (step 1 intermediate) (170 mg, 0.65 mmol) with di- tert-butyl dicarbonate (156 mg, 0.71 mmol) in the presence of DIPEA (168 mg, 1.30 mmol) in dichloromethane (5.0 mL) as per the procedure described in step 3 of Intermediate A1 to yield 240 mg of the product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.15 (d, J = 6.8 Hz, 6H), 1.57 (s, 9H), 4.47 (br s, 3H), 7.85 (s, 1H), 8.32 (s, 1H), 8.38 (s, 1H).

Step 3: tert-Butyl 3-amino-5-(trifluoromethyl)benzyl(isopropyl)carbamate

 

The titled compound was prepared by the reaction of tert-butyl isopropyl(3-nitro-5- (trifluoromethyl)benzyl)carbamate (step 2 intermediate ) (235 mg, 0.65 mmol) with iron powder (363 mg, 6.50 mmol) and ammonium chloride (278 mg, 5.20 mmol) in ethanol (5.0 mL) and water (1.5 mL) as per the procedure described in step 2 of Intermediate C28 to 170 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.05 (d, J = 6.8 Hz, 6H), 1.42 (s, 9H), 4.21 (br s, 3H), 5.56 (s, 2H), 6.62 (s, 1H), 6.67 (d, J = 8.4 Hz, 2H).

Intermediate C64

3-(1-Methyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline

Step 1: 4-(3-Nitro-5-(trifluoromethyl)phenyl)-1-(tetrahydro-2H-pyran -2-yl)-1H-pyrazole

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (1.0 g, 3.70 mmol) with 1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.12 g, 4.07 mmol) in the presence of cesium carbonate (1.80 g, 5.50 mmol), and 1,1'-bis(diphenylphosphino)ferrocene- palladium(II)dichloride dichloromethane complex (150 mg, 0.18 mmol) in 1,4-dioxane (10 mL) and water (2.0 mL) as per the procedure described in step 2 of Intermediate C34 to yield 730 mg of the desired compound ¹ H NMR (400 MHz, DMSO-d6) δ 1.53-1.68 (m, 2H), 1.69- 1.74 (m, 1H), 1.95-2.0 (m, 2H), 2.07-2.10 (m, 1H), 3.65-3.70 (m, 1H), 3.94-3.97 (m, 1H), 5.45 (dd , J1 = 2.4 Hz, J2 = 10.0 Hz, 1H), 7.86 (s, 1H), 8.32 (s, 1H), 8.51 (s, 1H), 8.75 (s, 1H), 8.87 (s, 1H).

Step 2: 4-(3-Nitro-5-(trifluoromethyl)phenyl)-1H-pyrazole

The titled compound was prepared by the reaction of 4-(3-nitro-5-(trifluoromethyl)phenyl)-1- (tetrahydro-2H-pyran-2-yl)-1H-pyrazole (step 1 intermediate) (350 mg, 1.03 mmol) with hydrochloric acid in ethyl acetate (10 mL) in methanol (5.0 mL) as per the procedure   described in step 1 of Intermediate C57 to yield 257 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.25 (s, 1H), 8.29 (s, 1H), 8.65 (s, 1H), 8.71 (s, 1H), 8.72 (d, J = 2.0 Hz, 1H), 13.24 (s, 1H).

Step 3: 1-Methyl-4-(3-nitro-5-(trifluorometh l hen l)-1H-pyrazole

The titled compound was prepared by the reaction of 4-(3-nitro-5-(trifluoromethyl)phenyl)- 1H-pyrazole (step 2 intermediate) (100 mg, 0.39 mmol) with methyl iodide (82 mg, 0.58 mmol) in the presence of sodium hydride (60% w/w, 17 mg, 0.42 mmol) in DMF (5.0 mL) as per the procedure described in step 2 of Intermediate C57 to yield 60 mg of the compound. 1H NMR (400 MHz, DMSO-d6) δ 3.89 (s, 3H), 8.24 (s, 2H), 8.41 (s, 1H), 8.59 (s, 1H), 8.66 (s, 1H).

Step 4: 3-(1-Methyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation of 1-methyl-4-(3-nitro-5- (trifluoromethyl)phenyl)-1H-pyrazole (step 3 intermediate) (55 mg, 0.20 mmol) in the presence of palladium on carbon (10% w/w, 50% wet, 20 mg) in methanol (5.0 mL) and THF (2.0 mL) as per the procedure described in step 3 of Intermediate C1 to yield 35 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.85 (s, 3H), 5.56 (s, 2H), 6.68 (s, 1H), 6.95 (s, 2H), 7.79 (s, 1H), 8.12 (s, 1H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above (step 1 and 4) are given below in Table 19.

Table 19: Chemical name, structure and analytical data of Intermediate C65

Intermediate C66

3-((1-Methylazetidin-3-yl)oxy)-5-(trifluoromethyl)aniline

 

Step 1: tert-Butyl 3-(3-nitro-5-(trifluorometh l henox )azetidine-1-carboxylate

The titled compound was prepared by the reaction of 3-nitro-5-(trifluoromethyl)phenol (1.0 g, 4.83 mmol) with N-Boc-3-hydroxyazetidine (1.0 g, 5.80 mmol) in the presence of triphenylphosphine (1.9 g, 7.20 mmol) and diisopropyl azodicarboxylate (DIAD) (1.35 mL, 7.20 mmol) in THF (10 mL) as per the procedure described in step 2 of Intermediate A2 to yield 1.52 g of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.39 (s, 9H), 3.84-3.88 (m, 2H), 4.36 (br s, 2H), 5.27-5.32 (m, 1H), 7.82 (s, 1H), 7.88-7.90 (m, 1H), 8.11 (s, 1H).

Step 2: 3-(3-Nitro-5-(trifluoromethyl)phenoxy)azetidine hydrochloride

The titled compound was prepared by the reaction of tert-butyl 3-(3-nitro-5- (trifluoromethyl)phenoxy)azetidine-1-carboxylate (step 1 intermediate) (1.50 g, 5.73 mmol) with hydrochloric acid in ethyl acetate (20 mL) in ethyl acetate (4.0 mL) as per the procedure described in step 1 of Intermediate C57 to yield 918 mg of the compound (isolated a hydrochloride salt). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 4.02-4.06 (m, 2H), 4.46-4.51 (m, 2H), 5.39-5.42 (m, 1H), 7.77 (d, J = 1.5 Hz, 1H), 7.95 (t, J = 2.0 Hz, 1H), 8.14 (s, 1H), 9.68 (br s, 2H); ESI-MS (m/z) 263 (M+H-HCl) ⁺ .

Step 3: 1-Methyl-3-(3-nitro-5-(trifluoromethyl)phenoxy)azetidine

Toa stirred solution of 3-(3-nitro-5-(trifluoromethyl)phenoxy)azetidine hydrochloride (step 2 intermediate) (900 mg, 3.01 mmol) in dichloroethane (10 mL) were added formaldehyde (37%, 135 mg, 4.52 mmol) and sodium triacetoxyborohydride (STAB) (958 mg, 4.52 mmol) and the mixture was stirred at RT for 18 h. The mixture was concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography to yield  

790 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.29 (s, 3H), 3.02-3.06 (m, 2H), 3.72-3.76 (m, 2H), 5.08 (t, J = 5.6 Hz, 1H), 7.67 (s, 1H), 7.85 (t, J = 2.0 Hz, 1H), 8.07 (s, 1H); ESI-MS (m/z) 277 (M+H) ⁺ .

Step 4: 3-((1-Methylazetidin-3-yl)oxy)-5-(trifluoromethyl)aniline

The titled compound was prepared by the reaction of 1-methyl-3-(3-nitro-5- (trifluoromethyl)phenoxy)azetidine (step 3 intermediate ) (780 mg, 2.82 mmol) with iron powder (780 mg, 14.1 mmol) and ammonium chloride (1.5 g, 28.2 mmol) in methanol (10 mL) and water (10 mL) as per the procedure described in step 2 of Intermediate C28 to 623 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.27 (s, 3H), 2.92-2.95 (m, 2H), 3.69 (dt, J1 =2.0 Hz, J1 = 6.0 Hz, 2H), 4.67-4.70 (m, 1H), 5.61 (s, 2H), 6.17 (s, 1H), 6.23 (t, J = 1.6 Hz, 1H), 6.45 (s, 1H); ESI-MS (m/z) 247 (M+H) ⁺ .

Intermediate C68

1-((4-Fluorophenyl)carbamoyl)cyclo ro anecarbox lic acid

To a stirred solution of cyclopropane-1,1-dicarboxylic acid (400 mg, 3.05 mmol) in dichloromethane (10 mL) were added thionyl chloride (222 µL, 3.05 mmol) followed by catalytic amount of DMF and the mixture was stirred at RT for 2 h. The mixture was concentrated under reduced pressure and the residue was dissolved in THF (10 mL). The solution was cooled to 0 °C; 4-fluoroaniline (0.29 mL, 3.05 mmol) was added to the reaction mixture followed by triethylamine (0.43 mL, 3.08 mmol). The resultant mixture was stirred overnight at RT. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic extracts were washed with saturated sodium bicarbonate solution, water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue thus obtained was purified by column chromatography to yield 270 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.41 (s, 4H), 7.12-7.17 (m, 2H), 7.61-7.64 (m, 2H), 10.51 (s, 1H), 12.81 (s, 1H).

Intermediate C71

3-((1S,4S)-5-Methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-5 -(trifluoromethyl)aniline

 

Step 1: (1S,4S)-tert-Butyl 5-(3-nitro-5-(trifluoromethyl)phenyl)-2,5- diazabicyclo[2.2.1]heptane-2-carboxylate

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (2.7 g, 13.6 mmol) with (1S,4S)-tert-butyl 2,5- diazabicyclo[2.2.1]heptane-2-carboxylate (500 mg, 2.52 mmol) in the presence of sodium tert-butoxide (350 mg, 3.64 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (138 mg, 0.15 mmol) and Xantphos (175 mg, 0.30 mmol) in toluene (10 mL) as per the procedure described in step 2 of Intermediate C34 to yield 840 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.60 (s, 9H), 2.06 (d, J = 5.2 Hz, 2H), 3.26-3.51 (m, 4H), 4.52 (s, 1H), 4.61-4.75 (m, 1H), 6.99 (s, 1H), 7.51 (s, 1H), 7.76 (s, 1H).

Step 2: (1S,4S)-2-(3-Nitro-5-(trifluoromethyl)phenyl)-2,5-diazabicyc lo[2.2.1]heptane hydrochloride

The titled compound was prepared by the reaction of (1S,4S)-tert-butyl 5-(3-nitro-5- (trifluoromethyl)phenyl)-2,5-diazabicyclo[2.2.1]heptane-2-ca rboxylate (step 1 intermediate) (400 mg, 1.13 mmol) with hydrochloric acid in 1,4-dioxane (4.0 mL) in ethanol (8.0 mL) as per the procedure described in step 1 of Intermediate C57 to yield 280 mg of the compound. The crude amine was as such carried forward to the next step.

Step 3: (1S,4S)-2-Methyl-5-(3-nitro-5-(trifluoromethyl)phenyl)-2,5- diazabicyclo[2.2.1]heptane

The titled compound was prepared by the reaction of (1S,4S)-2-(3-nitro-5- (trifluoromethyl)phenyl)-2,5-diazabicyclo[2.2.1]heptane hydrochloride (step 2 intermediate) (275 mg, 0.94 mmol) with methyl iodide (147 mg, 1.03 mmol) in the presence of potassium   carbonate (142 mg, 1.03 mmol) in acetonitrile (12 mL) as per the procedure described in step 2 of Intermediate C57 to yield 60 mg of the compound. The crude amine was as such carried forward to the next step.

Step 4: 3-((1S,4S)-5-Methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-5-(t rifluoromethyl)aniline The titled compound was prepared by the catalytic hydrogenation of (1S,4S)-2-methyl-5-(3- nitro-5-(trifluoromethyl)phenyl)-2,5-diazabicyclo[2.2.1]hept ane (step 3 intermediate) (60 mg, 0.19 mmol) in the presence of palladium on carbon (10% w/w, 50% wet, 20 mg) in methanol (10 mL) as per the procedure described in step 3 of Intermediate C1 to yield 60 mg (crude) of the compound. The crude amine was as such carried forward to the next step due to poor solubility and instability in solution form.

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 20.

Table 20: Chemical name, structure and analytical data of Intermediate C72-C73 and C75- C76

 

Intermediate C74

4-(3-Amino-5-(trifluoromethyl)phenyl)morpholin-3-one

Step 1: 4-(3-Nitro-5-(trifluoromethyl)phenyl)morpholine

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (2.0 g, 7.40 mmol) with morpholine (1.61 g, 18.5 mmol) in the presence of N,N-diisopropylethylamine (DIPEA) (2.7 mL, 14.8 mmol) in DMSO (10 mL) as per the procedure described in step 2 of Intermediate C1 to yield 1.1 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.36 (t, J = 5.2 Hz, 4H), 3.75 (t, J = 4.8 Hz, 4H), 7.64 (s, 1H), 7.78 (s, 1H), 7.91 (t, J = 2.4 Hz, 1H).

Step 2: 4-(3-Nitro-5-(trifluoromethyl)phenyl)morpholin-3-one

A suspension of 4-(3-nitro-5-(trifluoromethyl)phenyl)morpholine (step 1 intermediate) (1.1 g, 3.98 mmol), benzyltributylammonium chloride (7.5 g, 23.9 mmol) and KMnO4 (3.77 g, 23.9 mmol) in dichloromethane (70 mL) was heated overnight at 70 °C. The mixture was cooled to RT and diluted with ethyl acetate. The suspension was filtered through celite and the bed was washed with ethyl acetate. The combined filtrate and washings were concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography  

to yield 340 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.91-3.94 (m, 2H), 3.94-4.03 (m, 2H), 4.29 (s, 2H), 8.37 (d, J = 10.8 Hz, 2H), 8.66 (t, J = 1.6 Hz, 1H). Step 3: 4-(3-Amino-5-(trifluoromethyl)phenyl)morpholin-3-one

The titled compound was prepared by the reaction of 4-(3-nitro-5- (trifluoromethyl)phenyl)morpholin-3-one (step 2 intermediate ) (300 mg, 1.14 mmol) with iron powder (636 mg, 11.3 mmol) and ammonium chloride (487 mg, 9.10 mmol) in ethanol (10 mL) and water (4.0 mL) as per the procedure described in step 2 of Intermediate C28 to 150 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.69 (t, J = 5.2 Hz, 2H), 3.95 (t, J =4.8 Hz, 2H), 4.19 (s, 2H), 5.72 (s, 2H), 6.76 (s, 2H), 6.82 (d, J = 12.4 Hz, 2H). Intermediate C79

3'-Amino-5'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonitr ile

Step 1: 3'-Nitro-5'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonitrile

To a solution of 1-bromo-3-nitro-5-(trifluoromethyl)benzene (250 mg, 0.92 mmol) in 1,4- dioxane (10 mL) and water (1.0 mL) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)benzonitrile (215 mg, 0.94 mmol), [1,1′- bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (39 mg, 0.05 mmol) and potassium carbonate (269 mg, 1.95 mmol) and the mixture was heated at 100 °C for 18 h. The mixture was cooled to RT and diluted with ethyl acetate. The solution was filtered through celite. The filtrate was washed with water followed by brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by silica gel column chromatography to yield 350 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.03 (dd, J ₁ = 1.6 Hz, J ₂ = 3.6 Hz, 2H), 8.14 (dd, J ₁ = 2.0 Hz, J ₂ = 6.4 Hz, 2H), 8.60 (s, 2H), 8.81 (t, J = 1.6 Hz, 1H).

Step 2: 3'-Amino-5'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonitrile

  The titled compound was prepared by the reaction of 3'-nitro-5'-(trifluoromethyl)-[1,1'- biphenyl]-4-carbonitrile (step 2 intermediate ) (150 mg, 0.51 mmol) with iron powder (286 mg, 5.13 mmol) and ammonium chloride (219 mg, 4.10 mmol) in ethanol (5.0 mL) and water (2.0 mL) as per the procedure described in step 2 of Intermediate C28 to 50 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 5.80 (s, 2H), 6.92 (s, 1H), 7.08 (s, 1H), 7.12 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H).

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 21.

Table 21: Chemical name, structure and analytical data of Intermediate C80

Intermediate C81

4-(1-(4-Ethylpiperazin-1-yl)ethyl)-3-(trifluoromethyl)anilin e

Step1: 1-(4-Nitro-2-(trifluoromethyl)phenyl)ethanone

To a stirred solution of 1-bromo-4-nitro-2-(trifluoromethyl)benzene (500 mg, 1.85 mmol) and tributyl(1-ethoxyvinyl)tin (806 mg, 2.22 mmol) in DMF (5.0 mL) was added tetrakis(triphenylphosphine)palladium(0) (136 mg, 0.09 mmol) after purging argon for 15 min. The mixture was heated at 90 °C for 18 h. The mixture was cooled to RT and poured into dilute hydrochloric acid (50 mL). The solution was stirred at RT for 1 h. The aqueous mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica

 

gel column chromatography to yield 125 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.65 (s, 3H), 7.66 (d, J = 8.4 Hz, 1H), 8.50 (m, 1H), 78.60 (s, 1H).

Step 2: 1-(4-Nitro-2-(trifluoromethyl)phenyl)ethanol

To a solution of 1-(4-nitro-2-(trifluoromethyl)phenyl)ethanone (step 1 intermediate) (120 mg, 0.52 mmol) in methanol (5.0 mL) was added sodium borohydride (20 mg, 0.52 mmol) at 0 °C and the mixture was stirred at RT for 3 h. The reaction was quenched with acetone (2.0 mL) and diluted with water. The aqueous mixture was extracted twice with chloroform. The combined organic extracts were washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to yield 120 mg of the desired compound. ¹ H NMR (400 MHz, CDCl3) δ 1.53 (d, J = 6.4 Hz, 3H), 2.15 (d, J = 2.8 Hz, 1H), 5.42-5.43 (br s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 8.46 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 8.52 (s, 1H). Step 3: 1-Ethyl-4-(1-(4-nitro-2-(trifluoromethyl)phenyl)ethyl)pipera zine

To a stirred solution of 1-(4-nitro-2-(trifluoromethyl)phenyl)ethanol (step 2 intermediate) (600 mg, 2.56 mmol) in dichloromethane (5.0 mL) were added triethylamine (1.0 mL, 7.68 mmol) followed by methanesulfonyl chloride (Mesyl chloride) (587 mg, 5.12 mmol) at 0 °C and the mixture was stirred at RT for 1 h. The reaction was quenched with ice-water mixture and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure at 25 °C. The residue was dissolved in DMF (10 mL) and cooled to 0 °C. To that solution were added potassium carbonate (706 mg, 5.12 mmol) followed by N-ethylpiperazine (292 mg, 2.56 mmol) and the mixture was stirred overnight at RT. The reaction was quenched with ice-water mixture and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 320 mg of the desired compound. ¹ H NMR (400 MHz, CDCl ₃ ) δ 1.09 (t, J = 7.2 Hz, 3H), 1.32 (d, J = 10.4 Hz, 3H), 2.30-2.46 (m, 10H), 3.77 (q, J = 4.8 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 8.40 (dd, J ₁ = 2.4 Hz, J ₂ = 8.8 Hz, 1H), 8.51 (s, 1H).

Step 4: 4-(1-(4-Ethylpiperazin-1-yl)ethyl)-3-(trifluoromethyl)anilin e  

The titled compound was prepared by the reaction of 1-ethyl-4-(1-(4-nitro-2- (trifluoromethyl)phenyl)ethyl)piperazine (step 3 intermediate ) (220 mg, 0.66 mmol) with iron powder (371 mg, 6.64 mmol) and ammonium chloride (283 mg, 5.31 mmol) in ethanol (5.0 mL) and water (1.0 mL) as per the procedure described in step 2 of Intermediate C28 to 80 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.03 (t, J = 6.4 Hz, 3H), 1.17-1.23 (m, 5H), 2.51-2.92 (m, 7H), 3.36 (m, 2H), 5.76 (s, 2H), 6.78-6.81 (br s, 2H), 7.38 (d, J = 8.4 Hz, 1H).

Intermediate C82

N-(3-Amino-5-(trifluoromethyl)phenyl)propionamide

Step 1: N-(3-Bromo-5-(trifluoromethyl)phenyl)propionamide

To a stirred mixture of propionic acid (1.54 g, 20.8 mmol) and 3-bromo-5- (trifluoromethyl)aniline (5.0 g, 20.8 mmol) in dichloromethane (15 mL) were added EDCI. HCl (7.9 g, 41.6 mmol), HOBt (2.8 g, 20.8 mmol) and DIPEA (7.0 mL, 41.6 mmol) at 0 °C. The mixture was stirred at RT for 3 h. The mixture was diluted with water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with water and brine. The solution was dried over anhydrous sodium sulfate, fileted and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 2.3 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.90 (t, J = 7.2 Hz, 3H), 2.36 (q, J = 7.6 Hz, 2H), 7.60 (s, 1H), 7.98 (s, 1H), 8.11 (s, 1H), 10.35 (s, 1H).

Step 2: N-(3-((Diphenylmethylene)amino)-5-(trifluoromethyl)phenyl)pr opionamide

The titled compound was prepared by the reaction of N-(3-bromo-5- (trifluoromethyl)phenyl)propionamide (step 1 intermediate) (1.0 g, 3.37 mmol) with benzophenone imine (918 mg, 5.06 mmol) in the presence of cesium carbonate (2.2 g, 6.76  

mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (232 mg, 0.25 mmol) and (±)- 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (rac-BINAP) (105 mg, 0.16 mmol) in 1,4- dioxane (10 mL) as per the procedure described in step 2 of Intermediate C34 to yield 1.05 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.05 (t, J = 7.6 Hz, 3H), 2.30 (q, J = 7.6 Hz, 2H), 6.61 (s, 1H), 7.17-7.36 (m, 3H), 7.47-7.69 (m, 9H), 1.06 (s, 1H); ESI-MS (m/z) 397 (M+H) ⁺ .

Step 3: N-(3-Amino-5-(trifluoromethyl)phenyl)propionamide

The titled compound was prepared by the reaction of N-(3-((diphenylmethylene)amino)-5- (trifluoromethyl)phenyl)propionamide (step 2 intermediate) (1.0 g, 2.53 mmol) with hydrochloric acid in 1,4-dioxane (5.0 mL) in THF (10 mL) as per the procedure described in step 1 of Intermediate C57 to yield 431 mg of the compound. ¹ H NMR (400 MHz, DMSO- d6) δ 1.06 (t, J = 7.6 Hz, 3H), 2.29 (q, J = 7.6 Hz, 2H), 5.58 (s, 2H), 6.51 (s, 1H), 7.07 (s, 1H), 7.10 (s, 1H), 9.84 (s, 1H).

Intermediate C83

2-(4-Amino-2-(trifluoromethyl)phenyl)-2-methylpropanenitr ile

Step 1: 2-(4-Nitro-2-(trifluoromethyl)phenyl)acetonitrile

A mixture of 2-chloro-5-nitrobenzotrifluoride (3.0 g, 13.3 mmol), potassium carbonate (367 mg, 2.66 mmol), potassium iodide (3.3 g, 19.9 mmol) and ethyl cyanoacetate (1.80 g, 15.9 mmol) in DMF (20 mL) was stirred at RT for 72 h. The mixture was quenched with 10% aqueous citric acid solution and extracted twice with ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was dissolved in a mixture of water (25 mL) and acetic acid (10 mL) and was added 37% hydrochloric acid at RT. The mixture was heated at 100 °C for 30 h. The reaction mixture was cooled to RT and quenched with 10% aqueous potassium carbonate solution. The aqueous mixture was extracted twice with ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 2.3 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) ^

 

4.40 (s, 2H), 8.02 (d, J = 8.8 Hz, 1H), 8.49 (s, 1H), 8.60 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H) ; ESI-MS (m/z) 229 (M-H)-.

Step 2: 2-Methyl-2-(4-nitro-2-(trifluoromethyl)phenyl)propanenitrile

To a stirred solution of 2-(4-nitro-2-(trifluoromethyl)phenyl)acetonitrile (step 1 intermediate) (500 mg, 2.17 mmol) in THF (15 mL) were added methyl iodide (925 mg, 6.51 mmol) followed by potassium tert-butoxide solution (1M, 6.5 mL, 6.51 mmol) at 0 °C and the mixture was stirred at RT for 18 h. The mixture was diluted with aqueous ammonium chloride solution and extracted with ethyl acetate. The organic extract was washed with brine and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 70 mg of the desired compound (Fraction-1) along with 400 mg of 2-(4-nitro-2- (trifluoromethyl)phenyl)propanenitrile (Fraction-2). The fraction-2 (400 mg, 1.64 mmol) was further reacted with methyl iodide (465 mg, 3.27 mmol) in the presence of potassium tert- butoxide solution (1M, 3.27 mL, 3.27 mmol) in THF (20 mL) as per the procedure described above to yield 310 mg of the titled compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.88 (s, 6H), 8.11 (d, J = 9.2 Hz, 1H), 8.55 (s, 2H).

Step 3: 2-(4-Amino-2-(trifluoromethyl)phenyl)-2-methylpropanenitrile

The titled compound was prepared by the reaction of 2-methyl-2-(4-nitro-2- (trifluoromethyl)phenyl)propanenitrile (step 2 intermediate) (300 mg, 1.15 mmol) with iron powder (321 mg, 5.76 mmol) and ammonium chloride (308 mg, 5.76 mmol) in methanol (10 mL) and water (10 mL) as per the procedure described in step 2 of Intermediate C28 to 137 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.72 (s, 6H), 5.71 (s, 2H), 6.79 (dd, J ₁ = 1.6 Hz, J ₂ = 8.4 Hz, 1H), 6.99 (s, 1H), 7.35 (d, J = 8.8 Hz, 1H); ESI-MS (m/z) 229 (M+H) ⁺ .

Intermediate C84

N-(3-Amino-5-(trifluoromethyl)phenyl)-2-(dimethylamino)ac etamide

  The titled compound was prepared by the reaction of 5-(trifluoromethyl)benzene-1,3-diamine (1.1 g, 6.26 mmol) with N,N-dimethylglycine (644 mg, 6.25 mmol) in the presence of EDCI. HCl (2.39 mg, 12.5 mmol), HOBt (843 mg, 6.25 mmol) and DIPEA (2.15 mL, 12.5 mmol) in dichloromethane (10 mL) as per the procedure described in step 1 of Intermediate C82 to yield 230 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.62 (s, 6H), 3.04 (s, 2H), 5.60 (s, 2H), 6.52 (s, 1H), 7.13 (s, 1H), 7.18 (s, 1H), 9.72 (s, 1H); ESI-MS (m/z) 3261(M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 22.

Table 22: Chemical name, structure and analytical data of Intermediate C85

Intermediate C86

1-(3-Amino-5-(trifluoromethyl)phenyl)-N,N-dimethylazetidin-3 -amine

Step 1: N,N-Dimethyl-1-(3-nitro-5-(trifluorometh l henyl)azetidin-3-amine The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (200 mg, 0.74 mmol) with N,N-dimethylazetidin-3-amine hydrochloride (256 mg, 1.48 mmol) in the presence of sodium tert-butoxide (355 mg, 3.70 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ) (34 mg, 0.04 mmol) and Xantphos (30 mg, 0. 05 mmol) in 1,4-dioxane (4.0 mL) as per the procedure described in  

step 2 of Intermediate C34 to yield 155 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.13 (s, 6H), 3.24-3.27 (m, 1H), 3.79 (t, J = 5.2 Hz, 2H), 4.08 (t, J = 7.6 Hz, 2H), 7.07 (s, 1H), 7.37 (s, 1H), 7.63 (s, 1H); ESI-MS (m/z) 290 (M+H) ⁺ .

Step 2: 1-(3-Amino-5-(trifluoromethyl)phenyl)-N,N-dimethylazetidin-3 -amine

The titled compound was prepared by the reaction of N,N-dimethyl-1-(3-nitro-5- (trifluoromethyl)phenyl)azetidin-3-amine (step 1 intermediate) (150 mg, 0.52 mmol) with iron powder (290 mg, 5.19 mmol) and ammonium chloride (222 mg, 4.15 mmol) in ethanol (4.0 mL) and water (2.0 mL) as per the procedure described in step 2 of Intermediate C28 to 95 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.14 (s, 6H), 3.13-3.18 (m, 1H), 3.50 (t, J = 5.6 Hz, 2H), 3.86 (t, J = 7.2 Hz, 2H), 5.36 (s, 2H), 5.82 (s, 2H), 6.19 (s, 1H); ESI- MS (m/z) 260 (M+H) ⁺ .

Intermediate C87

(E)-3-(3-Amino-5-(trifluoromethyl)phenyl)acrylamide

Step 1: 3-(3-nitro-5-(trifluoromethyl)phenyl)propiolamide

To a solution of 1-bromo-3-nitro-5-(trifluoromethyl)benzene (1.0 g, 3.70 mmol) in degassed DMF (10 mL) were added propiolamide (511 mg, 7.40 mmol), bis(triphenylphosphine)palladium(II)dichloride (129 mg, 0.18 mmol), copper(II)iodide (71 mg, 0.37 mmol) and triethylamine (1.54 mL, 11.1 mmol) at RT. The resultant mixture was heated at 120 °C for 30 min in a microwave reactor. The mixture was cooled to RT and quenched with water. The product was extracted in ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 480 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.92 (br s, 2H), 8.32 (s, 1H), 8.46 (s, 1H), 8.57-8.66 (m, 1H).

Step 2: (E)-3-(3-Amino-5-(trifluoromethyl)phenyl)acrylamide

  To a stirred solution of 3-(3-nitro-5-(trifluoromethyl)phenyl)propiolamide (step 1 intermediate) (201 mg, 0.78 mmol) in a mixture of methanol and water (3:1, 10 mL) was added ammonium chloride (416 mg, 7.78 mmol) and the mixture was heated to 80 °C. Zinc dust (254 mg, 3.84 mmol) was added to the mixture in small portions and stirred at for 1 h at 80 °C. The mixture was cooled to RT and diluted with ethyl acetate. The solution was filtered through celite. The filtrate was washed with ethyl acetate and the combined organic layers were washed with water, followed by brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 65 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 5.73 (s, 2H), 6.58 (d, J = 16.0 Hz, 1H), 6.84 (s, 1H), 6.95 (d, J = 7.6 Hz, 2H), 7.16 (s, 1H), 7.30 (d, J = 15.6 Hz, 1H), 7.55 (s, 1H). The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 23.

Table 23: Chemical name, structure and analytical data of Intermediate C88, C93, C96, C102-103, and 105

 

Intermediate C89

3-(3-Amino-5-(trifluoromethyl)phenyl) ro iolamide

The titled compound was prepared by the reaction of 3-(3-nitro-5- (trifluoromethyl)phenyl)propiolamide (Intermediate C87-step 1 intermediate) (200 mg, 0.77 mmol) with iron powder (216 mg, 3.87 mmol) and ammonium chloride (42 mg, 0.77 mmol) in a mixture of ethanol, THF and water (2:1:1, 10 mL) as per the procedure described in step 2 of Intermediate C28 to yield 110 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 5.90 (s, 2H), 714-7.21 (m, 3H), 7.71 (s, 1H), 8.17 (s, 1H). Intermediate C90

(E)-3-(3-Amino-5-(trifluoromethyl)phenyl)acrylonitrile

To a solution of 3-bromo-5-(trifluoromethyl)aniline (502 mg, 2.09 mmol) in degassed DMF (5.0 mL) were added acrylonitrile (97 µL, 2.51 mmol), tetrakis(triphenylphosphine)palladium(0) (121 mg, 0.10 mmol), and triethylamine (0.87 mL, 6.27 mmol) at RT. The resultant mixture was heated at 130 °C for 30 min in a microwave

 

reactor. The mixture was cooled to RT and quenched with water. The product was extracted in ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 206 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 5.79 (s, 2H), 7.44 (d, J = 16.8 Hz, 1H), 7.92-7.96 (m, 2H), 7.12 (s, 1H), 7.59 (d, J = 16.8 Hz, 1H).

Intermediate C91

4-(5-Amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile

To a solution of 4-hydrazinylbenzonitrile (3.0 g, 22.55 mmol) in ethanol (30 mL) were added pivaloyl acetonitrile (3.38 g, 37.1 mmol) followed by PTSA (6.42 g, 33.8 mmol) and the mixture was heated to reflux using Dean-Stark apparatus for 16 h. The precipitated solid was filtered and dried to yield 3.5 g of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.22 (s, 9H), 5.50 (s, 2H), 7.83-7.93 (m, 5H); ESI-MS (m/z) 241 (M+H) ⁺ .

Intermediate C92

3-(5-Amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile

To a solution of 3-hydrazinylbenzonitrile (500 mg, 3.75 mmol) in hydrochloric acid in 1,4- dioxane (10 mL) was added pivaloyl acetonitrile (565 mg, 4.51 mmol) and the mixture was heated to reflux for 16 h. The mixture was cooled to RT and diluted with water. The solution was basified using saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to yield 830 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.22 (s, 9H), 5.45 (s, 2H), 7.62-7.73 (m, 2H), 7.94-8.03 (m, 3H); ESI-MS (m/z) 241 (M+H) ⁺ .

Intermediate C94

3-(methylsulfonyl)aniline  

Step 1: 1-(Methylsulfonyl)-3-nitrobenzene

To a solution of 1-iodo-3-nitrobenzene (500 mg, 2.01 mmol) in degassed DMSO (5.0 mL) were added sodium methanesulfinate (413 mg, 4.01 mmol), N,N-dimethylethylenediamine (106 mg, 1.20 mmol) copper(I)trifluoromethane sulfonate toluene complex (311 mg, 0.60 mmol) and the mixture was evacuated and flushed with nitrogen for thrice. The mixture was heated at 120 °C for 3 h. The mixture was cooled to RT, quenched with saturated ammonium chloride solution and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to yield 360 mg of the desired compound. The compound was as such taken forward to the next step without characterization.

Step 2: 3-(methylsulfonyl)aniline

The titled compound was prepared by the catalytic hydrogenation of 1-(methylsulfonyl)-3- nitrobenzene (step 1 intermediate) (351 mg, 1.74 mmol) in the presence of 10% palladium on carbon (50% wet, 150 mg) in methanol (5.0 mL) as per the procedure described in step 3 of Intermediate C1 to yield 230 mg of the compound. ESI-MS (m/z) 172 (M+H) ⁺ .

The chemical structure, name and analytical data of the intermediate prepared by following the procedure described above are given below in Table 24.

Table 24: Chemical name, structure and analytical data of Intermediate C98

Intermediate C95

3-(3-Methoxyprop-1-yn-1-yl)-5-(trifluoromethyl)aniline

 

Step 1: 3-(3-Nitro-5-(trifluoromethyl)phenyl)prop-2-yn-1-ol

The titled compound was prepared by the reaction of 1-bromo-3-nitro-5- (trifluoromethyl)benzene (502 mg, 1.86 mmol) with propargyl alcohol (219 µL, 3.72 mmol) in the presence of bis(triphenylphosphine)palladium(II)dichloride (65 mg, 0.09 mmol), copper iodide (35 mg, 0.18 mmol) and triethylamine (0.8 mL, 5.58 mmol) in DMF (5.0 mL) as per the procedure described in step 1 of Intermediate C87 to yield 205 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 4.37 (d, J = 6.0 Hz, 2H), 5.50 (t, J = 6.0 Hz, 1H), 8.26 (s, 1H), 8.46 (d, J = 2.0 Hz, 2H).

Step 2: 1-(3-Methoxyprop-1-yn-1-yl)-3-nitro-5-(trifluoromethyl)benze ne

The titled compound was prepared by the reaction of 3-(3-Nitro-5- (trifluoromethyl)phenyl)prop-2-yn-1-ol (step 1 intermediate) (738 mg, 3.01 mmol) with methyl iodide (0.37 mL, 6.02 mmol) in the presence of sodium hydride (60%w/w, 240 mg, 6.02 mmol) in THF (10 mL) as per the procedure described in step 2 of Intermediate C57 to yield 390 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.38 (s, 3H), 4.41 (s, 2H), 8.36 (s, 1H), 8.49 (s, 1H), 8.53 (s, 1H).

Step 3: 3-(3-Methoxyprop-1-yn-1-yl)-5-(trifluoromethyl)aniline

The titled compound was prepared by the reaction of 1-(3-methoxyprop-1-yn-1-yl)-3-nitro-5- (trifluoromethyl)benzene (step 2 intermediate) (465 mg, 1.79 mmol) with iron powder (501 mg, 8.97 mmol) and ammonium chloride (95 mg, 1.79 mmol) in a mixture of ethanol, THF and water (3:2:1, 12 mL) as per the procedure described in step 2 of Intermediate C28 to yield 312 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.34 (s, 3H), 4.31 (s, 2H), 5.78 (s, 2H), 7.546.81 (s, 1H), 6.86 (s, 1H), 6.87 (s, 1H); ESI-MS (m/z) 230 (M+H) ⁺ .  

Intermediate C97

3-(Difluoromethyl)aniline

Step 1: 1-(Difluoromethyl)-3-nitrobenzene

To a stirred solution of 3-nitrobenzaldehyde (506 mg, 3.35 mmol) in anhydrous dichloromethane (10 mL) was dropwise added DAST (1.32 mL, 10.04 mmol) at -78 °C. The mixture was gradually warmed up to RT and stirred for 3 h. The mixture was quenched with saturated sodium bicarbonate solution and extracted twice with ethyl acetate. The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography to yield 332 mg of the desired compound. The compound was as such taken forward to the next step without characterization.

Step 2: 3-(Difluoromethyl)aniline

The titled compound was prepared by the catalytic hydrogenation of 1-(difluoromethyl)-3- nitrobenzene (step 1 intermediate) (320 mg, 1.85 mmol) in the presence of 10% palladium on carbon (50% wet, 100 mg) in methanol (5.0 mL) as per the procedure described in step 3 of Intermediate C1 to yield 160 mg of the compound. ESI-MS (m/z) 144 (M+H) ⁺ .

Intermediate C100

2-(3-Aminophenyl)-2,2-difluoroethanol

Step 1: 2,2-Difluoro-2-(3-nitrophenyl)ethanol

The titled compound was prepared by the reaction of ethyl 2,2-difluoro-2-(3- nitrophenyl)acetate (Step 1 of C45) (408 mg, 1.66 mmol) with sodium borohydride (126 mg, 3.32 mmol) in ethanol (10 mL) as per the procedure described in step 2 of Intermediate C81 to yield 271 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 3.89-4.01 (m, 2H), 5.76 (t, J = 6.4 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 8.01 (dd, J ₁ = 0.4 Hz, J ₂ = 7.6 Hz, 1H), 8.31 (s, 1H), 8.36-8.41 (m, 1H).

 

Step 2: 2-(3-Aminophenyl)-2,2-difluoroethanol

The titled compound was prepared by the catalytic hydrogenation of 2,2-difluoro-2-(3- nitrophenyl)ethanol (step 1 intermediate) (260 mg, 1.28 mmol) in the presence of 10% palladium on carbon (50% wet, 100 mg) in methanol (8.0 mL) as per the procedure described in step 3 of Intermediate C1 to yield 75 mg of the compound. ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 3.70-3.77 (m, 2H), 5.31 (s, 2H), 5.53 (s, 1H), 6.58-6.65 (m, 2H), 6.68 (s, 1H), 7.05-7.10 (m, 1H); ESI-MS (m/z) 174 (M+H) ⁺ .

Intermediate C101

3-(3-Amino-5-(trifluoromethyl)phenyl)propiolonitrile

Step 1: 3-(3-Nitro-5-(trifluoromethyl)phenyl)propiolonitrile

To a stirred solution of 3-(3-nitro-5-(trifluoromethyl)phenyl)propiolamide (step 1 of C87) (100 mg, 0.38 mmol) in anhydrous dichloromethane (5.0 mL) was added Burgess reagent (110 mg, 0.46 mmol) at 0 °C and the mixture was stirred at RT for 2 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to yield 60 mg of the titled compound. ¹ H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 1.2 Hz, 1H), 8.77 (s, 1H), (d, J = 1.2 Hz, 1H), 8.99 (dd, J ₁ = 1.6 Hz, J ₂ = 2.4 Hz, 1H).

Step 2: 3-(3-Amino-5-(trifluoromethyl)phenyl)propiolonitrile

The titled compound was prepared by the reaction of 3-(3-nitro-5- (trifluoromethyl)phenyl)propiolonitrile (step 1 intermediate) (251 mg, 1.04 mmol) with iron powder (292 mg, 5.22 mmol) and ammonium chloride (56 mg, 1.04 mmol) in a mixture of ethanol, THF and water (2:2:1, 25 mL) as per the procedure described in step 2 of Intermediate C28 to yield 121 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 6.04 (s, 2H), 7.06-7.11 (m, 2H), 7.20 (s, 1H). Intermediate C106

3-(4-Methylpiperazin-1-yl)-5-(methylsulfonyl)aniline  

Step 1: 1-(3-Iodo-5-nitrophenyl)-4-methylpiperazine

The titled compound was prepared by the reaction of 1-fluoro-3-iodo-5-nitrobenzene (1.0 g, 3.75 mmol) 1-methylpiperazine (1.9 g, 18.7 mmol) in DMSO (10 mL) as per the procedure described in step 1 of Intermediate C24 to yield 901 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.20 (s, 3H), 2.40-2.45 (m, 4H), 3.25-3.30 (m, 4H), 7.61-7.67 (m, 2H), 7.77-7.80 (m, 1H); ESI-MS (m/z) 348 (M+H) ⁺ .

Step 2: 1-Methyl-4-(3-(methylsulfonyl)-5-nitrophenyl)piperazine

The titled compound was prepared by the reaction of 1-(3-iodo-5-nitrophenyl)-4- methylpiperazine (step 1 intermediate) (481 mg, 1.38 mmol) with sodium methanesulfinate (282 mg, 2.77 mmol), in the presence of N,N-dimethylethylenediamine (73 mg, 0.83 mmol) and copper(I)trifluoromethane sulfonate toluene complex (215 mg, 0.42 mmol) as per the procedure described in step 1 of Intermediate C94 to yield 310 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.20 (s, 3H), 2.44-2.49 (m, 4H), 2.54 (s, 3H), 3.38-3.42 (m, 4H), 7.74-7.77 (m, 1H), 7.89-7.94 (m, 2H); ESI-MS (m/z) 300 (M+H) ⁺ .

Step 3: 3-(4-Methylpiperazin-1-yl)-5-(methylsulfonyl)aniline

The titled compound was prepared by the catalytic hydrogenation of 1-methyl-4-(3- (methylsulfonyl)-5-nitrophenyl)piperazine (step 2 intermediate) (301 mg, 1.00 mmol) in the presence of 10% palladium on carbon (50% wet, 150 mg) in methanol (10 mL) as per the procedure described in step 3 of Intermediate C1 to yield 110 mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.22 (s, 3H), 2.41-2.47 (m, 4H), 3.08 (s, 3H), 3.09-3.14 (m, 4H), 5.45 (s, 2H), 6.36-6.39 (m, 1H), 6.51-6.57 (m, 2H); ESI-MS (m/z) 270 (M+H) ⁺ .

 

Examples

General procedures for the synthesis of the examples are described in Method A-R. All the examples were prepared by following either of the methods described below from the combination of appropriate intermediates. Name, structure, Intermediate/method used and characterization data for individual examples are given in Table 25.

M h A

Step 1: N-(4-((4-Ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)-3- hydroxybenzamide

A mixture of 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (Intermediate C1) (500 g, 1.74 mmol) and methyl 3-hydroxybenzoate (Intermediate B2) (317 mg, 1.91 mmol) in anhydrous THF (5.0 mL) was cooled to -20 °C and added potassium tert-butoxide (1M, 10 mL, 10.44 mmol). The mixture was stirred at RT for 3 h. The reaction mixture was cooled to -20 °C and quenched with saturated sodium bicarbonate solution. The aqueous mixture was extracted twice with ethyl acetate and the combined organic layers were washed with water followed by brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield 220 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.98 (t, J = 6.8 Hz, 3H), 2.31-2.50 (m, 8H), 2.51 (br s, 2H), 3.56 (s, 2H), 6.98-7.01 (br d, 1H), 7.31-7.35 (m, 2H), 7.39 (d, J = 6.4 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 9.79 (s, 1H), 10.42 (s, 1H); ESI-MS (m/z) 408 (M+H) ⁺ .

Step 2: tert-Butyl 4-(3-((4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-6H-pyrimido[5,4-b ][1,4]oxazine-8(7H)- carboxylate

 

To a stirred solution of N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)-3- hydroxybenzamide (step 1 intermediate) (200 mg, 0.49 mmol) and tert-butyl 4-chloro-6H- pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (134 mg, 0.49 mmol) in DMF (5.0 mL) was added cesium carbonate (241 mg, 0.74 mmol) and the mixture was stirred at 130 °C for 2 h. The mixture was cooled to RT and diluted with a mixture of ethyl acetate and water. The organic layer was separated and washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 245 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.98 (t, J = 7.2 Hz, 3H), 1.50 (s, 9H), 2.39-2.50 (m, 10H), 3.56 (s, 2H), 3.91 (t, J = 4.0 Hz, 2H), 4.39 (t, J = 4.0 Hz, 2H), 7.46 (dd , J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 8.20 (s, 1H), 10.52 (s, 1H); ESI-MS (m/z) 643 (M+H) ⁺ .

Step 3: 3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)-N-(4 -((4-ethylpiperazin-1- yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

To a stirred solution of tert-butyl 4-(3-((4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-6H-pyrimido[5,4-b ][1,4]oxazine-8(7H)- carboxylate (step 2 intermediate) (220 mg, 0.34 mmol) in ethanol (3.0 mL) was added hydrochloric acid in 1,4-dioxane (7.0 mL) at 0 °C and the mixture was stirred at RT for 3 h. The mixture was diluted with ethyl acetate and water. The organic layer was washed with water, saturated sodium bicarbonate solution followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 105 mg of the desired product.

Method B

Preparation of 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(4-(((2- (dimethylamino)ethyl)(methyl)amino)methyl)-3-(trifluoromethy l)phenyl)benzamide

 

Step 1: tert-Butyl 4-(2-chloro-5-(methoxycarbonyl)phenoxy)-6H-pyrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate

To a stirred solution of methyl 4-chloro-3-hydroxybenzoate (Intermediate B1) (226 mg, 1.21 mmol) and tert-butyl 4-chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (300 mg, 1.10 mmol) in DMF (10 mL) was added cesium carbonate (540 mg, 1.65 mmol) and the mixture was stirred at 130 °C for 3 h. The mixture was cooled to RT and quenched with saturated aqueous solution of sodium bicarbonate. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 334 mg of the desired product. ¹ H NMR (400 MHz, DMSO- d6) δ 1.50 (s, 9H), 3.86 (s, 3H), 3.94 (t, J = 4.4 Hz, 2H), 4.40 (t, J = 4.0 Hz, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.85-7.88 (m, 2H), 8.04 (s, 1H).

Step 2: 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(4-(((2- (dimethylamino)ethyl)(methyl)amino)methyl)-3-(trifluoromethy l)phenyl)benzamide

The titled compound was prepared by the reaction of tert-butyl 4-(2-chloro-5- (methoxycarbonyl)phenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8(7 H)-carboxylate (step 1 intermediate) (100 mg, 0.24 mmol) and N ¹ -(4-amino-2-(trifluoromethyl)benzyl)-N ¹ ,N ² ,N ² - trimethylethane-1,2-diamine (Intermediate C33) (58 mg, 0.22 mmol) in the presence of potassium tert-butoxide (1M, 1.42 mL, 1.42 mmol) in anhydrous THF (20 mL) as per the procedure described in step 1 of Method A to yield 20 mg of the product.

Method B’

Preparation of 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3- (piperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide

 

Step 1: tert-Butyl 4-(3-(4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin -4- yl)oxy)benzamido)-5-(trifluoromethyl)phenyl)piperazine-1-car boxylate

The titled compound was prepared by the reaction of tert-butyl 4-(2-chloro-5- (methoxycarbonyl)phenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8(7 H)-carboxylate (step 1- Method B) (150 mg, 0.34 mmol) and tert-butyl 4-(3-amino-5- (trifluoromethyl)phenyl)piperazine-1-carboxylate (Intermediate C39) (107 mg, 0.31 mmol) in the presence of potassium tert-butoxide (1M, 2.0 mL, 2.06 mmol) in anhydrous THF (10 mL) as per the procedure described in step 1 of Method A to yield 118 mg of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.43 (s, 9H), 3.19-3.21 (m, 4H), 3.46-3.52 (m, 6H), 4.21 (t, J = 4.0 Hz, 2H), 6.99 (s, 1H), 7.61-7.89 (m, 7H), 10.40 (s, 1H); ESI-MS (m/z) 635 (M+H) ⁺ . Step 2: 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3-(piperazin- 1-yl)-5-(trifluoromethyl)phenyl)benzamide

The titled compound was prepared by the reaction of tert-butyl 4-(3-(4-chloro-3-((7,8- dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamido)-5- (trifluoromethyl)phenyl)piperazine-1-carboxylate (step 1 intermediate) (110 mg, 0.17 mmol) with hydrochloric acid in 1,4-dioxane (4M, 20 mL) in ethanol (4.0 mL) as per the procedure described in step 3 of Method A to yield 54 mg of the product.

Method C

Preparation of 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) amino)-N-(4- ((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)be nzamide

Step 1: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- nitrobenzamide

 

To a stirred solution of 4-chloro-3-nitrobenzoic acid (1.0 g, 4.97 mmol) in dichloromethane (10 mL) were added oxalyl chloride (500 µL, 5.30 mmol) followed by catalytic amount of DMF and the mixture was stirred at RT for 2 h. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane. The solution was cooled to 0 °C; 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (Intermediate C1) (1.0 g, 3.30 mmol) was added to the reaction mixture followed by DIPEA (1.5 mL, 8.30 mmol). The resultant mixture was stirred overnight at RT. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic extracts were washed with saturated sodium bicarbonate solution, water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue thus obtained was purified by column chromatography to yield 1.43 g of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 0.98 (t, J = 7.2 Hz, 3H), 2.28-2.39 (m, 10H), 3.57 (s, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.98-8.04 (m, 2H), 8.16 (s, 1H), 8.27 (dd, J ₁ = 2.4 Hz, J ₂ = 8.4 Hz, 1H), 8.65 (s, 1H), 10.77 (s, 1H). Step 2: 3-Amino-4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)benzamide

To a stirred solution of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)-3-nitrobenzamide (step 1 intermediate) (1.4 g, 2.97 mmol) in a mixture of ethyl methanol and water (1:1, 60 mL) were added ammonium chloride (1.6 g, 29.7 mmol) followed by iron powder (830 mg. 14.8 mmol) in small portions at 80 °C. The mixture was stirred at 80 °C for 2 h. The mixture was cooled to RT, filtered and concentrated. The residue was diluted with a mixture of ethyl acetate and water. The organic layer was separated and washed with water followed by brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue obtained was purified by column chromatography to yield 732 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 0.98 (t, J = 7.2 Hz, 3H), 2.28-2.39 (m, 10H), 3.55 (s, 2H), 5.60 (s, 2H), 7.13 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 1H), 8.01 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1H), 8.18 (s, 1H), 10.41 (s, 1H); ESI-MS (m/z) 441 (M+H) ⁺ .

Step 3: tert-Butyl 4-((2-chloro-5-((4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)carbamoyl)phenyl)amino)-6H-pyrimido[ 5,4-b][1,4]oxazine-8(7H)- carboxylate

 

To a stirred solution of 3-amino-4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)benzamide (step 2 intermediate) (150 mg, 0.34 mmol) and tert-butyl 4-chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (102 mg, 0.37 mmol) in toluene (5.0 mL) were added sodium tert-butoxide (36 mg, 0.37 mmol), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (12 mg, 0.01 mmol) and (±)-2,2′- bis(diphenylphosphino)-1,1′-binaphthalene (rac-BINAP) (13 mg, 0.02 mmol) and the mixture was stirred at 140 °C for 25 h in a sealed tube. The mixture was diluted with ethyl acetate and the organic mixture was washed with water followed by brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 67 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 0.98 (t, J = 6.8 Hz, 3H), 1.49 (s, 9H), 2.30-2.39 (m, 10H), 3.57 (s, 2H), 3.90 (t, J = 8.0 Hz, 2H), 4.15 (t, J = 8.0 Hz, 2H), 7.72 (t, J = 7.2 Hz, 2H), 7.78 (m, 1H), 8.02- 8.04 (m, 2H), 8.20 (s, 1H), 8.43 (s, 1H), 8.55 (s, 1H), 10.56 (s, 1H); ESI-MS (m/z) 674 (M- H)-.

Step 4: 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) amino)-N-(4-((4- ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzam ide

The titled compound was prepared by the reaction of tert-Butyl 4-((2-chloro-5-((4-((4- ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)carbam oyl)phenyl)amino)-6H- pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (step 3 intermediate) (60 mg, 0.09 mmol) with hydrochloric acid in 1,4-dioxane (4.0 mL) in ethanol (4.0 mL) as per the procedure described in step 3 of Method A to yield 19 mg of the product.

Method D

Preparation of 4-chloro-N-(3-(cyanomethyl)phenyl)-3-((7,8-dihydro-6H-pyrimi do[5,4- b][1,4]oxazin-4-yl)oxy)benzamide

 

To a stirred solution of tert-butyl 4-(2-chloro-5-(methoxycarbonyl)phenoxy)-6H- pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Step 1-Method B) (100 mg, 0.24 mmol) in toluene (5.0 mL) were added 2-(3-aminophenyl)acetonitrile (31 mg, 0.24 mmol) and trimethyl aluminium solution (2M, 237 µL, 0.47 mmol) at 0 °C. The mixture was stirred at RT for 2 h. The mixture was poured into ice-water and extracted twice with ethyl acetate. The combined organic extracts were washed with brine and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 68 mg of the desired product. Method E

Preparation of 4-chloro-N-(3-(cyanodifluoromethyl)phenyl)-3-((7,8-dihydro-6 H- pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide

Step 1: Ethyl 2-(3-(3-((tert-butyldimethylsilyl)oxy)-4-chlorobenzamido)phe nyl)-2,2- difluoroacetate

To a stirred solution of 3-((tert-butyldimethylsilyl)oxy)-4-chlorobenzoic acid (600 mg, 2.09 mmol) in dichloromethane (10 mL) were added oxalyl chloride (364 µL, 4.19 mmol) followed by catalytic amount of DMF and the mixture was stirred at RT for 2 h. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane (10 mL). The solution was cooled to 0 °C; ethyl 2-(3-aminophenyl)-2,2-difluoroacetate (Intermediate C45) (495 mg, 2.30 mmol) was added to the reaction mixture followed by DIPEA (1.12 mL, 6.27 mmol). The resultant mixture was stirred overnight at RT. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic extracts were washed with saturated sodium bicarbonate solution, water and brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated. The residue thus obtained was purified by column chromatography to yield 655 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 0.27-0.35 (m, 6H), 1.02 (s, 9H), 1.27 (t, J = 6.8 Hz, 3H), 4.33 (q, J = 7.2 Hz, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.51-7.64 (m, 4H), 7.97 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 10.54 (s, 1H); ESI-MS (m/z) 484 (M+H) ⁺ .

 

Step 2: N-(3-(2-Amino-1,1-difluoro-2-oxoethyl)phenyl)-4-chloro-3-hyd roxybenzamide

A mixture of ethyl 2-(3-(3-((tert-butyldimethylsilyl)oxy)-4-chlorobenzamido)phe nyl)-2,2- difluoroacetate (step 1 intermediate) (650 mg, 1.34 mmol) and ammonia solution (7M in methanol, 10 mL) was heated at 80 °C for 4 h in a sealed tube. The reaction mixture was cooled and concentrated under reduced pressure to yield 560 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.32 (d, J = 8.0 Hz, 1H), 7.43-7.53 (m, 4H), 7.92 (d, J = 8.4 Hz, 1H), 8.03 (s, 2H), 8.37 (s, 1H), 10.47 (s, 1H), 10.51 (s, 1H); ESI-MS (m/z) 341 (M+H) ⁺ . Step 3: N-(3-(2-Amino-1,1-difluoro-2-oxoethyl)phenyl)-4-chloro-3-((7 ,8-dihydro-6H- pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide

To a stirred solution of N-(3-(2-amino-1,1-difluoro-2-oxoethyl)phenyl)-4-chloro-3- hydroxybenzamide (step 2 intermediate) (250 mg, 0.73 mmol) and tert-butyl 4-chloro-6H- pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (200 mg, 0.74 mmol) in DMF (5.0 mL) was added cesium carbonate (360 mg, 1.10 mmol) and the mixture was stirred at 130 °C for 3 h. The mixture was cooled to RT and quenched with saturated aqueous solution of sodium bicarbonate. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 45 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.51 (br s, 2H), 4.19-4.22 (m, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.66-7.91 (m, 6H), 7.94 (d, J = 8.0 Hz, 1H), 8.07 (m, 1H), 8.38 (s, 1H), 10.52 (s, 1H); ESI-MS (m/z) 476 (M+H) ⁺ .

Step 4: 4-Chloro-N-(3-(cyanodifluoromethyl)phenyl)-3-((7,8-dihydro-6 H-pyrimido[5,4- b][1,4]oxazin-4-yl)oxy)benzamide

To a solution of N-(3-(2-amino-1,1-difluoro-2-oxoethyl)phenyl)-4-chloro-3-((7 ,8-dihydro- 6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide (step 3 intermediate) (40 mg, 0.08 mmol) in dichloromethane (3.0 mL) was added Burgess reagent (76.8 mg, 0.32 mmol) and the mixture was stirred at RT for 3 h. The reaction mixture was concentrated under reduced

 

pressure and the residue obtained was purified by column chromatography to yield 7.0 mg of the desired compound.

Method F

Preparation of 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3- hydroxy-5-(trifluoromethyl)phenyl)benzamide

Step 1: 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3-methoxy-5- (trifluoromethyl)phenyl)benzamide

The titled compound was prepared by the reaction of tert-butyl 4-(2-chloro-5- (methoxycarbonyl)phenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8(7 H)-carboxylate (step 1- Method B) (100 mg, 0.24 mmol) and 3-methoxy-5-(trifluoromethyl)aniline (45 mg, 0.24 mmol) in the presence of potassium tert-butoxide (1M, 1.42 mL, 1.42 mmol) in anhydrous THF (3.0 mL) as per the procedure described in step 1 of Method A to yield 75 mg of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.51 (br s, 2H), 3.83 (s, 3H), 4.20 (t, J = 4.0 Hz, 2H), 7.00 (s, 1H), 7.69-7.90 (m, 7H), 10.56 (s, 1H); ESI-MS (m/z) 481 (M+H) ⁺ .

Step 2: 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3-hydroxy-5- (trifluoromethyl)phenyl)benzamide

To a solution of 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)-N-(3- methoxy-5-(trifluoromethyl)phenyl)benzamide (step 1 intermediate) (70 mg, 0.14 mmol) in dichloromethane (2.0 mL) was added boron tribromide (1M in dichloromethane, 1.2 mL, 1.16 mmol) at 0 °C and the mixture was stirred at RT for 4 h. The reaction mixture was quenched with methanol at 0 °C and concentrated under reduced pressure. The residue was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solution was filtered, concentrated and the residue was purified by flash column chromatography to yield 25 mg of the desired compound.

 

Method G

Preparation of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)oxy)be nzamide

Step 1: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- hydroxybenzamide

The titled compound was prepared by the reaction of methyl 4-chloro-3-hydroxybenzoate (Intermediate B1) (260 mg, 1.39 mmol) and 4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)aniline (Intermediate C1) (400 mg, 1.39 mmol) in the presence of potassium tert-butoxide (1M, 8.4 mL, 8.39 mmol) in anhydrous THF (8.0 mL) as per the procedure described in step 1 of Method A to yield 200 mg of the product. ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 0.98 (t, J = 7.2 Hz, 3H), 2.33-2.55 (m, 6H), 3.33 (br s, 4H), 3.56 (s, 2H), 7.42-7.53 (m, 3H), 7.70 (d, J = 8.4 Hz, 1H), 8.01 (dd, J ₁ = 2.0 Hz, J ₂ = 8.4 Hz, 1H),8.32 (s, 1H), 10.50 (s, 1H), 10.60 (br s, 1H).

Step 2: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((8-(4- methoxybenzyl)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidi n-4-yl)oxy)benzamide

To a stirred solution of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)-3-hydroxybenzamide (step 1 intermediate) (872 mg, 1.97 mmol) and 4-chloro-8-(4-methoxybenzyl)-7,8-dihydropyrido[2,3-d]pyrimid in-5(6H)-one (Intermediate A9) (600 mg, 1.97 mmol) in DMF (20 mL) was added cesium carbonate (3.2 g, 9.85 mmol) and the mixture was stirred at 50 °C for 3 h. The mixture was cooled to RT and quenched with water. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue  

thus obtained was purified by silica gel column chromatography to yield 400 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.99 (t, J = 7.2 Hz, 3H), 2.40-2.51 (m, 10H), 2.68 (t, J = 7.6 Hz, 2H), 3.57 (s, 2H), 3.65 (t, J = 6.8 Hz, 2H), 3.74 (s, 3H), 4.90 (s, 2H), 6.92 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.91-7.94 (br s, 2H), 8.04 (dd, J1 = 1.6 Hz, J2 = 8.4 Hz, 1H), 8.18 (s, 1H), 8.24 (s, 1H), 10.58 (s, 1H); ESI-MS (m/z) 709 (M+H) ⁺ .

Step 3: 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((5-oxo- 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)oxy)benzamide

A solution of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((8-(4-methoxybenzyl)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]p yrimidin-4-yl)oxy)benzamide (step 2 intermediate) (40 mg, 0.06 mmol) in a mixture of dichloroethane and trifluoroacetic acid (1:1, 600 µL) was stirred overnight at 80 °C. The mixture was cooled to RT and then to 0 °C before the addition of aqueous sodium bicarbonate solution till pH 7-8. The aqueous mixture was extracted twice with dichloromethane and the combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 12 mg of the desired product.

Method G’

Preparation of 4-chloro-N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)ph enyl)-3-((7-oxo- 7,8-dihydro-6H-pyrimido[5,4-b][1, amide

Step 1: 4-Chloro-3-hydroxy-N-(3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)benzamide

The titled compound was prepared by the reaction of methyl 4-chloro-3-hydroxybenzoate (Intermediate B1) (3.6 g, 19.3 mmol) and 3-(4-methylpiperazin-1-yl)-5-

 

(trifluoromethyl)aniline (Intermediate C19) (5.0 g, 19.3 mmol) in the presence of potassium tert-butoxide (1M, 115 mL, 115 mmol) in anhydrous THF (50 mL) as per the procedure described in step 1 of Method A to yield 2.6 g of the product. ¹ H NMR (400 MHz, DMSO- d6) δ 2.23 (s, 3H), 2.47 (s, 4H), 3.21 (br s, 4H), 6.95 (s, 1H), 7.42-7.44 (m, 1H), 7.51-7.53 (m, 2H), 7.60 (s, 1H), 7.65 (s, 1H), 10.32 (s, 1H), 10.60 (s, 1H).

Step 2: 4-Chloro-3-((8-(4-methoxybenzyl)-7-oxo-7,8-dihydro-6H-pyrimi do[5,4- b][1,4]oxazin-4-yl)oxy)-N-(3-(4-methylpiperazin-1-yl)-5-(tri fluoromethyl)phenyl)benzamide

To a stirred solution of 4-chloro-3-hydroxy-N-(3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)benzamide (step 1 intermediate) (80 mg, 0.19 mmol) and 4-chloro- 8-(4-methoxybenzyl)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one (Intermediate A2) (118 mg, 0.38 mmol) in DMSO (0.5 mL) was added cesium fluoride (88 mg, 0.58 mmol) and the mixture was stirred at 118 °C for 5-7 h. The mixture was cooled to RT and quenched with water. The precipitated solid was collected through filtration and dried under vacuum. The crude compound was purified by flash column chromatography to yield 110 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.24 (s, 3H), 2.52-2.56 (m, 4H), 3.18-3.22 (m, 4H),3.72 (s, 3H), 5.04 (s, 2H), 5.15 (s, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.97 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.64 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.93-7.98 (m, 2H), 8.22 (s, 1H), 10.41 (s, 1H).

Step 3: 4-Chloro-N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)ph enyl)-3-((7-oxo-7,8- dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide

The titled compound was prepared by the reaction of 4-chloro-3-((8-(4-methoxybenzyl)-7- oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)-N-(3 -(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)benzamide (step 2 intermediate) (100 mg, 0.15 mmol) with trifluoroacetic acid (2.0 mL) as per the procedure described in step 3 of Method G to yield 40 mg of the product.

Method H

Preparation of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)oxy)benzamid e

 

Step 1: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((5- hydroxy-8-(4-methoxybenzyl)-5,6,7,8-tetrahydropyrido[2,3-d]p yrimidin-4-yl)oxy)benzamide

To a solution of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((8-(4-methoxybenzyl)-5-oxo-5,6,7,8-tetrahydropyrido[2,3-d]p yrimidin-4-yl)oxy)benzamide (Step 2-Method G) (500 mg, 0.76 mmol) in THF (5.0 mL) at 0 °C were added sodium borohydride (54 mg, 0.71 mmol) followed by added methanol (1.0 mL) dropwise. The mixture was stirred for 3-4 h at RT. The reaction was quenched with water and extracted twice with chloroform. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 400 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ^^^^^^(t, J = 7.2 Hz, 3H), 1.70-1.74 (m, 1H), 1.92 (d, J = 2.8 Hz, 1H), 2.45-2.56 (m, 10H), 3.50-3.60 (m, 4H), 3.78 (s, 3H), 4.77 (d, J = 14.8 Hz, 1H), 4.89 (d, J = 15.2 Hz, 1H), 5.03 (br s, 1H), 5.26 (d, J = 4.4 Hz, 1H), 6.90 (dd, J ₁ = 2.0 Hz, J ₁ = 6.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.71-8.18 (m, 7H), 10.59 (s, 1H); ESI-MS (m/z) 711 (M+H) ⁺ .

Step 2: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((8-(4- methoxybenzyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl )oxy)benzamide

To a stirred mixture of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)-3-((5-hydroxy-8-(4-methoxybenzyl)-5 ,6,7,8-tetrahydropyrido[2,3- d]pyrimidin-4-yl)oxy)benzamide (step 1 intermediate) (400 mg, 0.56 mmol) in triethylsilane (16 mL) at 0 °C was added trifluoroacetic acid (8.0 mL) dropwise and the mixture was stirred

 

for 3-4 h at RT. The mixture was cooled to 0 °C and neutralized using aqueous sodium bicarbonate solution. The aqueous mixture was extracted twice with chloroform. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 200 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.09 (m, 3H), 1.89 (m, 2H), 2.50 (br s, 10H), 2.76 (m, 2H), 3.51-3.62 (m, 4H), 3.73 (s, 3H), 4.78 (s, 2H), 6.90 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 7.71-7.92 (m, 4H), 8.00 (s, 1H), 8.06 (d, J = 7.2 Hz, 1H), 8.18 (s, 1H), 10.57 (s, 1H); ESI-MS (m/z) 695 (M+H) ⁺ .

Step 3: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4-yl)oxy)benzamid e

The titled compound was prepared by the reaction of 4-chloro-N-(4-((4-ethylpiperazin-1- yl)methyl)-3-(trifluoromethyl)phenyl)-3-((8-(4-methoxybenzyl )-5,6,7,8-tetrahydropyrido[2,3- d]pyrimidin-4-yl)oxy)benzamide (step 2 intermediate) (100 mg, 0.14 mmol) with trifluoroacetic acid (2.0 mL) as per the procedure described in step 3 of Method G to yield 25 mg of the product.

Method I

Preparation of 1-(4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4- yl)oxy)phenyl)- 3-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)urea

Step 1: tert-Butyl 4-(5-amino-2-chlorophenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8 (7H)- carboxylate

The titled compound was prepared by the reaction of 5-amino-2-chlorophenol (80 mg, 0.55 mmol) and tert-butyl 4-chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (150 mg, 0.55 mmol) in the presence of cesium carbonate (540 mg, 1.65 mmol) in DMF (3.0 mL) as per the procedure described in step 2 of Method G to yield 130

 

mg of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.50 (s, 9H), 3.92 (t, J = 4.4 Hz, 2H), 4.37 (t, J = 4.0 Hz, 2H), 5.44 (s, 2H), 6.41 (d, J = 6.4 Hz, 1H), 6.47 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 1H), 8.04 (s, 1H); ESI-MS (m/z) 379 (M+H) ⁺ .

Step 2: tert-Butyl 4-(2-chloro-5-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)ureido)phenoxy)-6H-pyrimido[5,4-b][1 ,4]oxazine-8(7H)-carboxylate

To a stirred solution of 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (Intermediate C1) (150 mg, 0.52 mmol) in THF (20 mL) was added triphosgene (54 mg, 0.18 mmol) and the resulting mixture was stirred at 70 °C. After 1 h of heating the mixture was concentrated under reduced pressure and the residue was dissolved in THF (20 mL). That solution was added dropwise to a stirred mixture of tert-butyl 4-(5-amino-2-chlorophenoxy)- 6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (step 1 intermediate) (150 mg, 0.40 mmol) and triethylamine (172 µL, 1.19 mmol) in THF (20 mL) at RT. The resultant mixture was stirred for 2 h at 70 °C. The mixture was cooled to RT and quenched with water. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 100 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.09 (t, J = 7.2 Hz, 3H), 1.50 (s, 9H), 2.42-2.51 (m, 10H), 3.54 (br s, 2H), 3.94 (t, J = 8.4 Hz, 2H), 4.40 (t, J = 7.6 Hz, 2H), 7.29 (dd, J ₁ = 2.4 Hz, J ₂ = 8.0 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.55-7.94 (m, 3H), 7.95 (s, 1H), 8.05 (s, 1H), 9.15 (s, 1H), 9.20 (s, 1H). Step 3: 1-(4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4- yl)oxy)phenyl)-3-(4- ((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ur ea

The titled compound was prepared by the reaction of tert-butyl 4-(2-chloro-5-(3-(4-((4- ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ureido )phenoxy)-6H-pyrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate (step 2 intermediate) (100 mg, 0.14 mmol) with hydrochloric acid in 1,4-dioxane (2.0 mL) as per the procedure described in step 3 of Method A to yield 52 mg of the product.

Method I’

 

Preparation of 1-(4-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)ph enyl)-3-(3- (trifluoromethyl)phenyl)urea

Step 1: Benzyl (4-(3-(3-(trifluoromethyl)phenyl)ureido)phenyl) carbonate

The titled compound was prepared by the reaction of 3-(trifluoromethyl)aniline (218 mg, 1.34 mmol), triphosgene (138 mg, 0.47 mmol) and 4-aminophenylbenzylcarbonate (250 mg, 0.89 mmol) in the presence of triethylamine (361 mg, 3.57 mmol) in THF (5.0 mL) as per the procedure described in step 2 of Method I to yield 200 mg of the titled compound. ¹ H NMR (400 MHz, DMSO-d6) δ 5.07 (s, 2H), 6.95 (d, J = 2.0 Hz, 2H), 7.29-7.57 (m, 10H), 8.01 (s, 1H), 8.61 (s, 1H), 8.98 (s, 1H).

Step 2: 1-(4-Hydroxyphenyl)-3-(3-(trifluoromethyl)phenyl)urea

A solution of benzyl (4-(3-(3-(trifluoromethyl)phenyl)ureido)phenyl)carbonate (step 1 intermediate) (200 mg, 0.47 mmol) in methanol (5.0 mL) with catalytic amount of 10% palladium on carbon (50% wet, 200 mg) was hydrogenated at RT for 18 h. The mixture was diluted with chloroform filtered through celite and the celite bed. The filtrate was concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 48 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 6.69 (d, J = 6.8 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 7.48-7.53 (m, 2H), 8.0 (s, 1H), 8.44 (s, 1H), 8.91 (s, 1H), 9.11 (s, 1H); ESI-MS (m/z) 397 (M+H) ⁺ .

Step 3: tert-Butyl 4-(4-(3-(3-(trifluoromethyl)phenyl)ureido)phenoxy)-6H-pyrimi do[5,4- b][1,4]oxazine-8(7H)-carboxylate

 

The titled compound was prepared by the reaction of 1-(4-hydroxyphenyl)-3-(3- (trifluoromethyl)phenyl)urea (step 2 intermediate) (50 mg, 0.17 mmol) and tert-butyl 4- chloro-6H-pyrimido[5,4-b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (59 mg, 0.22 mmol) in the presence of cesium carbonate (109 mg, 0.34 mmol) in DMF (0.5 mL) as per the procedure described in step 2 of Method G to yield 30 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.48 (s, 9H), 3.34-3.39 (m, 1H), 3.91 (t, J = 4.4 Hz, 2H), 4.36 (t, J = 4.0 Hz, 2H), 7.10-7.12 (m, 2H), 7.50-7.58 (m, 4H), 8.02-8.04 (br s, 2H), 8.67 (s, 1H), 9.07 (s, 1H); ESI-MS (m/z) 432 (M+H) ⁺ .

Step 4: 1-(4-((7,8-Dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)ph enyl)-3-(3- (trifluoromethyl)phenyl)urea

The titled compound was prepared by the reaction of tert-butyl 4-(4-(3-(3- (trifluoromethyl)phenyl)ureido)phenoxy)-6H-pyrimido[5,4-b][1 ,4]oxazine-8(7H)-carboxylate (step 3 intermediate) (30 mg, 0.06 mmol) with hydrochloric acid in 1,4-dioxane (1.0 mL) as per the procedure described in step 3 of Method A to yield 11 mg of the product.

Method I”

Preparation of 1-(3-(tert-butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)-3-(4-(( 3,4-dihydro-2H- pyrido[3,2-b][1,4]oxazin-8-yl)oxy)phenyl)urea

Step 1: tert-Butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-2H-pyrido[3,2- b][1,4]oxazine-4(3H)-carboxylate

A suspension of tert-butyl 8-bromo-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate (Intermediate A7) (400 mg, 1.27 mmol), benzyl (4-hydroxyphenyl)carbamate (370 mg, 1.52 mmol) and tripotassium phosphate (966 mg, 4.56 mmol) in toluene (10 mL) was degassed and added palladium acetate (52 mg, 0.24 mmol) followed by 2-di-tert-butylphosphino-  

2′,4′,6′-triisopropylbiphenyl (t-BuXPhos) (129 mg, 0.30 mmol) at RT. The mixture was refluxed at 120 °C for 16 h. The mixture was cooled to RT, diluted with diethyl ether and filtered through celite. The filtrate was concentrated and the residue thus obtained was purified by flash column chromatography to yield 198 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.47 (s, 9H), 3.86 (t, J = 4.4 Hz, 2H), 4.27 (t, J = 4.4 Hz, 2H), 5.16 (s, 2H), 6.45 (d, J = 5.6 Hz, 1H), 7.04-7.09 (m, 2H), 7.34-7.46 (m, 5H), 7.52 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 5.6 Hz, 1H), 9.86 (s, 1H); ESI-MS (m/z) 478 (M+H) ⁺ .

Step 2: tert-Butyl 8-(4-aminophenoxy)-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carbox ylate

The titled compound was prepared by the hydrogenation reaction of tert-butyl 8-(4- (((benzyloxy)carbonyl)amino)phenoxy)-2H-pyrido[3,2-b][1,4]ox azine-4(3H)-carboxylate (step 1 intermediate) (230 mg, 0.48 mmol) I the presence of 10% palladium on carbon (50% wet, 40 mg) as per the procedure described in step 2 of Method I’ to yield 127 mg of the desired compound. The compound was as such taken forward to the next step without characterization.

Step 3: tert-Butyl 8-(4-(3-(3-(tert-butyl)-1-(4-cyanophenyl)-1H-pyrazol-5- yl)ureido)phenoxy)-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carbox ylate

To a stirred solution of 4-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile (Intermediate C91) (50 mg, 0.21 mmol) in a mixture of dichloromethane (6.0 mL) and saturated sodium bicarbonate solution (4.0 mL) at 0 °C was added diphosgene (100 µL, 0.83 mmol) and the mixture was stirred at RT for 1.5 h. The mixture was diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the isocyanate. The isocyanate intermediate was suspended in a mixture of THF (10 mL) and acetonitrile (1.0 mL) and cooled 0 °C. To that mixture were added tert-butyl 8-(4-

 

aminophenoxy)-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxyl ate (step 2 intermediate) (71 mg, 0.21 mmol) followed by DIPEA (142 µL, 0.83 mmol) and the resultant mixture was stirred at RT for 16 h. The mixture was diluted with ethyl acetate and washed with water followed by brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 52 mg of the desired compound. ESI-MS (m/z) 610 (M+H) ⁺ .

Step 3: 1-(3-(tert-Butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)-3-(4-(( 3,4-dihydro-2H- pyrido[3,2-b][1,4]oxazin-8-yl)oxy)phenyl)urea

The titled compound was prepared by the reaction of tert-butyl 8-(4-(3-(3-(tert-butyl)-1-(4- cyanophenyl)-1H-pyrazol-5-yl)ureido)phenoxy)-2H-pyrido[3,2-b ][1,4]oxazine-4(3H)- carboxylate (step 2 intermediate) (50 mg, 0.08 mmol) with hydrochloric acid in 1,4-dioxane (3.0 mL) in methanol (0.5 mL) as per the procedure described in step 3 of Method A to yield 7.0 mg of the product.

Method J

Preparation of 4-chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3- ((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)b enzamide

Step 1: Methyl 4-chloro-3-((8-(4-methoxybenzyl)-7-oxo-7,8-dihydro-6H-pyrimi do[5,4- b][1,4]oxazin-4-yl)oxy)benzoate

The titled compound was prepared by the reaction of 4-chloro-8-(4-methoxybenzyl)-6H- pyrimido[5,4-b][1,4]oxazin-7(8H)-one (Intermediate A2) (330 mg, 1.08 mmol) and 4-chloro- 3-hydroxybenzoate (Intermediate B1) (261 mg, 1.40 mmol) in the presence of cesium fluoride (328 mg, 2.16 mmol) in DMSO (2.0 mL) as per the procedure described in step 2 of Method G’ to yield 210 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.72 (s, 3H), 3.86 (s, 3H), 5.02 (s, 2H), 5.14 (s, 2H), 6.87 (dd, J ₁ = 2.0 Hz, J ₂ = 6.4 Hz, 2H), 7.31 (d, J = 6.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.88-7.90 (m, 2H), 8.19 (s, 1H).

 

Step 2: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((8-(4- methoxybenzyl)-7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxaz in-4-yl)oxy)benzamide

To a solution of methyl 4-chloro-3-((8-(4-methoxybenzyl)-7-oxo-7,8-dihydro-6H- pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzoate (step 1 intermediate) (210 mg, 0.46 mmol) and 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (Intermediate C1) (110 mg, 0.38 mmol) in toluene (3.0 mL) was dropwise added trimethyl aluminium solution (2M in toluene, 768 µL, 1.54 mmol) at RT. The mixture was stirred for 3-5 h at RT. The mixture was quenched with aqueous ammonium chloride solution and extracted twice in ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 115 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 0.99 (t, J = 7.2 Hz, 3H), 2.40-2.51 (m, 10H), 3.57 (s, 2H), 3.72 (s, 3H), 5.04 (s, 2H), 5.15 (s, 2H), 6.87 (dd, J1 = 2.0 Hz, J2 = 6.4 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.94-8.0 (m, 3H), 8.17 (s, 1H), 8.22 (s, 1H), 10.58 (s, 1H); ESI-MS (m/z) 711 (M+H) ⁺ .

Step 3: 4-Chloro-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluorome thyl)phenyl)-3-((7-oxo- 7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide

The titled compound was prepared by the reaction of 4-chloro-N-(4-((4-ethylpiperazin-1- yl)methyl)-3-(trifluoromethyl)phenyl)-3-((8-(4-methoxybenzyl )-7-oxo-7,8-dihydro-6H- pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide (step 2 intermediate) (100 mg, 0.14 mmol) with trifluoroacetic acid (3.0 mL) as per the procedure described in step 3 of Method G to yield 29 mg of the product.

Method J’

Preparation of N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)-4-methyl-3- ((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)b enzamide

 

Step 1: Methyl 3-((8-(4-methoxybenzyl)-7-oxo-7,8-dihydro-6H-pyrimido[5,4-b] [1,4]oxazin- 4-yl)oxy)-4-methylbenzoate

A suspension of 4-chloro-8-(4-methoxybenzyl)-6H-pyrimido[5,4-b][1,4]oxazin-7 (8H)-one (Intermediate A2) (370 mg, 1.21 mmol), methyl 3-hydroxy-4-methylbenzoate (Intermediate B3) (261 mg, 1.57 mmol) and tripotassium phosphate (513 mg, 2.42 mmol) in toluene (4.0 mL) was degassed and added palladium acetate (54 mg, 0.24 mmol) followed by 2-di-tert- butylphosphino-2′,4′,6′-triisopropylbiphenyl (t-BuXPhos) (77 mg, 0.18 mmol) at RT. The mixture was refluxed for 3-5 h. The mixture was cooled to RT, diluted with diethyl ether and filtered through celite. The filtrate was concentrated and the residue thus obtained was purified by flash column chromatography to yield 309 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.19 (s, 3H), 3.72 (s, 3H), 3.83 (s, 3H), 5.01 (s, 2H), 5.14 (s, 2H), 6.88 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.65 (s, 1H), 7.79 (dd, J ₁ = 2.0 Hz, J ₂ = 8.0 Hz, 1H), 8.17 (s, 1H).

Step 2: N-(4-((4-Ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)-3-((8-(4- methoxybenzyl)-7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxaz in-4-yl)oxy)-4- methylbenzamide

The titled compound was prepared by the reaction of methyl 3-((8-(4-methoxybenzyl)-7-oxo- 7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)-4-methyl benzoate (step 1 intermediate) (270 mg, 0.62 mmol) with 4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)aniline (Intermediate C1) (140 mg, 0.49 mmol) in the presence of trimethyl aluminium solution (2M in toluene, 970 µL, 1.94 mmol) in toluene (3.0 mL) as per the procedure described in step 2 of Method J to yield 210 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.98 (t, J = 8.4 Hz, 3H), 2.19 (s, 3H), 2.40-2.51 (m, 10H), 3.56 (br s, 2H), 3.72 (s, 3H), 5.02 (s, 2H), 5.15 (s, 2H), 6.88 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 1.2 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 7.2 Hz, 1H), 8.17-8.19 (br s, 2H), 10.44 (s, 1H).  

Step 3: N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phen yl)-4-methyl-3-((7-oxo- 7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzamide

The titled compound was prepared by the reaction of N-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)-3-((8-(4-methoxybenzyl)-7-oxo-7,8-d ihydro-6H-pyrimido[5,4- b][1,4]oxazin-4-yl)oxy)-4-methylbenzamide (step 2 intermediate) (150 mg, 0.22 mmol) with trifluoroacetic acid (3.0 mL) as per the procedure described in step 3 of Method G to yield 18 mg of the product.

Method K

Preparation of 4-chloro-N-(4-((4-(2-cyanoacetyl)piperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)-3-((7,8-dihydro-6H-pyrimido[5,4-b][ 1,4]oxazin-4- yl)oxy)benzamide

To a solution of 2-cyanoacetic acid (20 mg, 0.18 mmol) in acetonitrile (5.0 mL) were added DIPEA (0.1 mL, 0.56 mmol) followed by TBTU (50 mg, 0.18 mmol) at 0 °C and the mixture was stirred for 30 min. 4-Chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)- N-(4-(piperazin-1-ylmethyl)-3-(trifluoromethyl)phenyl)benzam ide (dihydrochloride salt) (80 mg, 0.14 mmol) was added to the mixture and stirred for 3 h at RT. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 6.0 mg of the desired product.

Method L

Preparation of 4-chloro-3-((8-methyl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxa zin-4-yl)oxy)- N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benz amide

Step 1: Methyl 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl) oxy)benzoate

 

The titled compound was prepared by the reaction of tert-butyl 4-(2-chloro-5- (methoxycarbonyl)phenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8(7 H)-carboxylate (step 1- Method B) (500 mg, 1.18 mmol) with hydrochloric acid in ethyl acetate (25 mL) in methanol (5.0 mL) as per the procedure described in step 3 of Method A to yield 307 mg of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 3.83-3.87 (m, 2H), 4.04 (s, 3H), 4.17-4.21 (m, 2H), 7.68-7.74 (m, 3H), 7.81 (br s, 2H).

Step 2: Methyl 4-chloro-3-((8-methyl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxa zin-4- yl)oxy)benzoate

To a stirred solution of methyl 4-chloro-3-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4- yl)oxy)benzoate (step 1 intermediate) (300 mg, 0.93 mmol) in DMF (5.0 mL) were added methyl iodide (132 mg, 0.93 mmol) followed by sodium hydride (60% w/w, 41 mg, 1.03 mmol) at 0 °C and the mixture was stirred overnight at RT. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. The solution was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 105 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.34 (s, 3H), 3.58 (t, J = 4.8 Hz, 2H), 3.85 (s, 3H), 4.27 (t, J = 4.4 Hz, 2H), 7.71-7.78 (m, 2H), 7.80-7.83 (m, 2H).

Step 3: 4-Chloro-3-((8-methyl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxa zin-4-yl)oxy)-N-(3- (4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide

The titled compound was prepared by the reaction of methyl 4-chloro-3-((8-methyl-7,8- dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)benzoate (step 2 intermediate) (100 mg, 0.30 mmol) and 3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)aniline (Intermediate C19) (78 mg, 0.30 mmol) in the presence of potassium tert-butoxide (1M, 1.9 mL, 1.8 mmol) in anhydrous THF (5.0 mL) as per the procedure described in step 1 of Method A to yield 81 mg of the product.

 

Method M

Preparation of N-(4-((7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)-3 -fluorophenyl)- N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

Step 1: tert-Butyl 4-(4-amino-2-fluorophenoxy)-6H-pyrimido[5,4-b][1,4]oxazine-8 (7H)- carboxylate

The titled compound was prepared by the reaction of tert-butyl 4-chloro-6H-pyrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate (Intermediate A1) (500 mg, 1.83 mmol) with 4-amino-2- fluorophenol (350 mg, 2.76 mmol) in the presence of cesium carbonate (1.2 g, 3.67 mmol) in DMF (10 mL) as per the procedure described in step 2 of Method A to yield 361 mg of the compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.49 (s, 9H), 3.90 (t, J = 4.4 Hz, 2H), 4.35 (t, J = 4.0 Hz, 2H), 5.36 (s, 2H), 6.36 (dd, J1 = 2.5 Hz, J2 = 6.8 Hz, 1H), 6.48 (dd, J1 = 2.8 Hz, J2 = 13.2 Hz, 1H), 6.92 (t, J = 8.8 Hz, 1H),7.95 (s, 1H); ESI-MS (m/z) 363 (M+H) ⁺ .

Step 2: tert-Butyl 4-(2-fluoro-4-(1-((4- fluorophenyl)carbamoyl)cyclopropanecarboxamido)phenoxy)-6H-p yrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate

To a stirred solution of tert-butyl 4-(4-amino-2-fluorophenoxy)-6H-pyrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate (step 1 intermediate) (161 mg, 0.45 mmol) in dichloromethane (5.0 mL) were added DIPEA (138 µL, 0.80 mmol), 1-((4- fluorophenyl)carbamoyl)cyclopropanecarboxylic acid (Intermediate C68) (100 mg, 0.45 mmol mmol) followed by HATU (305 mg, 0.80 mmol) at RT. The mixture was stirred at RT for 18 h before quenching it with water. The layers were separate and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with brine and

 

dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure to yield 75 mg of the titled compound. . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.45-1.46 (br s, 4H), 1.49 (s, 9H), 3.92 (t, J = 4.4 Hz, 2H), 4.38 (t, J = 3.6 Hz, 2H), 7.15 (t, J = 9.2 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.62- 7.89 (m, 3H), 8.03 (s, 1H), 10.02 (s, 1H), 10.30 (s, 1H).

Step 3: N-(4-((7,8-Dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)oxy)-3 -fluorophenyl)-N-(4- fluorophenyl)cyclopropane-1,1-dicarboxamide

The titled compound was prepared by the reaction of tert-Butyl 4-(2-fluoro-4-(1-((4- fluorophenyl)carbamoyl)cyclopropanecarboxamido)phenoxy)-6H-p yrimido[5,4- b][1,4]oxazine-8(7H)-carboxylate (step 2 intermediate) (65 mg, 0.11 mmol) with hydrochloric acid in 1,4-dioxane (8.0 mL) in ethanol (5.0 mL) as per the procedure described in step 3 of Method A to yield 20 mg of the product.

Method N

Preparation of 4-chloro-3-((3-methyl-2-oxo-1,2,3,4-tetrahydropyrido[2,3-d]p yrimidin-5- yl)oxy)-N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phe nyl)benzamide

Step 1: tert-Butyl (4-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-3-formylpyridin-2 -yl)carbamate

To a solution of 4-chloro-3-hydroxy-N-(3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)benzamide (Step 1-Method G’) (200 mg, 0.48 mmol) in DMF (3.0 mL) was added cesium carbonate (314 mg, 0.97 mmol) and the mixture was stirred for 30 min. The solution was cooled to 0 °C and added with tert-butyl (3-chloro-2- formylphenyl)carbamate (CAS# 1260667-07-9) (186 mg, 0.73 mmol) at the same temperature. The resultant mixture was stirred overnight at RT. The reaction was quenched with ice and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium  

sulfate, filtered and concentrated. The crude material was purified by silica gel column chromatography to yield 220 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.48 (s, 9H), 2.23 (s, 3H), 2.45-2.51 (m, 4H), 3.21 (t, J = 4.8 Hz, 4H), 6.45 (d, J = 6.0 Hz, 1H), 6.98 (s, 1H), 7.59 (s, 1H), 7.62 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 8.01 (dd, J ₁ = 2.0 Hz, J ₂ = 8.4 Hz, 1H), 8.08 (d, J = 2.0 Hz, 1H), 8.37 (d, J = 5.6 Hz, 1H), 10.40 (s, 2H), 10.48 (s, 1H); ESI-MS (m/z) 534 (M+H-BOC) ⁺ .

Step 2: 4-chloro-3-((3-methyl-2-oxo-1,2,3,4-tetrahydropyrido[2,3-d]p yrimidin-5-yl)oxy)-N- (3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzam ide

To a stirred solution of tert-butyl (4-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-3-formylpyridin-2 -yl)carbamate (step 1 intermediate) (50 mg, 0.08 mmol) in THF (1.0 mL) were added methylamine solution (33% in ethanol, 0.1 mL, 0.8 mmol) followed by a drop of acetic acid and the mixture was stirred overnight at RT. The precipitated solid was filtered, dried and purified by silica gel column chromatography to yield 12 mg of the desired compound.

Method O

Preparation of 4-chloro-3-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-8-yl)ox y)-N-(3-(4- methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide

Step 1: 8-Bromo-7-nitro-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (Nitro adduct)

To a stirred solution of 8-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (Step 2- Intermediate A7) (2.0 g, 9.30 mmol) in sulfuric acid (8 mL) was dropwise added a mixture of sulfuric acid and nitric acid (1:1, 14 mL) at 0 °C over a period of 15 min. The mixture was poured on crushed ice and stirred to give yellow solid. An aqueous solution of sodium hydroxide was added to the mixture till pH 8-9. The solid was filtered, washed with water and dried well. The crude solid was purified by recrystallization from acetone to yield 1.2 g of the desired product. ¹ H NMR (400 MHz, DMSO-d6) δ 4.50-4.52 (m, 2H), 4.56-4.59 (m, 2H), 8.76 (s, 1H).  

Step 2: 4-Chloro-N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)ph enyl)-3-((7-nitro-3,4- dihydro-2H-pyrido[3,2-b][1,4]oxazin-8- l ox benzamide

To a stirred solution of 8-bromo-7-nitro-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (Nitro adduct) (step 1 intermediate) (202 mg, 0.66 mmol) and 4-chloro-3-hydroxy-N-(3-(4- methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (Step 1-Method G’) (246 mg, 0.59 mmol) in DMF (12 mL) was added potassium carbonate (275 mg, 1.98 mmol) and the mixture was stirred at 105 °C for 2 h. The mixture was cooled to RT and quenched with water. The aqueous mixture was extracted twice with ethyl acetate. The combined organic layers were washed with water followed by brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by silica gel column chromatography to yield 368 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.32 (s, 3H), 2.51-2.59 (br s, 4H), 3.23 (br s , 4H), 4.40-4.42 (m, 2H), 4.46-4.48 (m, 2H), 6.97 (s, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.55 (s, 1H), 7.59 (s, 1H), 7.78-7.83 (m, 2H), 8.99 (s, 1H), 10.40 (s, 1H).

Step 3: tert-Butyl 8-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-7-nitro-2H-pyrido [3,2-b][1,4]oxazine-4(3H)- carboxylate

To a solution of 4-chloro-N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)ph enyl)-3-((7- nitro-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-8-yl)oxy)benza mide (step 2 intermediate) (361 mg, 0.66 mmol) in dichloromethane (15 mL) were added triethylamine (183 µL, 1.31 mmol) followed by di-tert-butyl dicarbonate (227 µL, 0.99 mmol) and the mixture was stirred for 5 min before the addition of DMAP (8 mg, 0.06 mmol) at RT. The resultant mixture was stirred for 30 min at RT. The reaction mixture was diluted with dichloromethane and washed with water and brine. The solution was dried over anhydrous sodium sulfate, filtered and

 

concentrated. The residue thus obtained was purified by silica gel column chromatography to yield 287 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.52 (s, 9H), 2.22 (s, 3H), 2.40-2.51 (m, 4H), 3.16-3.20 (m, 4H), 3.91 (d, J = 4.4 Hz, 2H), 4.28 (d, J = 4.0 Hz, 2H), 6.94 (s, 1H), 7.31 (d, J = 1.6 Hz, 1H), 7.55 (s, 1H), 7.57 (s, 1H), 7.77-7.81 (m, 2H), 8.80 (s, 1H), 10.36 (s, 1H).

Step 4: tert-Butyl 7-amino-8-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-2H-pyrido[3,2-b][ 1,4]oxazine-4(3H)- carboxylate

To a stirred solution of tert-butyl 8-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-7-nitro-2H-pyrido [3,2-b][1,4]oxazine-4(3H) carboxylate (step 3 intermediate) (280 mg, 0.40 mmol) in a mixture of methanol and water (3:1, 20 mL) was added ammonium chloride (215 mg, 4.05 mmol) and the mixture was heated to 80 °C. Zinc dust (135 mg, 2.06 mmol) was added in small portions and the mixture was stirred at for 1 h at 80 °C. The mixture was cooled to RT and poured in to aqueous sodium bicarbonate solution. The aqueous mixture was extracted twice with 5% mixture of methanol in chloroform. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 174 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.45 (s, 9H), 2.24 (s, 3H), 2.40 (br s, 4H), 3.21 (br s, 4H), 3.72 (t, J = 4.4 Hz, 2H), 4.08 (t, J = 4.0 Hz, 2H), 5.15 (s, 2H), 6.94 (s, 1H), 7.14 (s, 1H), 7.55-7.75 (m, 5H), 10.38 (s 1H).

Step 5: 4-Chloro-3-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-8-yl)ox y)-N-(3-(4- methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide

To a stirred solution of tert-butyl 7-amino-8-(2-chloro-5-((3-(4-methylpiperazin-1-yl)-5- (trifluoromethyl)phenyl)carbamoyl)phenoxy)-2H-pyrido[3,2-b][ 1,4]oxazine-4(3H)- carboxylate (step 4 intermediate) (119 mg, 0.18 mmol) in dichloromethane (5.0 mL) at -20 °C was added BF3-etherate (50%, 135 µL, 0.54 mmol) and the mixture was stirred for 5 min. Thereafter, a solution of tert-butyl nitrite (43 µL, 0.36 mmol) in DCM (5.0 mL) was added dropwise to the reaction mixture over a period of 10 min. The resultant mixture was stirred at -20 °C for 30 min. The mixture was allowed to warm up to 10 °C and the solvent was  

removed by blowing nitrogen gas. The crude black residue was dissolved in DMF (5.0 mL) and added to a stirred solution of iron sulfate (55 mg, 0.19 mmol) in DMF (5.0 mL) and the mixture was stirred for 5 min at RT. The poured in to aqueous sodium bicarbonate solution and the product was extracted twice in ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 28 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d6) δ 2.27 (s, 3H), 2.44-2.47 (br s, 4H), 3.19-3.21 (br s, 4H), 3.43 (br s, 2H), 4.10 (t, J = 4.0 Hz, 2H), 6.10 (d, J = 5.6 Hz, 1H), 6.96 (s, 2H), 7.53 (d, J = 5.6 Hz, 1H), 7.59 (s, 3H), 7.78-7.85 (m, 2H), 10.38 (s, 1H); ESI-MS (m/z) 548 (M+H) ⁺ .

Method P

Preparation of 1-(4-((7-cyano-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-8-yl) oxy)phenyl)-3- (4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl )urea

Step 1: tert-Butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-7-nitro-2H-pyrido[ 3,2- b][1,4]oxazine-4(3H)-carboxylate

The titled compound was prepared by the reaction of tert-butyl 8-bromo-7-nitro-2H- pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate (Intermediate A22) (1.5 g, 4.16 mmol) and benzyl (4-hydroxyphenyl)carbamate (1.11 mg, 4.58 mmol) in the presence of cesium fluoride (1.9 g, 12.5 mmol) in DMSO (40 mL) as per the procedure described in step 2 of Method G’ to yield 1.39 g of the compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.51 (s, 9H), 3.89 (t, J = 4.4 Hz, 2H), 4.25 (t, J = 4.0 Hz, 2H), 5.14 (s, 2H), 6.92 (d, J = 9.2 Hz, 2H), 7.34-7.43 (m, 7H), 8.69 (s, 1H), 9.72 (s, 1H).

Step 2: tert-Butyl 7-amino-8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-2H-pyrido[ 3,2- b][1,4]oxazine-4(3H)-carboxylate

 

The titled compound was prepared by the reaction of tert-Butyl 8-(4- (((benzyloxy)carbonyl)amino)phenoxy)-7-nitro-2H-pyrido[3,2-b ][1,4]oxazine-4(3H)- carboxylate (step 1 intermediate) (1.35 g, 2.58 mmol) with zinc dust (844 mg, 12.9 mmol) and ammonium chloride (1.38 g, 25.8 mmol) in a mixture of methanol and water (3:1, 40 mL) as per the procedure described in step 4 of Method O to yield 921 mg of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.45 (s, 9H), 3.73 (t, J = 4.4 Hz, 2H), 4.07 (t, J = 4.0 Hz, 2H), 4.94 (s, 2H), 5.13 (s, 2H), 6.79 (d, J = 9.2 Hz, 2H), 7.34-7.39 (m, 7H), 8.32 (s, 1H), 9.64 (s, 1H); ESI-MS (m/z) 492 (M+H) ⁺ .

Step 3: tert-Butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-7-iodo-2H-pyrido[3 ,2- b][1,4]oxazine-4(3H)-carboxylate

To a stirred suspension of tert-butyl 7-amino-8-(4-(((benzyloxy)carbonyl)amino)phenoxy)- 2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate (step 2 intermediate) (200 mg, 0.41 mmol) in acetonitrile (8.0 mL) was added PTSA (232 mg, 1.22 mmol) and stirred at RT for 10-15 min. To that mixture a solution of sodium nitrite (56 mg, 0.81 mmol) and potassium iodide (169 mg, 1.05 mmol) in water (1.0 mL) was added at 10-15 °C. The reaction mixture was gradually allowed to attain RT and poured on an aqueous solution of sodium bicarbonate. The aqueous mixture was extracted twice with ethyl acetate and the combined organic extracts were washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 102 mg of the desired compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.48 (s, 9H), 3.83 (t, J = 4.4 Hz, 2H), 4.16 (t, J = 3.6 Hz, 2H), 5.13 (m, 2H), 6.79 (d, J = 8.8 Hz, 2H), 7.34-7.43 (m, 7H), 8.27 (s, 1H),9.70 (s, 1H).

Step 4: tert-Butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-7-cyano-2H-pyrido[ 3,2- b][1,4]oxazine-4(3H)-carboxylate  

To a solution of tert-butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-7-iodo-2H- pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate (step 3 intermediate) (52 mg, 0.08 mmol) in DMF (5.0 mL) were added zinc cyanide (10 mg, 0.09 mmol), [1,1′- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (10 mg, 0.017 mmol) followed by water (10 µL) and the mixture was degassed for 5 min before adding tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) (8.0 mg, 0.008 mmol). The resultant mixture was heated to 145 °C for 15 min in a microwave reactor. The reaction mixture cooled to RT and poured on to water. The mixture was extracted twice with ethyl acetate. The combined organic extracts were washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue obtained was purified by column chromatography to yield 19 mg of the Boc-deprotected compound. The Boc-group was restored by the reaction of the compound with di-tert-butyl dicarbonate (1.5 eq.) in the presence of LiHMDS (1.5 eq.) in THF (5.0 vol.) as per the procedure described in step 3 of Intermediate A7 to yield the titled compound. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ δ 1.50 (s, 9H), 3.88 (t, J = 3.6 Hz, 2H), 4.21 (t, J = 4.4 Hz, 2H), 5.14 (m, 2H), 6.97 (d, J = 8.0 Hz, 2H), 7.33-7.44 (m, 7H), 8.41 (s, 1H), 9.78 (s, 1H).

Step 5: tert-Butyl 8-(4-aminophenoxy)-7-cyano-2H-pyrido[3,2-b][1,4]oxazine-4(3H )- carboxylate

A solution of tert-butyl 8-(4-(((benzyloxy)carbonyl)amino)phenoxy)-7-cyano-2H-pyrido[ 3,2- b][1,4]oxazine-4(3H)-carboxylate (step 4 intermediate) (120 mg, 0.24 mmol) in methanol (12 mL) with catalytic amount of 10% palladium on carbon (50% wet, 60 mg) was hydrogenated at RT for 1 h. The mixture was filtered through celite and the celite bed was rinsed with methanol. The combined filtrate and washings were concentrated and the residue thus obtained was purified by silica gel column chromatography to yield 48 mg of the desired

 

product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.50 (s, 9H), 3.87 (t, J = 4.4 Hz, 2H), 4.22 (t, J = 4.0 Hz, 2H), 6.92 (dd, J1 = 2.0 Hz, J2 = 6.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 9.34 (s, 1H). Step 6: tert-Butyl 7-cyano-8-(4-(3-(4-((4-ethylpiperazin-1-yl)methyl)-3- (trifluoromethyl)phenyl)ureido)phenoxy)-2H-pyrido[3,2-b][1,4 ]oxazine-4(3H)-carboxylate

The titled compound was prepared by the reaction of tert-butyl 8-(4-aminophenoxy)-7-cyano- 2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carboxylate (step 5 intermediate) (45 mg, 0.12 mmol) with 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (Intermediate C1) (35 mg, 0.12 mmol) by using triphosgene (13 mg, 0.04 mmol) and triethylamine (71 µL, 0.37 mmol) in THF (10 mL) as per the procedure described in step 2 of Method I to yield 53 mg of the compound. ESI-MS (m/z) 682 (M+H) ⁺ .

Step 7: 1-(4-((7-Cyano-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-8-yl) oxy)phenyl)-3-(4-((4- ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)urea

The titled compound was prepared by the reaction of tert-butyl 7-cyano-8-(4-(3-(4-((4- ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)ureido )phenoxy)-2H-pyrido[3,2- b][1,4]oxazine-4(3H)-carboxylate (step 6 intermediate) (52 mg, 0.07 mmol) with hydrochloric acid in 1,4-dioxane (2.5 mL) in 1,4-dioxane (2.0 mL) and methanol (0.5 mL) as per the procedure described in step 3 of Method A to yield 11 mg of the product. ¹ H NMR (400 MHz, DMSO-d6) δ 1.01 (t, J = 7.2 Hz, 3H), 2.42 (br s, 10H), 3.33 (br s, 2H), 3.54 (br s, 2H), 4.04 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 9.2 Hz, 2H), 7.41 (d, J = 9.2 Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 8.10 (s, 2H), 8.79 (s, 1H), 9.04 (s, 1H); ESI-MS (m/z) 582 (M+H) ⁺ .

Method Q

Preparation of 1-(3-(tert-butyl)-1-(3-cyanophenyl)-1H-pyrazol-5-yl)-3-(4-(( 3,4-dihydro-2H- pyrido[3,2-b][1,4]oxazin-8-yl)ox hen l urea

 

Step 1: Phenyl (3-(tert-butyl)-1-(3-cyanophenyl)-1H-pyrazol-5-yl)carbamate

To a stirred solution of 3-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile (Intermediate C92) (150 mg, 0.62 mmol) and pyridine (0.1 mL, 1.25 mmol) in THF (10 mL) was added phenyl chloroformate (0.12 mL, 0.94 mmol) at 0 °C. The mixture was stirred at RT for 2 h and diluted with ethyl acetate. The organic layer was washed with water, brine and dried over anhydrous sodium sulfate. The solution of filtered, concentrated and the residue obtained was purified by silica gel column chromatography to yield 79 mg of the desired product ESI-MS (m/z) 361 (M+H) ⁺ .

Step 2: tert-Butyl 8-(4-(3-(3-(tert-butyl)-1-(3-cyanophenyl)-1H-pyrazol-5- yl)ureido)phenoxy)-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carbox ylate

To a cooled (10 °C) solution of tert-butyl 8-(4-aminophenoxy)-2H-pyrido[3,2- b][1,4]oxazine-4(3H)-carboxylate (step 2 of Method I”) (60 mg, 0.17 mmol) in DMSO (5.0 mL) were added triethylamine (75 µL, 0.53 mmol) followed by phenyl (3-(tert-butyl)-1-(3- cyanophenyl)-1H-pyrazol-5-yl)carbamate (step 1 intermediate) (70 mg, 0.19 mmol) and the mixture was stirred at RT for 16 h. The mixture was diluted with ethyl acetate and washed with water, saturated ammonium chloride solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and the residue obtained was purified by silica gel column chromatography to yield 69 mg of the desired product. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.30 (s, 9H), 1.47 (s, 9H), 3.86 (t, J = 4.0 Hz, 2H), 4.27 (t, J = 4.0 Hz, 2H), 6.41 (s, 1H), 6.47 (d, J = 3.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.71-7.77 (m, 1H), 7.80 (d, J = 5.2 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 8.04 (s, 1H), 8.53 (s, 1H), 9.10 (s, 1H); ESI-MS (m/z) 610 (M+H) ⁺ .

Step 3: 1-(3-(tert-Butyl)-1-(3-cyanophenyl)-1H-pyrazol-5-yl)-3-(4-(( 3,4-dihydro-2H- pyrido[3,2-b][1,4]oxazin-8-yl)oxy)phenyl)urea

 

To a stirred solution of tert-butyl 8-(4-(3-(3-(tert-butyl)-1-(3-cyanophenyl)-1H-pyrazol-5- yl)ureido)phenoxy)-2H-pyrido[3,2-b][1,4]oxazine-4(3H)-carbox ylate (step 2 intermediate) (64 mg, 0.11 mmol) in acetonitrile (10 mL) was added PTSA (200 mg, 1.05 mmol) followed by few drops of methanol and the mixture was stirred at RT for 16 h. The mixture was diluted with ethyl acetate and washed with saturated sodium bicarbonate solution, water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and the residue obtained was purified by silica gel column chromatography to yield 21 mg of the desired product.

Method R

Preparation of 1-(3-(tert-butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)-3-(2-fl uoro-4-((2,3,4,5- tetrahydropyrido[3,2-b][1,4]oxazepin-9-yl)oxy)phenyl)urea (Example 228)

Step 1: tert-Butyl 9-(4-(3-(3-(tert-butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)ur eido)-3- fluorophenoxy)-3,4-dihydropyrido[3,2-b][1,4]oxazepine-5(2H)- carboxylate

To a solution of 4-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzonitrile (Intermediate 91) (120 mg, 0.34 mmol) in dichloromethane (10 mL) was added 1,1′-carbonyldiimidazole (CDI) (55 mg, 0.34 mmol) and the mixture was stirred at 50 °C for 1 h followed by overnight at RT. To that mixture was added 2-fluoro-4-((2,3,4,5-tetrahydropyrido[3,2-b][1,4]oxazepin-9- yl)oxy)aniline (prepared by procedure described in case of step 1 and 2 of method I”) (81 mg, 0.34 mmol) at RT and stirred for 16 h. The reaction was quenched with water and extracted twice with dichloromethane. The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column

  chromatography to yield 60 mg of the titled compound. ¹ H NMR (400 MHz, DMSO-d6) δ 1.29 (s, 9H), 1.36 (s, 9H), 1.90-1.93 (m, 2H), 3.60-3.68 (m, 2H), 3.96-4.02 (m, 2H), 6.42 (s, 1H), 6.66 (d, J = 5.6 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 9.2 Hz, 2H), 7.80 (d, J = 8.4 Hz, 2H), 7.97-8.02 (m, 2H), 8.58 (s, 1H), 9.14 (s, 1H); ESI-MS (m/z) 541 (M+H-Boc) ⁺ . Step 2: 1-(3-(tert-Butyl)-1-(4-cyanophenyl)-1H-pyrazol-5-yl)-3-(2-fl uoro-4-((2,3,4,5- tetrahydropyrido[3,2-b][1,4]oxazepin-9-yl)oxy)phenyl)urea

The titled compound was prepared by the reaction of tert-butyl 9-(4-(3-(3-(tert-butyl)-1-(4- cyanophenyl)-1H-pyrazol-5-yl)ureido)-3-fluorophenoxy)-3,4-di hydropyrido[3,2-b][1,4] oxazepine-5(2H)-carboxylate (step 1 intermediate) (50 mg, 0.08 mmol) with hydrochloric acid in 1,4-dioxane (5.0 mL) as per the procedure described in step 3 of Method A to yield 18 mg of the product.

Table 25: Structure, Chemical name, Method used and analytical data of Examples

   

 

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PHARMACOLOGICAL ACTIVITY

This is a one step binding assay based on the binding and displacement of the labeled tracer where compound addition is followed by addition of the anti-GST tagged europium (Eu) as the donor and Alexa Fluor labelled tracer as the acceptor. Simultaneous binding of both the tracer and GST-antibody to the kinase domain of HPK1 results in a high degree of FRET (fluorescence resonance energy transfer) from the anti-GST tagged europium (Eu) fluorophore to the Alexa Fluor® 647 fluorophore on the kinase tracer and this signal is reduced in presence of the inhibitor that can be measured.

Test compounds or reference compounds such as Sunitinib were dissolved in dimethylsulfoxide (DMSO) to prepare 10.0 mM stock solutions and diluted to the desired concentration. The final concentration of DMSO in the reaction was 3% (v/v). The assay mixture was prepared by mixing 6nM of the Eu‐Anti‐GST Antibody and 15nM MAP4K-1 enzyme in the Kinase buffer containing 50mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij‐35 with or without the desired concentration of the compound. The reaction was incubated on ice for 15mins. The pre-incubation step was terminated by addition of the 30nM Kinase Tracer 222 into the reaction mixture. After shaking for 5 min the reaction was further incubated for 1 hour at room temperature and this was kept and read at 4 ^o C on an ARTEMIS reader as per the kit instructions (Thermo). The inhibition of test compound was calculated based on the FRET ratio of 665 / 615. The activity was calculated as a percent of control reaction. IC50 values were calculated from dose response curve by nonlinear regression analysis using GraphPad Prism software.

The compounds prepared were tested using the above assay procedure and the results obtained are given in Table 26. Percentage inhibition at concentrations of 1.0 µM and 10.0 µM are given in the table along with IC50 (nM) details for selected examples.

The IC ₅₀ (nM) values are set forth in Table 26 wherein“A” refers to an IC ₅₀ value of less than 50 nM,“B” refers to IC50 value in range of 50.01 to 100.0 nM,“C” refers to IC50 values more than 100.01 to 500 nM and“D” refers to IC50 values more than 500 nM.

Table 26:

 

 

 

 

 

   

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as described above.

 

All publications and patent applications cited in this application are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference.