Inhibitors of ENL / AF9 yeats and FLT3

Abstract

Disclosed herein are compounds and pharmaceutical compositions comprising the compounds that inhibit ENL / AF9YEATS and FLT3. Also disclosed are methods of using the compounds and pharmaceutical compositions comprising the compounds to inhibit oncogene expression in cells or to treat acute leukemia. The compounds, pharmaceutical compositions and methods are useful for inhibiting cancer and key driver factors for cancer stem cell survival.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 404,659, filed September 8, 2022, which is incorporated herein in its entirety. Technical Field

[0003] The present application generally relates to compounds that inhibit ENL / AF9 YEATS and FLT3 and therapeutic methods using such compounds. The compounds and methods can be used to treat a variety of different diseases, including blood cancers such as leukemias. Background Art

[0004] Epigenome is the collection of chemical compounds adjacent to DNA, which is responsible for modifying genome without changing DNA sequence. It is dynamically regulated by the chemical changes of the histones wrapped around it by DNA, RNA and DNA. It has been shown that the mutation in the gene encoding epigenetic regulator works in acute myeloid leukemia (AML) pathogenesis (Shih AH, Abdel-Wahab O, Patel JP, et al. " The role of mutations in epigenetic regulators in myeloid malignancies. " Nat. Rev. Cancer 2012; 12: 599-612).

[0005] ENL is a chromatin reader protein with an amino-terminal YEATS domain (named after the earliest discovered members of the family: Yaf9, ENL, AF9, Taf14, Sas5) and a disordered carboxy-terminal protein-protein interaction (PPI) interface. YEATS is a family of histone acetyl-lysine readers that function as effectors by making chromatin more accessible to RNA polymerases and transcription factors. Erb et al. reported that a disproportionate number of leukemia oncogenes and genes are dependent on ENL at their promoters (Erb, MA et al., “Transcription control by the ENL YEATSdomain in acute leukaemia,” Nature 543, 270–274 (2017). Wan et al. found that ENL binds to acetylated histone H3 and then colocalizes with H3K27 and H3K9ac at the promoters of essential leukemia genes, and that ENL is required for AML maintenance (Wan L. et al., “ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia,” Nature 2017; 543: 265-9).

[0006] Given the role of ENL in leukemia proliferation, inhibitors of the YEATS domain of ENL are potential targets for treating blood cancers. For example, Moustakim et al. described small molecule inhibitors of the ENL YEATS domain (Moustakim, M. et al., "Discovery of an MLLT1 / 3 YEATS Domain Chemical Probe," Angew. Chem. Int. Ed. 2018, 57, 16302-16307). Moustakim's inhibitor compounds contain a cyclic nitrogen-containing heterocycle connected by a nitrogen atom to a methylene group attached to a benzimidazole core.

[0007] FLT3 (Fms-like tyrosine kinase 3, FLK2) is a class III receptor tyrosine kinase. It is activated by FLT3 ligand (FL) and transmits signals through PI3K, RAS and JAK / STAT pathways (Scholl C. et al., Semin. Oncol., 35:336-45 (2008); Meshinchi S. et al., Clin. Cancer Res., 15:4263-9 (2009)). FLT3 plays a role in early hematopoiesis, and FLT3-deficient mice have a reduced number of progenitor cells of multiple lymphoid lineages (Mackarehtschian et al., 2009). K et al., Immunity, 3:147-61 (1995). Activating mutations in FLT3 are found in approximately 30% of AML patients, representing the most frequent genetic alterations in the disease. Approximately 75% of activating mutations are internal tandem duplications (ITDs), and 25% are point mutations in the activation loop of the kinase domain. The most frequently identified activating point mutation is D835Y (Yamamoto et al., Blood, 97(8):2434-2439(2001)). However, mutations have also been found at N841I (Jiang, J. et al., Blood, 104(6):1855-1858(2004)) and Y842C (Kindler et al., Blood, 105(1):335-340(2005)). Additional point mutations have been identified in the juxtamembrane domain and the kinase domain, although these mutations have been shown to result in lower transforming potential (Reindel et al., Blood 107(9):3700-3707(2006)).

[0008] It has been shown that mouse bone marrow transplanted with a retrovirus expressing FLT3-ITD results in a lethal myeloproliferative disease in mice (Kelly et al., Blood 99:310-318 (2002)), characterized by a leukocytosis consisting of mature neutrophils. The disease does not show a block in differentiation as seen in human AML, suggesting that the FLT3 mutation confers a proliferation or survival advantage to the cells.

[0009] Many FLT3 inhibitors have been tested in clinical trials. Although they have shown preliminary clinical responses in AML, the observed reactions are short-lived and can rapidly develop drug resistance (Weisberg, E. et al., Oncogene, 29:5120-34 (2010)). The main drug resistance mechanism appears to be by obtaining the secondary mutations in FLT3, which can interfere with the combination of FLT3 inhibitors with FLT3 receptors (Weisberg, E. et al., Oncogene, 29:5120-34 (2010); Chu, SH et al., Drug Resist.Update, 12:8-16 (2009)). Although it is recognized that chemotherapy has poor tolerance, the combination of FLT3 inhibitors and chemotherapy is being tested in clinical trials. Other possible mechanisms for the lack of durable responses include inadequate target coverage (Pratz, KW et al., Blood, 139:3938-46 (2009)) and protection of AML cells in the bone marrow, where stromal growth factors may provide proliferative signals in addition to FLT3 activation (Tam, WF et al., Best Pract. Res. Clin. Haematol., 21:13-20 (2008)).

[0010] There remains a need for improved inhibitors that can be used to treat hematological cancers. SUMMARY OF THE INVENTION

[0012] The present invention relates to compounds, pharmaceutical compositions, and methods for inhibiting YEATS / ENL and FLT3, thereby treating various cancers, particularly hematological cancers such as leukemias. The compounds, pharmaceutical compositions, and methods disclosed herein can be used to inhibit key drivers of cancer and cancer stem cell survival, thereby providing enhanced anti-cancer activity. In particular, the compounds, pharmaceutical compositions, and methods disclosed herein can be used to inhibit tyrosine kinase activity via FLT3 and the epigenetic driver ENL-YEATS. Thus, cancer cells that rely on one or both of these pathways can be effectively treated with less reliance on molecular diagnostics.

[0013] In a first aspect, compounds of Formula I are provided, which in some embodiments inhibit both YEATS / ENL and FLT3:

[0014]

[0015] in:

[0016] R 1 and R 2 together to form pyrrolidine or piperidine;

[0017] R 3 Selected from hydrogen and C1-C8 alkyl;

[0018] R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and

[0019] R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

[0020] In a second aspect, compounds of Formula II are provided, which in some embodiments inhibit both YEATS / ENL and FLT3:

[0021]

[0022] in:

[0023] R 8 Selected from hydrogen and C1-C8 alkyl;

[0024] R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and

[0025] R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

[0026] In a third aspect, the present invention relates to a pharmaceutical composition comprising a compound of Formula I and / or Formula II and one or more pharmaceutically acceptable carriers. The pharmaceutical composition may further comprise one or more therapeutic agents. Exemplary therapeutic agents include Bcl-2 inhibitors, cyclin-dependent kinases 4 and 6 (CDK 4 / 6) inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone demethylase inhibitors, mTOR inhibitors, mutant isocitrate dehydrogenase (IDH1 and IDH2) inhibitors, glucocorticoids, epigenetic regulators, and chemotherapeutic agents.

[0027] In a fourth aspect, the present invention relates to a method for treating acute leukemia, the method including administering a therapeutically effective amount of a compound as described herein or a pharmaceutical composition comprising the compound to a subject in need. Acute leukemia can be acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML). The method may further include administering one or more other therapeutic agents, for example, Bcl-2 inhibitors, cyclin-dependent kinases 4 and 6 (CDK 4 / 6) inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone demethylase inhibitors, mTOR inhibitors, mutant isocitrate dehydrogenase (IDH1 and IDH2) inhibitors, glucocorticoids, epigenetic regulators, and chemotherapeutic agents. In a specific embodiment, one or more compounds of the present invention are administered simultaneously or sequentially with another FLT3 inhibitor. In another specific embodiment, one or more compounds of the present invention are administered simultaneously or sequentially with chemotherapeutic agents. In some embodiments, chemotherapeutic agents are medicines for treating AML, such as cytarabine, BCL-2 inhibitors (for example, venetoclax) or menin inhibitors. Detailed Description of the Invention

[0029] I. Definition

[0030] As used herein, "acyl" refers to saturated, unsaturated and aromatic groups of formyl and 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms in straight, branched, cyclic configurations and combinations thereof, which are attached to the parent structure through a carbonyl functional group. One or more carbons in the acyl residue can be replaced by nitrogen, oxygen or sulfur, as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, tert-butoxycarbonyl, benzyloxycarbonyl and the like. Lower acyl refers to groups containing one to four carbons. The double-bonded oxygen is referred to as "oxo" when it is mentioned as a substituent by itself.

[0031] As used herein, the term "alkyl" includes straight or branched hydrocarbon structures. Lower alkyl refers to an alkyl group having 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl. Preferred alkyl groups are those with C20 or lower, such as C1-C 10 Alkyl, C1-C8 alkyl and C1-C6 alkyl.

[0032] As used herein, "aryl" and "heteroaryl" refer to (i) phenyl (or benzene) or a monocyclic 5- or 6-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, N, or S; (ii) a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-4 heteroatoms selected from O, N, or S; or (iii) a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-5 heteroatoms selected from O, N, or S. Aromatic 6- to 14-membered carbocyclic rings include, for example, benzene, naphthalene, indane, tetralin, and fluorene, and 5- to 10-membered aromatic heterocyclic rings include, for example, imidazole, pyridine, indole, thiophene, chromenone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, and pyrazole. As used herein, aryl and heteroaryl refer to residues in which one or more rings are aromatic, but not all rings are necessarily aromatic.

[0033] As used herein, "arylalkyl" refers to a substituent in which the aryl residue is attached to the parent structure through an alkyl group. Examples are benzyl, phenethyl, and the like. "Heteroarylalkyl" refers to a substituent in which the heteroaryl residue is attached to the parent structure through an alkyl group. In one embodiment, the alkyl group of the arylalkyl or heteroarylalkyl group is an alkyl group having 1 to 6 carbon atoms. Examples include, for example, pyridinylmethyl, pyrimidinylethyl, and the like.

[0034] As used herein, “C1 to C 20 Hydrocarbons" or "C1 to C 20 Hydrocarbyl (as a substituent) includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl, and combinations thereof. Examples include cyclopropylmethyl, benzyl, phenethyl, cyclohexylmethyl, camphoryl, and naphthylethyl. Hydrocarbyl refers to any substituent containing hydrogen and carbon as the only elemental components. Cycloalkyl is a subset of hydrocarbyl and includes cyclic hydrocarbon groups having 3 to 8 carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, norbornyl, and the like.

[0035] "Alkoxy" or "alkoxyl" refers to a group of 1 to 8 carbon atoms attached to the parent structure through an oxygen in a linear, branched, or cyclic configuration, and combinations thereof. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like. Lower alkoxy refers to a group containing one to four carbon atoms. For the purposes of this application, alkoxy and lower alkoxy include methylenedioxy and ethylenedioxy.

[0036] As used herein, "carbocycle" includes ring systems in which the ring atoms are all carbon but in any oxidation state. Thus, (C3-C8)carbocycle refers to both non-aromatic and aromatic systems, including systems such as cyclopropane, benzene, and cyclohexene; (C8-C 12 ) Carbocyclic refers to systems such as norbornane, decahydronaphthalene, indane and naphthalene. If not otherwise limited, carbocyclic refers to monocyclic, bicyclic and polycyclic rings.

[0037] As used herein, the term "therapeutically effective amount" refers to any amount of a compound of the present invention or any other pharmaceutically active agent that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding patient not receiving such amount of a compound of the present invention or other pharmaceutically active agent.

[0038] As used herein, the term "fused bicyclic ring" refers to bicyclic carbocycles and bicyclic heterocycles in which each ring (carbocycle or heterocycle) shares two adjacent atoms with another ring (carbocycle or heterocycle). Each ring of the fused carbocycle can be selected from non-aromatic rings or aromatic rings. In a preferred embodiment, an aromatic ring (e.g., phenyl) can be fused to another aromatic ring. In other embodiments, an aromatic ring can be fused to a non-aromatic ring (e.g., cyclohexane, cyclopentane, or cyclohexene). Exemplary fused bicyclic rings include 6,6; 6,5; and 5,6 fused bicyclic ring systems, where each number represents the number of atoms in each ring. The fused bicyclic ring may be substituted at any one or more positions where it may have a hydrogen atom. The fused bicyclic ring is attached to the parent structure at the first numbered ring (e.g., the "6" ring of a fused 6,5 bicyclic ring).

[0039] As used herein, "heterocycle" refers to a cycloalkyl or aryl carbocyclic residue in which one to four carbon atoms are replaced by heteroatoms selected from N, O, and S. Nitrogen and sulfur heteroatoms may be optionally oxidized, and nitrogen heteroatoms may be optionally quaternized. Unless otherwise specified, heterocycles may be non-aromatic or aromatic. Examples of heterocycles falling within the scope of the present invention include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxane, benzodioxole (commonly referred to as methylenedioxyphenyl when appearing as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran, etc. It should be noted that heteroaryl is a subset of heterocycles in which the heterocycle is aromatic. Non-limiting examples of heteroaromatic rings include furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, triazole, tetrazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine, pyrimidine, quinazoline, pyridazine, cinnoline, phthalazine and triazine. Examples of heterocyclyl residues further include piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepine 2-oxoazepinyl, aza benzothiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, thienyl, benzothienyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, oxadiazolyl, triazolyl, and tetrahydroquinolinyl.

[0040] An oxygen heterocycle is a heterocycle containing at least one oxygen in the ring; it may contain additional oxygen atoms as well as other heteroatoms. A sulfur heterocycle is a heterocycle containing at least one sulfur in the ring; it may contain additional sulfur atoms as well as other heteroatoms. An oxygen heteroaryl is a subset of oxygen heterocycles; non-limiting examples include furan and oxazole. A sulfur heteroaryl is a subset of sulfur heterocycles; examples include thiophene and thiazine. A nitrogen heterocycle is a heterocycle containing at least one nitrogen in the ring; it may contain additional nitrogen atoms as well as other heteroatoms. Non-limiting examples include piperidine, piperazine, morpholine, pyrrolidine, and thiomorpholine. A nitrogen heteroaryl is a subset of nitrogen heterocycles; non-limiting examples include pyridine, pyrrole, and thiazole.

[0041] As used herein, the term "optionally substituted" is used interchangeably with "unsubstituted or substituted". The term "substituted" refers to the replacement of one or more hydrogen atoms in a specified group with a specified group. For example, substituted aryl, heterocyclic group, etc. refers to an aryl or heterocyclic group in which one or more hydrogen atoms in each residue are replaced by halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxy lower alkyl, carbonyl, phenyl, heteroaryl, phenylsulfonyl, hydroxy, lower alkoxy, haloalkoxy, oxaalkyl, carboxyl, alkoxycarbonyl [-C (= O) O-alkyl], amide [-C (= O) NH2], alkylaminocarbonyl [-C (= O) NH-alkyl], cyano, acetoxy, nitro, amino, Alkylamino, dialkylamino, dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclyloxy, arylalkyl, (cycloalkyl)alkyl, heterocyclyl, heterocyclylalkyl, alkylaminoalkyl, heterocyclylaminoalkyl, heterocyclylalkylaminoalkyl, cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl, arylaminoalkyl and arylalkylaminoalkyl, mercapto, alkylthio, alkylsulfinyl, benzyl, heterocyclyl, phenoxy, benzyloxy, heteroaryloxy, aminosulfonyl, amidino, guanidino and ureido. (C 1-6 )hydrocarbyl, -SO2alkyl, -SO2NH2 or -SO2NHalkyl.

[0042] As used herein, "oxaalkyl" refers to an alkyl residue in which one or more carbons (and their associated hydrogens) are replaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecanyl, and the like. Alkoxy is a subset of oxaalkyl in which the carbon at the point of attachment is replaced by oxygen. The term oxaalkyl is understood in the art [see "Naming and Indexing of Chemical Substances for Chemical Abstracts," 196, published by the American Chemical Society, but not limited to 127(a)], i.e., it refers to a compound in which oxygen is bound to its adjacent atoms by a single bond (forming an ether bond); it does not refer to a double-bonded oxygen, as would be found in a carbonyl group. Similarly, thiaalkyl and azaalkyl refer to alkyl residues in which one or more carbons have been replaced by sulfur or nitrogen, respectively. Non-limiting examples include ethylaminoethyl and methylthiopropyl.

[0043] As used herein, "solvate" refers to a compound in the solid state in which molecules of a suitable solvent are incorporated into the crystal lattice along with the compound. Suitable solvents for therapeutic administration are physiologically tolerable at the dose administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. Typically, solvates are formed by dissolving the compound in a suitable solvent and separating the solvate by cooling or using an antisolvent. Solvates are typically dry or azeotropic under ambient conditions.

[0044] As used herein, the terms "subject" or "subject in need thereof" are used interchangeably herein. These terms refer to a patient who has been diagnosed with the underlying condition to be treated. The subject may currently be experiencing symptoms associated with the condition, or may have experienced symptoms in the past. Additionally, a "subject in need thereof" may be a patient at risk for developing a particular disease, or a patient in whom one or more of the physiological systems of a disease has been reported, even though a diagnosis of the disease may not have been made.

[0045] As used herein, the terms "treatment" or "treating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desirable results, including, but not limited to, therapeutic benefit. Therapeutic benefit includes eradication or amelioration of the underlying condition being treated; it also includes eradication or amelioration of one or more symptoms associated with the underlying condition, such that an improvement is observed in the patient, although the patient may still be affected by the underlying condition.

[0046] II.Compounds

[0047] In one aspect, compounds of Formula I are provided:

[0048]

[0049] in:

[0050] R 1 and R 2 together to form pyrrolidine or piperidine;

[0051] R 3 Selected from hydrogen and C1-C8 alkyl;

[0052] R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and

[0053] R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

[0054] Exemplary R 4 Groups include, but are not limited to, benzene, pyridine, pyrimidine, pyridazine, and pyrazine.

[0055] Exemplary R 5 Groups include, but are not limited to, pyrrolidine; pyrroline; pyrazolidine; pyrazoline; imidazoline; imidazoline; pyrrole; pyrazole; imidazole; triazole; isoxazole; oxazole; 1,2,3-oxadiazole; 1,3,4-oxadiazole; furazan; 1,2,4-oxadiazole; 1,2,3,4-oxatriazole; 1,2,3,5-oxatriazole; isothiazole; thiazole; 1,2,3-thiadiazole; 1,3,4-thiadiazole; 1,2,5-thiadiazole; 1,2,4-thiadiazole; 1,2,3,4-thiatriazole; 1,2,3,5-thiatriazole; furan and thiophene.

[0056] In R 4 In certain embodiments where R is a heterocyclic ring, 4 The heteroatom is not directly adjacent to the amide carbon or R5 Combined. Similarly, in R 5 In certain embodiments where R is a heterocyclic ring, 5 The heteroatom does not directly contact R 4 combination.

[0057] In a more specific embodiment, there is provided a compound of Formula Ia':

[0058]

[0059] where R 3 、R 4 and R 5 As defined above for Formula I.

[0060] In one embodiment, R 3 In another embodiment, R 4 is benzene, which is optionally substituted as described above. In another embodiment, R 4 is pyrimidine, which is optionally substituted as described above. In yet another embodiment, R 5 is pyrazole, which is optionally substituted as described above. In a more specific embodiment, R 3 is methyl; R 4 is benzene or pyrimidine, optionally substituted as described above; and R 5 is a pyrazole, optionally substituted as described above. The compounds may have the R- or S-configuration at the chiral center (*).

[0061] In another more specific embodiment, there is provided a compound of Formula Ia":

[0062]

[0063] where R 3 、R 4 and R 5 As defined above for Formula I.

[0064] In one embodiment, R 3 In another embodiment, R 4 is benzene, which is optionally substituted as described above. In another embodiment, R 4 is pyrimidine, which is optionally substituted as described above. In yet another embodiment, R 5 is pyrazole, which is optionally substituted as described above. In a more specific embodiment, R 3 is methyl; R 4 is benzene or pyrimidine, optionally substituted as described above; and R 5is a pyrazole, optionally substituted as described above. The compounds may have the R- or S-configuration at the chiral center (*).

[0065] In another embodiment, a compound of Formula Ib is provided:

[0066]

[0067] where R 1 、R 2 、R 3 、R 5 and R 7 As defined above for Formula I; and

[0068] Each X is independently selected from CH and N.

[0069] In a more specific embodiment, there is provided a compound of Formula Ib':

[0070]

[0071] where R 3 、R 5 and R 7 As defined above for Formula I; and

[0072] Each X is independently selected from CH and N.

[0073] In a particular embodiment, R 3 In another specific embodiment, at least one X is N. In yet another specific embodiment, all X are C. In yet another specific embodiment, R 5 is pyrazole, which is optionally substituted as described above. In a more specific embodiment, R 3 is methyl, at least one X is N, and R 5 is pyrazole, which is optionally substituted as described above. In another more specific embodiment, R 3 is methyl, all X are C, and R 5 is a pyrazole, optionally substituted as described above. The compounds may be in the R- or S-configuration at the chiral center (*).

[0074] In another more specific embodiment, there is provided a compound of Formula Ib":

[0075]

[0076] where R 3 、R 5 and R 7 As defined above for Formula I; and

[0077] Each X is independently selected from CH and N.

[0078] In a particular embodiment, R 3 In another specific embodiment, at least one X is N. In yet another specific embodiment, all X are C. In yet another specific embodiment, R 5 is pyrazole, which is optionally substituted as described above. In a more specific embodiment, R 3 is methyl, at least one X is N, and R 5 is pyrazole, which is optionally substituted as described above. In another more specific embodiment, R 3 is methyl, all X are C, and R 5 is a pyrazole, optionally substituted as described above. The compounds may be in the R- or S-configuration at the chiral center (*).

[0079] In another more specific embodiment, there is provided a compound of Formula Ic:

[0080]

[0081] where R 1 、R 2 、R 3 、R 4 and R 6 As defined above for Formula I;

[0082] Each Z is independently selected from CH and N; and

[0083] Y is selected from NH and CH2.

[0084] In a more specific embodiment, there is provided a compound of Formula Ic':

[0085]

[0086] where R 3 、R 4 and R 6 As defined above for Formula I;

[0087] Each Z is independently selected from CH and N; and

[0088] Y is selected from NH and CH2.

[0089] In a particular embodiment, R 3 In another specific embodiment, at least one Z is N and Y is NH. In yet another specific embodiment, R 4 is benzene or pyrimidine, which is optionally substituted as described above. In a more specific embodiment, R 3is methyl, at least one Z is N, Y is NH, and R 4 is benzene or pyrimidine, optionally substituted as described above. The compounds may be in the R- or S-configuration at the chiral center (*).

[0090] In another more specific embodiment, there is provided a compound of Formula Ic":

[0091]

[0092] where R 3 、R 4 and R 6 As defined above for Formula I;

[0093] Each Z is independently selected from CH and N; and

[0094] Y is selected from NH and CH2.

[0095] In a particular embodiment, R 3 In another specific embodiment, at least one Z is N and Y is NH. In yet another specific embodiment, R 4 is benzene or pyrimidine, which is optionally substituted as described above. In a more specific embodiment, R 3 is methyl, at least one X is N, Y is NH, and R 4 is benzene or pyrimidine, optionally substituted as described above. The compounds may be in the R- or S-configuration at the chiral center (*).

[0096] In another embodiment, a compound of Formula Id is provided:

[0097]

[0098] where R 1 、R 2 、R 3 、R 6 and R 7 As defined above for Formula I;

[0099] Each X is independently selected from CH and N;

[0100] Each Z is independently selected from CH and N; and

[0101] Y is selected from CH2 and NH.

[0102] In another specific embodiment, there is provided a compound of Formula Id':

[0103]

[0104] where R 3 、R6 and R 7 As defined above for Formula I;

[0105] Each X is independently selected from CH and N;

[0106] Each Z is independently selected from CH and N; and

[0107] Y is selected from CH2 and NH.

[0108] In a particular embodiment, R 3 In another specific embodiment, at least one Z is N and Y is NH. In another specific embodiment, R 3 is methyl, at least one X is N, at least one Z is N, and Y is NH. In another specific embodiment, R 3 is methyl, all X's are C, at least one Z is N, and Y is NH. The compounds may be in the R- or S-configuration at the chiral center (*).

[0109] In another specific embodiment, a compound of Formula Id" is provided:

[0110]

[0111] where R 3 、R 6 and R 7 As defined above for Formula I;

[0112] Each X is independently selected from CH and N;

[0113] Each Z is independently selected from CH and N; and

[0114] Y is selected from CH and NH.

[0115] In a particular embodiment, R 3 In another specific embodiment, at least one Z is N and Y is NH. In another specific embodiment, R 3 is methyl, at least one X is N, at least one Z is N, and Y is NH. In another specific embodiment, R 3 is methyl, all X's are C, at least one Z is N, and Y is NH. The compounds may be in the R- or S-configuration at the chiral center (*).

[0116] As described above, the compounds described herein have a chiral center (*) and can be in R- or S- configuration. In one embodiment, the compound has the R- configuration, for example, Formula Ia' is:

[0117]

[0118] In another embodiment, the compound has the S-configuration, for example, Formula Ia" is:

[0119]

[0120] In another aspect, provided are compounds of Formula II:

[0121]

[0122] in:

[0123] R 8 Selected from hydrogen and C1-C8 alkyl;

[0124] R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and

[0125] R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

[0126] Exemplary R 4 Groups include, but are not limited to, benzene, pyridine, pyrimidine, pyridazine, and pyrazine.

[0127] Exemplary R 5 Groups include, but are not limited to, pyrrolidine; pyrroline; pyrazolidine; pyrazoline; imidazoline; imidazoline; pyrrole; pyrazole; imidazole; triazole; isoxazole; oxazole; 1,2,3-oxadiazole; 1,3,4-oxadiazole; furazan; 1,2,4-oxadiazole; 1,2,3,4-oxatriazole; 1,2,3,5-oxatriazole; isothiazole; thiazole; 1,2,3-thiadiazole; 1,3,4-thiadiazole; 1,2,5-thiadiazole; 1,2,4-thiadiazole; 1,2,3,4-thiatriazole; 1,2,3,5-thiatriazole; furan and thiophene.

[0128] In R 4 In certain embodiments where R is a heterocyclic ring, 4 The heteroatom is not directly adjacent to the amide carbon or R 5 Combined. Similarly, in R 5 In certain embodiments where R is a heterocyclic ring, 5 The heteroatom does not directly contact R 4 combination.

[0129] In one embodiment, a compound of Formula IIa is provided:

[0130]

[0131] where R 5 、R 7 and R 8 As defined above for Formula II; and

[0132] Each X is independently selected from CH and N.

[0133] In one embodiment, R 8 In another embodiment, at least one X is N. In yet another embodiment, all X are C. In yet another embodiment, R 5 is pyrazole, which is optionally substituted as described above. In a more specific embodiment, R 8 is methyl, at least one X is N, and R 5 is pyrazole, which is optionally substituted as described above. In another more specific embodiment, R 8 is methyl, all X's are C, and R5 is pyrazole, which is optionally substituted as described above.

[0134] In another specific embodiment, a compound of Formula lib is provided:

[0135]

[0136] where R 4 、R 6 and R 8 As defined above for Formula II;

[0137] Each Z is independently selected from CH and N; and

[0138] Y is selected from NH and CH2.

[0139] In one embodiment, R 8 In another embodiment, at least one Z is N. In yet another embodiment, R 4 is benzene or pyrimidine, which is optionally substituted as described above. In a particular embodiment, R 8 is methyl, at least one Z is N, Y is NH, and R 4 is benzene or pyrimidine, optionally substituted as described above.

[0140] In another embodiment, a compound of Formula IIc is provided:

[0141]

[0142] where R 6 、R 7 and R 8 As defined above for Formula II;

[0143] Each X is independently selected from CH and N;

[0144] Each Z is independently selected from CH and N; and

[0145] Y is selected from CH2 and NH.

[0146] In one embodiment, R 8 In another embodiment, at least one Z is N and Y is NH. In a specific embodiment, R 8 is methyl, at least one X is N, at least one Z is N, and Y is NH. In another specific embodiment, R 8 is methyl, all X's are C, at least one Z is N, and Y is NH. The compounds may be in the R- or S-configuration at the chiral center (*).

[0147] In another aspect, the compound of the present invention is selected from the group consisting of:

[0148]

[0149] As used herein, "compound" is intended to include salts of the compound unless expressly further limited. Thus, for example, the expression "a compound of Formula I" as shown above would include the following salts:

[0150]

[0151] Wherein X is any counterion. In a specific embodiment, the term "compound of Formula I" refers to the compound or a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base (including inorganic acids and bases and organic acids and bases). When the compounds of the present invention are basic (as they generally are), salts can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic acids and organic acids). Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid (besylate), benzoic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, ethanedisulfonic acid, ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalenesulfonic acid, nitric acid, oleic acid, pamoic acid, pantothenic acid, phosphoric acid, pivalic acid, polygalacturonic acid, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, teoclatic acid, p-toluenesulfonic acid, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium cations and carboxylate, sulfonate, and phosphonate anions attached to alkyl groups having 1 to 20 carbon atoms.

[0152] Compounds with R stereochemistry generally exhibit greater activity than the corresponding S enantiomer. In other embodiments, the compound has S stereochemistry at the chiral center.

[0153] Furthermore, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a 13 C or 14 Compounds having the structures of the present invention other than C-enriched carbon substitutions are within the scope of the present invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0154] III. Pharmaceutical Compositions

[0155] The present invention also provides pharmaceutical compositions comprising at least one compound described herein, including pharmaceutically acceptable salts and solvates thereof.

[0156] Pharmaceutical compositions comprise at least one compound described herein and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, one or more binders, fillers, buffers, stabilizers, surfactants, wetting agents, lubricants, diluents, disintegrants, viscosity-increasing or viscosity-reducing agents, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, taste-masking agents, fragrances, flavorings, diluents, polishing agents, polymer matrix systems, plasticizers, and other known additives that provide an elegant presentation of the drug or aid in the manufacture of a drug or drug product comprising the composition of the invention. Examples of carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.

[0157] Non-limiting examples of excipients include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth gum, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone, methylcellulose, pregelatinized starch, hydroxypropyl methylcellulose (e.g., Nos. 2208, 2906, 2910), hydroxypropyl cellulose, titanium dioxide, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, silicic acid, sorbitol, starch, pregelatinized starch Agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polycrilin potassium, sodium starch glycolate, potato or tapioca starch, other starch, pregelatinized starch, other starch, clay, other alginates, other celluloses, gums, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut, cottonseed, sunflower, sesame, olive, corn, and soybean), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid silica (manufactured by W.R. Grace of Baltimore, MD). Co.), solidified aerosols of synthetic silica (sold by Degussa Co. of Plano, TX), CAB-O-SIL (a fumed silica product sold by Cabot Co. of Boston, MA), colorants, and mixtures thereof.

[0158] The pharmaceutical compositions may optionally include one or more additional therapeutic agents.

[0159] Additional therapeutic agents include Bcl-2 inhibitors, cyclin-dependent kinase 4 and 6 (CDK 4 / 6) inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone demethylase inhibitors, mTOR inhibitors, mutant isocitrate dehydrogenase (IDH1 and IDH2) inhibitors, glucocorticoids, epigenetic modulators, and chemotherapeutic agents.

[0160] The standard of care for AML and ALL is currently chemotherapy using chemotherapeutic agents. Exemplary chemotherapeutic agents include, but are not limited to, daunorubicin, cytarabine, methotrexate, mitoxantrone, methotrexate, mafosamide, and vincristine.

[0161] Targeted therapeutics, such as those discussed below, can be used alone or in combination with chemotherapeutic agents.

[0162] Exemplary Bcl-2 inhibitors include, but are not limited to, oblimersen, navitoclax, and venetoclax, for example.

[0163] Exemplary cyclin-dependent kinase 4 and 6 (CDK 4 / 6) inhibitors include, but are not limited to, palbociclib, ribociclib, and abecilib.

[0164] Epigenetic modulators include, but are not limited to, menin-histone methyltransferase MLL (i.e., menin-MLL) inhibitors, FLT3 inhibitors, P-TEFb inhibitors, histone methyltransferase inhibitors (e.g., DOT1L and EZH2 inhibitors), bromodomain and extra-terminal domain (BET) inhibitors, and dihydroorotate dehydrogenase (DHODH) inhibitors.

[0165] Exemplary FLT3 inhibitors include, but are not limited to, sorafenib, lestaurinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, and ponatinib.

[0166] Combinations of epigenetic modulators, such as menin-MLL inhibitors and FLT3 inhibitors, are also being considered, as these have shown enhanced apoptosis induction in AML models.

[0167] In one embodiment, the additional therapeutic agent comprises a combination of at least one Bcl-2 inhibitor and at least one FLT3 inhibitor.

[0168] Exemplary DNA methyltransferase inhibitors include, but are not limited to, azacitidine and decitabine.

[0169] Exemplary HDAC inhibitors include, but are not limited to, panobinostat and vorinostat.

[0170] Exemplary mTOR inhibitors include, but are not limited to, everolimus.

[0171] Exemplary glucocorticoids include, but are not limited to, dexamethasone and prednisolone.

[0172] Exemplary mutant isocitrate dehydrogenase inhibitors include, but are not limited to, ivosidenib (IDH1) and ensidipine (IDH2).

[0173] In one embodiment, the additional therapeutic agent comprises a combination of at least one isocitrate dehydrogenase inhibitor and at least one CDK 4 / 6 inhibitor.

[0174] IV. How to use

[0175] The present invention also relates to methods of inhibiting oncogene expression in a cell using at least one compound described herein or pharmaceutical composition described herein. In one embodiment, the method of inhibiting oncogene expression in a cell comprises exposing the cell to at least one compound described herein. The present invention also relates to methods of treating acute leukemia using at least one compound described herein or pharmaceutical composition described herein. In one embodiment, the method of treating acute leukemia comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound described herein.

[0176] Acute leukemia is a rapidly progressive leukemia characterized by the replacement of normal bone marrow with cloned blasts resulting from the malignant transformation of hematopoietic cells. Acute leukemias include acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). ALL often involves the CNS, while acute promonocytic leukemia involves the gums, and AML involves localized collections (granulocytic sarcomas or chloromas) at any site.

[0177] In one embodiment, the acute leukemia is ALL. ALL is the most common malignancy in children, with a peak incidence between 3 and 5 years of age. It also occurs in adolescents, with a second, lower peak in adults. Typical treatment emphasizes early introduction of an intensive multidrug regimen that may include prednisone, vincristine, anthracyclines, or asparaginase. Other drugs and combinations include cytarabine and etoposide, as well as cyclophosphamide. Relapses usually occur in the bone marrow but may also occur in the CNS or testes, alone or in combination with the bone marrow. Although a second remission can be induced in many children, subsequent remissions are often short-lived.

[0178] In another embodiment, the acute leukemia is AML. The incidence of AML increases with age; it is the most common acute leukemia in adults. AML may be associated with chemotherapy or radiation (secondary AML). The remission induction rate is lower than ALL, and long-term disease-free survival is reported in only 20 to 40% of patients. The biggest difference in treatment from ALL is that AML responds to fewer drugs. The basic induction regimen includes cytarabine; and daunorubicin or idarubicin. Some regimens include 6-thioguanine, etoposide, vincristine and prednisone. The clinical aspects of AML are reviewed by C. Schiffer and R.M. Stone in Cancer Medicine, edited by David W. Kufe et al., 6th edition, BC Decker, 2003.

[0179] The French, American and British (FAB) classification system has been developed for the diagnosis and classification of acute myeloid leukemia. The diagnosis of acute myeloid leukemia requires that myeloblasts constitute 30% (or 20% based on the most recent World Health Organization (WHO) classification system) or more of the bone marrow cells or circulating white blood cells. The hematological characteristics of the disease define the various subtypes described below. The FAB nomenclature (M1 to M7) classifies the subtypes of acute myeloid leukemia according to the normal bone marrow elements to which the primitive cells are most similar. The following list includes the FAB classification system as well as other categories recognized by the WHO.

[0180] Minimally differentiated acute myeloid leukemia (MO)

[0181] Without mature acute myeloid leukemia (M1)

[0182] Acute myeloid leukemia with maturity (M2)

[0183] Mature acute myeloid leukemia with t(8;21)

[0184] Acute promyelocytic leukemia (M3)

[0185] Hypergranular type

[0186] Micro-particle type

[0187] Acute myelomonocytic leukemia (M4)

[0188] Acute myelomonocytic leukemia with bone marrow eosinophilia (M4E0)

[0189] Acute monocytic leukemia (M5)

[0190] Acute promonocytic leukemia (M5a)

[0191] Acute monocytic leukemia with maturation (M5b)

[0192] Erythroid / myeloid leukemia (M6a)

[0193] Pure erythroid malignancy (M6b)

[0194] Acute megakaryoblastic leukemia (M7)

[0195] Acute megakaryoblastic leukemia associated with t(1;22)

[0196] Acute basophilic leukemia

[0197] Acute myeloid fibrosis (acute myelodysplasia with myelofibrosis)

[0198] Acute leukemias and transient myeloproliferative disorders in Down syndrome

[0199] Low-cell acute myeloid leukemia

[0200] Myeloid sarcoma

[0201] In one embodiment, the method of treating the above-listed AML subtypes comprises administering to a subject in need thereof a therapeutically effective amount of at least one compound described herein.

[0202] At least one compound used in the present method may be provided in the form of a pharmaceutical composition as described above.

[0203] Routes of administration include enteral, eg, oral; and parenteral, eg, intravenous, intraarterial, intramuscular, intranasal, rectal, intraperitoneal, subcutaneous, and topical routes.

[0204] For parenteral administration, the active compound can be mixed with a suitable carrier or diluent, such as water, oil (particularly vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol or glycols, such as propylene glycol or polyethylene glycol. The solution for parenteral administration preferably contains a water-soluble salt of the active agent. Stabilizers, antioxidants and preservatives can also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and salts thereof and sodium EDTA. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben and chlorobutanol. Compositions for parenteral administration can take the form of aqueous or non-aqueous solutions, dispersions, suspensions or emulsions.

[0205] For oral administration, the active compound can be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, the active compound can be combined with at least one excipient, such as a filler, binder, humectant, disintegrant, solution retardant, absorption enhancer, wetting agent, absorbent or lubricant.

[0206] The specific dosage of the active compound used in the compositions and methods of the present invention to achieve therapeutic benefit will, of course, be determined by the individual patient's particular circumstances, including the patient's size, weight, age, and sex, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration.

[0207] For the compounds described herein, preferred daily doses are, for example, in the range of about 1 to about 10,000 mg, more preferably about 5 to about 5,000 mg, still more preferably about 10 to about 3,000 mg, and most preferably about 50 to about 1,000 mg. In certain embodiments, preferred daily doses are in the range of about 50 mg to about 4,000 mg, about 100 mg to about 3,000 mg, about 500 to about 2,000 mg, or about 750 mg to about 1,500 mg. In other embodiments, preferred daily doses are in the range of 2,000 mg to about 10,000 mg, about 3,000 to about 9,000 mg, about 4,000 mg to about 8,000 mg, or about 4,500 to about 7,500 mg.

[0208] One to four doses may be administered daily, e.g., once daily, as needed to provide therapeutic benefit. In certain embodiments, the therapeutic compounds of the invention are administered intravenously as a one-time dose or as part of a planned dosing regimen that may be distributed over several days, weeks, or months. As needed, the compounds of the invention may also be administered by regular injection to obtain therapeutic benefit.

[0209] The methods described herein may further include administering additional therapeutic agents, such as Bcl-2 inhibitors, cyclin-dependent kinases 4 and 6 (CDK 4 / 6) inhibitors, DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, histone demethylase inhibitors, mTOR inhibitors, mutant isocitrate dehydrogenase (IDH1 and IDH2) inhibitors, glucocorticoids, epigenetic regulators, and chemotherapeutic agents. Additional therapeutic agents can be administered simultaneously or sequentially with the compounds described herein. In some embodiments, the administration of the compounds described herein and additional therapeutic agents can produce a synergistic effect. Example

[0210] The following compounds have been prepared, isolated and characterized using the methods disclosed herein. They illustrate part of the scope of the invention and are not meant to limit the scope of the invention.

[0211] The compounds of the present invention are prepared by methods well known in the art of synthetic organic chemistry. During a synthetic sequence, it may sometimes be necessary or desirable to protect sensitive or reactive groups on any of the molecules involved. This is accomplished using conventional protecting groups, such as those described in TW Greene and PGM Wuts Greene's Protective Groups in Organic Synthesis, Fourth Edition, John Wiley and Sons, 2006. The protecting groups are removed at a convenient subsequent stage using methods well known in the art.

[0212] Unless otherwise stated, all reactions were carried out under a dry nitrogen atmosphere. The reaction temperatures shown refer to the reaction bath, while room temperature (rt) is considered to be 25°C. Commercial grade reagents and anhydrous solvents were used as received from the suppliers, and no further purification or drying was attempted on these components. Solvent removal under reduced pressure was accomplished with a Buchi rotary evaporator at approximately 28 mm Hg pressure using a Teflon-connected KNf vacuum pump. Flash column chromatography was performed using a Teledyne Isco CombiFlash Companion device with a RediSep Rf silica gel column. Proton NMR spectra were obtained on 300 MHz and 400 MHz Bruker nuclear magnetic resonance spectrometers. Chemical shifts (δ) are reported in parts per million (ppm), and coupling constant (J) values are given in Hz with the following spectral pattern labels: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet; m, multiplet; brs, broad singlet. Tetramethylsilane was used as an internal reference. Mass spectrometry analysis was performed on an Agilent 1200 system using positive and negative mode electron spray ionization (ESI). High pressure liquid chromatography (HPLC) purity analysis was performed using a Varian Pro Star HPLC system with a binary solvent system A and B using a gradient elution [A, H2O with 0.0284% NH4OAc and 0.0116% acetic acid; B, CH3CN] and a flow rate of 1 mL / min using a PDA scan for UV detection.

[0213] abbreviation

[0214] BINAP 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl

[0215] CH3CN acetonitrile

[0216] Cs2CO3 cesium carbonate

[0217] DCM dichloromethane

[0218] DIPEA Diisopropylethylamine

[0219] DMF dimethylformamide

[0220] DMP Dess-Martin Periodinane

[0221] EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0222] EA Ethyl acetate

[0223] EtOH

[0224] HCl

[0225] K2CO3 potassium carbonate

[0226] K3PO4 potassium phosphate

[0227] LiHMDS Lithium bis(trimethylsilyl)amide

[0228] MsCl methanesulfonyl chloride

[0229] MeOH methanol

[0230] NaHCO3 sodium bicarbonate

[0231] NaOAc sodium acetate

[0232] NH4Cl ammonium chloride

[0233] Pd(dppf)Cl2.CH2Cl2-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complexed with dichloromethane

[0234] Pd(dppf)Cl2 -bis(diphenylphosphino)ferrocene]dichloropalladium(II)

[0235] PE petroleum ether

[0236] RT Room temperature

[0237] SEMCl 2-(Trimethylsilyl)ethoxymethyl chloride

[0238] TFA trifluoroacetic acid

[0239] THF Tetrahydrofuran

[0240] THP Tetrahydropyran

[0241] TEA triethylamine

[0242] Intermediate 1: (R)-2-(1-methylpyrrolidin-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-c]pyridin-6-amine

[0243]

[0244] In a 20-L 4-neck round-bottom flask purged and maintained with a nitrogen inert atmosphere, 2-chloro-5-iodopyridin-4-amine (490.0 g, 1.90 mol, 1.00 equivalent), TEA (974 g, 9.60 mol, 5.00 equivalent) and DCM (12.30-L) were placed. Subsequently, at 0-5° C., a solution of MsCl (882 g, 7.70 mol, 4.00 equivalent) in DCM (7.4 L) was added dropwise under stirring. The resulting solution was stirred at 0-10° C. for 6 hours. The pH value of the solution was adjusted to 7-8 with NaHCO (1 mol / L). The resulting solution was extracted with 3×5 L of dichloromethane, and the organic layers were combined, dried over anhydrous sodium sulfate and concentrated. This gave 935 g (94.6%) of N-(2-chloro-5-iodopyridin-4-yl)-N-methylsulfonylmethanesulfonamide as a yellow oil.

[0245] LC-MS: (ES, m / z): [M+1] + =411.

[0246]

[0247] In a 10-L 4-neck round-bottom flask purged and maintained with a nitrogen inert atmosphere, N-(2-chloro-5-iodopyridin-4-yl)-N-methylsulfonyl methanesulfonamide (935.0 g, 2.28 mol, 1.00 equivalent), THF (4.70 L), H O (4.70 L) and NaOH (455 g, 11.4 mol, 5.00 equivalents) were placed. The resulting solution was stirred at room temperature for 16 hours. The resulting mixture was concentrated. The pH of the solution was adjusted to 3-4 with citric acid (1 mol / L). The solid was collected by filtration. This produced 438 g (57.9%) of N-(2-chloro-5-iodopyridin-4-yl) methanesulfonamide as a white solid.

[0248] LC-MS (ES, m / z): [M+1] + =333.

[0249]

[0250] In a 10-L 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere were placed tert-butyl (2R)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (530.00 g, 2.6 mol, 1.00 equiv), DCM (5.30 L), and DMP (1340 g, 3.16 mol, 1.20 equiv). The resulting solution was stirred at room temperature for 6 hours. The resulting solution was diluted with 5.3 L of H₂O. The resulting solution was extracted with 3×10 L of ethyl acetate and the organic layers were combined. The resulting mixture was washed with 3×5 L of Na₂S₂O₃ (aqueous) and 3×5 L of NaHCO₃ (aqueous). The resulting mixture was washed with 3×10 L of brine. The mixture was dried over anhydrous sodium sulfate and concentrated. This yielded 415 g (79.09%) of tert-butyl (2R)-2-formylpyrrolidine-1-carboxylate as a yellow oil.

[0251] LC-MS: (ES, m / z): [M+1] + =200.

[0252]

[0253] In a 10-L, 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere were placed KCO (348 g, 2.5 mol, 1.20 equiv), methanol (4.15 L), tert-butyl (2R)-2-formylpyrrolidine-1-carboxylate (415.00 g, 2.09 mol, 1.00 equiv), and dimethyl (1-diazo-2-oxopropyl)phosphonate (600 g, 3.1 mol, 1.50 equiv). The reaction mixture was stirred at room temperature for 16 hours and diluted with 4 L of H2O. The resulting solution was extracted with 3 x 4 L of petroleum ether, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated. This yielded 297 g (73.03%) of tert-butyl (2R)-2-ethynylpyrrolidine-1-carboxylate as a yellow oil.

[0254] LC-MS: (ES, m / z): [M+1] + =196.

[0255]

[0256] In a 10-L 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere were placed N-(2-chloro-5-iodopyridin-4-yl)methanesulfonamide (438.00 g, 1.32 mol, 1.00 equiv), TEA (533 g, 5.27 mol, 4.00 equiv), dimethylformamide (4.40 L), tert-butyl (2R)-2-ethynylpyrrolidine-1-carboxylate (283 g, 1.45 mol, 1.10 equiv), Pd(PPh 3 ) 2 Cl 2 (46 g, 0.066 mol, 0.05 equiv) and CuI (25 g, 0.13 mol, 0.10 equiv). The reaction mixture was stirred at 55° C. for 6 hours and diluted with 4.4 L of H 2 O. The resulting solution was extracted with 3 x 4.4 L of ethyl acetate, and the organic layers were combined and washed with 3 x 4.4 L of brine, dried over anhydrous sodium sulfate, and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:5). This yielded 363 g (68.9%) of tert-butyl (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]pyrrolidine-1-carboxylate as a white solid.

[0257] LC-MS: (ES, m / z): [M+1] + =400.

[0258]

[0259] In a 10-L 4-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere, (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]pyrrolidine-1-carboxylic acid tert-butyl ester (363.00 g, 0.91 mol, 1.00 equiv), MeOH (2.50 L), HO (1.10 L) and NaOH (109 g, 2.72 mol, 3.00 equiv). The reaction mixture was stirred at room temperature for 16 hours and concentrated. The solid was collected by filtration. This produced 259 g (88.67%) of (2R)-2-[6-chloro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyrrolidine-1-carboxylic acid tert-butyl ester as a white solid.

[0260] LC-MS: (ES, m / z): [M+1] + =322.

[0261]

[0262] In a 5-L 4-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere, (2R)-2-[6-chloro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyrrolidine-1-carboxylic acid tert-butyl ester (259.0 g, 0.80 mol, 1.00 equiv), Cs2CO3 (787 g, 2.4 mol, 3.00 equiv) and DMF (2.60 L) were placed. Subsequently, SEMCl (161 g, 0.97 mol, 1.20 equiv) was added dropwise at 0-5 ° C. with stirring. The reaction mixture was stirred at room temperature for 6 hours. The resulting solution was diluted with 2.6 L H2O and extracted with 3×2.6 L ethyl acetate, and the organic layers were combined and washed with 3×2 L brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The solid was collected by filtration. This resulted in 248 g (68.2%) of tert-butyl (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)pyrrolidine-1-carboxylate as a white solid.

[0263] LC-MS: (ES, m / z): [M+1] + =452.

[0264]

[0265] In a 10-L 4-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere, tert-butyl (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)pyrrolidine-1-carboxylate (248.00 g, 0.55 mol, 1.00 equiv), MeOH (2.40 L), and HCl (1.5 M) in MeOH (1.20 L). The reaction mixture was stirred at room temperature for 12 hours and concentrated. The resulting solution was diluted with 2.5 L of H2O. The pH of the solution was adjusted to 7-8 with NaHCO3 (1 mol / L) and extracted with 3 x 2.5 L of dichloromethane, then the organic layers were combined and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:2). This resulted in 177 g (91.7%) of (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)pyrrolidine as a white solid.

[0266] LC-MS (ES, m / z): [M+1] + =352.

[0267]

[0268] In a 10-L 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)pyrrolidine (177.00 g, 0.5 mol, 1.00 equiv), DCM (3.50 L), MeOH (1.77 L), paraformaldehyde (453 g, 5 mol, 10.00 equiv) and NaBH(OAc)3 (640 g, 3 mol, 6.00 equiv) were placed. The reaction mixture was stirred at room temperature for 12 hours. The pH of the solution was adjusted to 8-9 with NaHCO3 (1 mol / L) and the solid was filtered off. The filtrate was extracted with 3×1.7 L of dichloromethane, and the organic layers were combined and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:3). This resulted in 129 g (70.1%) of (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)-1-methylpyrrolidine as a yellow oil.

[0269] LC-MS: (ES, m / z): [M+1] + =366.

[0270]

[0271] In a 5 L 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)-1-methylpyrrolidine (129.00 g, 0.35 mol, 1.00 equiv), toluene (2.60 L), BINAP (22 g, 0.035 mol, 0.10 equiv), t-BuONa (101 g, 1.06 mol, 3.00 equiv), Pd2(dba)3 (16 g, 0.017 mol, 0.05 equiv) and diphenylformimine (192 g, 1.06 mol, 3.00 equiv) were placed. The reaction mixture was stirred at 110° C. for 16 h and diluted with 2.6 L of EA. The organic layer was collected and washed with 3 x 1 L of brine, dried over anhydrous sodium sulfate, and concentrated. The residue was applied to a silica gel column and eluted with THF / PE (1:3). This yielded 131 g (72.8%) of N-[2-[(2R)-1-methylpyrrolidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-yl]-1,1-diphenylmethanimine as a yellow oil.

[0272]

[0273] In a 10-L 4-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, N-[2-[(2R)-1-methylpyrrolidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-yl]-1,1-diphenylcarbamate (131.00 g, 0.26 mol, 1.00 equiv), THF (6.50 L), H₂O (1.10 L), and HCl (0.5 M) (88 g, 1.28 mol, 5.00 equiv) were placed. The resulting solution was stirred at room temperature for 12 hours. The resulting solution was diluted with 2.6 L of H₂O. The resulting solution was extracted with 3 x 1 L of dichloromethane, and the aqueous layers were combined. The pH of the solution was adjusted to 8-9 with NaHCO₃ (1 mol / L). The resulting solution was extracted with 3 x 2 L of dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was applied to a silica gel column using THF / PE (1:1). This yielded 53.1 g (59.7%) of 2-[(2R)-1-methylpyrrolidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-amine as a brown oil.

[0274] LC-MS: (ES, m / z): [M+1] + =347.

[0275] 1 H-NMR: (300MHz, CD3OD , ppm): δ8.18(d,J=1.0Hz,1H),6.68(d,J=1.0Hz,1H),6.46(s,1H),5.61-5.48(m,2H),3.61-3.49(m,3H),3.23 (t,J=7.9Hz,1H),2.46-2.35(m,2H),2.33(s,3H),2.02-1.84(m,3H),0.90(dd,J=8.8,7.4Hz,2H),0.0(s,9H)

[0276] SFC: Chiralpack IC-3 50×3.0 mm, 3 μm; IPA-hexane (Hex) = 1:1 (20 mM NH3); RT 1.27 (>98% ee)

[0277] Intermediate 2: (R)-2-(1-methylpiperidin-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-c]pyridin-6-amine

[0278]

[0279] In a 50-mL 3-necked round-bottom flask were placed tert-butyl (2R)-2-(hydroxymethyl)piperidine-1-carboxylate (5.00 g, 23.22 mmol, 1.00 equivalent), DMP (19.70 g, 46.45 mmol, 2.00 equivalent) and DCM (20.00 mL). The resulting solution was stirred at room temperature for 3 hours. The reaction was then quenched by the addition of Na2S2O3 (aqueous). The resulting solution was extracted with 2 x 50 mL of dichloromethane, and the organic layers were combined and concentrated. This produced 4.0 g (80.8%) of tert-butyl (2R)-2-formylpiperidine-1-carboxylate as a brown oil.

[0280]

[0281] In a 50-mL 3-necked round-bottom flask, (2R)-2-formylpiperidine-1-tert-butyl carboxylate (4.00 g, 18.755 mmol, 1.00 equivalent), K CO (3.11 g, 22.506 mmol, 1.20 equivalent) and MeOH (12.00 mL) were placed. Subsequently, at 0° C., a solution of dimethyl (1-diazo-2-oxopropyl)phosphonate (5.40 g, 0.028 mmol, 1.50 equivalent) in MeOH (6 mL) was added dropwise under stirring. The resulting solution was stirred at room temperature for 6 hours. The resulting solution was extracted with 2×50 mL petroleum ether, and the organic layers were combined and concentrated. This produced 2 g (51%) of (2R)-2-ethynylpiperidine-1-tert-butyl carboxylate as a yellow oil.

[0282] GC-MS: (ES, m / z): [M-81]=128.

[0283]

[0284] In the 50-mL 3-neck round-bottom flask purged and maintained with nitrogen inert nitrogen atmosphere, N-(2-chloro-5-iodopyridin-4-yl) methanesulfonamide (700.0mg, 2.1mmol, 1.00 equivalent), (2R)-2-ethynylpiperidine-1-tert-butyl formate (881.11mg, 4.210mmol, 2.00 equivalent), CuI (40.09mg, 0.211mmol, 0.10 equivalent), TEA (852.02mg, 8.420mmol, 4.00 equivalent), DMF (10.00mL) and Pd(PPh ) Cl (295.5mg, 0.421mmol, 0.20 equivalent). The resulting solution was stirred at 55°C for 2 hours. Solid was filtered out, and the resulting mixture was concentrated. Residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:10). This resulted in 540 mg (62%) of tert-butyl (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]piperidine-1-carboxylate as a brown solid.

[0285] LC-MS: (ES, m / z): [M+H]=414

[0286]

[0287] In a 50-mL round-bottom flask, (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]piperidine-1-carboxylic acid tert-butyl ester (430.00 mg) and HCl (gas) (10.00 mL) in ethyl acetate were placed. The resulting solution was stirred at room temperature for 6 hours. The resulting mixture was concentrated. This produced 380 mg of (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]piperidine hydrochloride as a brown solid.

[0288] LC-MS: (ES, m / z): [M+H-HCl]=314.

[0289]

[0290] In a 100-mL 3-necked round-bottom flask, (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]piperidine hydrochloride (380.0 mg, 1.089 mmol, 1.00 equiv), DCM (20.00 mL), MeOH (10.00 mL), paraformaldehyde (488.63 mg, 5.43 mmol, 5.00 equiv) and NaBH(OAc)3 (2299.37 mg, 10.85 mmol, 10.00 equiv) were placed. The resulting solution was stirred at room temperature for 12 hours. The reaction was then quenched by adding 20 mL of water. The resulting solution was extracted with 2×30 mL of dichloromethane, and the organic layers were combined and concentrated. The residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:1). This resulted in 201 mg (56.5%) of (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]-1-methylpiperidine as a white solid.

[0291] LC-MS: (ES, m / z): [M+H]=328.

[0292]

[0293] In a 50-mL round-bottom flask, (2R)-2-[6-chloro-1-methanesulfonylpyrrolo[3,2-c]pyridin-2-yl]-1-methylpiperidine (185.00 mg, 0.564 mmol, 1.00 equiv), NaOH (67.71 mg, 0.000 mmol, 3.00 equiv), H₂O (1.00 mL), and MeOH (5.00 mL) were placed. The resulting solution was stirred at room temperature for 2 hours. The resulting solution was extracted with 2×20 mL of ethyl acetate, and the organic layers were combined and concentrated. This yielded 120 mg (85.2%) of (2R)-2-[6-chloro-1H-pyrrolo[3,2-c]pyridin-2-yl]-1-methylpiperidine as a brown solid.

[0294] LC-MS: (ES, m / z): [M+H]=250.

[0295]

[0296] In a 50-mL 3-neck round-bottom flask, (2R)-2-[6-chloro-1H-pyrrolo[3,2-c]pyridin-2-yl]-1-methylpiperidine (120.0 mg, 0.480 mmol, 1.00 equivalent), Cs2CO3 (469.7 mg, 1.44 mmol, 3.00 equivalent), DMF (5.00 mL), SEM-Cl (120.16 mg, 0.720 mmol, 1.50 equivalent). The resulting solution was stirred at room temperature for 2 hours. The solid was filtered and the resulting solution was extracted with 2×20 mL of ethyl acetate. The organic layers were combined and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:2). This resulted in 85 mg (46.6%) of (2R)-2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-2-yl)-1-methylpiperidine as a light brown oil.

[0297] LC-MS: (ES, m / z): [M+H]=380.

[0298]

[0299] In an 8-mL sealed tube purged and maintained with an inert nitrogen atmosphere were placed 2-(6-chloro-1-[[2-(trimethylsilyl)ethoxy]methyl]-octahydropyrrolo[3,2-c]pyridin-2-yl)-1-methylpiperidine (80.00 mg, 0.206 mmol, 1.00 equiv), diphenylcarbamate (112.09 mg, 0.618 mmol, 3.00 equiv), t-BuONa (59.43 mg, 0.618 mmol, 3.00 equiv), toluene (3.00 mL), Pd2(dba)3.CHCl3 (23.71 mg, 0.041 mmol, 0.20 equiv), and BINAP (51.35 mg, 0.082 mmol, 0.40 equiv). The resulting solution was stirred at 100°C for 5 hours. The resulting mixture was concentrated. This resulted in 100 mg (crude) of N-[2-[(2R)-1-methylpiperidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-yl]-1,1-diphenylmethanimine as a brown oil.

[0300] LC-MS: (ES, m / z): [M+H]=525.

[0301]

[0302] In a 50-mL round-bottom flask, N-[2-[(2R)-1-methylpiperidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-yl]-1,1-diphenylcarbamidine (100.00 mg, 0.191 mmol, 1.00 equiv), THF (5.00 mL), and HCl (5.00 mL) were placed. The resulting solution was stirred at room temperature for 16 hours and extracted with 2 x 20 mL of ethyl acetate. The organic layers were then combined and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:1). This yielded 44 mg (34% over two steps) of 2-[(2R)-1-methylpiperidin-2-yl]-1-[[2-(trimethylsilyl)ethoxy]methyl]pyrrolo[3,2-c]pyridin-6-amine as a brown solid. The product was further purified by SFC using the following conditions (column: Lux 5 μm Amylose-1, 5×25 cm, 10 μm; mobile phase A: CO 2 , mobile phase B: IPA (0.5% 2 M NH 3 -MeOH); flow rate: 160 mL / min; gradient: isocratic 40% B; column temperature (° C.): 35; back pressure (bar): 100; wavelength: 220 nm; RT1 (min): 4.47; RT2 (min): 5.89; sample solvent: ACN; injection volume: 2 mL, and the major enantiomer was collected to obtain material with >98% ee.

[0303] LC-MS: (ES, m / z): [M+H]=361

[0304] 1 H-NMR (300MHz, methanol-d4, ppm): δ8.23-8.17(m,1H),6.68(s,1H),6.47(s,1H),3.56(t,J=8.2Hz,2H),3.08(d,J=11.9Hz,1H),2.24(d, J=14.1Hz,1H),2.15(s,3H),1.97-1.68(m,5H),1.48(d,J=10.5Hz,1H),1.17(d,J=6.2Hz,3H),0.91(t,J=8.1Hz,2H),-0.22(s,9H).

[0305] Acid 1: 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid

[0306]

[0307] In a 40-mL vial purged and maintained with an inert nitrogen atmosphere were placed methyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carboxylate (1.00 g, 3.801 mmol, 1.00 equiv), dioxane (20.00 mL), 4-bromo-1-(oxan-2-yl)pyrazole (0.97 g, 4.181 mmol, 1.1 equiv), Pd(dppf)Cl2 (0.28 g, 0.380 mmol, 0.1 equiv), and K3PO4 (2.42 g, 11.403 mmol, 3 equiv). The reaction mixture was stirred in a 100°C oil bath under a nitrogen atmosphere for 5 hours and concentrated under vacuum. The residue was diluted with 30 mL of H2O and extracted with 3×20 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1:1). This resulted in 800 mg (73.3%) of methyl 5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxylate as a yellow solid.

[0308] LC-MS: (ES, m / z): [M+H] + =288

[0309]

[0310] In a 50 mL round-bottom flask were placed methyl 5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxylate (800 mg, 2.784 mmol, 1.00 equiv), CH3OH (16 mL), H2O (5 mL), and sodium hydroxide (330 mg, 8.251 mmol, 2.96 equiv). The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The residue was diluted with 30 mL of H2O and extracted with 2×20 mL of ethyl acetate, and the aqueous layers were combined. The pH of the solution was adjusted to 3 with HCl (3 mol / L). The resulting solid was collected by filtration to yield 600 mg (78.85%) of 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid as a light yellow solid.

[0311] LC-MS: (ES, m / z): [M+H] + =274

[0312] Acid 2: 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]benzoic acid

[0313]

[0314] A solution of methyl 4-bromo-2H-pyrazole-3-carboxylate (3.0 g, 14.633 mmol, 1 eq) in DMF (30 mL) was treated with NaH (900 mg, 37.503 mmol, 2.56 eq) at 0 ° C under a nitrogen atmosphere for 10 minutes, followed by the dropwise addition of [2-(chloromethoxy)ethyl]trimethylsilane (3.7 g, 22.193 mmol, 1.52 eq) at 0 ° C. The mixture was stirred at RT for 4 hours. The reaction was quenched by the addition of water (20 mL) at 0 ° C. The resulting mixture was extracted with EA (50 mL × 3). The combined organic layers were washed with NaCl (aqueous) (50 mL × 3), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with EA / PE = 3 / 1 to give: Methyl 4-bromo-2-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole-3-carboxylate (3.5 g, 64.20%).

[0315] LC-MS (ES, m / z): [M+1] + =335.1,337.1

[0316]

[0317] To a solution of LiAlH4 (400 mg, 10.540 mmol, 2.94 equiv) in THF (10 mL, 123.428 mmol, 34.48 equiv) under a nitrogen atmosphere was added methyl 4-bromo-2-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole-3-carboxylate (1.2 g, 3.579 mmol, 1 equiv) in THF dropwise at -78 ° C. The mixture was stirred at -78 ° C to 0 ° C for 4 hours. The reaction was quenched by adding water / NaOH at -30 ° C and filtered, and the filter cake was washed with EA. The filtrate was extracted with EA (20 mL × 3). The combined organic layers were washed with NaCl (aqueous), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with EA / PE=1 / 2 to give (4-bromo-2-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-3-yl)methanol (270 mg, 22%) as a yellow oil.

[0318] LC-MS (ES, m / z): [M+1] + =306.9,308.9

[0319]

[0320] To a solution of (4-bromo-2-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-3-yl)methanol (250 mg, 0.814 mmol, 1 eq) in THF was added sodium hydride (60% in oil, NaH (25 mg, 1.042 mmol, 1.28 eq)) at 0°C. The mixture was stirred at 0°C for 15 minutes. CHI (200 mg, 1.409 mmol, 1.73 eq) was added and the mixture was warmed to RT and stirred for 1 hour. The reaction mixture was quenched with water and extracted with DCM (3×25 mL). The residue was purified by silica gel column chromatography eluting with EA / PE=1 / 4 to give 4-bromo-5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole (200 mg, 68.86%) as a yellow oil.

[0321] LC-MS (ES, m / z): [M+1] + =321.2,323.2

[0322]

[0323] To a solution of 4-bromo-5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole (200 mg, 0.622 mmol, 1 eq) and methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (250 mg, 0.954 mmol, 1.53 eq) in dioxane (8 mL, 94.432 mmol, 151.70 eq) and water (0.8 mL, 44.407 mmol, 71.34 eq) was added KCO (250 mg, 1.809 mmol, 2.91 eq) and Pd(dppf)Cl (20 mg, 0.027 mmol, 0.04 eq). After stirring at 80° C. under nitrogen atmosphere for 6 sec, the resulting mixture was concentrated under reduced pressure. The residue was purified by Prep-TLC / silica gel column chromatography eluting with EA / PE=1 / 3 to give methyl 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]benzoate (150 mg, 57.6%) as a yellow solid.

[0324] LC-MS (ES, m / z): [M+1] + =377.2

[0325]

[0326] To a solution of methyl 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]benzoate (150 mg, 0.398 mmol, 1 eq) in THF (8 mL, 98.742 mmol, 247.86 eq) and water (1 mL, 55.509 mmol, 139.34 eq) was added LiOH (100 mg, 4.175 mmol, 10.48 eq). The mixture was stirred at RT for 16 h and acidified to pH 5 with oxalic acid. The resulting mixture was extracted with EA (20 mL×3). The combined organic layers were washed with NaCl(aq), dried over anhydrous Na2SO4, and the filtrate was concentrated under reduced pressure to give 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]benzoic acid (100 mg, 58.86%) as a yellow solid.

[0327] LC-MS (ES, m / z): [M+1] + =363.2

[0328] Example 1: N-(2-methyl-1H-pyrrolo[3,2-c]pyridin-6-yl)-5-(1H-pyrazol-4-yl)picolinamide (Sample 1)

[0329]

[0330] To a solution of 6-chloro-2-iodo-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridine (550 mg, 1.346 mmol, 1.00 equiv) and trimethyl-1,3,5,2,4,6-trioxatriborane (1.69 g, 13.460 mmol, 10 equiv) in DMF (2 mL) was added KCO (557.91 mg, 4.038 mmol, 3 equiv) and Pd(dppf)Cl.CHCl (109.62 mg, 0.135 mmol, 0.1 equiv). After stirring at 80° C. under nitrogen atmosphere for 60 h, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (5:1) to give 6-chloro-2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridine (296 mg, 74.10%) as a yellow solid.

[0331]

[0332] To a solution of 6-chloro-2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridine (290 mg, 0.977 mmol, 1.00 equiv) and diphenylformimine (531.13 mg, 2.931 mmol, 3 equiv) in toluene (2 mL) and BINAP (121.65 mg, 0.195 mmol, 0.2 equiv) was added t-BuONa (281.64 mg, 2.931 mmol, 3 equiv) and Pd2(dba)3CHCl3 (101.11 mg, 0.098 mmol, 0.10 equiv). After stirring at 100°C under a nitrogen atmosphere for 16 hours, the resulting mixture was cooled and concentrated under reduced pressure. The reaction was diluted with water at room temperature. The resulting mixture was extracted with CHCl2 (2 x 50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This yielded N-(2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl)-1,1-diphenylmethanimine (300 mg, 41.72%) as a brown oil. The crude product / mixture was used directly in the next step without further purification.

[0333] LC-MS: (ES, m / z): [M+H]=442

[0334]

[0335] To a stirred solution of N-(2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl)-1,1-diphenylmethanimine (300 mg, 0.408 mmol, 1.00 eq., 60%) in THF (5 mL) was added HCl 1.0 M (2.45 mL, 2.448 mmol, 6 eq.) dropwise at room temperature. The resulting mixture was stirred at room temperature for 16 hours and extracted with THF (2×30 mL). The aqueous layer was basified to pH 7 with saturated NaHCO (aqueous) and extracted with CH2Cl2 (2×30 mL). The combined organic layers were washed with brine (2×20 mL) and dried over anhydrous Na2SO4. The filtrate was concentrated under reduced pressure. This resulted in 2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (180 mg, crude) as a brown solid.

[0336] LC-MS: (ES, m / z): [M+H]=278

[0337]

[0338] To a stirred solution of 2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (60 mg, 0.216 mmol, 1.00 equiv) and 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid (acid 1, 59.10 mg, 0.216 mmol, 1.0 equiv) in pyridine (1 mL) was added EDCI (82.92 mg, 0.432 mmol, 2.0 equiv) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The resulting mixture was diluted with water (30 mL) and extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (2×20 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to give N-(2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl)-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (120 mg, crude) as a brown solid.

[0339] LC-MS: (ES, m / z): [M+H]=533

[0340]

[0341] To a stirred solution of N-(2-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl)-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (120 mg, crude) in DCM (10 mL) was added CFCOOH (0.80 mL) dropwise at room temperature. The resulting mixture was stirred at room temperature for 16 hours and concentrated under vacuum. The residue was basified to pH 7 with NHOH (aqueous). The crude product was purified by Prep-HPLC using the following conditions: column, XBridgePrep C18 OBD Column, 5 μm, 19×150 mm; mobile phase, water (0.05% NH 3 H 2 O) and ACN (41% phase B in 7 minutes, up to 54%); detector, UV 254 nm) to produce N-{2-methyl-1H-pyrrolo[3,2-c]pyridin-6-yl}-5-(1H-pyrazol-4-yl)pyridine-2-carboxamide (17.8 mg) as a light yellow solid.

[0342] LC-MS: (ES, m / z): [M+H]=319

[0343] H-NMR (400MHz, DMSO-d6, ppm) δ13.22(s,1H),11.39(s,1H),10.26(s,1H),9.05(d,J=0.8 Hz, 1H), 8.46 (s, 1H), 8.35-8.18 (m, 4H), 8.18 (d, J = 8.0Hz, 1H), 6.24 (s, 1H), 2.40 (s, 3H).

[0344] Example 2: (R)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-5-(1H-pyrazol-4-yl)picolinamide (Sample 2)

[0345]

[0346] In an 8-mL vial purged and maintained with an inert nitrogen atmosphere were placed 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid (acid 1, 80 mg, 0.293 mmol, 1.00 equiv), pyridine (4 mL), EDCI (84.17 mg, 0.440 mmol, 1.5 equiv), and 2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (Intermediate 1, 101.44 mg, 0.293 mmol, 1 equiv). The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The residue was diluted with 20 mL of H2O and extracted with 3×10 mL of ethyl acetate. The organic layers were then combined and washed with 2×10 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 130 mg (73.7 g) of N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide as a brown oil.

[0347] LC-MS: (ES, m / z): [M+H] + =602

[0348]

[0349] In a 50-mL round-bottom flask, N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (130 mg, 0.216 mmol, 1.00 equiv), DCM (4.00 mL) and CF3COOH (4.00 mL) were placed. The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The resulting solution was diluted with 4 mL of DMF. The pH of the mixture was adjusted to 8 with NH3 / H2O. The crude product was purified by Prep-HPLC using the following conditions: column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; mobile phase, water (0.05% NH 3 H 2 O) and ACN (15% phase B, up to 31% over 7 minutes); detector, UV 254 nm. This yielded 22.2 mg (26.53%) of N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-5-(1H-pyrazol-4-yl)pyridine-2-carboxamide as a yellow solid.

[0350] LC-MS: (ES, m / z): [M+H] + =388

[0351] 1 H-NMR: (300 MHz, methanol-d 4, ppm)δ8.99(s,1H),8.51(d,J=0.9Hz,1H),8.38(s,1H),8.23(m,4H),6.52(s,1H) ,3.43(t,J=7.8Hz,1H),3.24-3.19(m,1H),2.41-2.27(m,5H),1.89-2.07(m,3H).

[0352] Example 3: (S)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-5-(1H-pyrazol-4-yl)picolinamide (Sample 3)

[0353]

[0354] At room temperature, an 8 mL vial was charged with 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid (acid 1, 60 mg, 0.220 mmol, 1.00 equiv), pyridine (4 mL), 2-[(2S)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (prepared according to Intermediate 1 using tert-butyl (2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate in Step 3, 76.08 mg, 0.220 mmol, 1 equiv) and EDCI (63.13 mg, 0.330 mmol, 1.5 equiv). The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The reaction mixture was diluted with 20 mL of H2O and extracted with 3×10 mL of ethyl acetate. The organic layers were combined and then washed with 2×10 mL of brine. The solution was dried over anhydrous sodium sulfate and concentrated under vacuum. This produced N-{2-[(2S)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxane-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (80 mg, crude) as a brown oil.

[0355] LC-MS: (ES, m / z): [M+H] + =602

[0356]

[0357] In a 50-mL round-bottom flask, N-{2-[(2S)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (80 mg), DCM (4 mL) and CF3COOH (4 mL) were placed. The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The residue was diluted with 4 mL of DMF. The pH value of the solution was adjusted to 8 with NH3 / H2O. The crude product was purified by Prep-HPLC using the following conditions: column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; mobile phase, water (0.05% NH3H2O) and ACN (15% B phase in 7 minutes, up to 31%); detector, UV 254 nm. This resulted in N-{2-[(2S)-1-methylpyrrolidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-5-(1H-pyrazol-4-yl)pyridine-2-carboxamide (7.8 mg, 15.14%) as a light yellow solid.

[0358] LC-MS: (ES, m / z): [M+H] + =388

[0359] 1 H-NMR: (300 MHz, methanol-d 4, ppm)δ8.98(s,1H),8.49(s,1H),8.38(s,1H),8.25-8.20(m,4H),6.51(s,1H),3.43(t,J=7.8Hz ,1H),3.28–3.16(m,1H),2.43-2.34(m,1H),2.29(s,3H),2.30–2.21(m,1H),2.09–1.94(m,3H).

[0360] Example 4: (R)-2-Fluoro-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-4-(1H-pyrazol-4-yl)benzamide (Sample 4)

[0361]

[0362] In an 8-mL vial purged and maintained with an inert nitrogen atmosphere were placed 2-fluoro-4-[1-(oxan-2-yl)pyrazol-4-yl]benzoic acid (prepared as for acid 1 using benzoic acid, 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-, methyl ester and -bromo-1-(oxan-2-yl)pyrazole, 100 mg, 0.344 mmol, 1.00 equiv), pyridine (4 mL) and 2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (Intermediate 1, 95.50 mg, 0.275 mmol, 0.8 equiv) and EDCI (198.11 mg, 1.032 mmol, 3 equiv). The resulting solution was stirred at room temperature for 16 hours and concentrated under vacuum. The resulting solution was diluted with 20 mL of H2O and extracted with 3 × 10 mL of ethyl acetate, and then the organic layers were combined. The solution was washed with 2 × 10 mL of brine and dried over anhydrous sodium sulfate and concentrated under vacuum. This produced 2-fluoro-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-(1H-pyrazol-4-yl)benzamide (100 mg, crude) as a brown oil.

[0363] LC-MS: (ES, m / z): [M+H] + =535

[0364]

[0365] In a 50-mL round-bottom flask, 2-fluoro-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-(1H-pyrazol-4-yl)benzamide (100 mg, crude product), DCM (4.00 mL) and CF3COOH (4.00 mL) were placed. The resulting solution was stirred at room temperature for 20 hours and concentrated under vacuum. The residue was diluted with 4 mL of DMF and the pH of the solution was adjusted to 8 with NH3H2O. The crude product (60 mg) was purified by Prep-HPLC using the following conditions: column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; mobile phase, water (0.05% NH 3 H 2 O) and ACN (15% phase B over 7 minutes, up to 31%); detector, UV 254 nm. This yielded 2-fluoro-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-4-(1H-pyrazol-4-yl)benzamide (19.4 mg) as a white solid.

[0366] LC-MS: (ES, m / z): [M+H] + =405

[0367] 1 H-NMR: (300 MHz, methanol-d 4, ppm)δ8.49(d,J=0.9Hz,1H),8.29(s,1H),8.21-8.05(m,2H),7.97(t,J=8.1Hz,1H),7.65–7.51(m, 2H), 6.51 (s, 1H), 3.43 (t, J = 7.9Hz, 1H), 3.25-3.20 (m, 1H), 2.43–2.24 (m, 5H), 2.07–1.94 (m, 3H).

[0368] F-NMR: (282 MHz, methanol-d4, ppm) δ-114.965

[0369] Example 5: (R)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-4-(1H-pyrazol-4-yl)benzamide (Sample 5)

[0370]

[0371] To a stirred solution of 4-[1-(oxan-2-yl)pyrazol-4-yl]benzoic acid (prepared according to WO 2021127166, acid AG, 50 mg, 0.184 mmol, 1.00 equiv) and 2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (Intermediate 1, 63.63 mg, 0.184 mmol, 1.0 equiv) in pyridine (2 mL) was added EDCI (70.40 mg, 0.368 mmol, 2.0 equiv) at room temperature. The resulting mixture was stirred at room temperature for 16 hours and concentrated under vacuum. This resulted in N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-[1-(oxan-2-yl)pyrazol-4-yl]benzamide as a brown oil (100 mg, crude).

[0372] LC-MS: (ES, m / z): [M+H]=601

[0373]

[0374] To a stirred solution / mixture of N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-[1-(oxan-2-yl)pyrazol-4-yl]benzamide (100 mg, crude) in DCM was added CF3COOH (2 mL, 26.926 mmol, 161.78 equiv) at room temperature. The resulting mixture was stirred at room temperature for 20 hours. The resulting mixture was concentrated under vacuum. The residue was basified to pH 8 with NH4OH (aqueous). The crude product was purified by Prep-HPLC using the following conditions: column, XBridge Shield RP18 OBD Column, 19×150 mm, 5 μm; mobile phase, water (0.05% NH 3 H 2 O) and ACN (18% ACN in 8 minutes, up to 35%); detector, UV 254 nm) to yield N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-4-(1H-pyrazol-4-yl)benzamide (30.6 mg, 43.1% for two steps) as a light yellow solid.

[0375] LC-MS: (ES, m / z): [M+H]=387

[0376] H-NMR: (400MHz, CD3OD-d4, ppm): δ8.51(s,1H),8.19(s,1H),8.09(s,2H),8.01(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,2H),6.51 (s,1H),3.45(t,J=8.0Hz,1H),3.27-3.22(m,1H),2.42(q,J=8.8Hz,1H),2.31(s,3H),2.32-2.24(m,1H),2.09-1.89(m,3H).

[0377] Example 6: (R)-N-(2-(1-methylpiperidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-4-(1H-pyrazol-4-yl)benzamide (Sample 6)

[0378]

[0379] 4-[1-(oxan-2-yl)pyrazol-4-yl]benzoic acid (prepared according to WO 2021127166, acid AG, 50 mg, 0.184 mmol, 1 eq) and EDCI (42.24 mg, 0.221 mmol, 1.2 eq) in pyridine (5 mL) were added to an 8 mL vial at room temperature. The resulting mixture was stirred for 10 minutes. To this mixture was added 2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (Intermediate 2, 66.21 mg, 0.184 mmol, 1 eq). The resulting mixture was stirred for an additional 16 hours, and the crude product was purified by reverse phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, 0.05% NH3.H2O in water, gradient from 5% to 60% MeCN over 10 minutes; detector, UV 220 nm). This resulted in N-{2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-[1-(oxan-2-yl)pyrazol-4-yl]benzamide (79 mg, 69.97%) as a white solid.

[0380] LC-MS (ES, m / z): [M+1] + =615.3

[0381]

[0382] N-{2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-4-[1-(oxan-2-yl)pyrazol-4-yl]benzamide (79 mg, 0.128 mmol, 1 equivalent) and trifluoroacetic acid (1 mL) in DCM (5 mL) were added to an 8 mL vial at room temperature. The resulting mixture was stirred at room temperature for another 1 hour and concentrated under reduced pressure. The residue was dissolved in DMF (1 mL) and adjusted to pH 10 with NH3.H2O. The crude product (50 mg) was purified by Prep-HPLC using the following conditions: (0.1% NH3.H2O in water and MeCN (20% in 8 minutes, up to 60%)) to give N-{2-[(2R)-1-methylpiperidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-4-(1H-pyrazol-4-yl)benzamide (10.3 mg, 20.02%) as a white solid.

[0383] LC-MS (ES, m / z): [M+1] + =401

[0384] 1 H-NMR: 1 H NMR (400 MHz, methanol-d4) δ 8.55 (s, 1H), 8.21 (s, 1H), 8.17 (m, 2H), 8.04 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H), 6.52 (s, 1H), 3.18 (m, 1H), 3.09 (s, 1H), 2.25-2.18 (s, 1H), 2.12 (s, 3H), 2.03-1.83 (m, 3H), 1.79-1.56 (m, 2H), 1.49-1.38 (m, 1H).

[0385] Example 7: (R)-N-(2-(1-methylpiperidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-5-(1H-pyrazol-4-yl)picolinamide (Sample 7)

[0386]

[0387] 5-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)picolinic acid (acid 1, 50 mg, 0.183 mmol, 1 eq) and EDCI (42.09 mg, 0.220 mmol, 1.2 eq) in pyridine (1 mL) were added to an 8 mL vial at room temperature. The resulting mixture was stirred at room temperature for 10 minutes. To the above mixture was added 2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (intermediate 2, 65.97 mg, 0.183 mmol, 1 eq). The resulting mixture was stirred at room temperature for another 16 hours. The crude product was purified by reverse phase flash chromatography using the following conditions (column, C18 silica gel; mobile phase, 0.05% NH3.H2O in water, gradient from 5% to 60% MeCN over 10 minutes; detector, UV 220 nm). This resulted in N-{2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (100 mg, 88.75%) as a white solid.

[0388]

[0389] N-{2-[(2R)-1-methylpiperidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}-5-[1-(oxan-2-yl)pyrazol-4-yl]pyridine-2-carboxamide (100 mg, 0.162 mmol, 1 eq) and trifluoroacetic acid (3 mL) in DCM (10 mL) were added to an 8 mL vial at room temperature. The resulting mixture was stirred for an additional 1 hour at room temperature and concentrated under reduced pressure. The residue was diluted with DMF (1 mL), adjusted to pH 10 with NH 3 .H 2 O, and the crude product (50 mg) was purified by Prep-HPLC using the following conditions (0.1% NH 3 .H 2 O in water and MeCN (20% in 8 minutes, up to 60%)) to yield N-{2-[(2R)-1-methylpiperidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}-5-(1H-pyrazol-4-yl)pyridine-2-carboxamide (14.3 mg, 21.94%) as a white solid.

[0390] LC-MS (ES, m / z): [M+1] + =402.2

[0391] 1 H-NMR 1H NMR (400 MHz, methanol-d4) δ 9.02 (s, 1H), 8.54 (s, 1H), 8.30 (s, 1H), 8.21-8.16 (m, 3H), 8.10 (m, 1H), 6.57 (s, 1H), 3.18 (m, 1H), 3.03 (m, 1H), 2.37 (m, 1H), 2.23-2.18 (s, 3H), 1.96-1.92 (m, 3H), 1.72-1.84 (m, 2H), 1.60-1.53 (m, 1H).

[0392] Example 8: (R)-4-(5-(methoxymethyl)-1H-pyrazol-4-yl)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)benzamide (Sample 8)

[0393]

[0394] A mixture of 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]benzoic acid (acid 2, 50 mg, 0.138 mmol, 1 eq) and 2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-amine (Intermediate 1, 75 mg, 0.216 mmol, 1.57 eq) and EDCI (50 mg, 0.261 mmol, 1.89 eq) in pyridine (3 mL, 0.038 mmol, 0.27 eq) was stirred at room temperature for 16 h. The reaction mixture was concentrated under vacuum to give 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}benzamide (60 mg), which was used directly in the next step.

[0395] LC-MS (ES, m / z): [M+1] + =691.3

[0396]

[0397] To a solution of DCM (6 mL) and 4-[5-(methoxymethyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl]-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}benzamide (60 mg, 0.087 mmol) was added trifluoroacetic acid (2 mL, 0.009 mmol, 0.10 equiv) at RT. The resulting mixture was stirred for 1 hour and concentrated under reduced pressure. The crude product was purified by Prep-HPLC using the following conditions: Column, SunFire Prep C18 OBD Column, 19×150 mm 5 μm 10 nm; Mobile phase, water (0.1% NH 3 H 2 O) and ACN (28% ACN over 9 minutes, up to 63%); Total flow rate, 20 mL / min; Detector, UV 220 nm. This yielded (R)-4-(5-(methoxymethyl)-1H-pyrazol-4-yl)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)benzamide (28.6 mg, 75.1%) as a white solid.

[0398] LC-MS (ES, m / z): [M+1] + =431.3

[0399] 1H NMR(400MHz,DMSO-d6)δ8.50(d,J=1.1Hz,1H),8.18(s,1H),8.02(m,3H),7.72(m,2H),6.50(s,1H),4.5 8(s,2H),3.41(m,4H),3.21(t,J=7.5Hz,1H),2.38(m,1H),2.28(m,4H),2.02(m,2H),1.95–1.86(m,1H).

[0400] Example 9: (R)-4-(5-fluoro-1H-pyrazol-4-yl)-N-(2-(1-methylpyrrolidin-2-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)benzamide (Sample 9)

[0401]

[0402] In a 40 mL vial were placed 4-bromo-3-fluoro-2H-pyrazole (100 mg, 0.606 mmol, 1 eq), [2-(chloromethoxy)ethyl]trimethylsilane (151.60 mg, 0.909 mmol, 1.5 eq), NaH (29.09 mg, 1.212 mmol, 2.0 eq), and THF (1 mL). The resulting solution was stirred at 25° C. for 4 hours. The reaction was monitored by LCMS. The residue was purified by reverse phase flash chromatography using the following conditions: column, C18 silica gel; mobile phase, water (0.1% FA) and ACN (5.0% ACN over 7 minutes, up to 45.0%); total flow rate, 20 mL / min; detector, UV 220 nm. The collected fractions were combined and concentrated under vacuum. This resulted in 4-bromo-5-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole as a light yellow oil (110 mg, 37.49%).

[0403]

[0404] In an 8 mL vial were placed 4-bromo-5-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazole (100 mg, 0.339 mmol, 1 eq), methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (115.42 mg, 0.441 mmol, 1.3 eq), Pd(dppf)Cl2 (12.39 mg, 0.017 mmol, 0.05 eq), K2CO3 (117.03 mg, 0.848 mmol, 2.5 eq), dioxane (1 mL), and H2O (0.1 mL). The resulting solution was stirred at 105°C for 16 hours. The reaction was cooled, diluted with water, and extracted with EA. The organic layer was washed with brine, dried over Na2SO4, and concentrated under vacuum. The residue was applied to a silica gel column and eluted with ethyl acetate / hexane (1 / 5). The collected fractions were combined and concentrated under vacuum. This produced methyl 4-(5-fluoro-1-{[(trimethylsilyl)methoxy]methyl}pyrazol-4-yl)benzoate (71 mg, 60.2%) as an off-white solid.

[0405]

[0406] In an 8mL vial, 4-(5-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl)methyl benzoate (70mg, 0.200mmol, 1 equivalent), 2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridine-6-amine (intermediate 1, 69.22mg, 0.200mmol, 1.0 equivalent) and THF (1mL) were added and stirred. LiHMDS (200.53mg, 1.200mmol, 6.0 equivalents) was added dropwise in an ice-water bath, and then stirred for 5 minutes. The reaction was warmed to room temperature and stirred for 2 hours. The reaction was then quenched by adding 50mL NH4Cl (aqueous) and extracted with EA. The resulting mixture was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. This resulted in 4-(5-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl)-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}benzamide (90 mg, 67.8%) as a brown oil.

[0407] LC-MS (ES, m / z): [M+1] + =665

[0408]

[0409] In an 8 mL vial, 4-(5-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrazol-4-yl)-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1-{[2-(trimethylsilyl)ethoxy]methyl}pyrrolo[3,2-c]pyridin-6-yl}benzamide (70 mg, 0.105 mmol, 1 eq), ethylenediamine (126.53 mg, 2.100 mmol, 20.0 eq), TBAF (412.85 mg, 1.575 mmol, 15.0 eq), and DMF (1 mL) were placed. The resulting solution was stirred at 70° C. for 8 hours. The reaction mixture was concentrated under vacuum. The crude product was purified by Prep-HPLC using the following conditions (Prep-HPLC-013): Column, SunFire Prep C18 OBD Column, 19×150 mm 5 μm; Mobile phase, water (0.1% FA) and ACN (5.0% ACN over 7 minutes, up to 45.0%); Total flow rate, 20 mL / min; Detector, UV 220 nm. Freeze drying yielded 4-(3-fluoro-2H-pyrazol-4-yl)-N-{2-[(2R)-1-methylpyrrolidin-2-yl]-1H-pyrrolo[3,2-c]pyridin-6-yl}benzamide (18.5 mg, 42.5%) as a white solid.

[0410] LC-MS (ES, m / z): [M+1] + =405

[0411] H-NMR (400MHz, DMSO-d6) δ12.73(s,1H),11.38(s,1H),10.44(s,1H),8.51(s,1H),8.33(t,J=2.0Hz,1H),8.21(d,J=1.0Hz,1H),8.14 –8.07(m,2H),7.73–7.67(m,2H),6.40(s,1H),3.29(s,1H),3.15(t,J=8.0Hz,1H),2.36–2.22(m,1H),2.18(s,4H),1.93–1.80(m,3H).

[0412] Example 10:

[0413] FRET assay:

[0414] Compounds of the present invention were tested in a TR-FRET ENL screening assay. TR-FRET (time-resolved fluorescence energy transfer) can be used to quantify ENL YEATS domain binding to crotonylated histone peptides (H3K9cr, aa1-20). Streptavidin-Eu (Eu) chelate binds to the biotinylated peptide, while anti-6×HIS ULight TM 6×HIS-ENL is bound. When the Eu chelate is excited at 320 nm, fluorescence resonance energy transfer (FRET) occurs when Eu and ULight are brought into proximity by the binding of ENL to the acyl peptide. ULight emission (FRET) is measured at 665 nm and normalized to Eu emission at 615 nm to reduce well-to-well variability.

[0415] FRET assay protocol

[0416] The compounds of the invention were dissolved in DMSO at a concentration of 3 mM and subsequently diluted in assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 0.05% BSA, 0.2% Pluronic F-127) to allow the assay to contain 1% DMSO. In a white 384 shallow well microplate (Proxiplate-384 Plus, PerkinElmer, 6008280), 150 nL of compound or vehicle (1% DMSO in assay buffer) for high control (HC) wells was mixed with 5 μL of 30 nM ENL protein (6×HIS ENLYEATS domain, EpiCypher, 15-0069) and incubated at room temperature for 15 minutes. Low control (LC) wells received 5 μL of assay buffer instead of ENL protein. Then 15nM H3K9cr peptide (H3aa1-20, biotinylated; EpiCypher, 12-0099) in 5 μL assay buffer was added and incubated at room temperature for 30 minutes. Finally, a mixture of 5 μL 45nM anti-6HIS ULight (PerkinElmer, TRF0105) and 1.5nM streptavidin-europium chelate (PerkinElmer, AD0060) was added and incubated at room temperature for another 30 minutes. TR-FRET signals (665nm signal / 615nm signal × 10,000) were measured using PerkinElmer 2104EnVision (xenon flash lamp excitation, 320nm ± 37.5nm excitation filter, 407nm cut-off dichroic mirror, 615nm ± 4.25 (europium) nm and 665nm ± 3.75nM (ULight) emission filter) were used. Compound concentration response curves were performed in duplicate over a concentration range of 0.15 nM to 30 μM. The response at each compound concentration minus the LC value was converted to the percent inhibition of the vehicle control group (HC-LC). The relationship between % inhibition and compound concentration was analyzed using a four-parameter logistic equation to estimate the lower and upper asymptotes, the compound concentration that produces 50% inhibition (IC50 value), and the slope at the midpoint.

[0417] Table 1: FRET assay results

[0418]

[0419]

[0420] Example 11: Cellular Assay

[0421] Cell-based assays were used to assess the ability of test compounds to reduce cell viability in both MV4:11 (MLL-AF4 MLL) and K562 cells cultured in Iscove's modified Dulbecco's medium (Gibco, 12440061) containing 10% FBS. Assays were performed over 12 days, and cells were split on days 4 and 8. Compound concentration response curves were performed in duplicate over a concentration range of 0.15 nM to 30 μM. On day 0, compounds or vehicles were plated directly into 96-well cell culture plates (Corning, 3599) at 300 nL, 5000 cells / well in a volume of 100 μL. Blank wells received cell culture medium. The plates were incubated at 37°C with 5% CO2 for 4 days. On days 4 and 8, cells were split and incubated for an additional 4 days, while aliquots of the cells were taken for CTG readings. For cell splits, 270 nL of compound or DMSO was added to a new 96-well cell culture plate containing 90 μL of culture medium plus 10 μL of cells from the original assay plate (after mixing) or 100 μL of culture medium (blank wells). This process was repeated on day 8.

[0422] use Homogeneous luminescence assay kit (Promega, G9243) was used to assess cell viability according to the manufacturer's instructions. This quantitative ATP indicates the presence of metabolically active cells. On days 4, 8, and 12, 20 μl of the remaining cell suspension was pipetted into a 384-well plate (Corning 3570), to which an equal volume of CellTiter-Glo reagent was added. The plate was incubated at room temperature for 10 minutes and then the luminescence signal was recorded using an EnVision plate reader (PE, 2104). The resulting data were analyzed as follows:

[0423] Inhibition (%) = 100% × (luminescence 媒介物 - Glow 样品 ) / (luminous 媒介物 - Glow 空白 )

[0424] The vehicle was cells treated with 0.3% DMSO, and the blank was culture medium. IC50 values were calculated by fitting the curve using XLfit (v5.3.1.3) as follows: Y = bottom + (top - bottom) / (1 + 10^((LogIC50 - X) * slope)).

[0425] Table 2: Cell assay results

[0426] Examples / Samples <![CDATA[MV4:11; IC on the 12th day 50 > 1 0.040 2 0.019 3 0.083 4 0.119 5 0.079 6 0.138 7 0.036 8 0.357 9 0.176

[0427] Example 12: FLT3 activity

[0428] FLT3 inhibition assay

[0429] reactants Merchants Catalog Number FLT3 Carna 08-154 Staurosporine BioAustralis BIA-S1086 FL peptide 2 PerkinElmer 760346

[0430] Solution 1

[0431] Components concentration <![CDATA[MgCl2]]> 10mM Brij-35 0.01％ DTT 2mM BSA 0.05％ EGTA 1mM HEPE (pH 7.5) 50mM

[0432] Starting from a 10 mM stock solution, each test compound was serially diluted into 10 concentrations by 3-fold dilution using a TECAN EVO 200. 60 nL of each stock solution was transferred to a 384-well plate using an Echo 550.

[0433] Solution 1 was prepared according to the table above and used to dilute FLT3, ATP and FL2 stock solutions to 0.9379 nM (1.33×), 400 μM (4×) and 6 μM (4×), respectively. FLT3 solution (15 μL) was added to each well at 25°C, shaken for 1 minute, and pre-incubated with the test compound and control for 30 minutes. 5 μL of ATP and FL-peptide 2 solution prepared as described above was added to each well, and the plate was shaken for 10 seconds, then briefly centrifuged at 1000 rpm and then incubated at room temperature for 90 minutes. The plate was read on a Caliper EZ reader and the IC was calculated using XLfit (the following equation) 50 value.

[0434] % inhibition = 100% × (luminescence HC - Glow 样品 ) / (luminous HC - Glow LC )

[0435] Luminescence 样品 : Test compound signal

[0436] Luminescence LC : Low control signal

[0437] Luminescence HC : High control signal

[0438] Table 3: FLT3 assay results

[0439]

[0440]

[0441] Not determined.

Claims

1. A compound of formula I: in: R 1 and R 2 together to form pyrrolidine or piperidine; R 3 Selected from hydrogen and C1-C8 alkyl; R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0- 3H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

2. The compound of claim 1, wherein the compound is of formula Ia': where R 3 、R 4 and R 5 As defined above for Formula I.

3. The compound of claim 1, wherein the compound is of formula Ia": where R 3 、R 4 and R 5 As defined above for Formula I.

4. The compound according to claim 2 or 3, wherein: R 4 is selected from benzene, pyridine, pyrimidine, pyridazine and pyrazine, which is optionally replaced by R as defined above for formula I 7 group substitution; and R 5 selected from pyrrolidine; pyrroline; pyrazolidine; pyrazoline; imidazoline; imidazoline; pyrrole; pyrazole; imidazole; triazole; isoxazole; oxazole; 1,2,3-oxadiazole; 1,3,4-oxadiazole; furazan; 1,2,4-oxadiazole; 1,2,3,4-oxatriazole; 1,2,3,5-oxatriazole; isothiazole; thiazole; 1,2,3-thiadiazole; 1,3,4-thiadiazole; 1,2,5-thiadiazole; 1,2,4-thiadiazole; 1,2,3,4-thiatriazole; 1,2,3,5-thiatriazole; furan and thiophene, optionally replaced by R as defined above for formula I 6 Group substitution.

5. The compound of any one of claims 2, 3 or 4, wherein R 3 It is a methyl group.

6. A compound of formula II: in: R 8 Selected from hydrogen and C1-C8 alkyl; R 4 is an aromatic 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 7 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0-3 H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 ) oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen; and R 5 is a 5-membered or 6-membered carbocyclic or heterocyclic ring, which is optionally substituted by one or more R 6 Group substitution: C1-C8 alkyl; C1-C 10 Haloalkyl; C3-C8 carbocycle; C1-C 10 Oxaalkyl, -SO2(C 1-6 )alkyl; -SO2NH(C 0-3 H 1-7 );-CONH(C 0- 3H 1-7 );-SO2NH(C 1-6 ) oxaalkyl; -CN; -CH2CN; -CH2NH2; -NH2, -NR 14 , where R 14 are independently selected from hydrogen, (C 1-6 ) fluoroalkyl and (C 1-3 )oxaalkyl, -CH2OH, benzyloxy, -C(=NH)-NH2; oxo; and halogen.

7. The compound of claim 6, wherein R 4 is selected from benzene, pyridine, pyrimidine, pyridazine and pyrazine, which is optionally replaced by R as defined above for formula I 7 group substitution; and R 5 selected from pyrrolidine; pyrroline; pyrazolidine; pyrazoline; imidazoline; imidazoline; pyrrole; pyrazole; imidazole; triazole; isoxazole; oxazole; 1,2,3-oxadiazole; 1,3,4-oxadiazole; furazan; 1,2,4-oxadiazole; 1,2,3,4-oxatriazole; 1,2,3,5-oxatriazole; isothiazole; thiazole; 1,2,3-thiadiazole; 1,3,4-thiadiazole; 1,2,5-thiadiazole; 1,2,4-thiadiazole; 1,2,3,4-thiatriazole; 1,2,3,5-thiatriazole; furan and thiophene, optionally replaced by R as defined above for formula I 6 Group substitution.

8. The compound of claim 6 or 7, wherein R 8 It is a methyl group.

9. The compound of claim 1 or claim 6, selected from the group consisting of:

10. A pharmaceutical composition comprising the compound of any one of claims 1 to 9 and one or more pharmaceutically acceptable carriers.

11. The pharmaceutical composition of claim 10, further comprising one or more therapeutic agents.

12. The pharmaceutical composition of claim 11, wherein the one or more therapeutic agents are selected from Bcl-2 inhibitors, cyclin-dependent kinases 4 and 6 (CDK 4 / 6 inhibitors), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, mTOR inhibitors, mutant isocitrate dehydrogenase (IDH1 and IDH2) inhibitors, glucocorticoids, epigenetic modulators, and chemotherapeutic agents.

13. A method for treating acute leukemia, comprising administering to a subject in need thereof a therapeutically effective amount of the compound according to any one of claims 1 to 9 or the pharmaceutical composition according to claims 10 to 12.

14. The method of claim 13, wherein the acute leukemia is acute lymphoblastic leukemia (ALL).

15. The method of claim 13, wherein the acute leukemia is acute myeloid leukemia (AML).

16. The method of claim 15, wherein the AML is a subtype selected from the group consisting of: minimally differentiated acute myeloid leukemia (MO), acute myeloid leukemia without maturation (M1), acute myeloid leukemia with maturation (M2), acute myeloid leukemia with maturation and t(8;21), acute promyelocytic leukemia (M3) - ultragranular, microgranular, acute myelomonocytic leukemia (M4), acute myelomonocytic leukemia with bone marrow eosinophilia (M4E0), acute monocytic leukemia (M5 ), acute promonocytic leukemia (M5a), acute monocytic leukemia with maturation (M5b), erythroid / myeloid erythroleukemia (M6a), pure erythroid malignancy (M6b), acute megakaryoblastic leukemia (M7), acute megakaryoblastic leukemia associated with t(1;22), acute basophilic leukemia, acute myelofibrosis (acute myelodysplasia with myelofibrosis), acute leukemias and transient myeloproliferative disorders in Down syndrome, low-cell acute myeloid leukemia, and myeloid sarcoma.

17. The method of claim 13, wherein the at least one compound is administered orally.

18. The method of claim 13, wherein the at least one compound is administered one to four times per day.

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