LIN28 inhibitors and methods of use thereof

Abstract

The present invention relates to LIN28 inhibitors and methods of use thereof. The present invention relates to compounds of formula (I) and compositions comprising said compounds. The invention also relates to methods of treating cancer. # imgabs0 #

Description

[0001] This application is a divisional application of the PCT international application PCT / US2020 / 064896 filed on December 14, 2020, which entered the Chinese national phase on August 10, 2022, and whose Chinese patent application number is 202080096261.1 and whose invention name is "LIN28 inhibitors and methods of use thereof".

[0002] Related Applications

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 949,873, filed on December 18, 2019, which is hereby incorporated by reference in its entirety.

[0004] Government support

[0005] This invention was made with government support under Grant No. TR001881 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The present invention relates to LIN28 inhibitors and methods of use thereof. Background Art

[0007] Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the clonal proliferation of myeloid blasts, resulting in a fatal outcome in the majority of affected adults (1). Even with very aggressive multidrug chemotherapy regimens, newer targeted therapies, and myeloablative allogeneic hematopoietic cell transplantation, the majority of patients succumb to AML within 5 years. Treatment-resistant leukemic stem cells (LSCs) are believed to be the underlying cause of high relapse rates and treatment failure (2-4). Therefore, the development of novel therapeutic strategies that can eradicate LSCs represents a major area of ​​unmet medical need.

[0008] Small molecules have been shown to be successful therapeutic agents in clinical applications targeting proteins involved in pathogenesis. However, currently FDA-approved drugs for G protein-coupled receptors, kinases, peptidases, nuclear receptors, proteases, ion channels, enzymes, etc. regulate less than 700 human genome-derived proteins (63). This means that less than ≤0.5% of the proteome and ≤0.05% of the genome have been explored as targets for therapeutic approaches. In addition, most small molecule drugs used clinically utilize structured binding pockets on the surface of proteins. Allosteric and / or conformational changes in remote catalytic or drug binding regions lead to drug resistance and ultimately drug ineffectiveness (64). Therefore, the development of new drugs that can target unexplored signaling pathways and overcome drug resistance mutations represents a major area of ​​unmet medical need. Summary of the invention

[0009] The present disclosure provides compounds of formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt thereof, wherein:

[0012] Selected from

[0013] Ring B is selected from phenyl and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

[0014] X is selected from N and C;

[0015] X 1 , X 3 and X 4 Each independently selected from N and CR x ;

[0016] R 1 is hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, and 5- to 6-membered heteroaryl rings having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

[0017] Each R 2 independently selected from hydrogen, halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 aliphatic group;

[0018] R 3 is selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

[0019] Each R x are independently selected from hydrogen, halogen and optionally substituted C 1-6 aliphatic group;

[0020] Each R is independently selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and

[0021] n is 0-3.

[0022] In some embodiments, the compound has Formula (Ia):

[0023]

[0024] or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the compound has Formula (Ib):

[0026]

[0027] or a pharmaceutically acceptable salt thereof.

[0028] In some embodiments, the compound has Formula (Iai) or Formula (Ia-ii):

[0029]

[0030] or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the compound has Formula (Ibi) or Formula (Ib-ii):

[0032]

[0033] or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the compound is not

[0035] In some embodiments, Ring B is a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Ring B is pyridinyl.

[0036] In some embodiments, the compound is selected from the group consisting of compounds of Formula (Ia-iii), (Ia-iv), (Iav), (Ib-iii), (Ib-iv), and (Ibv):

[0037]

[0038] In some embodiments, X 3 is N.

[0039] In some embodiments, R 1 In some embodiments, R 1 is an optionally substituted group selected from the following: C 1-6 aliphatic, phenyl, and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.1 C 1-6 Aliphatic group.

[0040] In some embodiments, R 1 In some embodiments, R 1 In some embodiments, R 1 It is phenyl.

[0041] In some embodiments, R 1 is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from oxygen, nitrogen and sulfur. 1 is a 5-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. 1 is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. 1 It is a 6-membered heteroaryl ring having 1-2 nitrogen atoms.

[0042] In some embodiments, R 1 Selected from

[0043] In some embodiments, R x In some embodiments, R x is halogen or optionally substituted C 1-6 In some embodiments, R x is an optionally substituted C 1-6 In some embodiments, R x C 1-6 In some embodiments, R x It is methyl.

[0044] In some embodiments, R 2 Selected from halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 Aliphatic group.

[0045] In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 NO 2 In some embodiments, R 2 In some embodiments, R 2 OCH 3 In some embodiments, R2 N(R) 2 In some embodiments, R 2 For NH 2 .

[0046] In some embodiments, R 2 is N(R)C(O)R. In some embodiments, R 2 Selected from NHC(O)CH 3 and N(CH 3 )C(O)CH 3 .

[0047] In some embodiments, R 2 For CO 2 R. In some embodiments, R 2 For CO 2 H.

[0048] In some embodiments, R 2 C(O)N(R) 2 In some embodiments, R 2 C(O)NHCH 3 .

[0049] In some embodiments, R 2 is an optionally substituted C 1-6 Aliphatic group.

[0050] In some embodiments, R 2 For CF 3 .

[0051] In some embodiments, R is hydrogen.

[0052] In some embodiments, R is an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0053] In some embodiments, R is optionally substituted C 1-6 In some embodiments, R is C 1-6 In some embodiments, R is methyl.

[0054] In some embodiments, R 3 In some embodiments, R 3 is an optionally substituted group selected from the following: C 1-6aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0055] In some embodiments, R 3 is an optionally substituted C 1-6 In some embodiments, R 3 C 1-6 In some embodiments, R 3 It is methyl.

[0056] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

[0057] In some embodiments, the compound is:

[0058] or a pharmaceutically acceptable salt thereof.

[0059] In certain aspects, the present disclosure provides compounds of formula (II) and pharmaceutically acceptable salts thereof:

[0060]

[0061] in:

[0062] R 1 C 1-6 Alkyl or C 3-6 Cycloalkyl;

[0063] R 2 is H, amino, nitro or amido; and

[0064] X 1 , X 3 and X 4 are each independently N or CH.

[0065] In some embodiments, X 1 , X 3 and X 4 At least one of is N. In some embodiments, X 1 , X 3 and X 4 At least two of are N. In some embodiments, X 1 , X 3 and X 4 Each of them is N.

[0066] In some embodiments, the compound is not

[0067] In some embodiments, R 1 For unsubstituted C 1-6 In some embodiments, R 1 is methyl optionally substituted by halogen. 1 is an unsubstituted methyl group. 1 C 2-6 Alkyl or C 3-6 Cycloalkyl.

[0068] In some embodiments, the compound is:

[0069] or a pharmaceutically acceptable salt thereof.

[0070] In some embodiments, the compound is JGJ002, JGJ003, JGJ004, JGJ005, JGJ007 or JGJ008, or a pharmaceutically acceptable salt thereof.

[0071] In some embodiments, R 2 is H, amino, nitro or –N(R 5 )C(O)R 6 , R 5 H or C 1-5 Alkyl, and R 6 C 1-6 alkyl.

[0072] In some embodiments, R 5 H or CH at each occurrence 3 .

[0073] In some embodiments, R 2 N(R 4 )C(O)R 5 ; R 5 is H; and R 6 C 1-6 alkyl.

[0074] In some embodiments, X 1 and X 3 Each is N; and X 4 For CH.

[0075] In some embodiments, R 2 is H, amino or nitro.

[0076] In some embodiments, R 2 NO 2or –N(R 5 )C(O)R 6 .

[0077] In some embodiments, the compound is

[0078]

[0079] or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, X 1 and X 3 Each is N, and X 4 For CH.

[0081] In some embodiments, R 2 N(R 5 )C(O)R 6 .

[0082] In some embodiments, the compound is:

[0083] or a pharmaceutically acceptable salt thereof.

[0084] In some embodiments, the compound is JGJ007 or JGJ088, or a pharmaceutically acceptable salt thereof. In certain aspects, the present disclosure relates to a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable excipient.

[0085] In certain aspects, the disclosure relates to methods of inhibiting Lin28 in a cell, the method comprising contacting a cell comprising Lin28 with a compound or composition disclosed herein.

[0086] In certain aspects, the disclosure relates to a method of inhibiting Lin28 in a cell, the method comprising contacting the cell with a compound or composition disclosed herein, the cell comprising Lin28.

[0087] In some embodiments, the cell is a cancer cell, such as an acute myeloid leukemia (AML) cell.

[0088] In certain aspects, the disclosure relates to methods of treating cancer comprising administering to a subject in need thereof a compound or composition disclosed herein.

[0089] In some embodiments, the subject has cancer, such as acute myeloid leukemia.

[0090] In certain aspects, the present disclosure relates to a method of treating cancer, comprising administering a compound or pharmaceutical composition disclosed herein to a subject suffering from cancer or exhibiting symptoms of cancer.

[0091] In some embodiments, treatment is or includes ameliorating one or more symptoms of cancer.

[0092] In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is acute myeloid leukemia.

[0093] In some embodiments, the compounds or pharmaceutical compositions disclosed herein are administered in an amount or according to a dosing regimen determined to achieve cancer cell inhibition and / or reduced cancer cell proliferation.

[0094] In some embodiments, the cancer cells comprise cancer stem cells. In some embodiments, the cancer stem cells comprise leukemia stem cells (LSC).

[0095] In certain aspects, the disclosure relates to a method of modulating splicing, the method comprising contacting a splicing-competent system with a compound disclosed herein.

[0096] In certain aspects, the present disclosure relates to a method comprising:

[0097] contacting a splicing-competent system with a compound disclosed herein; and

[0098] Evaluate in the system:

[0099] (i) the presence or level of splicing products (e.g., spliced ​​transcripts);

[0100] (ii) expression or localization of RNA; and / or

[0101] (iii) Expression or folding of polypeptides.

[0102] In certain aspects, the disclosure relates to a method of modulating splicing in a system having splicing capability by contacting the system with a compound disclosed herein, thereby observing one or more of the following:

[0103] (i) reduced RNA splicing;

[0104] (ii) altered RNA expression or localization; and / or

[0105] (iii) altered polypeptide expression or folding.

[0106] In certain aspects, the present disclosure relates to

[0107] In some embodiments, a method comprising contacting a splicing-competent system with a compound disclosed herein, wherein the compound is characterized in that when contacted with a cancer cell, the compound reduces proliferation of the cancer cell relative to proliferation observed in the absence of the compound.

[0108] In some embodiments, splicing is reduced when the compound is present compared to when the compound is not present.

[0109] In some embodiments, the method further comprises assessing splicing in the system compared to a reference condition.

[0110] In some embodiments, the reference condition is the absence of the compound.

[0111] In some embodiments, the reference condition is the presence of a control compound.

[0112] In some embodiments, the reference conditions are historical conditions.

[0113] In some embodiments, the compound inhibits one or more properties of a component of the splicing machinery and / or wherein the compound inhibits interactions between or among components of the splicing machinery.

[0114] In some embodiments, the compound binds directly to one or more splicing machinery components or complexes thereof.

[0115] In some embodiments, the splicing machinery component is an RNA component. In some embodiments, the splicing machinery component is a polypeptide component.

[0116] In some embodiments, the splicing machinery component is selected from the group consisting of an RNA component, a polypeptide component, and a complex thereof or a complex therebetween.

[0117] In some embodiments, the RNA component is or includes small nuclear RNA (snRNA).

[0118] In some embodiments, the snRNA is selected from the group consisting of: U1, U2, U4, U5, and U6.

[0119] In some embodiments, the polypeptide component is or includes a Sm polypeptide or a Lsm polypeptide.

[0120] In some embodiments, the polypeptide component is selected from the group consisting of: Prp3, Prp31, Prp4, CypH, 15.5K, Prp8, Brr2, Snu114, Prp6, Prp28, 40K, Dib1, Snu66, Sad1, and 27K.

[0121] In some embodiments, the splicing machinery component includes a Prp31 polypeptide.

[0122] In some embodiments, the splicing machinery components include U4 snRNA, U6 snRNA, and a Prp31 polypeptide.

[0123] In some embodiments, the compound inhibits the interaction between: U6 snRNA and Prp31 polypeptide; or U4 snRNA and Prp31 polypeptide.

[0124] In some embodiments, the compound inhibits the activity of a Prp31 polypeptide.

[0125] In some embodiments, contacting occurs in vitro, ex vivo, or in vivo.

[0126] In some embodiments, the splicing-competent system is a cancer cell.

[0127] In some embodiments, the cancer cells include cancer stem cells.

[0128] In some embodiments, the cancer stem cells comprise leukemia stem cells (LSCs). BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 The impact of the Lin28 / let-7 pathway on multiple pathways driving LSC proliferation is shown. Upregulation of let-7 miRNAs by inhibition of LIN28 exerts tumor suppressor functions by downregulating genes that promote LSC proliferation (MYC, RAS, IL-6, CCND) and survival (BCL-2) and indirectly inhibiting the NF-κB pathway.

[0130] Figures 2A-2D showed that Lin28b expression was increased in LSCs.

[0131] Figure 2A Shown is the Log2 expression of Lin28b in healthy HSCs compared to cells from various AML karyotypes, including inv(16), t(8;21), t(11q23) / MLL, complex karyotype, and normal karyotype.

[0132] Figure 2B Lin28b expression in non-DOX-induced LT-HSCs compared to DOX-induced MLL-AF9 WBM- and LT-HSC AML cells (→WBM-AML, LSCs) and relapses after Ara-C treatment (→rLSCs) is shown. Gene expression was normalized to Lin28b in non-DOX-induced LT-HSCs. n=5.

[0133] Figure 2C Shown are relative let-7a and -b miRNA expression in pre- and post-relapse LSCs normalized to non-induced LT-HSCs. n=3.

[0134] Figure 2DShown are CFC numbers of 1000WBM- or 100LT-HSC-derived AML cells treated with control 100 nM Ara-C or 30 μM 1632 (n=6) or transduced with shLin28b or shScramble (n=3) 7 days after seeding, ***P<0.001, **P<0.01, *P<0.05.

[0135] Figure 3A-3B It was shown that LN1632 inhibited LIN28B protein expression.

[0136] Figure 3A Western blots of Kasumi-1 and THP-1 cells treated with 1632 (120-160 μM) are shown.

[0137] Figure 3B Shown are TF1-α cells treated with 10 nM bortezomib (BZ), 120 μM 1632, or a combination thereof.

[0138] Figures 4A-4C Targeting Lin28 / let-7 inhibition was shown to abrogate AML growth.

[0139] Figure 4A Treatment with 100 mg / kg 1632 showed significant reduction in tumor growth (Figure). n = 5. SubQ implanted THP-1 cells (high LIN28B).

[0140] Figure 4B Treatment with 100 mg / kg 1632 showed minimal effect on tumor growth, n = 7. SubQ implanted MOLM-13 AML cells (no LIN28B).

[0141] Figure 4C Shown that treatment with 1632 at 100 mg / kg every other day prolonged survival (left) and reduced tumor burden (BLI, right panel, taken at d+26, as indicated by black arrows) in a systemic Kasumi-1 AML cell model. n = 5. Error bars represent SD.

[0142] Figures 5A-5C We show that targeted Lin28 inhibition downregulates LSC driver genes.

[0143] Figure 5A Heatmap shows the expression of direct (green) and indirect (black) let-7 target genes and pathways (MYC, NF-κB, JAK / STAT) downregulated in Kasumi-1 cells after treatment with 100 μM 1632 or control.

[0144] Figure 5B: Fold changes of miRNA and let-7 target genes evaluated in three primary AML patient samples after treatment with 80-120 μM 1632 or control, n=3. Error bars are SEM. ***P<0.001, **P<0.01, *P<0.05.

[0145] Figure 5C Figures showing gene set enrichment analysis (GSEA) evaluating changes in LSC gene signature in Kasumi-1 cells (GAL, top) and relapse prognosis in childhood AML (Yagi, bottom) after treatment with 100 μM 1632 or control. NES, normalized enrichment score; FDR q value, false discovery rate.

[0146] Figures 6A-6C We show that pharmacological LIN28 inhibition selectively abrogates LSC repopulation capacity in vivo.

[0147] Fig. 6A Shown are the CFC numbers of AML pt#13 and healthy donor CD34+ cells with and without treatment with 80-120 μM 1632, error bars represent SD, *P<0.05.

[0148] Figure 6B Shown is engraftment of primary human AML cells of pt#13 in NSGS 12 weeks after ex vivo treatment with 120 μM of control or 1632. ***P<0.001.

[0149] Figure 6C Representative flow cytometry gating scheme showing engraftment of human AML cells treated with 120 μM 1632 or control for 72 h 12 weeks after transplantation into NSGS.

[0150] Figure 7 The exemplary compounds of the present disclosure are shown to inhibit the binding of LIN28B to pre-let-7a. The compounds were bioscreened in triplicate at doses of 20, 5, and 1.25 μM. The signal response was corrected for compound autofluorescence. The dotted line represents the highest FRET signal achieved by the hit compound LN1632. All compounds above the dotted line have increased inhibitory activity on LIN28B / pre-let-7a-2 binding.

[0151] Figures 8A-8C Showing the binding of LN1632 to the ZKD motif of LIN28 and the upregulation of let-7.

[0152] Fig. 8A The predicted binding mode of LN1632 to ZKD of LIN28B. The red line indicates the close interaction and LN1632 is purple.

[0153] Figure 8B is the percentage (%) inhibition of LIN28B binding activity to pre-let-7a measured by increased FRET signal intensity. Values ​​were normalized to negative control treatment, n=3.

[0154] Figure 8C Relative levels of functional let-7 miRNA in HepG2 cells after treatment with LN1632 and analogs at concentrations of 3-10 μM are shown. Values ​​are normalized to total plasmid expression and control treatment, n = 6. **P < 0.01, error bars are SEM, ***P < 0.01.

[0155] Figures 9A-9C Delineating downregulation of cancer driver gene signatures by LN1632.

[0156] Fig.9A is a heat map showing the Marker_MYC_Target_V1 genes in Kasumi-1 cells after treatment with 40 μM LN1632 or control.

[0157] Fig. 9B Figures showing gene set enrichment analysis (GSEA) evaluating changes in LSC gene signature in Kasumi-1 cells (GAL, top) and relapse prognosis in childhood AML (Yagi, bottom) after treatment with 40 μM LN1632 or control. NES, normalized enrichment score; FDR q value, false discovery rate.

[0158] Fig. 9C Biological functional analysis of RNAseq data of Kasumi-1 cells after treatment with 40 μM LN1632 or control is shown. Ingenuity Pathway Analysis (IPA) predicts upstream inhibition of MYC and IL-6 pathways by differentially expressed genes in Kasumi-1 cells after treatment with 40 μM LN1632 or control (p-value: <0.05). The graph represents genes associated with specific biological functions that have changed in the uploaded dataset. Upregulated genes are shown in red nodes, and downregulated genes are shown in green nodes. The intensity of the color in the node indicates the degree of up (red) or down (green) regulation. The shape of the node reflects the functional category of each gene product: transcription regulator (horizontal ellipse), transmembrane receptor (vertical ellipse), enzyme (vertical diamond), cytokine / growth factor (square), kinase (inverted triangle), and complex / group / other (circle). Orange lines indicate predicted upregulation, while blue lines indicate predicted downregulation. Yellow lines indicate that expression contradicts the prediction. Gray lines indicate the direction in which no change was predicted. Solid or dotted lines indicate direct or indirect relationships, respectively.

[0159] Figures 10A-10BLN1632 was shown to be well tolerated in healthy C57BL / 6 female mice. Fig. 10A Is a series of graphs showing CBC (white blood cell count (WBC), neutrophils (NEU), lymphocytes (LYMPH), platelets (PLT), and hemoglobin (Hb)) levels in female C57Bl / 6 mice treated IP with 100 mg / kg of LN1632 daily for day +12, followed by every other day injections for day +9, n=5.

[0160] Fig. 10B Shows no significant changes in body weight gain after treatment with LN1632 or vehicle on day +21. n=5. Statistics: two-tailed Student's t-test, error bars are SEM. *P<0.05.

[0161] Figures 11A-11C Delineating the inhibition of cancer proliferation by LN1632 in vivo.

[0162] Fig.11A Daily treatment with 100 mg / kg LN1632 showed significant reduction in tumor growth, n = 5. Subcutaneously implanted THP-1 cells.

[0163] Fig. 11B Systemic Kasumi-1 AML xenografts are shown. Treatment with 100 mg / kg LN1632 every other day prolonged the survival and reduced tumor burden of systemic Kasumi-1 AML xenografts (pictures, taken on day +26). n=5.

[0164] Fig. 11C Subcutaneously implanted THP-1 cells exhibited inhibited proliferation when treated with LN1632, but to a lesser extent when treated with Ara-C. n = 3. Statistics: Two-tailed Student's t-test, ***P < 0.001, error bars are SEM.

[0165] Figures 12A-12C Target engagement of LN1632 is shown.

[0166] Fig. 12A Mass spectrometry cellular thermal shift assay (MS-CETSA) is shown: incubation with LN1632 induces a Tm shift of endogenous PRPF31 in Kasumi-1 cell lysates.

[0167] Fig. 12B Immunoprecipitation followed by mass spectrometry (IP-MS) of biotinylated LN1632 and competitive elution with non-labeled LN1632 captured PRPF31, n=3.

[0168] Fig. 12CCandidate targets of LN1632 identified by MS-CETSA and IP-MS are shown, sorted by abundance and overlapping factions.

[0169] Fig.13 The correlation of PRPF31 overexpression with poor prognosis is shown. Kaplan-Meier overall survival curves for different cancer patient cohort analyses. p-values ​​were calculated using the log-rank test. Vertical hash marks indicate censored data. Comparison of survival curves of patients with high (red) and low (black) PRPF31 expression.

[0170] Figures 14A-14D The dependence of TNBC proliferation on PRPF31 was shown.

[0171] Fig.14A The cell number of TNBC cells treated with pCMV-PRPF31 expression plasmid (red), control vector (pCMV-empty, black), shPRPF31 (green), or the combination of pCMV-PRPF31+100 μM LN1632, pCMV-GFP+100 μM LN1632, or shPRPF31+100 μM LN1632 is shown. n=3.

[0172] Fig. 14B Shown are % cell viability of MDA-MB-231 cells assessed by cell potency luminescence after 4 days of treatment with LN1632, JGJ023, JGJ034 or palbociclib at increasing doses, n=2.

[0173] Fig. 14C Shown are cell numbers of MDA-MB-231 cells incubated with control (DMSO), 16 μM JGJ023, or 16 μM palbociclib at days +6, +9, and +12 post-treatment, n=2.

[0174] Fig.14D Direct comparison of cell numbers of MDA-MB-231 cells at d+6 after treatment with 16 μM JGJ023 or 16 μM palbociclib is shown, n = 2. Statistics: Dose response curves for IC50 calculations were plotted as four-parameter linear regressions, two-tailed Student's t-tests for individual comparisons, error bars are SD, *P<0.05, **P<0.01.

[0175] Figures 15A-15C Apoptosis induction by LN1632 and its novel analogs in castration-resistant prostate cancer is depicted.

[0176] Fig.15A Shown are the % cell viability of CRPC LNCaP cells expressing wild-type androgen receptor after 4 days of treatment with LN1632, JGJ007, JGJ023, or standard of care enzalutamide.

[0177] Fig. 15B Shown is the % cell viability of metastatic CRPC 22Rv1 cells expressing mutant androgen receptor (ARV7) after 4 days of treatment with LN1632, JGJ007, JGJ023, or standard of care enzalutamide.

[0178] Fig. 15C Cell numbers are shown for 22Rv1 cells incubated with control (DMSO), 2 μM JGJ023, or 42 μM enzalutamide at days +5, +7, and +9 after treatment. Inset: JGJ023 induces apoptosis and reduces cell numbers in mCRPC compared to enzalutamide. n=3 for all experiments. Statistics: IC 50 The calculated dose-response curves were plotted as four-parameter linear regressions, and individual doses were compared with two-tailed Student's t-tests, error bars are SD, *P < 0.05, **P < 0.01.

[0179] Figures 16A-16C LN1632 and its novel analogs were shown to induce apoptosis and inhibit proliferation in colorectal cancer.

[0180] Fig.16A Shown is the cell viability % (87) of low MYC expressing epithelial CRC cells SW948 after 4 days of treatment with increasing doses of JGJ034 or standard of care cetuximab (EGFR monoclonal antibody).

[0181] Fig. 16B Shown is the cell viability % (88) of adenocarcinoma CRC cells SW480 with low MYC amplification after 4 days of treatment with escalating doses of JGJ034 or standard of care cetuximab.

[0182] Fig. 16C Shown is the % cell viability of cetuximab-resistant, metastatic adenocarcinoma CRC cells SW620 with high MYC amplification after 5 days of treatment with JGJ034 or standard of care cetuximab 39 n = 3 for all experiments. Statistics: Dose response curves for IC50 calculations were plotted as four-parameter linear regressions with error bars as SD. DETAILED DESCRIPTION

[0183] Compound

[0184] The present disclosure provides a compound of formula (I):

[0185]

[0186] or a pharmaceutically acceptable salt thereof, wherein:

[0187] Selected from

[0188] Ring B is selected from phenyl and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

[0189] X is selected from N and C;

[0190] X 1 , X 3 and X 4 Each independently selected from N and CR x ;

[0191] R 1 is hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, and 5- to 6-membered heteroaryl rings having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

[0192] Each R 2 independently selected from halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 aliphatic group;

[0193] R 3 is selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

[0194] Each R x are independently selected from hydrogen, halogen and optionally substituted C 1-6 aliphatic group;

[0195] Each R is independently selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and

[0196] n is 0-3.

[0197] In some embodiments of Formula (I), for Therefore, in some embodiments, the present disclosure provides a compound of formula (Ia):

[0198]

[0199] or a pharmaceutically acceptable salt thereof, wherein ring B, X 1 , X 3 , X 4 , R 1 , R 2 and n are each as defined above and described herein.

[0200] In some embodiments of Formula (I), for Therefore, in some embodiments, the present disclosure provides a compound of formula (Ib):

[0201]

[0202] or a pharmaceutically acceptable salt thereof, wherein ring B, X 1 , X 4 , R 1 , R 2 , R and n are each as defined above and described herein.

[0203] As generally defined above, X 1 Selected from N and CR x In some embodiments of any of Formulas (I), (Ia), and (Ib), X 1 is N. Therefore, in some embodiments, the present disclosure provides a compound of formula (Iai) or (Ibi):

[0204]

[0205] or a pharmaceutically acceptable salt thereof, wherein ring B, X 3 , X 4 , R 1 , R 2 , R and n are each as defined above and described herein.

[0206] In some embodiments of any of Formulas (I), (Ia), and (Ib), X 1 CR x Therefore, in some embodiments, the present disclosure provides a compound of formula (Ia-ii) or (Ib-ii):

[0207]

[0208] or a pharmaceutically acceptable salt thereof, wherein ring B, X 3 , X 4 , R 1 , R 2 , R, R x and n are each as defined above and described herein.

[0209] As generally defined above for formula (I), Ring B is selected from phenyl and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments of Formula (I), (Ia), (Iai), (Ia-ii), (Ib), (Ibi) and (Ib-ii), Ring B is phenyl. Thus, in some embodiments, the present disclosure provides a compound of Formula (Ia-iii), (Ia-iv), (Iav), (Ib-iii), (Ib-iv) and (Ibv):

[0210]

[0211]

[0212] or a pharmaceutically acceptable salt thereof, wherein X 1 , X 3 , X 4 , R 1 , R 2 , R, R x and n are each as defined above and described herein.

[0213] In some embodiments of Formula (I), (Ia), (Iai), (Ia-ii), (Ib), (Ibi), and (Ib-ii), Ring B is

[0214] In some embodiments of Formula (I), (Ia), (Iai), (Ia-ii), (Ib), (Ibi) and (Ib-ii), Ring B is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments of Formula (I), (Ia), (Iai), (Ia-ii), (Ib), (Ibi) and (Ib-ii), Ring B is a 5-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments of Formula (I), (Ia), (Iai), (Ia-ii), (Ib), (Ibi) and (Ib-ii), Ring B is a 6-membered heteroaryl ring having 1-2 nitrogen atoms, such as pyridinyl.

[0215] As generally defined above for formula (I), X 3 Selected from N and CR x In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), and (Iav), X 3is N. In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv) and (Iav), X 3 CR x .

[0216] As generally defined above for formula (I), X 4 Selected from N and CR x In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), X 4 is N. In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv) and (Ibv), X 4 CR x .

[0217] As generally defined above for formula (I), R 1 is hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 is an optionally substituted group selected from the following: C 1-6aliphatic, phenyl, and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 is an optionally substituted C 1-6 In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 is an optionally substituted C 1-3 Aliphatic groups, such as CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 or CH(CH 3 ) 2 In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 is an optionally substituted 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1is an optionally substituted 5-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R 1 is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms, for example pyridinyl or pyrimidinyl.

[0218] As generally defined above for formula (I), R 2 Selected from halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv) and (Ibv), at least one R 2 NO 2 In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 is OR, for example OMe.

[0219] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 N(R) 2In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 For NHR, such as NH 2 .

[0220] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 N(R)C(O)R, for example N(CH 3 )C(O)CH 3 In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 NHC(O)R, for example NHC(O)CH 3 .

[0221] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 For CO 2 R, such as CO 2 H.

[0222] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 C(O)N(R) 2In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 is C(O)N(H)R, for example C(O)NHCH 3 .

[0223] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 is an optionally substituted C 1-6 In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), at least one R 2 is an optionally substituted C 1-3 Aliphatic group.

[0224] As generally defined above for formula (I), each R x are independently selected from hydrogen, halogen and optionally substituted C 1-6 In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R x In other embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R x are independently selected from halogen and optionally substituted C 1-6In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R x is a halogen such as fluorine or chlorine.

[0225] In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R x is an optionally substituted C 1-6 In other embodiments, R x is an optionally substituted C 1-3 Aliphatic groups, such as CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 or CH(CH 3 ) 2 .

[0226] As generally defined above for formula (I), each R is independently selected from hydrogen or an optionally substituted group selected from: C 1-6 aliphatic; a 3- to 7-membered monocyclic carbocyclic ring; a 3- to 7-membered monocyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; a phenyl ring; a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R is hydrogen. In some embodiments of any one of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R is independently selected from an optionally substituted group selected from C 1-6aliphatic; a 3- to 7-membered monocyclic carbocyclic ring; a 3- to 7-membered monocyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; phenyl; a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R is an optionally substituted C 1-6 In some embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), R is an optionally substituted C 1-3 In some such embodiments, R is CH 3 or CH 2 CH 3 .

[0227] As generally defined above for Formula (I), n is 0-3. In some embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), n is 1-2. In other embodiments of any of Formulas (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), n is 0. In other embodiments of any of Formula (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), n is 1. In other embodiments of any of Formula (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), n is 2. In other embodiments of any of Formula (I), (Ia), (Iai), (Ia-ii), (Ia-iii), (Ia-iv), (Iav), (Ib), (Ibi), (Ib-ii), (Ib-iii), (Ib-iv), and (Ibv), n is 3.

[0228] In some embodiments of any of the disclosed compounds, R 1 C 1-6 In other embodiments, R 1 In other embodiments, R 1 is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from oxygen, nitrogen and sulfur. In other embodiments, wherein R 1 is a 5-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments, wherein R 1 is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R 1 is a 6-membered heteroaryl ring having 1-2 nitrogen atoms, for example

[0229] In some embodiments of any of the disclosed compounds, Rx In other embodiments, R x is halogen or optionally substituted C 1-6 In other embodiments, R x is an optionally substituted C 1-6 In other embodiments, R x For unsubstituted C 1-6 Aliphatic groups, such as methyl.

[0230] In some embodiments of any of the disclosed compounds, R 2 Selected from halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 In other embodiments, R 2 is halogen, such as fluorine. In other embodiments, R 2 NO 2 In other embodiments, R 2 is OR, such as OCH 3 In other embodiments, wherein R 2 N(R) 2 , such as NH 2 In other embodiments, R 2 N(R)C(O)R, for example NHC(O)CH 3 or N(CH 3 )C(O)CH 3 In other embodiments, R 2 For CO 2 R, such as CO 2 H. In other embodiments, R 2 C(O)N(R) 2 , such as C(O)NHCH 3 In other embodiments, R 2 is an optionally substituted C 1-6 Aliphatic groups, such as CF 3 .

[0231] In some embodiments of any of the disclosed compounds, R is hydrogen. In other embodiments, R is an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments, R is an optionally substituted C 1-6In other embodiments, R is unsubstituted C 1-6 Aliphatic groups, such as methyl.

[0232] In some embodiments of any of the disclosed compounds, R 3 In other embodiments, R 3 is an optionally substituted group selected from the following: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments, R 3 is an optionally substituted C 1-6 In other embodiments, R 3 For unsubstituted C 1-6 Aliphatic groups, such as methyl.

[0233] In some embodiments of any of the disclosed compounds, R 3 In other embodiments, R 3 is an optionally substituted group selected from the following: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In other embodiments, R 3 is an optionally substituted C 1-6 In some embodiments, R 3 For unsubstituted C 1-6 Aliphatic groups, such as methyl.

[0234] In some embodiments of any disclosed compound, n is 0. In other embodiments, n is 1. In other embodiments, n is 2. In some embodiments, the disclosure provides a compound selected from the following:

[0235]

[0236] or a pharmaceutically acceptable salt thereof.

[0237] In some embodiments, the present disclosure provides a compound selected from the group consisting of:

[0238]

[0239]

[0240] or a pharmaceutically acceptable salt thereof.

[0241] In some embodiments, the present disclosure provides a compound of formula (II):

[0242]

[0243] or a pharmaceutically acceptable salt thereof, wherein:

[0244] R 1 C 1-6 Alkyl or C 3-6 Cycloalkyl;

[0245] R 2 is H, amino, nitro or amido; and

[0246] X 1 , X 3 and X 4 are each independently N or CH.

[0247] In some embodiments, X 1 , X 3 and X 4 At least one of is N. In other embodiments, X 1 , X 3 and X 4 At least two of X are N. In other embodiments, X 1 , X 3 and X 4 Each of X is N. In other embodiments, X 1 and X 3 Each is N, and X 4 For CH.

[0248] In some embodiments, R 1 For unsubstituted C 1-6 In other embodiments, R 1 is methyl optionally substituted by halogen. In other embodiments, R 1 C 2-6 Alkyl or C 3-6 Cycloalkyl.

[0249] In some embodiments, R 2 is H, amino, nitro or –N(R 5 )C(O)R 6 ; R 5 H or C 1-5 alkyl; and

[0250] R 6 C 1-6 In other embodiments, R 2 N(R 5 )C(O)R6 , R 5 is H, and R 6 is C 1-6 alkyl. In other embodiments, R 2 is –N(R 5 )C(O)R 6 , R 5 is H, and R 6 is CH 3 . In other embodiments, R 2 is H, amino or nitro. In other embodiments, R 2 is NO 2 or –N(R 5 )C(O)R 6 .

[0251] In some embodiments, wherein the compound is:

[0252]

[0253] or a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, wherein the compound is JGJ002, JGJ003, JGJ004, JGJ005, JGJ007 or JGJ008, or a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, wherein the compound is JGJ007 or JGJ088, or a pharmaceutically acceptable salt thereof.

[0256] In some embodiments of formula (II), X 1 is N. Accordingly, in some embodiments, the present disclosure provides a compound of formula (II-a):

[0257]

[0258] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , X 3 and X 4 are each as defined above and described herein.

[0259] In some embodiments of formula (II), X 3 is N. Accordingly, in some embodiments, the present disclosure provides a compound of formula (I-b):

[0260]

[0261] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , X1 and X 4 Each is as defined above and described herein.

[0262] In some embodiments of Formula (II-a), X 3 is N. Therefore, in some embodiments, the present disclosure provides a compound of formula (Iai):

[0263]

[0264] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 and X 4 Each is as defined above and described herein.

[0265] As generally defined above for formula (II), R 1 C 1-6 Alkyl or C 3-6 In other embodiments of formula (II), (II-a), (II-b) and (II-ai), R 1 C 1-6 In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 1 C 1-3 Alkyl groups, such as R 1 CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 or CH(CH 3 ) 2 .

[0266] In other embodiments of any of Formulas (II), (II-a), (II-b), and (II-ai), R 1 C 3-6 In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 1 In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 1 It is cyclopentyl or cyclohexyl.

[0267] As generally defined above for formula (II), R 2is H, amino, nitro or acylamino. In some embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 2 is H. In other embodiments of any one of Formulas (II), (II-a), (II-b) and (II-ai), R 2 is amino, nitro or acylamino. In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 2 In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 2 In other embodiments of any one of formula (II), (II-a), (II-b) and (II-ai), R 2 is an acylamino group (e.g. ).

[0268] In some embodiments, the amino group is N(R) 2 .

[0269] In some embodiments, the amino group is N(R)C(O)R.

[0270] In some embodiments of any of the disclosed compounds, the compound is not

[0271] Pharmaceutical compositions and uses thereof

[0272] In some embodiments, the present disclosure provides the recognition that approaches targeting ribonucleic acid (RNA)-RNA binding protein (RBP) interactions constitute an emerging alternative approach to significantly expand the druggable proteome and genome and overcome intrinsic and acquired drug resistance.

[0273] In certain aspects, the disclosure further provides insights into the critical roles that RBPs play in cellular physiology by regulating RNA processing, translation, and turnover. In neoplasms, dysregulated expression of RBPs supports expression of alternatively spliced, modified, and stabilized RNA transcripts associated with cancer self-renewal, proliferation, and its adaptation to stress. In some embodiments, the disclosure provides compounds that modulate different RBP-protein interactions and thus represent a novel therapeutic approach for treating cancer and other diseases with dysfunctional RNA regulation.

[0274] MicroRNAs (miRNAs) are short noncoding RNAs of 19-22 nucleotides (nt) that hybridize to complementary mRNA targets and cause their decay, cleavage, or transcriptional repression (5-7). Aberrant miRNA expression has been shown to play an active role in malignant transformation, including leukemias (8-10). Specifically, AML let-7b and let-7c miRNAs were found to be significantly downregulated in core binding factor (CBF) leukemias with inv(16), t(8;21), and MLL / t(11q23) (11)(12). A systematic evaluation of the prognostic value of miRNA expression in many human cancers, including several AML subtypes, found that reduced expression of let-7 miRNA was generally associated with a poor prognosis (10, 13, 14). The let-7 tumor suppressor miRNA family contains 12 members that are differentially transcribed from eight chromosomal loci and inhibit several cancer stem cell oncogenes including KRAS, MYC, IL6, and HMGA1 / 2 as well as cell cycle regulators such as CCND1 / 2 and E2F( Figure 1 )(15, 16). In 2008, a large number of papers described LIN28A and its homolog LIN28B (hereafter referred to as LIN28) as key regulators of let-7 biogenesis by directly binding to pre-let-7 and / or pri-let-7, thereby impairing their processing into mature functional miRNAs (17-21). In fact, LIN28 is upregulated in more than 15% of human cancers (22) and cancer stem cells (CSCs) (23-27).

[0275] Structural studies have shown that the C-terminal zinc-knuckle domain (ZKD) of Lin28 binds to a highly conserved GGAG motif within the 3'-terminal loop of pri- / pre-let-7 (28-30). This binding allows the recruitment of the TUT enzyme to polyuridylate pre / pri-let-7, thereby preventing the maturation of let-7 miRNA (19, 31). As a result, reduced let-7 miRNA leads to overexpression of its directly regulated oncogenic target genes.

[0276] The RNA binding proteins LIN28A and LIN28B are overexpressed in many cancers, and high LIN28 protein is associated with decreased patient survival (54). LIN28A / B (hereafter referred to as Lin28) impairs the processing of functional mature let-7 microRNA (miRNA) by binding its C-terminal zinc knuckle domain (ZKD) to the highly conserved GGAG motif within the 3' terminal loop of pri- / pre-let-7 (17-21, 28-30). As a result, in some embodiments, reduced let-7 miRNA leads to overexpression of its direct oncogenic target genes, such as MYC, KRAS, and CCND1. In addition to its ability to inhibit let-7 miRNA biogenesis, Lin28 has been shown to bind to mRNA transcripts of the insulin-like growth factor 2 protein (Igf2), thereby affecting their abundance and / or translation (69, 70).

[0277] In various cancers, increasing evidence indicates that LIN28 overexpression (32-34) and let-7 loss (35-37) are associated with CSC resistance to radiotherapy and chemotherapy, ultimately leading to reduced overall survival. Specifically, in AML, dysregulated LIN28 / let-7 has been shown to promote leukemogenesis through an LSC-like transcriptional program and is associated with poor clinical outcome (38). In bone marrow aspirates of refractory AML patients, let-7a has been found to confer Ara-C chemotherapy resistance through the BCL-2 family member BCL-XL (39). Importantly, several studies have highlighted that specific overexpression of BCL-2 and BCL-XL in AML and LSC is associated with chemotherapy resistance and poor overall / disease-free survival (40-43). In addition, let-7 miRNA targets IL6 and RAS, two well-known genetic drivers of the NF-κB pathway, another important regulator of LSC homeostasis (44)( Figure 1 ).

[0278] Emerging evidence suggests that NF-κB and BCL-2 are activated in LSCs, but not in hematopoietic stem cells (HSCs), as core components of the proinflammatory cellular stress response (45, 46). Therefore, therapeutic inhibition of LIN28 and, consequently, upregulation of let-7 may selectively kill LSCs. Given the fundamental role of Lin28 / let-7 in leukemia and other CSCs and its relevance to therapy resistance, it is conceivable that targeted inhibition of LIN28 may represent a novel approach for precision AML therapy. Notably, studies of conditional Lin28a and Lin28b knockout mice revealed that embryonic, but not neonatal or adult, Lin28 deficiency resulted in growth defects (47), suggesting that Lin28 has heterochronic effects. Furthermore, in mice, Lin28b expression was found to be reduced in hematopoietic stem cells (48, 49), consistent with the accumulation of mature let-7 in common myeloid progenitors during hematopoietic maturation (50). Therefore, therapeutic inhibition of LIN28 and the resulting upregulation of let-7 miRNA could selectively kill LSCs but would likely be highly tolerated by healthy tissues.

[0279] To date, five high-throughput screens (HTS) have been reported with the goal of identifying pharmacologically active compounds that disrupt LIN28 binding to pre-let-7 miRNAs. We used FRET-HTS to screen 16,000 drug-like organic compounds and identified the first hit compound 501632 (51) (hereafter referred to as LN1632) as binding to LIN28B and selectively upregulating let-7 miRNA levels and inducing differentiation in mouse embryonic stem cells (51). Lim et al. (52) screened an in-house library and identified a benzopyranylpyrazole-based compound as the main hit molecule, while Lightfoot et al. used a biophysical assay to identify 6-hydroxy-DL-DOPA and benzo[a]phenoxazine, which inhibited Lin28 / let-7 interaction in vitro. The Sliz group developed a fluorescence polarization HTS and identified LI71 and TPEN, the latter being a potent ZKD domain inhibitor (53). Despite the increasing number of reported small-molecule inhibitors of the Lin28 / let-7 interaction, the pharmacological inhibition of LIN28 in vivo for targeted AML and LSC therapies has not yet been established. In addition, small-molecule inhibitors with high specificity for LIN28 that inhibit its activity have not yet been developed.

[0280] The present disclosure reports the in vitro and in vivo inhibition of Lin28 and Lin28 / let-7 by compounds of formula (I) or (II):

[0281] As described herein, compounds of formula (I) and (II) exhibit Lin28 / let-7 inhibitory activity in an in vitro FRET assay and in LSC and LSC-like Kasumi-1 cells. FRET assays were performed as previously described (51).

[0282] Similarly, compounds of formula (I) and (II) were shown to inhibit protein-RNA interactions, particularly Lin28 / let-7 and PRPF31 / U4, both in vitro and in vivo.

[0283] The present disclosure provides a method for treating cancer, the method comprising administering a compound or composition as described herein to a subject suffering from cancer or showing symptoms of cancer. In some embodiments, the method comprises treating or improving one or more symptoms of cancer. In some embodiments, cancer is a blood cancer, such as acute myeloid leukemia. In some embodiments, the method comprises administering a compound or composition with an amount of cancer cell proliferation determined to achieve cancer cell inhibition and / or reduction or according to a dosing regimen for cancer cell proliferation determined to achieve cancer cell inhibition and / or reduction. In some embodiments, cancer cells include cancer stem cells. In some embodiments, cancer stem cells include leukemia stem cells (LSC). In some embodiments, the method comprises administering a compound or composition with an amount of cancer cell proliferation determined to achieve cancer cell inhibition and / or reduction or according to a dosing regimen for cancer cell proliferation determined to achieve cancer cell inhibition and / or reduction, wherein the assay shown in Example 3 or 5 or a similar assay is used to assess cancer cell inhibition and / or reduced cancer cell proliferation.

[0284] In some embodiments, the present disclosure provides a method of modulating splicing, the method comprising contacting a system having splicing capability with a compound described herein.

[0285] In some embodiments, the present disclosure provides a method comprising:

[0286] contacting a splicing-competent system with a compound as described herein; and assessing in said system:

[0287] (i) the presence or level of splicing products (e.g., spliced ​​transcripts);

[0288] (ii) expression or localization of RNA; and / or

[0289] (iii) Expression or folding of polypeptides

[0290] In some embodiments, the disclosure provides a method of modulating splicing in a system having splicing capability by contacting the system with a compound described herein, thereby observing one or more of the following:

[0291] (i) reduced RNA splicing;

[0292] (ii) altered RNA expression or localization; and / or

[0293] (iii) altered polypeptide expression or folding.

[0294] In some embodiments, the present disclosure provides a method comprising contacting a system having splicing ability with a compound described herein, wherein the compound is characterized in that when contacted with a cancer cell, the compound reduces the proliferation of the cancer cell relative to the proliferation observed when it is not present. In some embodiments, splicing is reduced when the compound is present compared to when the compound is not present. In some embodiments, the method also includes evaluating splicing in the system compared to a reference condition. In some embodiments, the reference condition is the absence of the compound. In some embodiments, the reference condition is the presence of a control compound. In some embodiments, the reference condition is a historical condition. In some embodiments, the compound inhibits one or more properties of a splicing machinery component and / or wherein the compound inhibits interactions between or among splicing machinery components. In some embodiments, the compound directly binds to one or more splicing machinery components or a complex thereof. In some embodiments, the splicing machinery component is an RNA component. In some embodiments, the splicing machinery component is a polypeptide component. In some embodiments, the splicing machinery component is selected from an RNA component, a polypeptide component, and a complex thereof or therebetween. In some embodiments, the RNA component is or includes a small nuclear RNA (snRNA). In some embodiments, the snRNA is selected from U1, U2, U4, U5 and U6. In some embodiments, the polypeptide component is or includes an Sm polypeptide or an Lsm polypeptide. In some embodiments, the polypeptide component is selected from Prp3, Prp31, Prp4, CypH, 15.5K, Prp8, Brr2, Snu114, Prp6, Prp28, 40K, Dib1, Snu66, Sad1 and 27K. In some embodiments, the splicing machinery component includes a Prp31 polypeptide. In some embodiments, the splicing machinery component includes U4 snRNA, U6 snRNA and a Prp31 polypeptide component. In some embodiments, the compound inhibits the interaction between: U6 snRNA and Prp31 polypeptide; or U4 snRNA and Prp31 polypeptide. In some embodiments, the compound inhibits the activity of the Prp31 polypeptide.

[0295] In some embodiments, contacting occurs in vitro, ex vivo, or in vivo. In some embodiments, the splicing-competent system is a cancer cell. In some embodiments, the splicing-competent cancer cell comprises a cancer stem cell. In some embodiments, the splicing-competent cancer stem cell comprises a leukemia stem cell (LSC).

[0296] The compositions and methods of the present invention can be used to treat individuals in need. In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When applied to an animal such as a human, the composition or compound is preferably applied in the form of a pharmaceutical composition, which comprises, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions, such as water or physiologically buffered saline or other solvents or vehicles, such as glycols, glycerol, oils such as olive oil or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are applied to humans, particularly for invasive routes of administration (i.e., such as injections or implants that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of an agent or selectively target one or more cells, tissues or organs. Pharmaceutical compositions can be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, lyophilized agents for reconstruction, powders, solutions, syrups, suppositories, injections, etc. The composition may also be presented in a transdermal delivery system, such as a skin patch. The composition may also be presented in a solution suitable for topical administration, such as a lotion, cream or ointment.

[0297] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent, which, for example, acts to stabilize a compound (such as a compound of the present invention), increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, into which, for example, a compound of the present invention may be incorporated. For example, liposomes comprising phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0298] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0299] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0300] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of routes of administration, including, for example, oral administration (e.g., an infusion in water or non-aqueous solutions or suspensions for application to the tongue, tablets, capsules (including dispersible capsules and gelatin capsules), boluses, powders, granules, pastes); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment or spray applied to the skin). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0301] The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. In general, in one hundred parts, this amount will be in the range of about 1% to about 99% active ingredient, preferably about 5% to about 70%, and most preferably about 10% to about 30%.

[0302] The methods for preparing these formulations or compositions include the step of bringing into association the active compound (e.g., a compound of the present invention) with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0303] Formulations of the invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilizates, powders, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or as a mouthwash, etc., each containing a predetermined amount of the compound of the invention as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

[0304] To prepare solid dosage forms for oral administration (capsules (including dispersible capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) humectants, such as glycerol Oils; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including dispersible capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose (lactose / milk sugar) and high molecular weight polyethylene glycols.

[0305] Tablets can be prepared by compression or molding, optionally containing one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium carboxymethyl starch or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0306] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules may be optionally scored or prepared with coatings and shells, such as enteric coatings or other coatings well known in the art of pharmaceutical formulation. They may also be formulated to provide slow release or controlled release of the active ingredients contained therein using, for example, hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions for providing the desired release characteristics. They may be sterilized by, for example, filtering through a filter that retains bacteria or by incorporating a sterilizing agent in the form of a sterile solid composition soluble in sterile water or some other sterile injectable medium just before use. These compositions may also optionally contain an opacifier and may have a composition in which they release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, with one or more of the above-mentioned excipients, where appropriate.

[0307] Liquid dosage forms that can be used for oral administration include pharmaceutically acceptable emulsions, lyophilized agents for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0308] Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0309] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0310] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0311] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0312] Powders and sprays may contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0313] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of this invention to the body. Such dosage forms can also be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or a gel.

[0314] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0315] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0316] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc., may be added to ensure that the effects of microorganisms are prevented. It may also be necessary to include isotonic agents, such as sugar, sodium chloride, etc. in the composition. In addition, extended absorption of injectable drug forms may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0317] In some cases, in order to prolong the effect of the drug, it is necessary to slow down the absorption of the subcutaneous or intramuscular injection of the drug. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenteral drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0318] Injectable depot forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the properties of the specific polymer used, the release rate of the drug can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by embedding the drug in liposomes or microemulsions compatible with body tissues.

[0319] For use in the methods of the invention, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0320] The method of introduction can also be provided by a rechargeable or biodegradable device. Regarding the controlled delivery of drugs (including protein biopharmaceuticals), various slow-release polymer devices have been developed and tested in vivo in recent years. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants to continuously release compounds at specific target sites.

[0321] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0322] The selected dosage level will depend upon a variety of factors including the activity of the specific compound or combination of compounds employed, or the esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the specific compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0323] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means the concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the subject's weight, sex, age and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the condition being treated, the stability of the compound, and (if necessary) the stability of another type of therapeutic agent administered with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th Edition, 1814-1882, incorporated herein by reference).

[0324] In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0325] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. In certain embodiments of the invention, the active compound may be administered twice or three times a day. In a preferred embodiment, the active compound will be administered once a day.

[0326] Patients receiving such treatment are any animal in need thereof, including primates, particularly humans; other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0327] In certain embodiments, the compounds of the invention may be used alone or in combination with another type of therapeutic agent.

[0328] The disclosure includes the pharmaceutically acceptable salts of the compounds of the present invention for use in the compositions and methods of the present invention. In certain embodiments, the salts considered by the present invention include but are not limited to alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the salts considered by the present invention include but are not limited to L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc salts. In certain embodiments, the salts considered by the present invention include but are not limited to Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, the salts contemplated by the present invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxy-ethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid / decanoic acid, caproic acid / hexanoic acid, caprylic acid / octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, acid), dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, phenylglycolic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, niacin, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0329] Pharmaceutically acceptable acid addition salts may also exist in the form of various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or insoluble in such solvents.

[0330] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0331] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0332] definition

[0333] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and techniques used in conjunction with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0334] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th Edition", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th Edition", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th Edition", Sinauer Associates, Inc., Sunderland, MA (2000).

[0335] Unless otherwise defined herein, chemical terms used herein are used according to conventional usage in the art as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., ed., McGraw-Hill, San Francisco, CA (1985).

[0336] All of the above, as well as any other publications, patents, and published patent applications mentioned in this application, are expressly incorporated herein by reference. In the event of a conflict, the present specification, including its specific definitions, will control.

[0337] The term "agent" as used herein refers to a compound (such as an organic or inorganic compound, a mixture of compounds), a biomacromolecule (such as a nucleic acid, an antibody, including parts thereof and humanized, chimeric and human antibodies and monoclonal antibodies, a protein or a part thereof, such as a peptide, a lipid, a carbohydrate) or an extract made from a biological material such as a bacterium, a plant, a fungus or an animal (particularly a mammal) cell or tissue. Agents include, for example, agents with known structures and agents with unknown structures.

[0338] "Patient", "subject" or "individual" are used interchangeably and refer to human or non-human animals. These terms include mammals, such as humans, primates, livestock animals (including cattle, pigs, etc.), companion animals (such as canines, felines, etc.), and rodents (such as mice and rats).

[0339] "Treatment" of a disease or patient refers to taking steps to obtain beneficial or desired results, including clinical results. As used herein and as well understood in the art, "treatment" is a means for obtaining beneficial or desired results (including clinical results). Beneficial or desired clinical results may include, but are not limited to, the mitigation or improvement of one or more symptoms or diseases, the reduction of the extent of the disease, the stabilization (i.e., no deterioration) of the disease state, the prevention of disease spread, the delay or slowing of the disease process, the improvement or alleviation of the disease state, and relief (whether partial relief or total relief), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to the survival expected when not receiving treatment.

[0340] The term "prevention" is art-recognized and when used with respect to a condition such as a local recurrence (e.g., pain), a disease such as cancer, a symptom such as heart failure, or any other medical condition, is well known in the art and includes administering a composition that reduces the frequency of symptoms of the medical condition, or delays the onset thereof, relative to a subject not receiving the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a patient population receiving prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0341] "Administering" a substance, compound or agent to a subject or "administering" a substance, compound or agent can be implemented using one of the various methods known to those skilled in the art. For example, a compound or agent can be administered by intravenous, intraarterial, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, oral (by ingestion), intranasal (by inhalation), intraspinal, intracerebral, and transdermal (by absorption, for example, through a skin tube). A compound or agent can also be appropriately introduced by a rechargeable or biodegradable polymer device or other device (such as a patch and pump) or a preparation that provides extended, slow or controlled release of a compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0342] The appropriate method of administering a substance, compound or agent to a subject will also depend on, for example, the age and / or physical condition of the subject and the chemical and biological properties (e.g., solubility, digestibility, bioavailability, stability and toxicity) of the compound or agent. In some embodiments, the compound or agent is orally administered to the subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a prolonged release or slow release formulation, or is administered using a device for this slow or prolonged release.

[0343] As used herein, the phrase "combined administration" refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while the previously administered therapeutic agent is still effective in vivo (e.g., both agents are effective in the patient simultaneously, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effects of the different therapeutic agents.

[0344] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administering a single dose, but may only occur after a series of doses are administered. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required for a subject will depend, for example, on the subject's size, health, and age, and the nature and extent of the condition being treated (such as cancer or MDS). A skilled artisan can readily determine the effective amount for a given situation by routine experimentation.

[0345] Association: If the presence, level, degree, type and / or form of one event or entity is related to the presence, level, degree, type and / or form of another, then two events or entities are "associated" with each other, as the term is used herein. For example, if the presence, level and / or form of a particular entity (e.g., a polypeptide, a genetic feature, a metabolite, a microorganism, etc.) is related to the incidence and / or susceptibility of a disease, disorder or condition (e.g., in a relevant population), it is considered to be associated with a particular disease, disorder or condition. In some embodiments, if two or more entities interact directly or indirectly so that they are physically close to each other and / or remain in physical proximity, they are physically "associated" with each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0346] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition sets that may be different from each other, but are similar enough to allow comparisons between them, so that those skilled in the art will understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, comparable conditions, environments, individuals or population sets are characterized by a plurality of substantially identical features and one or a small amount of different features. In the context, those of ordinary skill in the art will understand that in any given case, two or more such agents, entities, situations, condition sets require what degree of identity to be considered comparable. For example, those of ordinary skill in the art will understand that when characterized by a sufficient number and type of substantially identical features, environments, individuals or population sets are comparable to each other to ensure that the differences in the results or observed phenomena obtained according to or using different environments, individuals or population sets are caused by changes in those different features or indicate a reasonable conclusion of the changes.

[0347] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0348] Inhibitor: As used herein, the term "inhibitor" (inhibitory agent) refers to an entity, condition, or event whose presence, level, or extent is associated with a reduced level or activity of a target. In some embodiments, an inhibitor can act directly (in which case it exerts its effect on its target directly, such as by binding to the target); in some embodiments, an inhibitor can act indirectly (in which case it exerts its effect by interacting with and / or otherwise altering a modulator of the target, thereby reducing the level and / or activity of the target). In some embodiments, an inhibitor is an inhibitor whose presence or level is associated with a reduced level or activity of a target relative to a particular reference level or activity (e.g., a level or activity observed under appropriate reference conditions, such as the presence of a known inhibitor, or the absence of the inhibitor in question).

[0349] Reference: As used herein, describes a standard or control with which it is compared. For example, in some embodiments, an agent, animal, individual, colony, sample, sequence or value of interest is compared to a reference or control agent, animal, individual, colony, sample, sequence or value. In some embodiments, the test and / or determination of the reference or control is performed substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is measured or characterized under conditions or environments comparable to those being evaluated. Those skilled in the art will understand when there is enough similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0350] Small molecule: As used herein, the term "small molecule" means a low molecular weight organic and / or inorganic compound. Typically, a "small molecule" is a molecule of less than about 5 kilodaltons (kD). In some embodiments, a small molecule is less than about 4kD, 3kD, about 2kD, or about 1kD. In some embodiments, a small molecule is less than about 800 daltons (D), about 600D, about 500D, about 400D, about 300D, about 200D, or about 100D. In some embodiments, a small molecule is less than about 2000g / mol, less than about 1500g / mol, less than about 1000g / mol, less than about 800g / mol, or less than about 500g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric portion. In some embodiments, a small molecule is not and / or does not include a protein or polypeptide (e.g., not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not include a polynucleotide (e.g., not an oligonucleotide). In some embodiments, the small molecule is not and / or does not contain a polysaccharide; for example, in some embodiments, the small molecule is not a glycoprotein, a proteoglycan, a glycolipid, etc.). In some embodiments, the small molecule is not a lipid. In some embodiments, the small molecule is a modulator (e.g., an inhibitor / inhibitory agent or an activator). In some embodiments, the small molecule has biological activity. In some embodiments, the small molecule is detectable (e.g., comprising at least one detectable portion). In some embodiments, the small molecule is a therapeutic agent. Those of ordinary skill in the art who read this disclosure will understand that certain small molecule compounds described herein can be provided and / or used in any of a variety of forms, such as crystalline forms, salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc. Those skilled in the art will understand that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms. In some embodiments, such small molecules can be used in accordance with the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or can be in the form of a mixture of stereoisomers; in some embodiments, such small molecules can be used in accordance with the present disclosure in the form of a racemic mixture. Those skilled in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules can be used in accordance with the present disclosure in the form of individual tautomers or in a form that can be interconverted between tautomeric forms. Those skilled in the art will appreciate that certain small molecule compounds have structures that allow isotopic substitution (e.g., 2 H or 3 H replaces H; 11 C. 13 C or 14 C replaces 12C; 13 N or 15N replaces 14N; 17 O or 18 O replaces 16O; 36 Cl replaces XXC; 18 In some embodiments, such small molecules may be used in accordance with the present disclosure in one or more isotopically modified forms or mixtures thereof. In some embodiments, reference to a particular small molecule compound may relate to a particular form of the compound. In some embodiments, a particular small molecule compound may be provided and / or used in salt form (e.g., in acid addition salt or base addition salt form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, where the small molecule compound is a compound that exists or is found in nature, the compound may be provided and / or used in accordance with the present disclosure in a form different from that in which it exists or is found in nature. One of ordinary skill in the art will appreciate that, in some embodiments, the preparation of a particular small molecule compound is different from the compound present in a reference preparation or source, and the particular small molecule compound contains an absolute or relative amount of the compound or its particular form, which is different from the absolute or relative (with respect to another component of the preparation, including, for example, another form of the compound) amount of the compound or form present in a reference preparation of interest (e.g., present in a primary sample from a source of interest, such as a biological or environmental source). Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound can be considered a different form of the compound than a racemic mixture of the compound; a particular salt of a small molecule compound can be considered a different form than another salt form of the compound; a preparation of a compound form containing only one conformer ((Z) or (E)) comprising a double bond can be considered a different form of the compound than a compound containing another conformer ((E) or (Z)) of the double bond; a preparation in which one or more atoms differ from the isotopes present in a reference preparation can be considered a different form; and so forth.

[0351] Splicing component: A person skilled in the art will understand upon reading this disclosure that a "splicing component" is an agent or entity that participates in a splicing reaction. In some embodiments, a splicing component is or comprises a component of a spliceosome. In some embodiments, a splicing component is or comprises a splicing regulator. In some embodiments, a splicing component is or comprises an RNA, a polypeptide, and / or a complex thereof or therebetween. In some embodiments, one or more of U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, U6 snRNA, Sm polypeptide, Lsm polypeptide, Prp3 polypeptide, Prp31 polypeptide, Prp4 polypeptide, CypH polypeptide, 15.5K polypeptide, Prp8 polypeptide, Brr2 polypeptide, Snu114 polypeptide, Prp6 polypeptide, Prp28 polypeptide, 40K polypeptide, Dib1 polypeptide, Snu66 polypeptide, Sad1 polypeptide, or 27K polypeptide may be a splicing component, or may be part of a splicing component.

[0352] System with splicing competence: Those skilled in the art who read this disclosure will understand that a "system with splicing competence" is a system that includes all components necessary to complete one or more splicing events (e.g., of one or more specific RNAs). In some embodiments, the system with splicing competence can be an in vitro or ex vivo system. In some embodiments, the system with splicing competence can be or include one or more cells (e.g., in culture, in a tissue, or in an organism).

[0353] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0354] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group, and may be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0355] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0356] The term "alkoxy" refers to an alkyl group attached to an oxygen. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0357] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, and may be represented by the general formula alkyl-O-alkyl.

[0358] The term "alkyl" refers to a saturated aliphatic group, including straight chain alkyl, branched chain alkyl, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl and cycloalkyl substituted alkyl. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., for a straight chain alkyl having 30 carbon atoms or less, for a straight chain alkyl having 30 carbon atoms or less, for a branched ...1-30 , for the branched chain C 3-30 ), and more preferably 20 or less.

[0359] In addition, the term "alkyl" as used throughout the specification, examples and claims is intended to include unsubstituted and substituted alkyl groups, the latter referring to alkyl moieties having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon backbone, including halogenated alkyl groups, such as trifluoromethyl and 2,2,2-trifluoroethyl, and the like.

[0360] The term "aliphatic" as used herein for compounds of formula (I) refers to a straight chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon that is completely saturated or contains one or more unsaturated units but is not aromatic (also referred to herein as "carbocyclic" or "alicyclic")

[0361] Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle") refers to a monocyclic C3-C8 hydrocarbon or a bicyclic C8 hydrocarbon that is completely saturated or contains one or more unsaturated units but is not aromatic. 7 -C 10 Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, and hybrids thereof.

[0362] As described herein, compounds of formula (I) may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogen atoms of the designated moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogen atoms that are explicitly or implicitly replaced in the structure (e.g., means at least and means at least ). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be the same or different at each position. The combinations of substituents contemplated by the present invention are preferably those that form stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when subjected to conditions that allow their production, detection, and in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0363] Suitable monovalent substituents on the substitutable carbon atoms of an "optionally substituted" group are independently halogen; -(CH 2 ) 0– 4 R o ; –(CH 2 ) 0–4 OR o ; -O(CH 2 ) 0-4 R o 、-O–(CH 2 ) 0–4 C(O)OR o ; –(CH 2 ) 0–4 CH(OR o ) 2 ; –(CH 2 ) 0–4 SR o ; You can use R o Substituted –(CH 2 ) 0–4 Ph; can use R o Substituted –(CH 2 ) 0–4 O(CH 2 ) 0–1 Ph; can use R o Substituted –CH=CHPh; can be replaced by R o Substituted –(CH 2 ) 0–4 O(CH 2 ) 0–1 -pyridyl; –NO 2 ;–CN;–N 3 ;-(CH 2 ) 0–4 N(R o ) 2 ; –(CH 2 ) 0–4 N(R o )C(O)Ro ;–N(R o )C(S)R o ;–(CH 2 ) 0–4 N(R o )C(O)NR o 2 ;-N(R o )C(S)NR o 2 ;–(CH 2 ) 0–4 N(R o )C(O)OR o ;–N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2 ;-N(R o )N(R o )C(O)OR o ;–(CH 2 ) 0–4 C(O)R o ;–C(S)R o ;–(CH 2 ) 0–4 C(O)OR o ;–(CH 2 ) 0–4 C(O)SR o ;-(CH 2 ) 0–4 C(O)OSiR o 3 ;–(CH 2 ) 0–4 OC(O)R o ;–OC(O)(CH 2 ) 0– 4 SR o ;–(CH 2 ) 0–4 SC(O)R o ;–(CH 2 ) 0–4 C(O)NR o 2 ;–C(S)NR o 2 ;–C(S)SR o ;–SC(S)SR o 、-(CH 2 ) 0–4OC(O)NR o 2 ; -C(O)N(OR o )R o ; –C(O)C(O)R o ; –C(O)CH 2 C(O)R o ; –C(NOR o )R o ; -(CH 2 ) 0–4 SSR o ; -(CH 2 ) 0–4 S(O) 2 R o ; -(CH 2 ) 0–4 S(O)(NH)R o ; –(CH 2 ) 0–4 S(O) 2 OR o ; –(CH 2 ) 0–4 OS(O) 2 R o ; –S(O) 2 NR o 2 ; -(CH 2 ) 0–4 S(O)R o ; -N(R o )S(O) 2 NR o 2 ; –N(R o )S(O) 2 R o ; –N(OR o )R o ; –C(NH)NR o 2 ; –P(O) 2 R o ; -P(O)R o 2 ; -OP(O)R o 2 ; –OP(O)(OR o ) 2 ; SiR o 3 ; –(C 1–4 linear or branched alkylene)O–N(R o ) 2 ; or –(C 1–4Straight or branched alkylene) C(O)O–N(R o ) 2 , where each R o may be substituted as defined below and are independently hydrogen, C 1–6 Aliphatic group, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, -CH 2 -(5- to 6-membered heteroaryl ring), a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or an 8- to 10-membered bicyclic aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or notwithstanding the above definitions, two independent occurrences of R o Together with their central atoms they form a 3 to 12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which heteroatoms may be substituted as defined below.

[0364] R o (or two independent occurrences of R o Suitable monovalent substituents on the ring formed by the middle atom thereof are independently halogen, -(CH 2 ) 0–2 R ● 、–(halogen R ● ),–(CH 2 ) 0–2 OH, –(CH 2 ) 0–2 OR ● ,–(CH 2 ) 0–2 CH(OR ● ) 2 ; -O(halogen R ● ),–CN,–N 3 ,–(CH 2 ) 0–2 C(O)R ● ,–(CH 2 ) 0–2 C(O)OH, –(CH 2 ) 0–2 C(O)OR ● ,–(CH 2 ) 0–2 SR ● ,–(CH 2 ) 0– 2 SH, –(CH 2 ) 0–2 NH 2 ,–(CH 2 )0–2 NHR ● ,–(CH 2 ) 0–2 NR ● 2 ,–NO 2 、–SiR ● 3 ,–OSiR ● 3 、-C(O)SR ● ,–(C 1–4 Straight or branched alkylene)C(O)OR ● or –SSR ● , where each R ● unsubstituted or, when preceded by "halogen", substituted only by one or more halogens, and independently selected from C 1–4 Aliphatic group, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, or a 3- to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0365] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =0 ("oxo"), =S, =NNR * 2 、=NNHC(O)R * 、=NNHC(O)OR * 、=NNHS(O) 2 R * , =NR * , =NOR * , –O(C(R * 2 )) 2– 3 O–or–S(C(R * 2 )) 2–3 S–, where each independent occurrence of R * is selected from hydrogen, C which may be substituted as defined below 1-6 aliphatic, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bonded to the ortho-substitutable carbon of the "optionally substituted" group include: -O(CR * 2 ) 2–3 O–, where each independent occurrence of R * is selected from hydrogen, C which may be substituted as defined below1–6 aliphatic, or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0366] R * Suitable substituents on the aliphatic group include halogen, -R ● 、-(halogen R ● ), -OH, –OR ● 、–O(halogen R ● ), –CN, –C(O)OH, –C(O)OR ● , –NH 2 , –NHR ● ,–NR ● 2 or –NO 2 , where each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens, and is independently C 1–4 Aliphatic group, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0367] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include or Each of these is independently hydrogen, C which may be substituted as defined below 1–6 aliphatic, unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independent occurrences of Together with its middle atoms, it forms an unsubstituted 3- to 12-membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0368] Suitable substituents on the aliphatic group are independently halogen, -R ● 、-(halogen R ● ), –OH, –OR ● 、–O(halogen R ● ), –CN, –C(O)OH, –C(O)OR ● , –NH 2 , –NHR ● ,–NR ● 2or-NO 2 , where each R ● is unsubstituted or, when preceded by "halo", is substituted only by one or more halogens, and is independently C 1–4 Aliphatic group, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0369] When used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C x-y " or "C x -C y " is intended to include groups containing from x to y carbons in the chain. 0 Alkyl represents hydrogen where the group is in terminal position and if internal it is a bond. For example, C 1-6 Alkyl groups contain 1 to 6 carbon atoms in the chain.

[0370] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0371] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, and may be represented by the general formula alkylS-.

[0372] As used herein, the term "amide" refers to a group

[0373]

[0374] Where R 9 and R 10 Each independently represents hydrogen or a hydrocarbon group, or R 9 and R 10 Together with the N atom to which they are attached they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0375] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, such as the moiety represented by the formula

[0376]

[0377] Where R 9 , R 10 and R 10 ' each independently represents hydrogen or a hydrocarbon group, or R 9 and R 10 Together with the N atom to which they are attached they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0378] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0379] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0380] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon. Preferably, the ring is a 5-7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes a polycyclic system with two or more rings, wherein two or more carbons are common to two adjacent rings, wherein at least one ring is aromatic, for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic radicals. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0381] The term "carbamate" is art-recognized and refers to the group

[0382]

[0383] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0384] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0385] As used herein, the terms "carbocycle", "carbocyclyl" and "carbocyclic" refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably, the carbocycle contains 3 to 10 atoms, more preferably 5 to 7 atoms.

[0386] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0387] The term "carbonate" is art-recognized and refers to the group -OCO 2 -.

[0388] As used herein, the term "carboxyl" refers to a group of the formula -CO 2 The group represented by H.

[0389] As used herein, the term "ester" refers to the group -C(O)OR 9 , where R 9 It represents a hydrocarbon group.

[0390] As used herein, the term "ether" refers to a hydrocarbyl group connected to another hydrocarbyl group through an oxygen. Thus, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. An ether may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

[0391] As used herein, the terms "halo" and "halogen" mean halogen and include chlorine, fluorine, bromine and iodine.

[0392] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a heteroaryl group.

[0393] The terms "heteroaryl" and "heteroaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5 to 7 rings, more preferably 5 to 6 rings, whose ring structure includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "heteroaryl" also include polycyclic systems with two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is heteroaromatic, for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic radicals. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine etc.

[0394] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0395] The term "heterocyclylalkyl" as used herein refers to an alkyl group substituted with a heterocyclyl group.

[0396] The terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10-membered rings, more preferably 3 to 7-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more rings, wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is heterocyclic, for example, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Heterocyclyl includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc.

[0397] As used herein, the term "hydrocarbyl" refers to a group bonded by a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a main chain that is primarily carbon, but may optionally contain heteroatoms. Therefore, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl, but substituents such as acetyl (which has a =O substituent on the connecting carbon) and ethoxy (which is connected by oxygen rather than carbon) are not. Hydrocarbyl includes, but is not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0398] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0399] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, the term "lower" is meant to include groups having ten or fewer atoms, preferably six or fewer atoms in the substituent. For example, "lower alkyl" refers to an alkyl group containing ten or fewer, preferably six or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents defined herein are respectively low acyl, low acyloxy, low alkyl, low alkenyl, low alkynyl or low alkoxy, whether they occur alone or in combination with other substituents, such as in narrating hydroxyalkyl and aralkyl (in this case, for example, when calculating the carbon atoms in the alkyl substituent, the atoms in the aryl are not counted).

[0400] The terms "polycyclic group", "polycyclic" and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl) wherein two or more atoms are common to two adjacent rings, e.g., the ring is a "fused ring". Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0401] The term "sulfate" is art-recognized and refers to the group -OSO 3 H or a pharmaceutically acceptable salt thereof.

[0402] The term "sulfonamide" is art-recognized and refers to a group represented by the general formula

[0403]

[0404] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0405] The term "sulfoxide" is art-recognized and refers to the group -S(O)-.

[0406] The term "sulfonate" is art-recognized and refers to the group SO 3 H or a pharmaceutically acceptable salt thereof.

[0407] The term "sulfone" is art-recognized and refers to the group -S(O) 2 -.

[0408] The term "substituted" refers to a substituent having a substituent replacing hydrogen on one or more carbons of the main chain. It should be understood that "substituted" or "substituted by ... " includes implicit conditions, i.e., such substitution is based on the allowed valence of the substituted atom and the substituent, and the substitution produces a stable compound, for example, it will not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" considers all allowed substituents of organic compounds. In a broad sense, allowable substituents include non-cyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Allowable substituents can be one or more substituents and are the same or different for appropriate organic compounds. For purposes of the present invention, heteroatoms such as nitrogen can have any allowable substituents that satisfy the valence of heteroatoms of hydrogen substituents and / or organic compounds described herein. Substituents may include any of the substituents described herein, for example, halogen, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted if appropriate.

[0409] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0410] As used herein, the term "thioester" refers to the group -C(O)SR 9 or –SC(O)R 9

[0411] Where R 9 It represents a hydrocarbon group.

[0412] As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

[0413] The term "urea" is art-recognized and can be represented by the general formula

[0414]

[0415] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0416] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation) as well as enhancing a function or activity.

[0417] The phrase "pharmaceutically acceptable" is recognized in the art. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0418] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt that is suitable for use in or compatible with the treatment of a patient.

[0419] As used herein, the term "pharmaceutically acceptable acid addition salt" means any non-toxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids forming suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids forming suitable salts include monocarboxylic acids, dicarboxylic acids and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid and salicylic acid and sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Monobasic or dibasic acid salts can be formed, and such salts can exist in hydrated form, solvated form or substantially anhydrous form. Generally, the acid addition salts of the compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally exhibit higher melting points compared to their free base forms. The selection of suitable salts is known to those skilled in the art. Other non-pharmaceutically acceptable salts, such as oxalates, may be used, for example, to isolate a compound of formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0420] As used herein, the term "pharmaceutically acceptable base addition salt" means any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any intermediate thereof. Exemplary inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline or ammonia. The selection of appropriate salts will be known to those skilled in the art.

[0421] Many compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter can exist in the R or S configuration, with the R and S symbols being used according to the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereomeric forms (including all possible mixtures of stereoisomers) of compounds, salts, prodrugs, or mixtures thereof.

[0422] Additionally, certain alkenyl-containing compounds may exist as Z (same) or E (hetero) isomers. In each case, the disclosure includes both the mixture and the separate individual isomers.

[0423] Some compounds may also exist as tautomeric forms. Although not explicitly indicated in the formulas described herein, such forms are intended to be encompassed within the scope of the present disclosure.

[0424] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, such as hydrolyzed or oxidized, in a host to form a compound of the present disclosure (e.g., a compound of formula I) after administration. Typical examples of prodrugs include compounds having biologically unstable or cleavable (protective) groups on the functional portion of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramidates as biologically unstable or cleavable (protective) groups are disclosed in U.S. Patents 6,875,751, 7,585,851 and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to produce compounds of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. General procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs", editor H. Bundgaard, Elsevier, 1985.

[0425] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter aid, diluent, excipient, solvent or encapsulating material, which can be used to formulate a medicament for medical or therapeutic use.

[0426] As used herein, the term "logarithm of solubility", "LogS" or "logS" is used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is often associated with poor absorption. The LogS value is the unit exfoliation logarithm (base 10) of the solubility measured in moles / liter.

[0427] Example

[0428] Having generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0429] Embodiment 1: synthesis

[0430] General Experimental Procedures. Unless otherwise stated, all reactions were performed under an argon atmosphere. Tetrahydrofuran (THF) was distilled from benzoquinone ketyl radical under an argon atmosphere. Dichloromethane and triethylamine were distilled from calcium hydride under an argon atmosphere. All other solvents and reagents were purified according to literature procedures or purchased from Sigma-Aldrich, Acros, Oakwood, and Fisher Scientific Co. 1 H NMR spectra were recorded at 400 or 500 MHz and reported relative to the deuterated solvent signal. 1 Data for H NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. The resolution pattern is designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. Recordings were made at 100 or 125 MHz. 13 C NMR spectroscopy. 13 C NMR spectral data are reported as chemical shifts. Chemical shifts are reported in parts per million (ppm, δ). Thin layer chromatography (TLC) was performed using pre-coated silica gel sheets. Visual detection was performed using potassium permanganate or ceric ammonium nitrate staining. Flash chromatography was performed using SilicaFlash P60 (60A, 40-63 μm) silica gel with compressed air.

[0431]

[0432] 3-Chloro-6-hydrazinopyridazine.

[0433] To a solution of 3,6-dichloropyridazine (400 mg, 2.686 mmol) in EtOH (8 mL) was added hydrazine monohydrate (148 mg, 2.954 mmol) and the mixture was stirred at 100° C. for 3 hours. After the mixture was cooled to 23° C., the resulting solid was collected and eluted with EtOH.2 O. The mother liquor was concentrated and washed with Et 2 The precipitate was washed with 4% CO 0. The combined solids were washed with dichloromethane to give the desired product (light yellow, 320.2 mg, 2.216 mmol, 82%) and used in the next step without further purification. 1 H NMR (400 MHz, DMSO-d 6 )δ8.24(br s,1H),7.41(d,J=9.6Hz,1H),7.09(d,J=9.2Hz,1H),4.37(br s,2H); 13 C NMR (100 MHz, DMSO-d 6 )δ161.8,145.4,128.7,116.1. The spectral data are consistent with the literature data. [Reference: Heterocycles, 2009, 78 (4) 961-975]

[0434]

[0435] 6-Chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine.

[0436] A mixture of 3-chloro-6-hydrazinopyridazine (300 mg, 2.075 mmol) in AcOH (1.5 mL) was heated at 100 °C for 2 h. After the reaction mixture was cooled to 23 °C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with brine and anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The resulting off-white crude solid (238.5 mg, 68%) was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 )δ8.04(d,J=9.6Hz,1H),7.09(d,J=9.6Hz,1H),2.81(s,3H).

[0437]

[0438] 3-Methyl-6-phenyl-[1,2,4]triazolo[4,3-b]pyridazine, JGJ002. 6-Chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (20 mg, 0.119 mmol), phenylboronic acid (14.5 mg, 0.119 mmol), K 2 CO 3 (24.6 mg, 0.178 mmol) and Pd(PPh 3 ) 4A mixture of (13.6 mg, 0.012 mmol) in 1,4-dioxane (0.3 mL) and water (30 uL) was heated at 120 °C for 18 hours. After the reaction mixture was cooled to 23 °C, it was diluted with water and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous MgSO 4 Dried, filtered and concentrated under reduced pressure. The obtained crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to obtain the desired product JGJ002 (20.4 mg, 0.098 mmol, 82%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ.8.13(d,J=9.2Hz,1H),7.98-8.01(m,2H),7.54-7.56(4H,m),2.88(s,3H) 13 C NMR (100 MHz, CDCl 3 )δ153.4,147.5,143.4,134.4,130.9,129.2,127.2,124.9,118.8,9.8.

[0439]

[0440] 3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)aniline, JGJ003. Using the same procedure as above, 6-chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (30 mg, 0.178 mmol), 3-nitrophenylboronic acid (35.6 mg, 0.214 mmol), K 2 CO 3 (36.9 mg, 0.267 mmol) and Pd(PPh 3 ) 4 (20.6 mg, 0.018 mmol) in 1,4-dioxane (0.3 mL) and water (30 uL) gave 3-methyl-6-(3-nitrophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (19.7 mg, 0.077 mmol, 43%). 1 H NMR (400 MHz, CDCl 3 )δ.8.86(t,J=2.0Hz,1H),8.39(m,2H),8.24(d,J=9.6Hz,1H),7.71(t,J=8.0Hz,1H),7.62(d,J=9.6Hz,1H),2.91(s,3H) 13 CNMR (100MHz, CDCl 3)δ151.1,148.8,147.7,143.2,136.1,132.8,130.4,125.8,125.4,122.2,118.0,9.9. Then the nitro compound (19.4 mg, 0.076 mmol) and SnCl 2 A mixture of (72.1 mg, 0.380 mmol) in EtOH (0.2 mL) was heated under reflux for 1 h. After the mixture was cooled to 23 °C, it was filtered through a celite pad and washed with EtOAc. To the mixture was added saturated NaHCO 3 The solution was extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dried, filtered and concentrated under reduced pressure. The obtained crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to obtain the desired product JGJ003 (10 mg, 0.044 mmol, 63%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.10(d,J=10.0Hz,1H),7.51(d,J=10.0Hz,1H),7.26-7.32(m,3H),6.83-6.86(m,1H),2.86(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ153.5,147.3(two peaks overlap),143.4,135.2,130.0,124.4,119.1,117.4,117.2,113.1,9.7.

[0441]

[0442] N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide, JGJ004. To a solution of 3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)aniline (JGJ003, 20 mg, 0.088 mmol) in dichloromethane (0.5 mL) was added trimethylamine (10.8 mg, 0.106 mmol) and acetyl chloride (7.6 mg, 0.099 mmol). The mixture was stirred at 23 °C for 6 hours. To this mixture was added water and extracted with dichloromethane. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (dichloromethane:MeOH=6:1) to give the desired product JGJ004 (21.1 mg, 0.079 mmol, 89%) as an ivory solid. 1H NMR (400 MHz, CDCl 3 )δ8.32(s,1H),8.09(d,J=9.6Hz,1H),7.88(br s,1H),7.70(d,J=7.6Hz,1H),7.65(d,J=8.0Hz,1H),7.54(d,J=10.0Hz,1H),7.48(t,J=8.0Hz,1H),2.86(s,3H),2.25(s,3H). 13 C NMR (125MHz, CD 3 OD)δ172.8,156.1,149.9,145.8,141.8,137.0,131.5,126.3,124.8,124.2,122.6,120.5,24.8,10.4.

[0443]

[0444] N-methyl-N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide, JGJ001. To a solution of N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide (JGJ004, 16.5 mg, 0.062 mmol) was added a 60% dispersion of NaH in mineral oil (5 mg, 0.124 mmol) at 0°C and stirred for 30 minutes. Then iodomethane (17.5 mg, 0.124 mmol) was added and the reaction mixture was stirred at 23°C for 2 hours. After the reaction was complete, water was added and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to give the desired product JGJ001 (9.8 mg, 0.035 mmol, 56%) as an ivory solid. 1 H NMR (500 MHz, CDCl 3 )δ8.17(d,J=9.5Hz,1H),7.95(d,J=7.5Hz,1H),7.88(s,1H),7.62(dd,J=8.0,7.5Hz ,1H),7.54(d,J=10.0Hz,1H),J=8.0Hz,1H),3.35(s,3H),2.89(s,3H),1.94(s,3H); 13 C NMR (125 MHz, CDCl 3)δ170.3,152.1,147.6,145.6,143.3,136.2,130.7,129.5,126.4,125.9,125.4,118.4,37.3,22.6,9.9.

[0445]

[0446] 6-Chloropyridazin-3-amine. A mixture of 3,6-dichloropyridazine (200 mg, 2.342 mmol) and ammonium hydroxide (1.5 mL) in a sealed tube was heated at 100 °C for 16 hours. After the mixture was cooled to 23 °C, dichloromethane was added and the precipitate was separated, washed with dichloromethane to give the desired product as a light yellow solid (quantitative). 1 H NMR (400 MHz, DMSO-d 6 )δ7.32(d,J=8.0Hz,1H),6.81(d,J=8.0Hz,1H),6.59(s,2H).

[0447]

[0448] 2-Bromopropanal. To a solution of propanal (2.91 mL, 40 mol) in dichloromethane (40 mL) was added a solution of bromine (2.05 mL, 40 mol) in dichloromethane (10 mL) dropwise at 0 ° C over 1.5 hours. The mixture was warmed to 23 ° C and stirred for 30 minutes. After adding water to the reactant, the resulting organic layer was separated and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted with dichloromethane (30 mL) and then the combined organic layers were washed with brine and washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude product (dark yellow oil, quantitative) was used in the next step without any purification. 1 H NMR (400 MHz, CDCl 3 )δ9.35(br s,1H),4.34(qd,J=6.8,2.0Hz,1H),1.75(d,J=6.8Hz,3H). The spectral data are consistent with the literature data. [Reference: Bull. Korean Chem. Soc. 2013, 34(1), 271-274.

[0449]

[0450] 6-Chloro-3-methylimidazo[1,2-b]pyridazine. A mixture of 6-chloropyridazin-3-amine (238.3 mg, 1.839 mmol) and 2-bromopropanal (crude, 503.9 mg, 3.679 mmol) in EtOH was heated under reflux for 4 hours. After the mixture was cooled to 23 °C, it was concentrated and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-Hex:EtOAc:MeOH=1:1:0.1) to give the desired product (55.2 mg, 0.329 mmol, 18%) as a light brown solid. 1 H NMR (400 MHz, CDCl 3 )δ7.87(d,J=9.6Hz,1H),7.56(s,1H),6.99(1H,J=9.6Hz,1H),2.55(s,3H).

[0451]

[0452] 3-Methyl-6-(3-nitrophenyl)imidazo[1,2-b]pyridazine, JGJ005. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (55.2 mg, 0.329 mmol), 3-nitrophenylboronic acid (60.5 mg, 0.362 mmol), K 2 CO 3 (68.3 mg, 0.494 mmol) and Pd(PPh 3 ) 4 Reaction of (38.1 mg, 0.033 mmol) in 1,4-dioxane (0.5 mL) and water (150 μL) gave the desired product JGJ005 (61.9 mg, 0.244 mmol, 74%) as a yellow solid. 1 H NMR (500 MHz, CDCl 3 )δ8.88(dd,J=2.0,1.5Hz,1H),8.38(ddd,J=7.5,1.5,1.0Hz,1H),8.35(ddd,J=8.0,2.0,1.0Hz,1 H),8.07(d,J=9.5Hz,1H),7.73(t,J=8.0Hz,1H),7.67(s,1H),7.50(d,J=9.5Hz,1H),2.67(s,3H); 13 C NMR (125 MHz, CDCl 3)δ148.8 (two peaks overlap), 138.1, 137.7, 133.3, 132.7, 130.0, 126.0, 125.8, 124.4, 122.0, 113.7, 8.8.

[0453]

[0454] 3-(3-Methylimidazo[1,2-b]pyridazin-6-yl)aniline, JGJ006. Using the same procedure as described for JGJ003, 3-methyl-6-(3-nitro-phenyl)imidazo[1,2-b]pyridazine (54.4 mg, 0.214 mmol) and SnCl 2 Reaction of (202.8 mg, 1.070 mmol) in EtOH (0.5 mL) gave the desired product JGJ006 (27.2 mg, 0.107 mmol, 50%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ7.92(d,J=9.2Hz,1H),7.56(d,J=0.8Hz,1H),7.38(d,J=9.6Hz,1H),7.28-7.34(m,3H),6.79(ddd,J=7.7,2.0,1.2Hz,1H),3.87(br s,2H),2.61(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ151.3,147.0,138.1,137.0,132.0,129.8,125.3,125.1,117.3,116.5,114.8,113.3,8.7.

[0455]

[0456] N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ007. Using the same procedure as described for JGJ004, the reaction of 3-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (JGJ006, 23.3 mg, 0.104 mmol), triethylamine (12.6 mg, 0.125 mmol) and acetyl chloride (9 mg, 0.114 mmol) in dichloromethane (0.5 mL) gave the desired product JGJ007 (16.5 mg, 0.067 mmol, 60%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ8.44(s,1H),8.21(s,1H),7.89(br s,1H),7.61-7.69(m,3H),7.41(t,J=8.0Hz,1H),7.37(br d,J=8.4Hz,1H),2.57(s,3H),2.22(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.8,150.8,138.9,136.6,132.2,129.5,125.2,122.6,121.2,118.3,114.6,24.5,8.7 (two low-field carbons were not observed).

[0457]

[0458] N-Methyl-N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ008. Using the same procedure as described for JGJ001, the reaction of N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide (JGJ007, 26.4 mg, 0.099 mmol), a 60% dispersion of NaH in mineral oil (8 mg, 0.199 mmol) and iodomethane (28.2 mg, 0.199 mmol) in dimethylformamide (DMF, 0.3 mL) gave the desired product JGJ008 (17.5 mg, 0.062 mmol, 63%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.00(d,J=9.6Hz,1H),7.96(d,J=8.0Hz,1H),7.89(dd,J=2.0,1.6Hz,1H),7.62(s,1H),7.58(dd,J=8. 0,7.6Hz,1H),7.43(d,J=9.2Hz,1H),7.32(dd,J=7.6,1.2Hz,1H),3.34(s,3H),2.64(s,3H),1.95(s,3H); 13 CNMR (125MHz, CDCl 3 )δ170.5,149.9,145.4,138.1,137.8,132.8,130.4,128.3,126.2,125.7,125.6,114.0,37.2,22.6,8.8 (not a single low-field carbon was observed).

[0459]

[0460] 3-Methyl-6-(2-nitrophenyl)imidazo[1,2-b]pyridazine, JGJ009. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (67.1 mg, 0.400 mmol), 2-nitrophenylboronic acid (73.5 mg, 0.440 mmol), NaOH (48 mg, 1.201 mmol) and Pd(PPh 3 ) 4 Reaction of (46.3 mg, 0.040 mmol) in THF (0.4 mL) and water (0.2 mL) gave the desired product JGJ009 (16.3 mg, 0.064 mmol, 16%) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 )8.02(dd,J=8.0,0.8Hz,1H),7.99(d,J=9.6Hz,1H),7.75(m,1H),7.64-7.70(m,2H),7.63(d,J=1.2Hz,1H),7.10(d,J=9.2Hz,1H),2.54(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ149.6,149.0,137.7,132.9,132.8,131.7,131.4,130.2,125.6,125.5,124.7,115.8,8.6.

[0461]

[0462] 2-(3-Methylimidazo[1,2-b]pyridazin-6-yl)aniline, JGJ010. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (25.4 mg, 0.152 mmol), 2-aminophenylboronic acid (22.8 mg, 0.167 mmol), K 2 CO 3 (31.4 mg, 0.227 mmol) and Pd(PPh 3 ) 4 Reaction of (17.5 mg, 0.015 mmol) in 1,4-dioxane (0.4 mL) and water (80 μL) gave the desired product JGJ010 (26.2 mg, 0.117 mmol, 70%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)7.97(d,J=9.6Hz,1H),7.57(s,1H),7.67(m,1H),7.42(d,J=9.6Hz,1H),7.24(m,1H),6.82-6.87(m,2H),2.59(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ152.8,145.9,137.3,131.8,130.7,129.7,125.6,124.9,118.6,118.0,117.4,116.5,8.8.

[0463]

[0464] N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ011. Using the same procedure as described for JGJ004, the reaction of 2-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (JGJ010, 39.4 mg, 0.176 mmol), triethylamine (21.3 mg, 0.211 mmol) and acetyl chloride (16.5 mg, 0.211 mmol) in dichloromethane (0.8 mL) gave the desired product JGJ011 (35 mg, 0.131 mmol, 75%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ10.57(br s,NH),8.47(d,J=8.4Hz,1H),7.99(d,J=9.6Hz,1H),7.61(s,1H),7.60(dd,J=8.0,0.8Hz,1H),7.44(ddd,J =8.8,7.2,0.8Hz,1H),7.34(d,J=9.2Hz,1H),7.20(ddd,J=8.0,7.2,0.8Hz,1H),2.60(s,3H),2.17(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.1,152.0,137.3,136.4,132.8,130.6,129.5,126.3,124.6,124.0,123.5,122.4,116.7,25.1,8.9.

[0465]

[0466] N-methyl-N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ012. Using the same procedure as described for JGJ001, the reaction of N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide (JGJ011, 19.1 mg, 0.072 mmol), sodium hydride (NaH, 60% dispersion in mineral oil, 5.7 mg, 0.143 mmol) and iodomethane (20.4 mg, 0.143 mmol) in dimethylformamide (DMF, 0.3 mL) gave the desired product JGJ012 (12.8 mg, 0.046 mmol, 64%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.98(d,J=9.2Hz,1H),7.66(m,1H),7.60(s,1H)7.52(m,2H),7.34(m,1H),7.10(d,J=9.6Hz,1H),3.01(s,3H),2.54(s,3H),1.90(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ170.9,150.1,142.5,137.4,134.5,132.8,131.0,130.9,130.7,129.5,128.7,125.7,116.0,36.7,22.7,8.7.

[0467]

[0468] 3-(3-Methylimidazo[1,2-b]pyridazin-6-yl)benzoic acid, JGJ013. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (50 mg, 0.299 mmol), 3-carboxyphenylboronic acid (54.5 mg, 0.328 mmol), K 2 CO 3 (82.5 mg, 0.597 mmol) and Pd(PPh 3 ) 4 Reaction of (34.5 mg, 0.030 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ013 (32.4 mg, 0.128 mmol, 43%) as a white solid. 1 H NMR (400MHz, CD 3OD)8.73(dd,J=1.6,1.2Hz,1H),8.25(d,J=8.0Hz,1H),8.16(ddd,J=7.6,1.6,1.2Hz,1H),8.03(d,J=9.6 Hz, 1H), 7.75 (d, J = 9.6Hz, 1H), 7.62 (dd, J = 8.0, 7.6Hz, 1H), 7.58 (d, J = 0.4Hz, 1H), 2.63 (d, J = 0.4Hz, 3H).

[0469]

[0470] 6-(2,3-Dimethoxyphenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ014. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (42 mg, 0.251 mmol), 2,3-dimethoxyphenylboronic acid (50.2 mg, 0.276 mmol), K 2 CO 3 (52 mg, 0.376 mmol) and Pd(PPh 3 ) 4 Reaction of (29 mg, 0.025 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ014 (39.6 mg, 0.147 mmol, 59%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )7.92(d,J=9.6Hz,1H),7.58(s,1H),7.46(d,J=9.2Hz,1H),7.29(dd,J=7.6,0.8Hz,1H),7.1 9(t,J=8.0Hz,1H),7.05(ddd,J=8.0,7.6,0.8Hz,1H),3.93(s,3H),3.76(s,3H),2.60(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ153.2,150.7,147.5,138.0,131.7,131.1,125.2,124.4,124.2,122.2,118.4,113.6,61.4,56.0,8.8.

[0471]

[0472] 6-(3-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ015. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (51.5 mg, 0.307 mmol), 3-fluorophenylboronic acid (47.3 mg, 0.338 mmol), K 2 CO 3 (63.7 mg, 0.461 mmol) and Pd(PPh 3 ) 4 Reaction of (35.5 mg, 0.031 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ015 (38.2 mg, 0.168 mmol, 55%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )7.98(d,J=9.2Hz,1H),7.75(m,2H),7.61(s,1H),7.48(m,1H),7.41(d,J=9.2Hz,1H),7.18(m,1H),2.63(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ163.2(d,J=244.9Hz),149.8(d,J=2.6Hz),138.2,138.1,132.6,130.5(d,J=8.1Hz),125.5,122.6(d,J=2.9Hz),116.7(d,J=21.2Hz),114.2,113.9(d,J=23.1Hz),8.7.(Not a single low-field carbon was observed).

[0473]

[0474] N-methyl-3-(3-methylimidazo[1,2-b]pyridazin-6-yl)benzamide, JGJ016. Hydroxybenzotriazole (HOBT, 16.1 mg, 0.159 mmol), (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl, 30.4 mg, 0.159 mmol) and N,N-diisopropylethylamine (DIPEA, 102.6 mg, 0.794 mmol) were added to a solution of JGJ013 (20.1 mg, 0.079 mmol) and methylamine hydrochloride (10.7 mg, 0.159 mmol) in dichloromethane (0.3 mL) and DMF (0.5 mL). The mixture was stirred at 23 ° C for 12 hours. After water was added to the reactant, it was extracted with ethyl acetate (10 mL X3). The combined organic layer was washed with brine and purified by anhydrous MgSO 4Dry, filter and concentrate under reduced pressure. The crude residue was purified by flash column chromatography (dichloromethane:MeOH=6:1) to give the desired product JGJ016 (8.6 mg, 0.032 mmol, 41%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.39(t,J=1.6Hz,1H),8.10(dddd,J=8.0,1.6,1.2,0.8Hz,1H),7.91(d,J=9.6Hz,1H),7.86(dddd,J=7.6,1.6,1.2Hz,1H), 7.58(s,1H),7.55(dd,J=8.0,7.6Hz,1H),7.41(d,J=9.6Hz,1H),6.75(m,NH),3.06(d,J=4.8Hz,3H),2.59(d,J=0.4Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ167.7,153.3,138.0,136.3,135.5,132.3,129.7,129.2,128.0,125.7,125.5,125.4,114.4,26.9,8.7.

[0475]

[0476] 3-Methyl-6-(pyridin-3-yl)imidazo[1,2-b]pyridazine, JGJ017. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (58.8 mg, 0.351 mmol), 3-pyridineboronic acid (47.4 mg, 0.386 mmol), K 2 CO 3 (72.7 mg, 0.526 mmol) and Pd(PPh 3 ) 4 Reaction of (40.6 mg, 0.035 mmol) in 1,4-dioxane / water (5:1 v / v, 0.6 mL) gave the desired product JGJ017 (37.2 mg, 0.177 mmol, 50%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)9.20(d,J=1.6Hz,1H),8.69(dd,J=4.8,1.6Hz,1H),8.29(ddd,J=8.0,2.0,1.6Hz,1H),7.98(d,J=9.2Hz,1 H),7.60(d,J=0.4Hz,1H),7.42(ddd,J=8.0,4.8,0.8Hz,1H),7.41(d,J=9.6Hz,1H),2.60(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ150.6,148.6,148.2,137.9,134.2,132.7,131.6,125.7,125.5,123.6,113.7,8.6.

[0477]

[0478] 6-(2-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ018. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (27.5 mg, 0.164 mmol), 2-fluorophenylboronic acid (25.3 mg, 0.181 mmol), K 2 CO 3 (34.0 mg, 0.246 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (19.0 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ018 (18.1 mg, 0.080 mmol, 49%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )7.96(d,J=9.6Hz,1H),7.91(ddd,J=8.0,7.6,2.0Hz,1H),7.60(s,1H),7.43-7.49(m,2H),7 .30(ddd,J=8.0,7.6,1.2Hz,1H),7.21(ddd,J=11.2,8.4,0.8Hz,1H),2.61(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3)δ160.4(d,J=249.3Hz),148.2,137.9,132.2,131.4(d,J=8.5Hz),130.7(d,J=2.6Hz),125.3, 124.7, 124.6 (d, J = 3.6Hz), 124.3 (d, J = 11.7Hz), 117.5 (d, J = 7.9Hz), 116.4 (d, J = 22.2Hz), 8.7.

[0479]

[0480] 6-Chloromidazo[1,2-b]pyridazine. To a solution of 6-chloropyridazine-3-amine (400 mg, 3.088 mmol) in EtOH (6 mL) and water (4 mL) was added bromoacetaldehyde diethyl acetal (930 μL, 6.175 mmol) and HBr (280 μL). The resulting mixture was heated at 103 ° C overnight. After the mixture was cooled to 23 ° C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude residue was used in the next step without further purification. (Brown solid; 394.5 mg, 2.569 mmol, 83%) 1 H NMR (400 MHz, CDCl 3 )δ7.92(s,1H),7.90(d,J=9.6Hz,1H),7.76(s,1H),7.04(d,J=9.6Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ146.9,137.5,134.4,127.0,118.9,117.2.

[0481]

[0482] N-(3-(Imidazolo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ019. Using the same procedure as described for JGJ002, 6-chloro-imidazo[1,2-b]pyridazine (71.6 mg, 0.427 mmol), 3-aminophenylboronic acid (69.5 mg, 0.449 mmol), K 2 CO 3 (88.6 mg, 0.641 mmol) and Pd(PPh 3 ) 4(49.3 mg, 0.043 mmol) in 1,4-dioxane / water (5:1 v / v, 1.0 mL) gave 3-(imidazo[1,2-b]pyridazin-6-yl)aniline (87.9 mg, 0.392 mmol, 92%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ019 (49.6 mg, 0.221 mmol, 69%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.19(s,1H),8.13(br s,1H),7.96(m,2H),7.76(s,1H),7.61-7.65(m,2H),7.43(d,J=9.6Hz,1H),7.39-7.43(m,1H),2.22(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.9,151.8,138.9,138.2,136.1,133.6,129.7,125.4,122.7,121.4,118.4,117.1,116.7,24.6.

[0483]

[0484] 6-(3-Fluorophenyl)imidazo[1,2-b]pyridazine, JGJ020. Using the same procedure as described for JGJ002, 6-chloroimidazo[1,2-b]pyridazine (50 mg, 0.326 mmol), 3-fluorophenylboronic acid (50.1 mg, 0.358 mmol), K 2 CO 3 (67.5 mg, 0.488 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (18.8 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ020 (36.9 mg, 0.173 mmol, 53%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.96-7.99(m,2H),7.77(s,1H),7.62-7.68(m,2H),7.42-7.46(m,1H),7.39(d,J=9.6Hz,1H),7.14(m,1H); 13 C NMR (100 MHz, CDCl 3)δ163.1(d,J=245.1Hz),150.4(d,J=2.6Hz),137.5(d,J=7.8Hz),134.2,131.9(d,J=9.8Hz),130.5(d,J= 8.1Hz),128.4(d,J=12.1Hz),125.7,122.5(d,J=2.9Hz),116.8(d,J=21.1Hz),115.7,113.8(d,J=23.2Hz)

[0485]

[0486] 6-Chloro-2-methylimidazo[1,2-b]pyridazine. To a solution of 6-chloropyridazin-3-amine (100 mg, 0.772 mmol) in EtOH (2 mL) were added trimethylamine (78 mg, 0.772 mmol) and chloroacetone (142.8 mg, 1.544 mmol), and the mixture was stirred at 120 ° C overnight. After the mixture was cooled to 23 ° C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc=1:1) to give the desired product (87.2 mg, 0.520 mmol, 67%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 )δ7.72(dd,J=9.2,0.4Hz,1H),7.65(s,1H),6.93(d,J=9.2Hz,1H),2.44(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ145.8,144.8,137.0,125.6,117.9,114.5,14.7.

[0487]

[0488] N-(3-(2-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ021. Using the same procedure as described for JGJ002, 6-chloro-2-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 3-aminophenylboronic acid (35.9 mg, 0.232 mmol), K 2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4(24.4 mg, 0.021 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave 3-(2-methylimidazo[1,2-b]pyridazin-6-yl)aniline (49.6 mg, quantitative) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ021 (27.2 mg, 0.102 mmol, 46%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.92(s,1H),8.16(s,1H),7.73(d,J=9.6Hz,1H),7.63(m,2H),7.54(d,J=7.6H z,1H),7.33(t,J=8.0Hz,1H),7.27(d,J=10.0Hz,1H),2.44(s,3H),2.19(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ169.2,150.7,143.8,139.0,137.7,136.1,129.4,123.9,122.3,121.1,118.2,115.7,114.3,24.4,14.5.

[0489]

[0490] 6-(3-Fluorophenyl)-2-methylimidazo[1,2-b]pyridazine, JGJ022. Using the same procedure as described for JGJ002, 6-chloro-2-methylimidazo[1,2-b]pyridazine (21.4 mg, 0.128 mmol), 3-fluorophenylboronic acid (17.9 mg, 0.128 mmol), K 2 CO 3 (26.5 mg, 0.192 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (7.4 mg, 0.006 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave the desired product JGJ022 (13.7 mg, 0.060 mmol, 47%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.89(d,J=9.2Hz,1H),7.78(s,1H),7.65-7.70(m,2H),7.43-7.49(m,1H),7.38(d,J=9.2Hz,1H),7.16(m,1H),2.52(d,J=0.4Hz,3H); 13C NMR (100 MHz, CDCl 3 )δ163.2(d,J=245.0Hz),149.8(d,J=2.7Hz),144.5,137.9(d,J=8.0Hz),130.5(d,J=8.2Hz),124.5,122.5(d,J=3.0Hz),116.7(d,J=21.1Hz),115.3,114.4,113.9(d,J=23.2Hz),14.8.(No low-field carbon was observed)

[0491]

[0492] 6-Chloro-3-phenylimidazo[1,2-b]pyridazine. To a solution of 6-chloroimidazo[1,2-b]pyridazine (394.5 mg, 2.569 mmol) in DMF (6 mL) was added N-iodosuccinimide (635.8 mg, 2.826 mmol) and the mixture was stirred at 23 °C for 48 hours. After the reaction was complete, it was evacuated to remove the solvent. The residue was diluted with dichloromethane and saturated with Na 2 S 2 CO 3 The organic layer was separated and washed with brine, and then washed with anhydrous MgSO 4 The mixture was dried, filtered and concentrated under reduced pressure to give 6-chloro-3-iodoimidazo[1,2-b]pyridazine in quantitative yield. Then 6-chloro-3-iodoimidazo[1,2-b]pyridazine (107.2 mg, 0.326 mmol), phenylboronic acid (43.7 mg, 0.358 mmol), K 2 CO 3 (54.0 mg, 0.391 mmol) and Pd(PPh 3 ) 4 A mixture of (18.8 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 2 mL) was heated at 90 °C overnight. After the reaction was cooled to 23 °C, it was diluted in water and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc=2:1) ​​to give the desired product (28.4 mg, 0.124 mmol, 38%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.06(s,1H),8.03(m,2H),7.98(d,J=9.6Hz,1H),7.52(m,2H),7.39(m,1H),7.08(d,J=9.2Hz,1H);13 C NMR (100 MHz, CDCl 3 )δ146.8,138.5,133.1,129.1,128.7,128.4,127.6,127.1,126.8,118.3.

[0493]

[0494] N-(3-(3-phenylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ023. Using the same procedure as described for JGJ002, 6-chloro-3-phenylimidazo[1,2-b]pyridazine (15.5 mg, 0.068 mmol), 3-aminophenylboronic acid (11.5 mg, 0.074 mmol), K 2 CO 3 (14.0 mg, 0.101 mmol) and Pd(PPh 3 ) 4 (3.9 mg, 0.003 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave 3-(3-phenylimidazo[1,2-b]pyridazin-6-yl)aniline (17.5 mg, 0.061 mmol, 91%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ023 (10.9 mg, 0.033 mmol, 54%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.18(s,1H),8.12(m,2H),8.04(s,1H),7.99(d,J=9.6Hz,1H),7.93(brs,1H),7.6 4-7.70(m,2H),7.50(m,2H),7.46(d,J=9.6Hz,1H),7.35-7.44(m,2H),2.22(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.7,151.1,138.8,136.4,133.0,129.6,128.8,128.7,128.6,127.9,126.8,125.8,122.7,121.3,118.3,115.6,24.6. (No low-field carbon was observed)

[0495]

[0496] 6-(3-Fluorophenyl)-3-phenylimidazo[1,2-b]pyridazine, JGJ024. Using the same procedure as described for JGJ002, 6-chloro-3-phenylimidazo[1,2-b]pyridazine (12.9 mg, 0.056 mmol), 3-fluorophenylboronic acid (8.6 mg, 0.062 mmol), K 2 CO 3 (11.7 mg, 0.084 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (3.2 mg, 0.003 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave the desired product JGJ024 (9.5 mg, 0.033 mmol, 58%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.10-8.14(m,4H),7.72-7.79(m,2H),7.48-7.56(m,4H),7.42(m,1H),7.20(m,1H); 13 C NMR (100 MHz, CDCl 3 )δ163.2(d,J=245.0Hz),150.5(d,J=2.7Hz),137.8(d,J=7.8Hz),133.0,130.6(d,J=8.2Hz),129.1,128.8 ,128.4,128.1,127.1,126.9,126.1,122.7(d,J=2.9Hz), 117.0(d,J=21.2Hz), 115.3,114.0(d,J=23.2Hz).

[0497]

[0498] 3-Methyl-6-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine, JGJ025. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.9 mg, 0.214 mmol), 3-trifluoromethylphenylboronic acid (42.7 mg, 0.225 mmol), K 2 CO 3 (44.4 mg, 0.321 mmol) and Pd(PPh 3 ) 4 Reaction of (12.4 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave the desired product JGJ018 (29.2 mg, 0.105 mmol, 49%) as a white solid. 1H NMR (400 MHz, CDCl 3 )δ8.27(s,1H),8.20(d,J=8.0Hz,1H),8.09(d,J=9.2Hz,1H),7.76(d,J=8.0Hz,1H),7.65-7.69(m,2H),7.51(d,J=9.2Hz,1H),2.66(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ149.8,136.7,132.6,132.1(d,J=9.8Hz),131.5(q,J=32.4Hz),130.2,129.5,128.4(d,J=12.0Hz),126.4(q,J=3.5Hz),125.7,123.9(q,J=270.8Hz),123.8(q,J=3.8Hz),114.1,8.7. (Due to the presence of some impurities, it will be tested again 13 C NMR)

[0499]

[0500] N-(3-Fluoro-5-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ026. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 3-fluoro-5-aminophenylboronic acid (34.3 mg, 0.221 mmol), K 2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4 (12.2 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave 3-fluoro-5-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (25 mg, 0.103 mmol, 49%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ026 (8 mg, 0.028 mmol, 28%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.38(br s,1H),8.00(d,J=9.2Hz,1H),7.89(s,1H),7.65(d,J=9.2Hz,1H),7.60(s,1H),7.43(s,1H),7.41(s,1H),2.60(s,3H),2.24(s,3H);

[0501] N-(4-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ027. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 4-aminophenylboronic acid (38.4 mg, 0.221 mmol), K 2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4 (12.2 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave 4-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (31.4 mg, 0.140 mmol, 66%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ026 (7.2 mg, 0.027 mmol, 19%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.99(d,J=8.8Hz,2H),7.95(d,J=9.2Hz,1H),7.68(d,J=8.4Hz,2H),7.58(s,1H),7.47(br s,1H),7.43(d,J=9.6Hz,1H),2.62(s,3H),2.23(s,3H);

[0502]

[0503] 6-Chloro-3-(pyridin-3-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (82.6 mg, 0.297 mmol), pyridine-3-boronic acid (40 mg, 0.325 mmol), K 2 CO 3 (61.3 mg, 0.443 mmol) and Pd(PPh 3 ) 4 Reaction of (17.1 mg, 0.015 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave the desired product (41.5 mg, 0.180, 61%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)δ9.21(s,1H),8.62(s,1H),8.40(m,1H),8.11(s,1H),7.98(d,J=9.6Hz,1H),7.43(dd,J=7.6,0.8Hz,1H),7.12(d,J=9.2Hz,1H); 13 CNMR (100MHz, CDCl 3 )δ149.0,147.6,147.2,139.1,133.6,133.5,127.4,126.0,124.3,123.6,118.9.

[0504]

[0505] N-(3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ028. Using the same procedure as described for JGJ002, 6-chloro-3-(pyridin-3-yl)imidazo[1,2-b]pyridazine (41.5 mg, 0.180 mmol), 3-aminophenylboronic acid (30.7 mg, 0.198 mmol), K 2 CO 3 (37.3 mg, 0.270 mmol) and Pd(PPh 3 ) 4 (10.4 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) afforded 3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (50.0 mg, 0.174 mmol, 96%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to afford the desired product JGJ028 (18.2 mg, 0.055 mmol, 32%) as a light yellow solid. 1 H NMR (400MHz, CD 3 OD)δ9.29(d,J=1.2Hz,1H),8.60(ddd,J=8.0,2.0,1.6Hz,1H),8.49(d,J=4.0Hz,1H),8.25(dd,J=2.0,1.6Hz,1H),8.18(s,1H),8 .02(d,J=9.6Hz,1H),7.68(d,J=9.6Hz,1H),7.60-7.65(m,2H),7.55(dd,J=8.0,4.8Hz,1H),7.37(t,J=8.0Hz,1H),2.16(s,3H); 13 C NMR (100MHz, CD 3OD)δ172.1,153.6,149.2,148.0,141.5,141.2,137.1,135.9,134.1,130.8,127.1,127.0,126.9,125.7,123.8,123.0,119.5,118.6,24.3.

[0506]

[0507] 6-Chloro-3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (83.6 mg, 0.299 mmol), pyrimidine-5-boronic acid (40.8 mg, 0.329 mmol), K 2 CO 3 (62 mg, 0.449 mmol) and Pd(PPh 3 ) 4 Reaction of (17.3 mg, 0.015 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave the desired product (9.8 mg, 0.042 mmol, 14%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.42(s,2H),9.23(s,1H),8.18(s,1H),8.04(d,J=9.6Hz,1H),7.20(d,J=9.6Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ157.7,154.0,147.7,133.7,132.1,128.5,127.7,123.0,119.8.

[0508]

[0509] N-(3-(3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ029. Using the same procedure as described for JGJ002, 6-chloro-3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazine (9.8 mg, 0.042 mmol), 3-aminophenylboronic acid (7.2 mg, 0.047 mmol), K 2 CO 3 (8.8 mg, 0.064 mmol) and Pd(PPh 3 ) 4(4.9 mg, 0.004 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave 3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (6.7 mg, 0.023 mmol, 55%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ029 (5.1 mg, 0.015 mmol, 67%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 +5% v / v CD 3 OD)δ9.58(s,2H),9.18(s,1H),8.23(s,1H),8.19(d,J=9.6Hz,1H),8.10(s,1H),7.99(d,J=8.0 Hz,1H)7.65(d,J=9.2Hz,1H),7.63(d,J=8.0Hz,1H),7.46(dd,J=8.4,7.6Hz,1H),2.19(s,3H); 13 C NMR (125 MHz, CDCl 3 +5% v / v CD 3 OD)δ169.7,156.8,153.9,152.5,139.6,134.7,131.9,129.9,125.9,123.7,122.4,122.2,122.1,118.0,117.7,117.6,24.0.

[0510]

[0511] 6-Bromoimidazo[1,2-a]pyridine. To a solution of 2-amino-5-bromopyridine (500 mg, 2.89 mmol) in EtOH (6 mL) and water (4 mL) was added bromoacetaldehyde diethyl acetal (870 μL, 5.78 mmol) and HBr (260 μL) at 23 °C. The resulting mixture was heated at 103 °C overnight. After the mixture was cooled to 23 °C, it was diluted in water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude residue was used in the next step without further purification. (brown solid; 331.7 mg, 1.68 mmol, 58%) 1 H NMR (400 MHz, CDCl 3)δ8.09(dd,J=2.0,0.8Hz,1H),7.46(d,J=0.8Hz,1H),7.39(s,1H),7.32(d,J=9.6Hz,1H),7.00(dd,J=9.6,2.0Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ143.2,133.8,127.3,125.4,117.8,112.3,106.5.

[0512]

[0513] N-(3-(Imidazolo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ030. Using the same procedure as described for JGJ002, 6-bromoimidazo[1,2-a]pyridine (50 mg, 0.254 mmol), 3-aminophenylboronic acid (43.3 mg, 0.279 mmol), K 2 CO 3 (52.6 mg, 0.381 mmol) and Pd(PPh 3 ) 4 (29.3 mg, 0.025 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave 3-(imidazo[1,2-a]pyridin-6-yl)aniline (22.3 mg, 0.107 mmol, 42%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ030 (13.6 mg, 0.054 mmol, 51%) as a white solid. 1 H NMR (400MHz, CD 3 OD)δ8.68(s,1H),7.89-7.94(m,2H),7.56-7.62(m,3H),7.51(ddd,J=7.6,2.0,1.2Hz,1H),7.35-7.43(m,2H),2.16(s,3H); 13 C NMR (100MHz, CD 3 OD)δ170.3,139.2,137.4,132.1,129.1,126.7,125.7,123.8,122.1,119.1,118.0,115.8,113.5,22.4.(No low field carbon was observed)

[0514]

[0515] 6-Bromo-3-methylimidazo[1,2-a]pyridine. A mixture of 2-amino-5-bromopyridine (200 mg, 1.156 mmol) and 2-bromopropanal (purity>95%, 318 mg, 2.312 mmol) in EtOH (5 mL) was heated under reflux overnight. After the mixture was cooled to 23 °C, it was concentrated and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc=3:2) to give the desired product (86.9 mg, 0.412 mmol, 36%) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.00(d,J=1.2Hz,1H),7.49(d,J=9.2Hz,1H),7.40(s,1H),7.20(dd,J=9.6,2.0Hz,1H),2.46(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ143.5,132.1,126.5,123.0,120.3,118.3,106.9.9.0.

[0516]

[0517] N-(3-(3-methylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ031. Using the same procedure as described for JGJ002, 6-bromo-3-methylimidazo[1,2-a]pyridine (35 mg, 0.166 mmol), 3-aminophenylboronic acid (28.3 mg, 0.182 mmol), K 2 CO 3 (34.4 mg, 0.249 mmol) and Pd(PPh 3 ) 4 (9.6 mg, 0.008 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-(3-methylimidazo[1,2-a]pyridin-6-yl)aniline (28.1 mg, 0.106 mmol, 64%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ031 (15.8 mg, 0.060 mmol, 56%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ8.30(br s,1H),8.12(s,1H),7.87(s,1H),7.65(d,J=8.0Hz,1H),7.54(d,J=8.0Hz,1H),7.36-7.43(m,3H),7.27(m,1H),2.49(s,3H),2.23(s,3H);

[0518]

[0519] 3-(3-phenylimidazo[1,2-a]pyridin-6-yl)aniline, JGJ032. 2-amino-5-bromo-pyridine (100 mg, 0.508 mmol), 3-aminophenylboronic acid (76.5 mg, 0.558 mmol), triphenylphosphine (26.6 mg, 0.102 mmol) and K were added to a microwave tube. 2 CO 3 Pd(OAc) (140.3 mg, 1.015 mmol) was added to a mixture of toluene:EtOH mixture (2:1 v / v, 1.7 mL) 2 (11.4 mg, 0.059 mmol) and filled with argon. The mixture was sealed with a silicon septum and irradiated in a microwave at 140 ° C for 30 minutes under stirring. After the mixture was cooled to 23 ° C, bromobenzene (119.5 mg, 0.761 mmol) was injected into the tube with a syringe, and the mixture was again subjected to microwave irradiation at 140 ° C for 2.5 hours under stirring. The reaction vessel was cooled to 23 ° C and the mixture was diluted with water and extracted with dichloromethane. The combined organic layer was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc:MeOH=1:1:0.1) to give the desired product (28.8 mg, 0.101 mmol, 20%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.46(s,1H),7.83(d,J=9.2Hz,1H),7.73(s,1H),7.45-7.61(m,6H),7.23 (d,J=8.0Hz,1H),6.90(d,J=8.0Hz,1H),6.81(t,J=2.0Hz,1H),6.71(m,1H);

[0520]

[0521] N-(3-(3-phenylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ033. Using the same procedure as described for JGJ004, the reaction of 3-(3-phenylimidazo[1,2-a]pyridin-6-yl)aniline (JGJ032, 22.8 mg, 0.080 mmol), triethylamine (12.1 mg, 0.120 mmol) and acetyl chloride (9.4 mg, 0.120 mmol) in dichloromethane (2 mL) gave the desired product JGJ033 (12.2 mg, 0.037 mmol, 47%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.48(s,1H),7.80(dd,J=2.0,1.6Hz,1H),7.73(s,1H),7.51-7.65(m,7H),7.43(m,1H),7.35(dd,J=8.0Hz,1H),7.27(m,1H),2.12(s,3H); 13 C NMR (100MHz, CD 3 OD)δ170.3,139.2,137.4,131.2,129.2,129.0,128.4,128.2,127.7,127.1,126.5,125.6,122.0,120.5,119.0,117.8,116.5,22.4.(No low field carbon was observed)

[0522]

[0523] 5-Chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine. 2-Chloro-5-hydrazinopyridine (71.3 mg, 0.5 mmol) was added to a 4% w / w H 2 SO 4 To the solution in aqueous solution (5 mL) was added (2,2-dimethoxyethyl)benzene (87.3 mg, 0.525 mmol). The reaction vessel was sealed with a silicon septum and stirred at 23 °C for 1 minute, then irradiated in a microwave at 160 °C for 5 minutes. After the mixture was cooled to 23 °C, it was slowly poured into 40% w / w KOH solution (5 mL). The mixture was extracted with EtOAc and the combined organic layers were washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The obtained crude residue was purified by flash column chromatography (n-hexane: EtOAc = 3:2) to give the desired product (71.3 mg, 0.312 mmol, 62%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)δ8.96(br s,1H),7.99(d,J=7.2Hz,2H),7.59(s,1H),7.57(d,J=8.8Hz,1H),7.39(t,J=7.6Hz,2H),7.23(dd,J=7.6,7.2Hz,1H),7.12(d,J=8.9Hz,1H). The spectral data are consistent with the literature data. [Reference: Eur.J.Org.Chem.2013,3328-3336.

[0524]

[0525] N-(3-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ034. Using the same procedure as described for JGJ002, 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (40 mg, 0.175 mmol), 3-aminophenylboronic acid (29.8 mg, 0.192 mmol), K 2 CO 3 (36.3 mg, 0.262 mmol) and Pd(PPh 3 ) 4 (20.2 mg, 0.018 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave 3-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.8 mg, 0.066 mmol, 38%) as a white solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ034 (13.5 mg, 0.041 mmol, 63%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.29(s,1H),8.24(d,J=7.2Hz,2H),7.88(s,1H),7.83(d,J=7.6Hz,1H),7.82(d,J=8.8Hz,1H),7.6 4(d,J=8.4Hz,1H),7.62(d,J=7.6Hz,1H),7.39-7.44(m,3H),7.21(dd,J=7.6,7.2Hz,1H),2.17(s,3H); 13 C NMR (100MHz, CD 3 OD)δ170.3,150.1,143.3,141.1,138.7,134.5,129.3,128.5,127.9,126.3,126.2,125.1,122.4,119.3 (two peaks),118.3,115.7,114.0,22.4.

[0526]

[0527] 5-Chloro-3-propyl-1H-pyrrolo[3,2-b]pyridine. Using the same procedure as described for 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine, 2-chloro-5-hydrazinopyridine (71.8 mg, 0.5 mmol) and valeraldehyde (45.1 mg, 0.524 mmol) were mixed in a 4% w / w H 2 SO 4 The reaction in aqueous solution (5 mL) afforded the desired product as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.01(br s,1H),7.61(d,J=8.0Hz,1H),7.26(s,1H),7.08(d,J=8.0Hz,1H),2.77(t,J=7.6Hz,2H),1.73(m,2H),0.94(t,J=7.2Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ145.0,143.4,127.8,126.3,120.9,117.2,116.6,26.8,23.0,14.0.

[0528]

[0529] 3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline, JGJ035. Using the same procedure as described for JGJ002, 5-chloro-3-propyl-1H-pyrrolo[3,2-b]pyridine (40 mg, 0.206 mmol), 3-aminophenylboronic acid (31 mg, 0.226 mmol), K 2 CO 3 (42.6 mg, 0.308 mmol) and Pd(PPh 3 ) 4 Reaction of (23.8 mg, 0.021 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ035 (42.5 mg, 0.169 mmol, 82%) as a white solid. 1 H NMR (400MHz, CD 3OD)δ7.72(d,J=8.4Hz,1H),7.44(d,J=8.8Hz,1H),7.34(dd,J=2.0,1.6Hz,1H),7.30(s,1H),7.24(ddd,J=7.6,1.6,1.2Hz ,1H),7.19(t,J=7.6Hz,1H),6.76(ddd,J=7.6,2.0,1.2Hz,1H),2.85(t,J=7.6Hz,2H),1.79(m,2H),1.02(t,J=7.2Hz,3H); 13 C NMR (100MHz, CD 3 OD)δ152.4,128.8,146.2,143.4,130.2,130.1,127.6,120.3,118.8,117.4,116.3,115.8,27.1,24.6,14.5.(No low field carbon was observed)

[0530]

[0531] N-(3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ036. Using the same procedure as described for JGJ004, the reaction of 3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (JGJ035, 34.5 mg, 0.137 mmol), triethylamine (20.8 mg, 0.206 mmol) and acetyl chloride (16.2 mg, 0.206 mmol) in dichloromethane (3 mL) gave the desired product JGJ036 (28.8 mg, 0.098 mmol, 72%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.11(dd,J=2.0,1.6Hz,1H),7.75(d,J=8.4Hz,1H),7.64-7.67(m,2H),7.49(d,J=8.8Hz,1H),7.39 (t,J=8.0Hz,1H),7.32(s,1H),2.85(t,J=7.2Hz,2H),2.15(s,3H),1.80(m,2H),1.01(t,J=7.2Hz,3H); 13 C NMR (100MHz, CD 3 OD)δ171.8,151.4,146.4,143.1,140.1,130.3,129.9,127.9,124.2,120.6,120.4,120.2,117.4,115.7,27.1,24.5,23.9,14.5.

[0532]

[0533] N-(3-Fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ037. Using the same procedure as described for JGJ002, 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (19.4 mg, 0.085 mmol), 3-fluoro-5-aminophenylboronic acid (14.5 mg, 0.093 mmol), K 2 CO 3 (17.6 mg, 0.127 mmol) and Pd(PPh 3 ) 4 (9.8 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) afforded 3-fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.1 mg, 0.060 mmol, 70%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to afford the desired product JGJ037 (13.8 mg, 0.040 mmol, 67%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.25(m,2H),7.98(t,J=1.6Hz,1H),7.88(s,1H),7.79(d,J=8.4Hz,1H),7.61(d,J=8 .8Hz,1H),7.58(m,2H),7.43(t,J=7.6Hz,2H),7.21(td,J=7.6,1.2Hz,1H),2.15(s,3H); 13 C NMR (100MHz, CD 3 OD)δ171.9,164.6(d,J=239.6Hz),150.2(d,J=2.9Hz),145.1,144.7(d,J=8.9Hz),141.7(d,J=11.5Hz),136.0,130.9, 129.4, 127.8, 127.6, 126.6, 120.5, 117.3, 115.3, 114.7 (d, J = 3.2Hz), 109.8 (d, J = 23.1Hz), 107.3 (d, J = 27.0Hz), 24.0.

[0534]

[0535] N-(3-Fluoro-5-(3-methylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ038. Using the same procedure as described for JGJ002, 6-bromo-3-methylimidazo[1,2-a]pyridine (23.4 mg, 0.111 mmol), 3-fluoro-5-aminophenylboronic acid (18.9 mg, 0.122 mmol), K 2 CO 3 (23.0 mg, 0.166 mmol) and Pd(PPh 3 ) 4 (12.8 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-fluoro-5-(3-methylimidazo[1,2-a]pyridin-6-yl)aniline (13.2 mg, 0.055 mmol, 49%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ038 (8.3 mg, 0.029 mmol, 64%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.41(s,1H),7.56-7.63(m,3H),7.52(dt,J=10.8,2.0Hz,1H),7.40(s,1H),7.23(dt,J=9.6,2.0Hz,1H),2.57(s,3H),2.17(s,3H);

[0536]

[0537] 6-Chloro-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (90.5 mg, 0.324 mmol), 4-pyridineboronic acid (43.8 mg, 0.356 mmol), K 2 CO 3 (67.1 mg, 0.486 mmol) and Pd(PPh 3 ) 4 Reaction of (37.4 mg, 0.032 mmol) in 1,4-dioxane / water (5:1 v / v, 0.7 mL) gave the desired product (15.3 mg, 0.066 mmol, 20%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)δ8.72(d,J=5.2Hz,2H),8.23(s,1H),7.98-8.02(m,3H),7.18(d,J=9.2Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ150.2,147.3,139.8,135.2,134.9,127.5,126.1,119.9,119.4.

[0538]

[0539] N-(3-Fluoro-5-(3-(pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ039. Using the same procedure as described for JGJ002, 6-chloro-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine (15.3 mg, 0.066 mmol), 3-fluoro-5-aminophenylboronic acid (11.3 mg, 0.073 mmol), K 2 CO 3 (13.7 mg, 0.100 mmol) and Pd(PPh 3 ) 4 (7.7 mg, 0.007 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-fluoro-5-(3-(pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (10.7 mg, 0.035 mmol, 53%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ039 (3.8 mg, 0.011 mmol, 31%) as a light yellow solid. 1 H NMR (400MHz, CD 3 OD)δ8.69(s,2H),8.46(s,1H),8.36(d,J=5.2Hz,2H),8.20-8.23(m,2H),7.88(d,J =9.2Hz,1H),7.64(dt,J=10.8,1.6Hz,1H),7.55(dt,9.6,1.6Hz,1H),2.20(s,3H).

[0540] Embodiment 2: LIN28 is significantly overexpressed in human and mouse AML and drives MLL leukemogenesis.

[0541] Analysis of the AML and healthy hematopoietic cell (HSC, Blood Spot (55)) databases showed that Lin28b expression was significantly enriched in multiple AML karyotypes when compared with healthy HSCs ( Figure 2A). In addition, Lin28 has been found to be a key driver of MLL-related leukemia (56). To further characterize the role of Lin28 / let-7 in regulating AML, LSC proliferation and treatment resistance in vivo, we used a doxycycline (DOX)-inducible transgenic mouse model for MLL-AF9-driven AML (iMLL-AF9 (57)). In this model, long-term HSCs (LT-HSCs, Lin - CD34 - Sca-1 - c-Kit + CD150 + CD48 - )-derived AML blasts are closely associated with an LSC-like phenotype, giving rise to particularly aggressive cytarabine (Ara-C)-resistant AML (57). We transplanted whole bone marrow (WBM) cells or fluorescence-activated cell sorted (FACS) LT-HSCs from non-induced iMLL-AF9 mice into congenic mice (B6.SJL, CD 45.1) and maintained the recipients on DOX. mRNA analysis of AML cells harvested at day +35 (d) demonstrated that expression of Lin28b was significantly increased in WBM- and LT-HSC-derived AML cells (LSCs) compared with healthy non-DOX-induced LT-HSCs ( Figure 2B In addition, relapsed AML cells generated from rLSCs after treatment with Ara-C (100 mg / kg) on ​​d+60 were further enriched in Lin28b expression ( Figure 2B In addition, the levels of both let-7a and let-7b miRNAs were negatively correlated with the increased Lin28b in rLSCs ( Figure 2C )(10, 39). Our results are consistent with papers reporting that increased Lin28 is associated with disease recurrence after chemotherapy in colon cancer and liver cancer stem cells (22, 58).

[0542] Embodiment 3: Lin28 inhibition overcomes treatment resistance in relapsed AML.

[0543] Since Lin28 is overexpressed in human AML, LSCs, and rLSCs, we sought to determine whether genetic Lin28b or pharmacological Lin28 / let-7 inhibition by LN1632 could abrogate the proliferation of LSCs and thereby overcome their therapeutic resistance. We isolated LT-HSCs by FACS and incubated 500 cells with DOX and simultaneously transduced them with shLin28b or its corresponding control shScramble or treated cells with 200 nM Ara-C, 30 μM 1632, or control for 48 h. While Ara-C did not alter the number of colony-forming competent cells (CFC), genetic silencing of (shLin28b) or pharmacological inhibition of Lin28 by LN1632 treatment significantly abrogated the CFC of LSCs ( Figure 2D ).

[0544] Embodiment 4: Targeted LIN28 / let-7 inhibition reduces tumor burden in AML in vivo.

[0545] Given that genetic Lin28b inhibition and LN1632 treatment abolished CFCs of LSCs, we sought to explore the role of LN1632 in human AML. By Western blotting, we confirmed that the Lin28 inhibitor LN1632 dose-dependently reduced LIN28B protein levels in AML in the context of t(8;21) (Kasumi-1) and MLL rearrangements (THP-1). Figure 3A ). Notably, the proteasome inhibitor bortezomib was able to suppress the decrease in LIN28B protein levels in TF1-α cells after 1632 treatment ( Figure 3B ), suggesting that 1632 may directly target LIN28B, leading to its proteasomal degradation. Therefore, we investigated the inhibitory effect of targeting Lin28 / let-7 in AML in vivo. We established intermittent dosing of 100 mg / kg every other day for 21 days in healthy C57BL / 6 mice, which was non-toxic and well tolerated because they showed normal weight gain, complete blood count (CBC) and behavior. Therefore, we implanted THP-1 (high LIN28B) cells or MOLM-13 (no LIN28B) subcutaneously (subQ) into NSG mice and after 12 days (tumor size = 40 mm 2 ) was started with 1632 at 100 mg / kg IP every other day. Our results showed a significant reduction in tumor growth in THP-1 but not MOLM-13 xenografts ( Figure 4A We further evaluated 1632 in systemic Kasumi-1 cell line xenografts (LSC-like CD34 +CD38-, high LIN28B, AML t(8;21)). IP injection of 100 mg / kg of 1632 every other day for 21 days significantly prolonged the survival of the animals ( Figure 4B Bioluminescence imaging (BLI) confirmed that tumor burden was reduced in 1632-treated mice compared with vehicle ( Figure 4C ,picture).

[0546] Embodiment 5: Targeted inhibition of LIN28 downregulates NF-κB and BCL-2 in primary AML.

[0547] To measure the full extent to which LN1632 regulates gene expression, we performed RNA sequencing (RNAseq) in LSC-like Kasumi-1 cells. Figure 5A As shown in the heat map in Figure 2, we found that a whole set of direct let-7 target genes (including CCND1 / 2, E2F2, HMGA1, LIN28B, MYC, NFKB1, MRAS, IL6, and STAT5) were significantly downregulated (44) (green, Figure 5A Importantly, we confirmed this gene expression pattern in primary AML cells from three relapsed patients (LIN28B overexpression was verified compared to healthy WBM). 72 hours after treatment, we found a significant dose-dependent upregulation of mature let-7a / b and downregulation of multiple let-7 target genes including NFκB1 ( Figure 5B This is important because NFκB1 is regulated by let-7 through IL-6 (23), in conjunction with other BCL-2 family members (BAX, BCL2L15, and BMF, Figure 5A ) are well-characterized genes associated with unique properties of LSC survival and AML relapse (45, 59, 60). Consistent with this, gene set enrichment analysis revealed global changes in a gene expression signature previously shown to distinguish LSC from non-self-renewing leukemic cell populations (61) and poor prognosis in pediatric AML relapse (62) ( Figure 5C We next explored the effect of LN1632 on primary AML cells. Fig. 6A CFC assays in the cytotoxic T cells showed that treatment with LN1632 affected CD34 + Colony formation of AML pt. #13 cells is significantly more abundant than healthy CD34 + BM cells. In addition, ex vivo treatment of AML pt#13 cells with 1632 or control inhibited AML repopulation in vivo ( Figure 6B -C). Therefore, our results suggest that LN1632 has a greater effect on LSCs than on HSCs.

[0548] Embodiment 6: Lin28 / let-7 inhibitory activity of exemplary compounds.

[0549] To improve the binding and inhibitory ability of compounds to LIN28b, we predicted the binding mode of LN1632 to LIN28B. A close-up of the LIN28 protein crystal structure revealed the possible binding mode of LN1632 to the GGAG-RNA sequence binding pocket of the CCHC domain of LIN28 (not shown). With this model, we synthesized novel compounds with improved binding ability to Lin28b (JGJ002–JGJ008, Figure 8). The compounds were screened using a previously described FRET assay with EGFP-tagged LIN28B as the donor and BHQ-1 quencher-tagged pre-let-7a-2 (pre-let-7a-2-BHQ1) as the acceptor (51). Briefly, recombinant LIN28B-EGFP was harvested from stably transduced HEK cells and diluted with binding buffer (300mM NaCl, 25mM HEPES pH 7.2, 10μM ZnCl2, 1% Odyssey blocking buffer, 0.05% Tween 20, 0.5mM TCEP) to adjust the ideal FRET quenching signal intensity. Protein lysate and compounds (JGJ001–JGJ008) were pre-incubated for 20 minutes in 100uL of diluted protein lysate at doses ranging from 1.25uM–20uM. Pre-let-7a-2-BHQ1 was then added to the mixture (at 6.25nM) and the EGFP-LIN28B donor emission was measured using a Tecan Spark Plate Reader (20nM band, 488nM excitation, 545nM emission readout, 30 flashes / second). The results showed that compounds JGJ005, JGJ007, and JGJ008 in particular inhibited the FRET signal intensity to a greater extent than the original hit compound LN1632. Based on these results, we concluded that JGJ005, JGJ007, and JGJ008 have a greater inhibitory effect on LIN28B / pre-let-7a2 binding than the original compound LN1632 ( Figure 7 ), and thus are expected to inhibit LIN28 binding to pre-let-7 microRNAs, thereby preventing their degradation. Increased endogenous let-7 miRNA levels can target a full set of LCS and cancer stem cell marker genes, thereby inhibiting tumor growth.

[0550] Example 7: Evaluation of in vitro and in vivo activity of LN1632

[0551] Using targeted high-throughput fluorescence resonance electron transfer (FRET) screening, triazolopyridazines were identified as a class of small molecules that block the interaction of RBP LIN28 with pre-let-7 miRNA (51). To investigate how LN1632 interacts with the LIN28 protein, a computer-simulated molecular docking study was performed using the crystal structure of the LIN28B pre-let-7a complex (PDB ID: 5UDZ) (28). Based on the ability of LN1632 to compete for the LIN28B-pre-let-7 complex in the FRET assay, it was hypothesized that the binding site might be shared with the ZKD RNA binding motif of LIN28. Docking results showed that LN1632 binds to the pocket originally occupied by the GGAG motif of pre-let-7a. The results also indicated that the amide group of the phenyl ring of LN1632 is located in a pocket near the zinc ion binding site through hydrogen bonding interactions with LIN28B. ( Fig. 8A ).

[0552] To test the structure-activity relationship, 39 LN1632-related analogs (JGJ001-39) were synthesized and their potency and specificity in inhibiting LIN28B-RNA binding activity and upregulating mature let-7 miRNA levels were measured. By performing a previously published FRET assay, it was observed that JGJ023, JGJ026, JGJ032, and JGJ034 inhibited the RNA binding ability of LIN28 significantly more than compound LN1632 ( Figure 8B In addition, in HepG2 cells, JGJ023, JGJ026, and JGJ034 upregulated mature let-7 miRNA at doses significantly lower than LN1632, as measured by a dual-luciferase reporter assay ( Figure 8C Dual-luciferase reporter assays were performed as described previously (89).

[0553] To determine the extent to which LN1632 modulates gene expression, RNA sequencing was performed in human Kasumi-1 AML cells. Figures 9A-9B The data in the study showed that cells treated with LN1632 significantly downregulated genes in the signature_MYC-target_V1 gene signature (70), leukemia stem cell, and relapse prognostic signatures (61, 62). In addition, ingenuity pathway analysis predicted inhibition of upstream signaling molecules IL6 and MYC ( Fig. 9C ).

[0554] Next, the tumor inhibitory effect of LN1632 in vivo was investigated. The maximum tolerated dose (MTD) was evaluated in healthy female C57Bl / 6 mice. Daily dosing of 100 mg / kg for +12 days, followed by an every other day dosing regimen for +9 days was well tolerated, and mice had normal complete blood count (CBC) curves, without any leukopenia or thrombocytopenia, only mild anemia and normal weight gain ( Fig. 10A -B).

[0555] Subsequently, the tumor suppressive effect of LN1632 in cancer in vivo was evaluated. THP-1 AML cells (2x10 6 cells) were implanted into NSGS mice (cell suspension in Matrigel, 3:1) and on d+12 or d+8 (tumor size = 50 mm 2 ) began daily IP injection of 100 mg / kg LN1632. The results showed that tumor growth was significantly reduced 19 days after injection ( Fig.11A ). These results are consistent with a recent report showing that LN1632 selectively inhibits LIN28B-expressing Ewing's sarcoma (EwS) but not LIN28B-depleted EwS (72) and LIN28B-expressing TNBC cells (73). The effect of LN1632 was also evaluated in systemic Kasumi-1 xenografts. IP injections of 100 mg / kg of LN1632 every other day for 21 days significantly prolonged animal survival ( Fig. 11B Bioluminescence imaging (BLI) confirmed that tumor burden was reduced in LN1632-treated mice compared with vehicle ( Fig. 11B , Figure). The effects of LN1632 on cytarabine chemotherapy (Ara-C) were also compared as previously described (74). THP-1 AML cells (1.5x10 6 cells, high LIN28B) in NSGS mice. Daily IP injections of 100 mg / kg LN1632, 60 mg / kg cytarabine chemotherapy (Ara-C) or vehicle were started on d3 after AML cell implantation and continued until the vehicle group reached the maximum allowed tumor size (250 mm 2 ). Compared with Ara-C or vehicle-treated mice, mice treated with LN1632 showed increased inhibition of AML tumor proliferation ( Fig. 11C ).

[0556] Since LN1632 showed significant antiproliferative effects in in vivo cancer models, other functional interaction partners of LN1632 were evaluated. Mass spectrometry cellular thermal shift assay (MS-CETSA, Fig. 12A), and immunoprecipitation using biotinylated LN1632 ( Fig. 12B These experiments demonstrated that LN1632 interacts with other RNA-binding proteins, especially pre-mRNA processing factor 31 ( Fig. 12C , PRPF31). PRPF31 is a component of the spliceosomal complex and is significantly overexpressed in embryonic stem cells (76) and downregulated during differentiation (77). PRPF31 is recruited to the intron where its highly conserved Nop domain coordinates U4 snRNA–15.5K protein interactions. Subsequently, PRPF31 stabilizes the U4 / U6.U5 tri-snRNP by simultaneously interacting with PRPF6 and induces the spliceosomal complex to transition to an activated state (78).

[0557] like Fig.13 As shown in Figure 2, PRPF31 overexpression is associated with poor prognosis in various tumors, including lung and gastric adenocarcinomas and triple-negative breast cancer (TNBC) ( Fig.13 ). Dysregulation of components of the U4 / U6.U5 tri-snRNP complex has been shown to drive tumorigenesis in colorectal cancer (79), TNBC (80-82), hepatocellular carcinoma (83), and lung cancer. Dysfunctional RNA splicing and overexpression of splicing factors are important mechanisms for tumor cell survival and intersect with many hallmarks of cancer (84-86). Emerging studies have shown that, in some embodiments, components of the spliceosome are essential for the oncoprotein MYC to drive cancer progression. Without wishing to be bound by any particular theory, because MYC is the most commonly amplified oncogene in human cancers and plays a key role in malignant transformation, in some embodiments, therapies that exploit the spliceosome and particularly target PRPF31 and the U4 / U6 spliceosome complex would be very attractive.

[0558] MDA-MB-231TNBC cells were used to evaluate whether LN1632 targets PRPF31. Overexpression of PRPF31 increased cell proliferation, while genetic silencing of PRPF31 significantly reduced the number of cells assessed over 7 days ( Fig.14A Importantly, PRPF31 overexpression (pLenti-C-mGFP-P2A-Puro-PRPF31, Origene) rescued the antiproliferative effect of LN1632, indicating that LN1632 targets PRPF31. In addition, gene silencing of PRPF31 by short hairpin-mediated RNA (shRNA, ThermoFisher Scientific, TRCN0000001180) abolished the pro-apoptotic effect of LN1632. Taken together, these results suggest that LN1632 targets PRPF31 ( Fig.14A ).

[0559] To test whether LN1632 and its novel analogs affect cancer cell growth, we performed a double-blind, double-blind, double-dose ... Fig. 14B -D), castration-resistant prostate cancer cells (CRPC, Fig.15A -C) and colorectal cancer cells (CRC, Fig.15A -C) Cell viability and cell counting assays were performed. The data showed that LN1632 and the novel analogs JGJ034 and JGJ037 preferentially reduced proliferation and induced apoptosis in MYC-driven cancers, including TNBC, CRPC, lung and colorectal adenocarcinoma cells (Table 1).

[0560] In order to measure cell viability, cell titer luminescence (CTG, Promega CellTiter-Glo 2.0 assay) and MTT assay (SigmaAldrich, cell proliferation kit I) were performed. In short, cells were serum starved overnight before being seeded in 96-well plates. After incubation for 24 hours, cells were treated with JGJ compounds with increasing concentrations for 96 hours. When the assay reads out, CellTiter-Glo reagent is added and luminescence is measured after incubation at room temperature for 10 minutes. For MTT assays, MTT labeling reagents are added and incubated for 4 hours. Subsequently, the culture medium is removed and 50 μL DMSO is added to dissolve the crystals, and the absorbance is measured at 570nm. Cell viability is calculated as (sample-background) / (control-background). Current standard care drugs enzalutamide, palbociclib and cetuximab are used as comparative controls.

[0561] Table 1

[0562]

[0563]

[0564]

[0565] The in vitro ADME characteristics of selected analogs are summarized in Tables 2 and 3.

[0566] Table 2.

[0567]

[0568] Table 3.

[0569]

[0570] Example 8: Synthesis of LN1632 analogs (JGJ compounds)

[0571] General experimental methods

[0572] Unless otherwise noted, all reactions were performed under an argon atmosphere. Tetrahydrofuran (THF) was distilled from benzoquinone ketyl radical under an argon atmosphere. Dichloromethane and triethylamine were distilled from calcium hydride under an argon atmosphere. All other solvents and reagents were purified according to literature procedures or purchased from Sigma-Aldrich, Acros, Oakwood, and Fisher Scientific Co. 1 H NMR spectra were recorded at 400 or 500 MHz and reported relative to the deuterated solvent signal. 1 HNMR spectral data are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. The resolution pattern is designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. Recordings were made at 100 or 125 MHz. 13 CNMR spectroscopy. 13 C NMR spectral data are reported as chemical shifts. Chemical shifts are reported in parts per million (ppm, δ). Thin layer chromatography (TLC) was performed using pre-coated silica gel sheets. Visual detection was performed using potassium permanganate or ceric ammonium nitrate staining. Flash chromatography was performed using SilicaFlash P60 (60A, 40-63 μm) silica gel with compressed air.

[0573]

[0574] 3-Chloro-6-hydrazinopyridazine.

[0575] To a solution of 3,6-dichloropyridazine (400 mg, 2.686 mmol) in EtOH (8 mL) was added hydrazine monohydrate (148 mg, 2.954 mmol) and the mixture was stirred at 100° C. for 3 hours. After the mixture was cooled to 23° C., the resulting solid was collected and eluted with EtOH. 2 O. The mother liquor was concentrated and washed with Et 2 The precipitate was washed with 4% CO 0. The combined solids were washed with dichloromethane to give the desired product (light yellow, 320.2 mg, 2.216 mmol, 82%) and used in the next step without further purification. 1 H NMR (400 MHz, DMSO-d 6 )δ8.24(br s,1H),7.41(d,J=9.6Hz,1H),7.09(d,J=9.2Hz,1H),4.37(br s,2H); 13 C NMR (100 MHz, DMSO-d 6)δ161.8,145.4,128.7,116.1. The spectral data are consistent with the literature data. [Reference: Heterocycles, 2009, 78 (4) 961-975]

[0576]

[0577] 6-Chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine.

[0578] A mixture of 3-chloro-6-hydrazinopyridazine (300 mg, 2.075 mmol) in AcOH (1.5 mL) was heated at 100 °C for 2 h. After the reaction mixture was cooled to 23 °C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with brine and anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The resulting off-white crude solid (238.5 mg, 68%) was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 )δ8.04(d,J=9.6Hz,1H),7.09(d,J=9.6Hz,1H),2.81(s,3H).

[0579]

[0580] 3-Methyl-6-phenyl-[1,2,4]triazolo[4,3-b]pyridazine, JGJ002. 6-Chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (20 mg, 0.119 mmol), phenylboronic acid (14.5 mg, 0.119 mmol), K 2 CO 3 (24.6 mg, 0.178 mmol) and Pd(PPh 3 ) 4 A mixture of (13.6 mg, 0.012 mmol) in 1,4-dioxane (0.3 mL) and water (30 μL) was heated at 110 °C for 18 hours. After the reaction mixture was cooled to 23 °C, it was diluted with water and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous MgSO 4 Dried, filtered and concentrated under reduced pressure. The obtained crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to obtain the desired product JGJ002 (20.4 mg, 0.098 mmol, 82%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ.8.13(d,J=9.2Hz,1H),7.98-8.01(m,2H),7.54-7.56(4H,m),2.88(s,3H) 13 C NMR (100 MHz, CDCl 3 )δ153.4,147.5,143.4,134.4,130.9,129.2,127.2,124.9,118.8,9.8.

[0581]

[0582] 3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)aniline, JGJ003. Using the same procedure as above, 6-chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazine (30 mg, 0.178 mmol), 3-nitrophenylboronic acid (35.6 mg, 0.214 mmol), K 2 CO 3 (36.9 mg, 0.267 mmol) and Pd(PPh 3 ) 4 (20.6 mg, 0.018 mmol) in 1,4-dioxane (0.3 mL) and water (30 μL) gave 3-methyl-6-(3-nitrophenyl)-[1,2,4]triazolo[4,3-b]pyridazine (19.7 mg, 0.077 mmol, 43%). 1 H NMR (400 MHz, CDCl 3 )δ.8.86(t,J=2.0Hz,1H),8.39(m,2H),8.24(d,J=9.6Hz,1H),7.71(t,J=8.0Hz,1H),7.62(d,J=9.6Hz,1H),2.91(s,3H) 13 CNMR (100MHz, CDCl 3 )δ151.1,148.8,147.7,143.2,136.1,132.8,130.4,125.8,125.4,122.2,118.0,9.9. Then the nitro compound (19.4 mg, 0.076 mmol) and SnCl 2 A mixture of (72.1 mg, 0.380 mmol) in EtOH (0.2 mL) was heated under reflux for 1 h. After the mixture was cooled to 23 °C, it was filtered through a celite pad and washed with EtOAc. To the mixture was added saturated NaHCO 3 The solution was extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4Dried, filtered and concentrated under reduced pressure. The obtained crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to obtain the desired product JGJ003 (10 mg, 0.044 mmol, 63%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.10(d,J=10.0Hz,1H),7.51(d,J=10.0Hz,1H),7.26-7.32(m,3H),6.83-6.86(m,1H),2.86(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ153.5,147.3(two peaks overlap),143.4,135.2,130.0,124.4,119.1,117.4,117.2,113.1,9.7.

[0583]

[0584] N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide, JGJ004. To a solution of 3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)aniline (JGJ003, 20 mg, 0.088 mmol) in dichloromethane (0.5 mL) was added trimethylamine (10.8 mg, 0.106 mmol) and acetyl chloride (7.6 mg, 0.099 mmol). The mixture was stirred at 23 °C for 6 hours. To this mixture was added water and extracted with dichloromethane. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (dichloromethane:MeOH=6:1) to give the desired product JGJ004 (21.1 mg, 0.079 mmol, 89%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.32(s,1H),8.09(d,J=9.6Hz,1H),7.88(br s,1H),7.70(d,J=7.6Hz,1H),7.65(d,J=8.0Hz,1H),7.54(d,J=10.0Hz,1H),7.48(t,J=8.0Hz,1H),2.86(s,3H),2.25(s,3H). 13 C NMR (125MHz, CD 3OD)δ172.8,156.1,149.9,145.8,141.8,137.0,131.5,126.3,124.8,124.2,122.6,120.5,24.8,10.4.

[0585]

[0586] N-methyl-N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide, JGJ001. To a solution of N-(3-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl)acetamide (JGJ004, 16.5 mg, 0.062 mmol) was added a 60% dispersion of NaH in mineral oil (5 mg, 0.124 mmol) at 0°C and stirred for 30 minutes. Then iodomethane (17.5 mg, 0.124 mmol) was added and the reaction mixture was stirred at 23°C for 2 hours. After the reaction was complete, water was added and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (dichloromethane:MeOH=10:1) to give the desired product JGJ001 (9.8 mg, 0.035 mmol, 56%) as an ivory solid. 1 H NMR (500 MHz, CDCl 3 )δ8.17(d,J=9.5Hz,1H),7.95(d,J=7.5Hz,1H),7.88(s,1H),7.62(dd,J=8.0,7.5Hz ,1H),7.54(d,J=10.0Hz,1H),J=8.0Hz,1H),3.35(s,3H),2.89(s,3H),1.94(s,3H); 13 C NMR (125 MHz, CDCl 3 )δ170.3,152.1,147.6,145.6,143.3,136.2,130.7,129.5,126.4,125.9,125.4,118.4,37.3,22.6,9.9.

[0587]

[0588] 6-Chloropyridazin-3-amine. A mixture of 3,6-dichloropyridazine (200 mg, 2.342 mmol) and ammonium hydroxide (1.5 mL) in a sealed tube was heated at 100 °C for 16 hours. After the mixture was cooled to 23 °C, dichloromethane was added and the precipitate was separated, washed with dichloromethane to give the desired product as a light yellow solid (quantitative).1 H NMR (400 MHz, DMSO-d 6 )δ7.32(d,J=8.0Hz,1H),6.81(d,J=8.0Hz,1H),6.59(s,2H).

[0589]

[0590] 2-Bromopropanal. To a solution of propanal (2.91 mL, 40 mol) in dichloromethane (40 mL) was added a solution of bromine (2.05 mL, 40 mol) in dichloromethane (10 mL) dropwise at 0 ° C over 1.5 hours. The mixture was warmed to 23 ° C and stirred for 30 minutes. After adding water to the reactant, the resulting organic layer was separated and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted with dichloromethane (30 mL) and then the combined organic layers were washed with brine and washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude product (dark yellow oil, quantitative) was used in the next step without any purification. 1 H NMR (400 MHz, CDCl 3 )δ9.35(br s,1H),4.34(qd,J=6.8,2.0Hz,1H),1.75(d,J=6.8Hz,3H). The spectral data are consistent with the literature data. [Reference: Bull. Korean Chem. Soc. 2013, 34(1), 271-274.

[0591]

[0592] 6-Chloro-3-methylimidazo[1,2-b]pyridazine. A mixture of 6-chloropyridazin-3-amine (500 mg, 3.860 mmol) and 2-bromopropanal (crude, 793 mg, 5.789 mmol) in EtOH (10 mL) was heated under reflux for 4 hours. After the mixture was cooled to 23 °C, it was concentrated and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (dichloromethane:MeOH=15:1) to give the desired product (172 mg, 1.026 mmol, 27%) as a light brown solid. 1 H NMR (400 MHz, CDCl 3 )δ7.87(d,J=9.6Hz,1H),7.56(s,1H),6.99(1H,J=9.6Hz,1H),2.55(s,3H). The spectral data are consistent with the literature data. [Reference: Chem.Pharm.Bull.1996,44(1),122-131.

[0593]

[0594] 3-Methyl-6-(3-nitrophenyl)imidazo[1,2-b]pyridazine, JGJ005. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (55.2 mg, 0.329 mmol), 3-nitrophenylboronic acid (60.5 mg, 0.362 mmol), K 2 CO 3 (68.3 mg, 0.494 mmol) and Pd(PPh 3 ) 4 Reaction of (38.1 mg, 0.033 mmol) in 1,4-dioxane (0.5 mL) and water (150 μL) gave the desired product JGJ005 (61.9 mg, 0.244 mmol, 74%) as a yellow solid. 1 H NMR (500 MHz, CDCl 3 )δ8.88(dd,J=2.0,1.5Hz,1H),8.38(ddd,J=7.5,1.5,1.0Hz,1H),8.35(ddd,J=8.0,2.0,1.0Hz,1 H),8.07(d,J=9.5Hz,1H),7.73(t,J=8.0Hz,1H),7.67(s,1H),7.50(d,J=9.5Hz,1H),2.67(s,3H); 13 C NMR (125 MHz, CDCl 3 )δ148.8 (two peaks overlap), 138.1, 137.7, 133.3, 132.7, 130.0, 126.0, 125.8, 124.4, 122.0, 113.7, 8.8.

[0595]

[0596] 3-(3-Methylimidazo[1,2-b]pyridazin-6-yl)aniline, JGJ006. Using the same procedure as described for JGJ003, 3-methyl-6-(3-nitro-phenyl)imidazo[1,2-b]pyridazine (54.4 mg, 0.214 mmol) and SnCl 2 Reaction of (202.8 mg, 1.070 mmol) in EtOH (0.5 mL) gave the desired product JGJ006 (27.2 mg, 0.107 mmol, 50%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)δ7.92(d,J=9.2Hz,1H),7.56(d,J=0.8Hz,1H),7.38(d,J=9.6Hz,1H),7.28-7.34(m,3H),6.79(ddd,J=7.6,2.0,1.2Hz,1H),3.86(br s,2H),2.61(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ151.3,147.0,138.1,137.0,132.0,129.8,125.3,125.1,117.3,116.5,114.8,113.3,8.7.

[0597]

[0598] N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ007. Using the same procedure as described for JGJ004, the reaction of 3-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (JGJ006, 23.3 mg, 0.104 mmol), triethylamine (12.6 mg, 0.125 mmol) and acetyl chloride (9 mg, 0.114 mmol) in dichloromethane (0.5 mL) gave the desired product JGJ007 (16.5 mg, 0.067 mmol, 60%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ9.12(br s,NH),8.23(s,1H),7.82(d,J=9.6Hz,1H),7.61-7.69(m,2H),7.53(s,1H ),7.36(t,J=8.0Hz,1H),7.30(d,J=9.6Hz,1H),2.51(s,3H),2.21(s,3H); 13 CNMR (100MHz, CDCl 3 )δ169.2,150.8,139.1,137.8,136.3,131.7,129.4,125.4,124.8,122.4,121.2,118.3,114.7,24.4,8.5.

[0599]

[0600] N-Methyl-N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ008. Using the same procedure as described for JGJ001, the reaction of N-(3-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide (JGJ007, 26.4 mg, 0.099 mmol), a 60% dispersion of NaH in mineral oil (8 mg, 0.199 mmol) and iodomethane (28.2 mg, 0.199 mmol) in dimethylformamide (DMF, 0.3 mL) gave the desired product JGJ008 (17.5 mg, 0.062 mmol, 63%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.00(d,J=9.6Hz,1H),7.96(d,J=8.0Hz,1H),7.89(dd,J=2.0,1.6Hz,1H),7.62(s,1H),7.58(dd,J=8. 0,7.6Hz,1H),7.43(d,J=9.2Hz,1H),7.32(dd,J=7.6,1.2Hz,1H),3.34(s,3H),2.64(s,3H),1.95(s,3H); 13 CNMR (125MHz, CDCl 3 )δ170.5,149.9,145.4,138.1,137.8,132.8,130.4,128.3,126.2,125.7,125.6,114.0,37.2,22.6,8.8 (not a single low-field carbon was observed).

[0601]

[0602] 3-Methyl-6-(2-nitrophenyl)imidazo[1,2-b]pyridazine, JGJ009. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (67.1 mg, 0.400 mmol), 2-nitrophenylboronic acid (73.5 mg, 0.440 mmol), NaOH (48 mg, 1.201 mmol) and Pd(PPh 3 ) 4 Reaction of (46.3 mg, 0.040 mmol) in THF (0.4 mL) and water (0.2 mL) gave the desired product JGJ009 (16.3 mg, 0.064 mmol, 16%) as a yellow solid. 1 H NMR (400 MHz, CDCl 3)8.02(dd,J=8.0,0.8Hz,1H),7.99(d,J=9.6Hz,1H),7.75(m,1H),7.64-7.70(m,2H),7.63(d,J=1.2Hz,1H),7.10(d,J=9.2Hz,1H),2.54(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ149.6,149.0,137.7,132.9,132.8,131.7,131.4,130.2,125.6,125.5,124.7,115.8,8.6.

[0603]

[0604] 2-(3-Methylimidazo[1,2-b]pyridazin-6-yl)aniline, JGJ010. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (25.4 mg, 0.152 mmol), 2-aminophenylboronic acid (22.8 mg, 0.167 mmol), K 2 CO 3 (31.4 mg, 0.227 mmol) and Pd(PPh 3 ) 4 Reaction of (17.5 mg, 0.015 mmol) in 1,4-dioxane (0.4 mL) and water (80 μL) gave the desired product JGJ010 (26.2 mg, 0.117 mmol, 70%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )7.97(d,J=9.6Hz,1H),7.57(s,1H),7.67(m,1H),7.42(d,J=9.6Hz,1H),7.24(m,1H),6.82-6.87(m,2H),2.59(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ152.8,145.9,137.3,131.8,130.7,129.7,125.6,124.9,118.6,118.0,117.4,116.5,8.8.

[0605]

[0606] N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ011. Using the same procedure as described for JGJ004, the reaction of 2-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (JGJ010, 39.4 mg, 0.176 mmol), triethylamine (21.3 mg, 0.211 mmol) and acetyl chloride (16.5 mg, 0.211 mmol) in dichloromethane (0.8 mL) gave the desired product JGJ011 (35 mg, 0.131 mmol, 75%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ10.57(br s,NH),8.47(d,J=8.4Hz,1H),7.99(d,J=9.6Hz,1H),7.61(s,1H),7.60(dd,J=8.0,0.8Hz,1H),7.44(ddd,J =8.8,7.2,0.8Hz,1H),7.34(d,J=9.2Hz,1H),7.20(ddd,J=8.0,7.2,0.8Hz,1H),2.60(s,3H),2.17(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.1,152.0,137.3,136.4,132.8,130.6,129.5,126.3,124.6,124.0,123.5,122.4,116.7,25.1,8.9.

[0607]

[0608] N-methyl-N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ012. Using the same procedure as described for JGJ001, the reaction of N-(2-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide (JGJ011, 19.1 mg, 0.072 mmol), sodium hydride (NaH, 60% dispersion in mineral oil, 5.7 mg, 0.143 mmol) and iodomethane (20.4 mg, 0.143 mmol) in dimethylformamide (DMF, 0.3 mL) gave the desired product JGJ012 (12.8 mg, 0.046 mmol, 64%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ7.98(d,J=9.2Hz,1H),7.66(m,1H),7.60(s,1H)7.52(m,2H),7.34(m,1H),7.10(d,J=9.6Hz,1H),3.01(s,3H),2.54(s,3H),1.90(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ170.9,150.1,142.5,137.4,134.5,132.8,131.0,130.9,130.7,129.5,128.7,125.7,116.0,36.7,22.7,8.7.

[0609]

[0610] 3-(3-Methylimidazo[1,2-b]pyridazin-6-yl)benzoic acid, JGJ013. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (50 mg, 0.299 mmol), 3-carboxyphenylboronic acid (54.5 mg, 0.328 mmol), K 2 CO 3 (82.5 mg, 0.597 mmol) and Pd(PPh 3 ) 4 Reaction of (34.5 mg, 0.030 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ013 (32.4 mg, 0.128 mmol, 43%) as a white solid. 1 H NMR (400MHz, CD 3 OD)8.73(dd,J=1.6,1.2Hz,1H),8.25(d,J=8.0Hz,1H),8.16(ddd,J=7.6,1.6,1.2Hz,1H),8.03(d,J=9.6 Hz, 1H), 7.75 (d, J = 9.6Hz, 1H), 7.62 (dd, J = 8.0, 7.6Hz, 1H), 7.58 (d, J = 0.4Hz, 1H), 2.63 (d, J = 0.4Hz, 3H).

[0611]

[0612] 6-(2,3-Dimethoxyphenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ014. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (42 mg, 0.251 mmol), 2,3-dimethoxyphenylboronic acid (50.2 mg, 0.276 mmol), K 2 CO 3 (52 mg, 0.376 mmol) and Pd(PPh 3 ) 4 Reaction of (29 mg, 0.025 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ014 (39.6 mg, 0.147 mmol, 59%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )7.92(d,J=9.6Hz,1H),7.58(s,1H),7.46(d,J=9.2Hz,1H),7.29(dd,J=7.6,0.8Hz,1H),7.1 9(t,J=8.0Hz,1H),7.05(ddd,J=8.0,7.6,0.8Hz,1H),3.93(s,3H),3.76(s,3H),2.60(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ153.2,150.7,147.5,138.0,131.7,131.1,125.2,124.4,124.2,122.2,118.4,113.6,61.4,56.0,8.8.

[0613]

[0614] 6-(3-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ015. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (51.5 mg, 0.307 mmol), 3-fluorophenylboronic acid (47.3 mg, 0.338 mmol), K 2 CO 3 (63.7 mg, 0.461 mmol) and Pd(PPh 3 ) 4 Reaction of (35.5 mg, 0.031 mmol) in 1,4-dioxane (0.5 mL) and water (100 μL) gave the desired product JGJ015 (38.2 mg, 0.168 mmol, 55%) as an ivory solid. 1H NMR (400 MHz, CDCl 3 )7.98(d,J=9.2Hz,1H),7.75(m,2H),7.61(s,1H),7.48(m,1H),7.41(d,J=9.2Hz,1H),7.18(m,1H),2.63(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ163.2(d,J=244.9Hz),149.8(d,J=2.6Hz),138.2,138.1,132.6,130.5(d,J=8.1Hz),125.5,122.6(d,J=2.9Hz),116.7(d,J=21.2Hz),114.2,113.9(d,J=23.1Hz),8.7.(Not a single low-field carbon was observed).

[0615]

[0616] N-methyl-3-(3-methylimidazo[1,2-b]pyridazin-6-yl)benzamide, JGJ016. Hydroxybenzotriazole (HOBT, 16.1 mg, 0.159 mmol), (3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl, 30.4 mg, 0.159 mmol) and N,N-diisopropylethylamine (DIPEA, 102.6 mg, 0.794 mmol) were added to a solution of JGJ013 (20.1 mg, 0.079 mmol) and methylamine hydrochloride (10.7 mg, 0.159 mmol) in dichloromethane (0.3 mL) and DMF (0.5 mL). The mixture was stirred at 23 ° C for 12 hours. After water was added to the reactant, it was extracted with ethyl acetate (10 mL X3). The combined organic layer was washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude residue was purified by flash column chromatography (dichloromethane:MeOH=6:1) to give the desired product JGJ016 (8.6 mg, 0.032 mmol, 41%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3)δ8.39(t,J=1.6Hz,1H),8.10(dddd,J=8.0,1.6,1.2,0.8Hz,1H),7.91(d,J=9.6Hz,1H),7.86(dddd,J=7.6,1.6,1.2Hz,1H), 7.58(s,1H),7.55(dd,J=8.0,7.6Hz,1H),7.41(d,J=9.6Hz,1H),6.75(m,NH),3.06(d,J=4.8Hz,3H),2.59(d,J=0.4Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ167.7,153.3,138.0,136.3,135.5,132.3,129.7,129.2,128.0,125.7,125.5,125.4,114.4,26.9,8.7.

[0617]

[0618] 3-Methyl-6-(pyridin-3-yl)imidazo[1,2-b]pyridazine, JGJ017. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (58.8 mg, 0.351 mmol), 3-pyridineboronic acid (47.4 mg, 0.386 mmol), K 2 CO 3 (72.7 mg, 0.526 mmol) and Pd(PPh 3 ) 4 Reaction of (40.6 mg, 0.035 mmol) in 1,4-dioxane / water (5:1 v / v, 0.6 mL) gave the desired product JGJ017 (37.2 mg, 0.177 mmol, 50%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )9.20(d,J=1.6Hz,1H),8.69(dd,J=4.8,1.6Hz,1H),8.29(ddd,J=8.0,2.0,1.6Hz,1H),7.98(d,J=9.2Hz,1 H),7.60(d,J=0.4Hz,1H),7.42(ddd,J=8.0,4.8,0.8Hz,1H),7.41(d,J=9.6Hz,1H),2.60(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3)δ150.6,148.6,148.2,137.9,134.2,132.7,131.6,125.7,125.5,123.6,113.7,8.6.

[0619]

[0620] 6-(2-Fluorophenyl)-3-methylimidazo[1,2-b]pyridazine, JGJ018. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (27.5 mg, 0.164 mmol), 2-fluorophenylboronic acid (25.3 mg, 0.181 mmol), K 2 CO 3 (34.0 mg, 0.246 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (19.0 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ018 (18.1 mg, 0.080 mmol, 49%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )7.96(d,J=9.6Hz,1H),7.91(ddd,J=8.0,7.6,2.0Hz,1H),7.60(s,1H),7.43-7.49(m,2H),7 .30(ddd,J=8.0,7.6,1.2Hz,1H),7.21(ddd,J=11.2,8.4,0.8Hz,1H),2.61(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ160.4(d,J=249.3Hz),148.2,137.9,132.2,131.4(d,J=8.5Hz),130.7(d,J=2.6Hz),125.3, 124.7, 124.6 (d, J = 3.6Hz), 124.3 (d, J = 11.7Hz), 117.5 (d, J = 7.9Hz), 116.4 (d, J = 22.2Hz), 8.7.

[0621]

[0622] 6-Chloromidazo[1,2-b]pyridazine. To a solution of 6-chloropyridazine-3-amine (400 mg, 3.088 mmol) in EtOH (6 mL) and water (4 mL) was added bromoacetaldehyde diethyl acetal (930 μL, 6.175 mmol) and HBr (280 μL). The resulting mixture was heated at 103 ° C overnight. After the mixture was cooled to 23 ° C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude residue was used in the next step without further purification. (Brown solid; 394.5 mg, 2.569 mmol, 83%) 1 H NMR (400 MHz, CDCl 3 )δ7.92(s,1H),7.90(d,J=9.6Hz,1H),7.76(s,1H),7.04(d,J=9.6Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ146.9,137.5,134.4,127.0,118.9,117.2.

[0623]

[0624] N-(3-(Imidazolo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ019. Using the same procedure as described for JGJ002, 6-chloro-imidazo[1,2-b]pyridazine (71.6 mg, 0.427 mmol), 3-aminophenylboronic acid (69.5 mg, 0.449 mmol), K 2 CO 3 (88.6 mg, 0.641 mmol) and Pd(PPh 3 ) 4 (49.3 mg, 0.043 mmol) in 1,4-dioxane / water (5:1 v / v, 1.0 mL) gave 3-(imidazo[1,2-b]pyridazin-6-yl)aniline (87.9 mg, 0.392 mmol, 92%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ019 (49.6 mg, 0.221 mmol, 69%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ8.19(s,1H),8.13(br s,1H),7.96(m,2H),7.76(s,1H),7.61-7.65(m,2H),7.43(d,J=9.6Hz,1H),7.39-7.43(m,1H),2.22(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.9,151.8,138.9,138.2,136.1,133.6,129.7,125.4,122.7,121.4,118.4,117.1,116.7,24.6.

[0625]

[0626] 6-(3-Fluorophenyl)imidazo[1,2-b]pyridazine, JGJ020. Using the same procedure as described for JGJ002, 6-chloroimidazo[1,2-b]pyridazine (50 mg, 0.326 mmol), 3-fluorophenylboronic acid (50.1 mg, 0.358 mmol), K 2 CO 3 (67.5 mg, 0.488 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (18.8 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ020 (36.9 mg, 0.173 mmol, 53%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.96-7.99(m,2H),7.77(s,1H),7.62-7.68(m,2H),7.42-7.46(m,1H),7.39(d,J=9.6Hz,1H),7.14(m,1H); 13 C NMR (100 MHz, CDCl 3 )δ163.1(d,J=245.1Hz),150.4(d,J=2.6Hz),137.5(d,J=7.8Hz),134.2,131.9(d,J=9.8Hz),130.5(d,J= 8.1Hz),128.4(d,J=12.1Hz),125.7,122.5(d,J=2.9Hz),116.8(d,J=21.1Hz),115.7,113.8(d,J=23.2Hz)

[0627]

[0628] 6-Chloro-2-methylimidazo[1,2-b]pyridazine. To a solution of 6-chloropyridazin-3-amine (100 mg, 0.772 mmol) in EtOH (2 mL) were added trimethylamine (78 mg, 0.772 mmol) and chloroacetone (142.8 mg, 1.544 mmol), and the mixture was stirred at 120 ° C overnight. After the mixture was cooled to 23 ° C, it was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane: EtOAc = 1: 1) to give the desired product (87.2 mg, 0.520 mmol, 67%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 )δ7.72(dd,J=9.2,0.4Hz,1H),7.65(s,1H),6.93(d,J=9.2Hz,1H),2.44(d,J=0.8Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ145.8,144.8,137.0,125.6,117.9,114.5,14.7.

[0629]

[0630] N-(3-(2-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ021. Using the same procedure as described for JGJ002, 6-chloro-2-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 3-aminophenylboronic acid (35.9 mg, 0.232 mmol), K 2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4 (24.4 mg, 0.021 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave 3-(2-methylimidazo[1,2-b]pyridazin-6-yl)aniline (49.6 mg, quantitative) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ021 (27.2 mg, 0.102 mmol, 46%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3)δ8.92(s,1H),8.16(s,1H),7.73(d,J=9.6Hz,1H),7.63(m,2H),7.54(d,J=7.6H z,1H),7.33(t,J=8.0Hz,1H),7.27(d,J=10.0Hz,1H),2.44(s,3H),2.19(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ169.2,150.7,143.8,139.0,137.7,136.1,129.4,123.9,122.3,121.1,118.2,115.7,114.3,24.4,14.5.

[0631]

[0632] 6-(3-Fluorophenyl)-2-methylimidazo[1,2-b]pyridazine, JGJ022. Using the same procedure as described for JGJ002, 6-chloro-2-methylimidazo[1,2-b]pyridazine (21.4 mg, 0.128 mmol), 3-fluorophenylboronic acid (17.9 mg, 0.128 mmol), K 2 CO 3 (26.5 mg, 0.192 mmol) and Pd(PPh 3 ) 4 Reaction of 4-nitropropene (7.4 mg, 0.006 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave the desired product JGJ022 (13.7 mg, 0.060 mmol, 47%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.89(d,J=9.2Hz,1H),7.78(s,1H),7.65-7.70(m,2H),7.43-7.49(m,1H),7.38(d,J=9.2Hz,1H),7.16(m,1H),2.52(d,J=0.4Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ163.2(d,J=245.0Hz),149.8(d,J=2.7Hz),144.5,137.9(d,J=8.0Hz),130.5(d,J=8.2Hz),124.5,122.5(d,J=3.0Hz),116.7(d,J=21.1Hz),115.3,114.4,113.9(d,J=23.2Hz),14.8.(No low-field carbon was observed)

[0633]

[0634] 6-Chloro-3-phenylimidazo[1,2-b]pyridazine. To a solution of 6-chloroimidazo[1,2-b]pyridazine (394.5 mg, 2.569 mmol) in DMF (6 mL) was added N-iodosuccinimide (635.8 mg, 2.826 mmol) and the mixture was stirred at 23 °C for 48 hours. After the reaction was complete, it was evacuated to remove the solvent. The residue was diluted with dichloromethane and saturated with Na 2 S 2 CO 3 The organic layer was separated and washed with brine, and then washed with anhydrous MgSO 4 The mixture was dried, filtered and concentrated under reduced pressure to give 6-chloro-3-iodoimidazo[1,2-b]pyridazine in quantitative yield. Then 6-chloro-3-iodoimidazo[1,2-b]pyridazine (107.2 mg, 0.326 mmol), phenylboronic acid (43.7 mg, 0.358 mmol), K 2 CO 3 (54.0 mg, 0.391 mmol) and Pd(PPh 3 ) 4 A mixture of (18.8 mg, 0.016 mmol) in 1,4-dioxane / water (5:1 v / v, 2 mL) was heated at 90 °C overnight. After the reaction was cooled to 23 °C, it was diluted in water and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc=2:1) ​​to give the desired product (28.4 mg, 0.124 mmol, 38%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.06(s,1H),8.03(m,2H),7.98(d,J=9.6Hz,1H),7.52(m,2H),7.39(m,1H),7.08(d,J=9.2Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ146.8,138.5,133.1,129.1,128.7,128.4,127.6,127.1,126.8,118.3.

[0635]

[0636] N-(3-(3-phenylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ023. Using the same procedure as described for JGJ002, 6-chloro-3-phenylimidazo[1,2-b]pyridazine (15.5 mg, 0.068 mmol), 3-aminophenylboronic acid (11.5 mg, 0.074 mmol), K 2 CO 3 (14.0 mg, 0.101 mmol) and Pd(PPh 3 ) 4 (3.9 mg, 0.003 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave 3-(3-phenylimidazo[1,2-b]pyridazin-6-yl)aniline (17.5 mg, 0.061 mmol, 91%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ023 (10.9 mg, 0.033 mmol, 54%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.18(s,1H),8.12(m,2H),8.04(s,1H),7.99(d,J=9.6Hz,1H),7.93(brs,1H),7.6 4-7.70(m,2H),7.50(m,2H),7.46(d,J=9.6Hz,1H),7.35-7.44(m,2H),2.22(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ168.7,151.1,138.8,136.4,133.0,129.6,128.8,128.7,128.6,127.9,126.8,125.8,122.7,121.3,118.3,115.6,24.6. (No low-field carbon was observed)

[0637]

[0638] 6-(3-Fluorophenyl)-3-phenylimidazo[1,2-b]pyridazine, JGJ024. Using the same procedure as described for JGJ002, 6-chloro-3-phenylimidazo[1,2-b]pyridazine (12.9 mg, 0.056 mmol), 3-fluorophenylboronic acid (8.6 mg, 0.062 mmol), K 2 CO 3 (11.7 mg, 0.084 mmol) and Pd(PPh 3 ) 4Reaction of 4-nitropropene (3.2 mg, 0.003 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave the desired product JGJ024 (9.5 mg, 0.033 mmol, 58%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.10-8.14(m,4H),7.72-7.79(m,2H),7.48-7.56(m,4H),7.42(m,1H),7.20(m,1H); 13 C NMR (100 MHz, CDCl 3 )δ163.2(d,J=245.0Hz),150.5(d,J=2.7Hz),137.8(d,J=7.8Hz),133.0,130.6(d,J=8.2Hz),129.1,128.8 ,128.4,128.1,127.1,126.9,126.1,122.7(d,J=2.9Hz), 117.0(d,J=21.2Hz), 115.3,114.0(d,J=23.2Hz).

[0639]

[0640] 3-Methyl-6-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine, JGJ025. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.9 mg, 0.214 mmol), 3-trifluoromethylphenylboronic acid (42.7 mg, 0.225 mmol), K 2 CO 3 (44.4 mg, 0.321 mmol) and Pd(PPh 3 ) 4 Reaction of (12.4 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave the desired product JGJ018 (29.2 mg, 0.105 mmol, 49%) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.27(s,1H),8.20(d,J=8.0Hz,1H),8.09(d,J=9.2Hz,1H),7.76(d,J=8.0Hz,1H),7.65-7.69(m,2H),7.51(d,J=9.2Hz,1H),2.66(s,3H); 13 C NMR (100 MHz, CDCl 3)δ149.8,136.7,132.6,132.1(d,J=9.8Hz),131.5(q,J=32.4Hz),130.2,129.5,128.4(d,J=12.0Hz),126.4(q,J=3.5Hz),125.7,123.9(q,J=270.8Hz),123.8(q,J=3.8Hz),114.1,8.7. (Due to the presence of some impurities, it will be tested again 13 C NMR)

[0641]

[0642] N-(3-Fluoro-5-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ026. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 3-fluoro-5-aminophenylboronic acid (34.3 mg, 0.221 mmol), K 2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4 (12.2 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave 3-fluoro-5-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (25 mg, 0.103 mmol, 49%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ026 (8 mg, 0.028 mmol, 28%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.38(br s,1H),8.00(d,J=9.2Hz,1H),7.89(s,1H),7.65(d,J=9.2Hz,1H),7.60(s,1H),7.43(s,1H),7.41(s,1H),2.60(s,3H),2.24(s,3H);

[0643]

[0644] N-(4-(3-methylimidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ027. Using the same procedure as described for JGJ002, 6-chloro-3-methylimidazo[1,2-b]pyridazine (35.3 mg, 0.211 mmol), 4-aminophenylboronic acid (38.4 mg, 0.221 mmol), K2 CO 3 (43.7 mg, 0.316 mmol) and Pd(PPh 3 ) 4 (12.2 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) gave 4-(3-methylimidazo[1,2-b]pyridazin-6-yl)aniline (31.4 mg, 0.140 mmol, 66%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ026 (7.2 mg, 0.027 mmol, 19%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ7.99(d,J=8.8Hz,2H),7.95(d,J=9.2Hz,1H),7.68(d,J=8.4Hz,2H),7.58(s,1H),7.47(br s,1H),7.43(d,J=9.6Hz,1H),2.62(s,3H),2.23(s,3H);

[0645]

[0646] 6-Chloro-3-(pyridin-3-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (82.6 mg, 0.297 mmol), pyridine-3-boronic acid (40 mg, 0.325 mmol), K 2 CO 3 (61.3 mg, 0.443 mmol) and Pd(PPh 3 ) 4 Reaction of (17.1 mg, 0.015 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave the desired product (41.5 mg, 0.180, 61%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.21(s,1H),8.62(s,1H),8.40(m,1H),8.11(s,1H),7.98(d,J=9.6Hz,1H),7.43(dd,J=7.6,0.8Hz,1H),7.12(d,J=9.2Hz,1H); 13 CNMR (100MHz, CDCl 3)δ149.0,147.6,147.2,139.1,133.6,133.5,127.4,126.0,124.3,123.6,118.9.

[0647]

[0648] N-(3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ028. Using the same procedure as described for JGJ002, 6-chloro-3-(pyridin-3-yl)imidazo[1,2-b]pyridazine (41.5 mg, 0.180 mmol), 3-aminophenylboronic acid (30.7 mg, 0.198 mmol), K 2 CO 3 (37.3 mg, 0.270 mmol) and Pd(PPh 3 ) 4 (10.4 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.4 mL) afforded 3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (50.0 mg, 0.174 mmol, 96%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to afford the desired product JGJ028 (18.2 mg, 0.055 mmol, 32%) as a light yellow solid. 1 H NMR (400MHz, CD 3 OD)δ9.29(d,J=1.2Hz,1H),8.60(ddd,J=8.0,2.0,1.6Hz,1H),8.49(d,J=4.0Hz,1H),8.25(dd,J=2.0,1.6Hz,1H),8.18(s,1H),8 .02(d,J=9.6Hz,1H),7.68(d,J=9.6Hz,1H),7.60-7.65(m,2H),7.55(dd,J=8.0,4.8Hz,1H),7.37(t,J=8.0Hz,1H),2.16(s,3H); 13 C NMR (100MHz, CD 3 OD)δ172.1,153.6,149.2,148.0,141.5,141.2,137.1,135.9,134.1,130.8,127.1,127.0,126.9,125.7,123.8,123.0,119.5,118.6,24.3.

[0649]

[0650] 6-Chloro-3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (83.6 mg, 0.299 mmol), pyrimidine-5-boronic acid (40.8 mg, 0.329 mmol), K 2 CO 3 (62 mg, 0.449 mmol) and Pd(PPh 3 ) 4 Reaction of (17.3 mg, 0.015 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave the desired product (9.8 mg, 0.042 mmol, 14%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.42(s,2H),9.23(s,1H),8.18(s,1H),8.04(d,J=9.6Hz,1H),7.20(d,J=9.6Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ157.7,154.0,147.7,133.7,132.1,128.5,127.7,123.0,119.8.

[0651]

[0652] N-(3-(3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ029. Using the same procedure as described for JGJ002, 6-chloro-3-(pyrimidin-5-yl)imidazo[1,2-b]pyridazine (9.8 mg, 0.042 mmol), 3-aminophenylboronic acid (7.2 mg, 0.047 mmol), K 2 CO 3 (8.8 mg, 0.064 mmol) and Pd(PPh 3 ) 4 (4.9 mg, 0.004 mmol) in 1,4-dioxane / water (5:1 v / v, 0.2 mL) gave 3-(3-(pyridin-3-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (6.7 mg, 0.023 mmol, 55%) as a pale yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ029 (5.1 mg, 0.015 mmol, 67%) as an ivory solid. 1H NMR (400 MHz, CDCl 3 +5% v / v CD 3 OD)δ9.58(s,2H),9.18(s,1H),8.23(s,1H),8.19(d,J=9.6Hz,1H),8.10(s,1H),7.99(d,J=8.0 Hz,1H)7.65(d,J=9.2Hz,1H),7.63(d,J=8.0Hz,1H),7.46(dd,J=8.4,7.6Hz,1H),2.19(s,3H); 13 C NMR (125 MHz, CDCl 3 +5% v / v CD 3 OD)δ169.7,156.8,153.9,152.5,139.6,134.7,131.9,129.9,125.9,123.7,122.4,122.2,122.1,118.0,117.7,117.6,24.0.

[0653]

[0654] 6-Bromoimidazo[1,2-a]pyridine. To a solution of 2-amino-5-bromopyridine (500 mg, 2.89 mmol) in EtOH (6 mL) and water (4 mL) was added bromoacetaldehyde diethyl acetal (870 μL, 5.78 mmol) and HBr (260 μL) at 23 °C. The resulting mixture was heated at 103 °C overnight. After the mixture was cooled to 23 °C, it was diluted in water and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO 3 The solution was washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The crude residue was used in the next step without further purification. (brown solid; 331.7 mg, 1.68 mmol, 58%) 1 H NMR (400 MHz, CDCl 3 )δ8.09(dd,J=2.0,0.8Hz,1H),7.46(d,J=0.8Hz,1H),7.39(s,1H),7.32(d,J=9.6Hz,1H),7.00(dd,J=9.6,2.0Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ143.2,133.8,127.3,125.4,117.8,112.3,106.5.

[0655]

[0656] N-(3-(Imidazolo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ030. Using the same procedure as described for JGJ002, 6-bromoimidazo[1,2-a]pyridine (50 mg, 0.254 mmol), 3-aminophenylboronic acid (43.3 mg, 0.279 mmol), K 2 CO 3 (52.6 mg, 0.381 mmol) and Pd(PPh 3 ) 4 (29.3 mg, 0.025 mmol) in 1,4-dioxane / water (5:1 v / v, 1 mL) gave 3-(imidazo[1,2-a]pyridin-6-yl)aniline (22.3 mg, 0.107 mmol, 42%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ030 (13.6 mg, 0.054 mmol, 51%) as a white solid. 1 H NMR (400MHz, CD 3 OD)δ8.68(s,1H),7.89-7.94(m,2H),7.56-7.62(m,3H),7.51(ddd,J=7.6,2.0,1.2Hz,1H),7.35-7.43(m,2H),2.16(s,3H); 13 C NMR (100MHz, CD 3 OD)δ170.3,139.2,137.4,132.1,129.1,126.7,125.7,123.8,122.1,119.1,118.0,115.8,113.5,22.4.(No low field carbon was observed)

[0657]

[0658] 6-Bromo-3-methylimidazo[1,2-a]pyridine. A mixture of 2-amino-5-bromopyridine (200 mg, 1.156 mmol) and 2-bromopropanal (purity>95%, 318 mg, 2.312 mmol) in EtOH (5 mL) was heated under reflux overnight. After the mixture was cooled to 23 °C, it was concentrated and extracted with EtOAc. The combined organic layers were washed with brine and purified by anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc=3:2) to give the desired product (86.9 mg, 0.412 mmol, 36%) as a white solid. 1 H NMR (400 MHz, CDCl 3)δ8.00(d,J=1.2Hz,1H),7.49(d,J=9.2Hz,1H),7.40(s,1H),7.20(dd,J=9.6,2.0Hz,1H),2.46(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ143.5,132.1,126.5,123.0,120.3,118.3,106.9.9.0.

[0659]

[0660] N-(3-(3-methylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ031. Using the same procedure as described for JGJ002, 6-bromo-3-methylimidazo[1,2-a]pyridine (35 mg, 0.166 mmol), 3-aminophenylboronic acid (28.3 mg, 0.182 mmol), K 2 CO 3 (34.4 mg, 0.249 mmol) and Pd(PPh 3 ) 4 (9.6 mg, 0.008 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-(3-methylimidazo[1,2-a]pyridin-6-yl)aniline (28.1 mg, 0.106 mmol, 64%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ031 (15.8 mg, 0.060 mmol, 56%) as an ivory solid. 1 H NMR (400 MHz, CDCl 3 )δ8.30(br s,1H),8.12(s,1H),7.87(s,1H),7.65(d,J=8.0Hz,1H),7.54(d,J=8.0Hz,1H),7.36-7.43(m,3H),7.27(m,1H),2.49(s,3H),2.23(s,3H);

[0661]

[0662] 3-(3-phenylimidazo[1,2-a]pyridin-6-yl)aniline, JGJ032. 2-amino-5-bromo-pyridine (100 mg, 0.508 mmol), 3-aminophenylboronic acid (76.5 mg, 0.558 mmol), triphenylphosphine (26.6 mg, 0.102 mmol) and K were added to a microwave tube. 2 CO 3Pd(OAc) (140.3 mg, 1.015 mmol) was added to a mixture of toluene:EtOH mixture (2:1 v / v, 1.7 mL) 2 (11.4 mg, 0.059 mmol) and filled with argon. The mixture was sealed with a silicon septum and irradiated in a microwave at 140 ° C for 30 minutes under stirring. After the mixture was cooled to 23 ° C, bromobenzene (119.5 mg, 0.761 mmol) was injected into the tube with a syringe, and the mixture was again subjected to microwave irradiation at 140 ° C for 2.5 hours under stirring. The reaction vessel was cooled to 23 ° C and the mixture was diluted with water and extracted with dichloromethane. The combined organic layers were washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure.The crude residue was purified by flash column chromatography (n-hexane:EtOAc:MeOH=1:1:0.1) to give the desired product (28.8 mg, 0.101 mmol, 20%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.46(s,1H),7.83(d,J=9.2Hz,1H),7.73(s,1H),7.45-7.61(m,6H),7.23 (d,J=8.0Hz,1H),6.90(d,J=8.0Hz,1H),6.81(t,J=2.0Hz,1H),6.71(m,1H);

[0663]

[0664] N-(3-(3-phenylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ033. Using the same procedure as described for JGJ004, the reaction of 3-(3-phenylimidazo[1,2-a]pyridin-6-yl)aniline (JGJ032, 22.8 mg, 0.080 mmol), triethylamine (12.1 mg, 0.120 mmol) and acetyl chloride (9.4 mg, 0.120 mmol) in dichloromethane (2 mL) gave the desired product JGJ033 (12.2 mg, 0.037 mmol, 47%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.48(s,1H),7.80(dd,J=2.0,1.6Hz,1H),7.73(s,1H),7.51-7.65(m,7H),7.43(m,1H),7.35(dd,J=8.0Hz,1H),7.27(m,1H),2.12(s,3H); 13 C NMR (100MHz, CD 3OD)δ170.3,139.2,137.4,131.2,129.2,129.0,128.4,128.2,127.7,127.1,126.5,125.6,122.0,120.5,119.0,117.8,116.5,22.4.(No low field carbon was observed)

[0665]

[0666] 5-Chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine. 2-Chloro-5-hydrazinopyridine (71.3 mg, 0.5 mmol) was added to a 4% w / w H 2 SO 4 To the solution in aqueous solution (5 mL) was added (2,2-dimethoxyethyl)benzene (87.3 mg, 0.525 mmol). The reaction vessel was sealed with a silicon septum and stirred at 23 °C for 1 minute, then irradiated in a microwave at 160 °C for 5 minutes. After the mixture was cooled to 23 °C, it was slowly poured into 40% w / w KOH solution (5 mL). The mixture was extracted with EtOAc and the combined organic layers were washed with anhydrous MgSO 4 Dry, filter and concentrate under reduced pressure. The obtained crude residue was purified by flash column chromatography (n-hexane: EtOAc = 3:2) to give the desired product (71.3 mg, 0.312 mmol, 62%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.96(br s,1H),7.99(d,J=7.2Hz,2H),7.59(s,1H),7.57(d,J=8.8Hz,1H),7.39(t,J=7.6Hz,2H),7.23(dd,J=7.6,7.2Hz,1H),7.12(d,J=8.9Hz,1H). The spectral data are consistent with the literature data. [Reference: Eur.J.Org.Chem.2013,3328-3336.

[0667]

[0668] N-(3-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ034. Using the same procedure as described for JGJ002, 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (40 mg, 0.175 mmol), 3-aminophenylboronic acid (29.8 mg, 0.192 mmol), K 2 CO 3 (36.3 mg, 0.262 mmol) and Pd(PPh3 ) 4 (20.2 mg, 0.018 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave 3-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.8 mg, 0.066 mmol, 38%) as a white solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ034 (13.5 mg, 0.041 mmol, 63%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.29(s,1H),8.24(d,J=7.2Hz,2H),7.88(s,1H),7.83(d,J=7.6Hz,1H),7.82(d,J=8.8Hz,1H),7.6 4(d,J=8.4Hz,1H),7.62(d,J=7.6Hz,1H),7.39-7.44(m,3H),7.21(dd,J=7.6,7.2Hz,1H),2.17(s,3H); 13 C NMR (100MHz, CD 3 OD)δ170.3,150.1,143.3,141.1,138.7,134.5,129.3,128.5,127.9,126.3,126.2,125.1,122.4,119.3 (two peaks),118.3,115.7,114.0,22.4.

[0669]

[0670] 5-Chloro-3-propyl-1H-pyrrolo[3,2-b]pyridine. Using the same procedure as described for 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine, 2-chloro-5-hydrazinopyridine (71.8 mg, 0.5 mmol) and valeraldehyde (45.1 mg, 0.524 mmol) were added in 4% w / w H 2 SO 4 Reaction in aqueous solution (5 mL) afforded the desired product (56.7 mg, 0.291 mmol, 58%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.01(br s,1H),7.61(d,J=8.0Hz,1H),7.26(s,1H),7.08(d,J=8.0Hz,1H),2.77(t,J=7.6Hz,2H),1.73(m,2H),0.94(t,J=7.2Hz,3H); 13C NMR (100 MHz, CDCl 3 )δ145.0,143.4,127.8,126.3,120.9,117.2,116.6,26.8,23.0,14.0.

[0671]

[0672] 3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline, JGJ035. Using the same procedure as described for JGJ002, 5-chloro-3-propyl-1H-pyrrolo[3,2-b]pyridine (40 mg, 0.206 mmol), 3-aminophenylboronic acid (31 mg, 0.226 mmol), K 2 CO 3 (42.6 mg, 0.308 mmol) and Pd(PPh 3 ) 4 Reaction of (23.8 mg, 0.021 mmol) in 1,4-dioxane / water (5:1 v / v, 0.5 mL) gave the desired product JGJ035 (42.5 mg, 0.169 mmol, 82%) as a white solid. 1 H NMR (400MHz, CD 3 OD)δ7.72(d,J=8.4Hz,1H),7.44(d,J=8.8Hz,1H),7.34(dd,J=2.0,1.6Hz,1H),7.30(s,1H),7.24(ddd,J=7.6,1.6,1.2Hz ,1H),7.19(t,J=7.6Hz,1H),6.76(ddd,J=7.6,2.0,1.2Hz,1H),2.85(t,J=7.6Hz,2H),1.79(m,2H),1.02(t,J=7.2Hz,3H); 13 C NMR (100MHz, CD 3 OD)δ152.4,128.8,146.2,143.4,130.2,130.1,127.6,120.3,118.8,117.4,116.3,115.8,27.1,24.6,14.5.(No low field carbon was observed)

[0673]

[0674] N-(3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ036. Using the same procedure as described for JGJ004, the reaction of 3-(3-propyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (JGJ035, 34.5 mg, 0.137 mmol), triethylamine (20.8 mg, 0.206 mmol) and acetyl chloride (16.2 mg, 0.206 mmol) in dichloromethane (3 mL) gave the desired product JGJ036 (28.8 mg, 0.098 mmol, 72%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.11(dd,J=2.0,1.6Hz,1H),7.75(d,J=8.4Hz,1H),7.64-7.67(m,2H),7.49(d,J=8.8Hz,1H),7.39 (t,J=8.0Hz,1H),7.32(s,1H),2.85(t,J=7.2Hz,2H),2.15(s,3H),1.80(m,2H),1.01(t,J=7.2Hz,3H); 13 C NMR (100MHz, CD 3 OD)δ171.8,151.4,146.4,143.1,140.1,130.3,129.9,127.9,124.2,120.6,120.4,120.2,117.4,115.7,27.1,24.5,23.9,14.5.

[0675]

[0676] N-(3-Fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)phenyl)acetamide, JGJ037. Using the same procedure as described for JGJ002, 5-chloro-3-phenyl-1H-pyrrolo[3,2-b]pyridine (19.4 mg, 0.085 mmol), 3-fluoro-5-aminophenylboronic acid (14.5 mg, 0.093 mmol), K 2 CO 3 (17.6 mg, 0.127 mmol) and Pd(PPh 3 ) 4(9.8 mg, 0.009 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) afforded 3-fluoro-5-(3-phenyl-1H-pyrrolo[3,2-b]pyridin-5-yl)aniline (18.1 mg, 0.060 mmol, 70%) as an ivory solid. This was followed by acetylation using the same procedure as described for JGJ004 to afford the desired product JGJ037 (13.8 mg, 0.040 mmol, 67%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.25(m,2H),7.98(t,J=1.6Hz,1H),7.88(s,1H),7.79(d,J=8.4Hz,1H),7.61(d,J=8 .8Hz,1H),7.58(m,2H),7.43(t,J=7.6Hz,2H),7.21(td,J=7.6,1.2Hz,1H),2.15(s,3H); 13 C NMR (100MHz, CD 3 OD)δ171.9,164.6(d,J=239.6Hz),150.2(d,J=2.9Hz),145.1,144.7(d,J=8.9Hz),141.7(d,J=11.5Hz),136.0,130.9, 129.4, 127.8, 127.6, 126.6, 120.5, 117.3, 115.3, 114.7 (d, J = 3.2Hz), 109.8 (d, J = 23.1Hz), 107.3 (d, J = 27.0Hz), 24.0.

[0677]

[0678] N-(3-Fluoro-5-(3-methylimidazo[1,2-a]pyridin-6-yl)phenyl)acetamide, JGJ038. Using the same procedure as described for JGJ002, 6-bromo-3-methylimidazo[1,2-a]pyridine (23.4 mg, 0.111 mmol), 3-fluoro-5-aminophenylboronic acid (18.9 mg, 0.122 mmol), K 2 CO 3 (23.0 mg, 0.166 mmol) and Pd(PPh 3 ) 4(12.8 mg, 0.011 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-fluoro-5-(3-methylimidazo[1,2-a]pyridin-6-yl)aniline (13.2 mg, 0.055 mmol, 49%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ038 (8.3 mg, 0.029 mmol, 64%) as an ivory solid. 1 H NMR (400MHz, CD 3 OD)δ8.41(s,1H),7.56-7.63(m,3H),7.52(dt,J=10.8,2.0Hz,1H),7.40(s,1H),7.23(dt,J=9.6,2.0Hz,1H),2.57(s,3H),2.17(s,3H);

[0679]

[0680] 6-Chloro-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine. Using the same procedure as described for 6-chloro-3-phenylimidazo[1,2-b]pyridazine, 6-chloro-3-iodoimidazo[1,2-b]pyridazine (90.5 mg, 0.324 mmol), 4-pyridineboronic acid (43.8 mg, 0.356 mmol), K 2 CO 3 (67.1 mg, 0.486 mmol) and Pd(PPh 3 ) 4 Reaction of (37.4 mg, 0.032 mmol) in 1,4-dioxane / water (5:1 v / v, 0.7 mL) gave the desired product (15.3 mg, 0.066 mmol, 20%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ8.72(d,J=5.2Hz,2H),8.23(s,1H),7.98-8.02(m,3H),7.18(d,J=9.2Hz,1H); 13 C NMR (100 MHz, CDCl 3 )δ150.2,147.3,139.8,135.2,134.9,127.5,126.1,119.9,119.4.

[0681]

[0682] N-(3-Fluoro-5-(3-(pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)phenyl)acetamide, JGJ039. Using the same procedure as described for JGJ002, 6-chloro-3-(pyridin-4-yl)imidazo[1,2-b]pyridazine (15.3 mg, 0.066 mmol), 3-fluoro-5-aminophenylboronic acid (11.3 mg, 0.073 mmol), K 2 CO 3 (13.7 mg, 0.100 mmol) and Pd(PPh 3 ) 4 (7.7 mg, 0.007 mmol) in 1,4-dioxane / water (5:1 v / v, 0.3 mL) gave 3-fluoro-5-(3-(pyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl)aniline (10.7 mg, 0.035 mmol, 53%) as a light yellow solid. This was followed by acetylation using the same procedure as described for JGJ004 to give the desired product JGJ039 (3.8 mg, 0.011 mmol, 31%) as a light yellow solid. 1 H NMR (400MHz, CD 3 OD)δ8.69(s,2H),8.46(s,1H),8.36(d,J=5.2Hz,2H),8.20-8.23(m,2H),7.88(d,J =9.2Hz,1H),7.64(dt,J=10.8,1.6Hz,1H),7.55(dt,9.6,1.6Hz,1H),2.20(s,3H).

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[0773] Incorporated by Reference

[0774] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0775] Equivalent solutions

[0776] Although specific embodiments of the present invention have been discussed, the above description is illustrative and not restrictive. After reading this specification and the following claims, many variations of the present invention will become clear to those skilled in the art. The full scope of the present invention should be determined by reference to the claims together with the full scope of their equivalents, and this specification together with such variations.

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt thereof, in: Selected from Ring B is selected from phenyl and a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; X is selected from N and C; X 1 , X 3 and X 4 Each independently selected from N and CR x ; R 1 is hydrogen or an optionally substituted group selected from: C 1-6 aliphatic, phenyl, and 5- to 6-membered heteroaryl rings having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R 2 Selected from hydrogen, halogen, NO 2 、N(R) 2 、OR、N(R)C(O)R、CO 2 R、C(O)N(R) 2 and optionally substituted C 1-6 aliphatic group; R 3 is selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R x are independently selected from hydrogen, halogen or optionally substituted C 1-6 aliphatic group; Each R is independently selected from hydrogen and an optionally substituted group selected from: C 1-6 aliphatic; 3 to 7 membered monocyclic carbocyclic ring; 3 to 7 membered monocyclic heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; phenyl; and 5 to 6 membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and n is 0-3.

2. The compound according to claim 1, wherein the compound has formula (Ia): or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound has formula (Ib): or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 2, wherein the compound has formula (Iai) or formula (Ia-ii): or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 3, wherein the compound has formula (Ibi) or formula (Ib-ii): or a pharmaceutically acceptable salt thereof.

6. A compound according to any one of claims 1 to 5, wherein the compound is not 7. The compound according to any one of claims 1 to 6, wherein ring B is a 5- to 6-membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur.

8. The compound according to any one of claims 1 to 6, wherein ring B is pyridyl.

9. A compound according to any one of claims 1 to 6, wherein the compound is selected from the group consisting of compounds of formula (Ia-iii), (Ia-iv), (Iav), (Ib-iii), (Ib-iv) and (Ibv):

10. A compound according to any one of claims 1, 2, 4 and 7 to 9, wherein X 3 is N.

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