EA040307B1 - NLRP3 MODULATORS - Google Patents

Abstract

The present invention provides compounds of formula (I): wherein all variables are as defined herein. These compounds are NLRP3 modulators that can be used as therapeutic agents.

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/532,932, filed July 14, 2017, U.S. Provisional Patent Application No. 62/662,405, filed April 25, 2018, and U.S. Provisional Patent Application No. 62/689,412, filed June 25,

2018, the contents of which are incorporated herein by reference in their entirety.

Field of technology

The present disclosure relates to chemical compounds (e.g., a compound or a pharmaceutically acceptable salt of a compound) that modulate (e.g., agonize or partially agonize) NLRP3, which are useful, for example, for treating a condition, disease, or disorder in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity that contributes to the pathology and/or symptoms and/or progression, and/or treating a refractory state of the disorder, disease, or disorder (e.g., low-T-cell infiltrating malignancies) in a subject (e.g., a human). Furthermore, the present disclosure relates to compositions, as well as other methods for using and preparing the same.

State of the art

Nucleotide-binding oligomerizing domain-like receptors (NLRs) comprise a family of intracellular receptors that detect pathogen-associated molecular patterns (PAMPs) and endogenous molecules (see, e.g., Ting, J. P. Y. et al., The NLR gene family: a standard nomenclature, Immunity, 28(3):285–287, (2008)).

NLRPs are a subfamily of NLRs that contain a pyrin domain and consist of proteins such as NLRP1, NLRP3, NLRP4, NLRP6, NLRP7, and NLRP12. NLRPs are thought to be involved in the formation of multiprotein complexes called inflammasomes (see, e.g., Chaput, C. et al., NOD-like receptors in lung diseases, Frontiers in Immunology, 4: article 393, (2013)). These complexes typically include one or two NLR proteins, the apoptosis-associated Speck-like adaptor molecule containing a CARD domain (ASC), and procaspase-1 F (see, e.g., Bauernfeind, F and Hornung, V. Of inflammasomes and pathogens—sensing of microbes by the inflammasome, EMBO Molecular Medicine, 5(6):814–826, (2013)).

One such inflammasome consists of the NLRP3 scaffold, the adaptor molecule ASC, and procaspase-1 (see, e.g., Hirota, J.A., et al., The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter, Journal of Allergy and Clinical Immunology, 129(4):1116.e6-1 125.e6, (2012)), and its expression is thought to be induced by inflammatory cytokines and TLR agonists in myeloid cells and human bronchial epithelial cells (Id.). The NLRP3 inflammasome is thought to mediate caspase-1-dependent conversion of pro-IL-1β and pro-IL-18 to IL-1e and IL-18. Furthermore, GE-1β and IL-18 play important roles in the treatment of various types of cancer (see, e.g., Chen, L-C. et al., EMBO Mol Med., 4(12): 1276–1293 (2012) and Tse, B. W-C. et al., PLoS One, 6(9):e24241 (2011)). IL-18 has been shown to suppress resistance to immune checkpoint inhibitors in animal models of colorectal cancer (see, e.g., Ma, Z. et al., Clin. Cancer Res. Jan 11. (2016) DOI: 10.1158/1078-0432.CCR-15-1655).

The essence of the invention

The present disclosure relates to chemical compounds (e.g., a compound or a pharmaceutically acceptable salt of a compound) that modulate (e.g., agonize or partially agonize) NLRP3, which are useful, for example, for treating a condition, disease, or disorder in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity that contributes to the pathology and/or symptoms and/or progression, and/or treating a refractory state of the disorder, disease, or disorder (e.g., low-T-cell infiltrating malignancies) in a subject (e.g., a human). The present disclosure also relates to compositions, as well as other methods for using and preparing the same.

The term NLRP3 agonist includes compounds that directly bind or modify NLRP3 at the protein level such that NLRP3 activity is increased, such as by activation, stabilization, altered distribution, or otherwise.

Some compounds described in this paper, which agonize NLRP3 to a lesser extent than a full NLRP3 agonist, can function in assays as both antagonists and agonists. These compounds counteract NLRP3 activation by a full NLRP3 agonist because they prevent the full effect of NLRP3 interaction.

However, the compounds themselves also activate NLRP3 activity to some extent, typically less than the corresponding amount of a full NLRP3 agonist. Such compounds may be referred to as partial NLRP3 agonists. In some embodiments, the compounds described herein are agonists (e.g., full agonists) of NLRP3. In other embodiments, the compounds described herein are partial agonists of NLRP3. Typically, the receptor exists in an active (Ra) and an inactive (Ri) conformation. Some compounds that act on the receptor can alter the ratio of Ra to Ri (Ra/Ri). For example, a full

- An agonist increases the Ra/Ri ratio and can produce a maximal, saturating effect. A partial agonist, when bound to a receptor, produces a response that is less than the response elicited by a full agonist (e.g., an endogenous agonist). Thus, the Ra/Ri ratio for a partial agonist is less than that for a full agonist. However, the efficacy of a partial agonist may be greater or less than that of a full agonist.

In one aspect, the compounds of formula (I) or a pharmaceutically acceptable salt thereof are

where W' represents H;

W is independently selected from -O-CH2CH(OH)(CH2OH), -NH-(CH2)3-4-OH, -NH-(CH2)1-2-CH(CH3)OH, -NH( _CH2 ) _1-2 -C( _CH3 ) ₂ OH, -O-( _CH2 ) _1-2- (pyrazolyl), -NH-( _CH2 ) _1-2- (pyrazolyl), -N[H-( _CH2 ) _1-2- (pyrimidinyl),

-NH-(CH ₂ ) _1-2 -(pyridazinyl), -NH-(CH ₂ ) _1-2 -CF ₂ (pyridyl),

R ³ is independently pyrazolyl or thienyl; and R ⁴ is independently H or F.

In another aspect

W' represents H;

W is independently selected from -O-CH2CH(OH)(CH2OH), -NH-(CH2)3-4-OH, -NH-(CH2)1-2-CH( _{CH3)OH, -NH-(CH2)1-2 -C(CH3)2OH , -O- ( CH2} ) _1-2- (pyrazolyl), -NH-( _CH2 ) _1-2- (pyrazolyl), -NH-( _CH2 ) _1-2- (pyrimidinyl),

Kd _x x λα

-NH-(СН ₂ ) _1-2 -(pyridazinyl), -NH-(СН ₂ ) _1-2 -CF ₂ (pyridyl), ^0H , ^0H , ^{NH NHχ} ^ and '\ Upi- ^NH \ g

R ³ independently represents or ; and

R ⁴ independently represents H or F.

In another aspect

W' represents H;

W is independently selected from: -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) _3-4 -OH, χΰ A:

-NH-(CH ₂ ) _1-2 -CH(CH ₃ )OH, -NH-(CH ₂ ) _1-2 -C(CH ₃ ) ₂ OH, -NH-(CH ₂ ) _1-2 -(pyrazolyl), ^c - ^0H ;

R ³ independently represents

R ⁴ independently represents H or F.

In another aspect

W' represents H;

W is independently selected from: -O-CH2CH(OH)(CH2OH), -NH-(CH2)3-4-OH,

-NH-(CH2)1-2-CH(CH3)OH, -NH-(CH2)1.2-C(CH ₃ )2OH, -NH-(СН2)1-2-(nurazolyl) and ° ⁿ

N \

R ³ is ; and

R ⁴ independently represents H or F.

In another aspect, the compound is selected from:

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or a pharmaceutically acceptable salt thereof. In another aspect, the compound is selected from:

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In another aspect, the compound is or a pharmaceutically acceptable salt thereof. In another aspect, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In another aspect, the compound is or a pharmaceutically acceptable salt thereof.

In another aspect, the compound is

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or its pharmaceutically acceptable salt.

In another aspect, the compound is

In another aspect, a pharmaceutical composition is provided comprising a compound or a pharmaceutically acceptable salt or stereoisomer thereof and one or more pharmaceutically acceptable excipients.

In another aspect, there is provided the use of the compound or a pharmaceutically acceptable salt or stereoisomer thereof for the treatment of cancer.

In another aspect, the cancer is selected from acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, malignant tumor of the anal region, malignant tumor of the appendix, teratoid rhabdoid tumor, basal cell carcinoma, malignant tumor of the bile ducts, malignant tumor of the bladder, malignant tumor of the bone, malignant tumor of the brain, malignant tumor of the breast, bronchial tumor, carcinoid tumor, cardiac tumor, cervical malignancy, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, malignant tumor of the colon, malignant tumor of the rectum and colon, craniopharyngioma, malignant tumor of the endometrium, ependymoma, malignant tumor of the esophagus, esthesioneuroblastoma, Ewing's sarcoma, malignant tumor of the eye, malignant tumor of the fallopian tubes, malignant tumor of the gallbladder bladder, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, oral and lip cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, perioral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestinal cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine, vaginal malignancy and vulvar malignancy.

In another aspect, the malignant tumor is a refractory malignant tumor.

In another aspect, the cancer is selected from breast cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

In another aspect, the cancer is selected from hormone receptor-positive breast cancer, microsatellite-stable colon or rectal cancer, pancreatic cancer, and prostate cancer.

In another aspect, the compound is administered in combination with one or more additional cancer treatments.

In another aspect, one or more additional methods of treating cancer include surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy, or a combination thereof.

In another aspect, the adjunctive cancer therapy comprises one or more agents selected from nivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, TSR042, GLS-010, AM-0001, STI-1110, AGEN2034, MGD013, IBI308, BMS-936559, atezolizumab, durvalumab, avelumab, STI-1014, CX-072, LY3300054, CK-301, urelumab, PF-05082566, MEDI6469, TRX518, varlilumab, CP-870893, BMS-986016, MGA271, lirilumab, IPH2201, emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, bavituximab, CC-90002, bevacizumab, MNRP1685A, ipilimumab, MK-1308, AGEN-1884, and tremelimumab.

In another aspect, the adjunctive cancer therapy comprises one or more agents selected from

- 5 040307 nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab and avelumab.

In another aspect, the use of the pharmaceutical composition for the treatment of cancer is proposed.

In another aspect, the cancer is selected from acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, malignant tumor of the anal region, malignant tumor of the appendix, teratoid rhabdoid tumor, basal cell carcinoma, malignant tumor of the bile ducts, malignant tumor of the bladder, malignant tumor of the bone, malignant tumor of the brain, malignant tumor of the breast, bronchial tumor, carcinoid tumor, cardiac tumor, cervical malignancy, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, malignant tumor of the colon, malignant tumor of the rectum and colon, craniopharyngioma, malignant tumor of the endometrium, ependymoma, malignant tumor of the esophagus, esthesioneuroblastoma, Ewing's sarcoma, malignant tumor of the eye, malignant tumor of the fallopian tubes, malignant tumor of the gallbladder bladder, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, oral and lip cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, perioral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestinal cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine, vaginal malignancy and vulvar malignancy.

In another aspect, the malignant tumor is a refractory malignant tumor.

In another aspect, the cancer is selected from breast cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

In another aspect, the cancer is selected from hormone receptor-positive breast cancer, microsatellite-stable colon or rectal cancer, pancreatic cancer, and prostate cancer.

In another aspect, the compound is administered in combination with one or more additional cancer treatments.

In another aspect, one or more additional methods of treating cancer include surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy, or a combination thereof.

In another aspect, the adjunctive cancer therapy comprises one or more agents selected from nivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, TSR042, GLS-010, AM-0001, STI-1110, AGEN2034, MGD013, IBI308, BMS-936559, atezolizumab, durvalumab, avelumab, STI-1014, CX-072, LY3300054, CK-301, urelumab, PF-05082566, MEDI6469, TRX518, varlilumab, CP-870893, BMS-986016, MGA271, lirilumab, IPH2201, emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, bavituximab, CC-90002, bevacizumab, MNRP1685A, ipilimumab, MK-1308, AGEN-1884, and tremelimumab.

In another aspect, the adjunctive cancer therapy comprises one or more agents selected from nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab, and avelumab.

In one aspect, methods of modulating (e.g., agonizing, partially agonizing, antagonizing) NLRP3 activity are provided as comprising contacting NLRP3 with a chemical compound described herein (e.g., a compound described herein generally or specifically, or a pharmaceutically acceptable salt thereof, or compositions comprising thereof). In preferred embodiments, the methods of modulating NLRP3 activity are agonizing and partially agonizing. In some embodiments, the methods of modulating NLRP3 activity are agonizing. In some embodiments, the methods of modulating NLRP3 activity are partially agonizing. The methods include in vitro methods, such as contacting a sample that includes one or more cells containing NLRP3 (e.g., THP-1 cells) with the chemical compound. The methods can also include in vivo methods; for example, administering a chemical compound to a subject (e.g., a human) having a disease in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity that contributes to the pathology and/or symptoms and/or progression of the disease (e.g., a malignant tumor; e.g., a refractory malignant tumor).

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In some embodiments, the compounds of the invention are useful for treating a condition, disease, or disorder in which decreased NLRP3 activity (e.g., a condition, disease, or disorder associated with suppressed or impaired NLRP3 signaling) contributes to the pathology and/or symptoms and/or progression of the condition, disease, or disorder (e.g., a cancer) in a subject (e.g., a human).

Cancer is considered refractory if it does not respond to (or is resistant to) cancer treatment. Refractory cancer is also known as resistant cancer.

In another aspect, methods for treating cancer are provided that comprise administering to a subject in need of such treatment an effective amount of a chemical compound described herein (e.g., a compound described generally or specifically herein, or a pharmaceutically acceptable salt thereof, or compositions containing thereof). In some embodiments, the cancer may be refractory cancer.

In a further aspect, methods are provided for treating a disease in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity that causes pathology and/or symptoms and/or progression of the disease, which comprise administering to a subject in need of such treatment an effective amount of a chemical compound described herein (e.g., a compound described herein generally or specifically, or a pharmaceutically acceptable salt thereof, or compositions containing thereof). In another aspect, methods are provided for treating, which comprise administering to a subject having a disease in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity that causes pathology and/or symptoms and/or progression of the disease an effective amount of a chemical compound described herein (e.g., a compound described herein generally or specifically, or a pharmaceutically acceptable salt thereof, or compositions containing thereof).

In a further aspect, methods of treatment are provided that comprise administering to a subject a chemical compound described herein (e.g., a compound described herein generally or specifically, or a pharmaceutically acceptable salt thereof, or compositions containing thereof), wherein the chemical compound is administered in an amount effective to treat a disease in which enhancing NLRP3 signaling can correct the deficiency in innate immune activity causing the pathology and/or symptoms and/or progression of the disease, thereby resulting in treatment of the disease.

Embodiments may include one or more of the following features. The chemical compound may be administered in combination with one or more additional cancer therapies (e.g., surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy, or a combination thereof; for example, with cancer therapies that involve the administration of one or more (e.g., two, three, four, five, six or more) additional anticancer agents. Non-limiting examples of additional anticancer agents (chemotherapeutic agents) are selected from an alkylating agent (e.g., cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, and/or oxaliplatin); an antimetabolite (e.g., azathioprine and/or mercaptopurine); a terpenoid (e.g., a vinca alkaloid and/or a taxane; for example, vincristine, vinblastine, vinorelbine, and/or vindesine, taxol, paclitaxel and/or docetaxel); a topoisomerase (e.g., type I topoisomerase and/or type 2 topoisomerase; e.g., camptothecins such as irinotecan and/or topotecan; amsacrine, etoposide, etoposide phosphate and/or teniposide); a cytotoxic antibiotic (e.g., actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and/or mitomycin); a hormone (e.g., a luteinizing hormone-releasing hormone agonist; e.g., leuprolidine, goserelin, triptorelin, histrelin, bicalutamide, flutamide and/or nilutamide); an antibody (e.g., abciximab, adalimumab, alemtuzumab, atlizumab, basiliximab, belimumab, bevacizumab, bretuximab vedotin, canakinumab, cetuximab, certolizumab pegol, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tositumomab and/or trastuzumab); an antiangiogenic agent; a cytokine; an antithrombotic agent; a growth inhibitory agent; an anthelmintic agent; and an immune checkpoint inhibitor that targets an immune checkpoint receptor selected from CTLA-4, PD-1, PD-L1, PD-1- PD-L1, PD-1- PD-L2, T-cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), galectin 9 - TIM3, phosphatidylserine - TIM3, lymphocyte activation gene protein 3 (LAG3), MHC class II - LAG3, 4-1BB-4-1BB ligand, 0X40-0X40 ligand, GITR, GITR ligand - GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEMLIGHT-LTA, HVEM, HVEM-BTLA, HVEM-CD160, HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80PDL-1, PDL2-CD80, CD244, CD48-CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2, butyrophilins, including BTNL2, the Siglec family, representatives of the TIGIT and PVR families, KIR, ILT and LIR, NKG2D and NKG2A, MICA and MICB, CD244, CD28, CD86-CD28,

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CD86-CTLA, CD80-CD28, phosphatidylserine, TIM3, phosphatidylserine - TIM3, SIRPA-CD47, VEGF, neutrophilin, CD160, CD30 and CD155 (e.g. CTLA-4 or PD1 or PD-L1) and other immunomodulatory agents such as interleukin-2 (IL-2), indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFe), CD39, CD73 adenosine-CD39-CD73 and CXCR4-CXCL12.

The subject may have a malignant tumor; for example, the subject has undergone, and/or is undergoing, and/or will undergo, one or more cancer treatments. Non-limiting examples of malignant tumor include acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendiceal cancer, teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, colon cancer, rectal cancer, craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, Gallbladder tumor, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, oral and lip cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, perioral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestinal cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, a vaginal malignancy and a vulvar malignancy. In other embodiments, the mammal has been identified as having a malignancy or an infectious disease. Common infectious diseases include, but are not limited to, Acinobacter infection, actinomycosis, African sleeping sickness, acquired immunodeficiency syndrome, amebiasis, anaplasmosis, anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidiasis, Baylisascaris infection, BK virus infection, black piedra, Blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever, botulism, Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderi infection, Buruli ulcer, Calicivirus infection, Campylobacteriosis, Candidiasis, Cat scratch disease, Cellulitis, Chagas disease, Chancroid, Varicella, Chikungunya, Chlamydia infection, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Viral upper respiratory tract infection, Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous wandering larva syndrome, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Desmodesmus infection, Dientamoebiasis, Diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis, Enterococcus infection, Enterovirus infection, epidemic typhus, erythema infectiosum, exanthema succedaneum, fasciolopsiasis, fascioliasis, fatal familial insomnia, filariasis, Clostridium myonecrosis food poisoning, free-living ameba infection, Fusobacterium infection, gas gangrene, geotrichosis, Gerstmann-Sträussler-Scheinker syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale, group A streptococcal infection, group B streptococcal infection, Haemophilus infection influenza, hand-foot-and-mouth disease, hantavirus pulmonary syndrome, heartland virus disease, heliobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm, human bocavirus infection, human ehrlichiosis, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, hymenolepiasis, infectious mononucleosis Epstein-Barr, influenza, isosporiasis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, Lassa fever, Legionnaires' disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, Middle East respiratory syndrome, melioidosis, meningitis, meningococcal disease, metagonimiasis, microsporidiosis, molluscum contagiosum, monkeypox, mumps, murine typhus, mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis, variant Creutzfeldt-Jakob disease, nocardiosis, onchocerciasis, paracoccidioidomycosis, paragonimiasis, pasteurellosis, pediculosis capitis, pediculosis pubis, pelvic inflammatory disease, whooping cough, plague, pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsialpox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, severe acute respiratory syndrome, scabies, schistosomiasis, septicemia, shigellosis, herpes zoster, smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute sclerosing Panencephalitis, syphilis, taeniasis, tetanus, tinea barbae, trichophytosis profunda, tinea corporis, tinea cruris, tinea mani, lichen nigricans, tinea pedis, tinea pedis, tinea versicolor, toxocariasis, trachoma, toxoplasmosis, trichinosis, trichomoniasis, trichuriasis, tuberculosis, tularemia, typhoid fever, Ureaplasma urealyticum infection, valley fever, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white piedra, Yersinia psuedotuberculosis infection, yersiniosis, yellow fever, and zygomycosis.

The chemical compound can be injected into the tumor.

The chemical compound may be administered systemically (including, but not limited to, orally, subcutaneously, intramuscularly, intravenously).

The methods may additionally include identification of the subject.

Other embodiments include those described in the detailed description and/or the claims.

Additional definitions

To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures of organic chemistry, medicinal chemistry, and pharmacology described herein are well known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Unless otherwise stated herein, references made in the singular may also include the plural.

Unless otherwise specified, any heteroatom with unsaturated valences is assumed to contain sufficient hydrogen atoms to saturate the valences. Throughout the specification and the appended claims, a given chemical formula or name shall encompass all stereo and optical isomers and their racemates, where such isomers exist. Unless otherwise specified, all chiral (enantiomeric and diastereomeric) and racemic forms are within the scope of the invention. Many geometric isomers of C=C double bonds, C=N double bonds, ring systems, and the like may also be present in the compounds, and all such stable isomers are contemplated by the present invention. Cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separate isomeric forms. The present compounds may be isolated in optically active or racemic forms. Optically active forms can be obtained by resolution of racemic forms or by synthesis from optically active starting materials. All processes used to prepare the compounds of the present invention and the intermediates obtained herein are considered part of the present invention. When enantiomeric or diastereomeric products are obtained, they can be separated by conventional techniques, for example, by chromatography or fractional crystallization. Depending on the process conditions, the final products of the present invention are obtained either in free (neutral) form or in salt form. Both the free form and the salts of these final products are within the scope of the present invention. If desired, one form of a compound can be converted into another form. A free base or acid can be converted into a salt; a salt can be converted into the free compound or another salt; a mixture of isomeric compounds of the present invention can be separated into individual isomers. The compounds of the present invention, their free forms, and their salts, can exist in multiple tautomeric forms, in which hydrogen atoms are relocated to other parts of the molecule, and consequently, the chemical bonds between the atoms of the molecule are rearranged. It is understood that all tautomeric forms, if they can exist, are included in the invention.

As used herein, the term NLRP3 includes, but is not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide strands, complementary sequences, peptides, polypeptides, proteins, homologous and/or orthologous NLRP3 molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, various particles, and active fragments thereof.

The term "acceptable" when used in relation to a composition, formulation, or ingredient means that it does not have a persistent detrimental effect on the general health of the subject being treated. The term "API" refers to an active pharmaceutical ingredient. The terms "effective amount" or "therapeutically effective amount" as used herein refer to a sufficient amount of a chemical compound (e.g., a compound exhibiting activity as a mitochondrial uncoupling agent, or a pharmaceutically acceptable salt and/or hydrate and/or co-crystal thereof; for example, a compound such as niclosamide or a pharmaceutically acceptable salt and/or hydrate and/or co-crystal thereof; for example, a compound such as a niclosamide analogue or a pharmaceutically acceptable salt and/or hydrate and/or co-crystal thereof) to be administered that will alleviate to some extent one or more symptoms of the disease or condition being treated. The result includes a reduction and/or amelioration of the signs, symptoms, or causes of the disease, or any other desired change in the biological system. For example, the term "effective amount" for therapeutic applications is the amount of a composition containing a compound disclosed herein required to provide a clinically significant reduction in disease symptoms. The corresponding effective amount in any given case is determined using any suitable methodology, such as a dose-escalation study.

The term "excipient" or "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition, or carrier medium, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. According to one embodiment, each component is pharmaceutically acceptable in the sense that it is compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with the tissue or organ of humans and animals without undue toxicity, irritation, allergic reaction, immunogenicity, or other problems or complications commensurate with a reasonable benefit-risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, UK (2012); Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al, Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: (2009); Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: (2007); Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, (2009).

The term "pharmaceutically acceptable salt" refers to a composition of a compound that does not significantly irritate the host or inhibit the biological activity or properties of the compound. In some cases, pharmaceutically acceptable salts are prepared by reacting the compound described herein with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. In some cases, pharmaceutically acceptable salts are prepared by reacting a compound containing an acid group described herein with a base to form a salt, such as an ammonium salt, an alkali metal salt, such as a sodium or potassium salt, an alkaline earth metal salt, for example, a potassium or magnesium salt, salts of organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, tri(hydroxymethyl)methylamine, and salts of amino acids, such as arginine, lysine, etc., or by other methods defined previously. A pharmacologically acceptable salt is not particularly limited as long as it can be used in medicinal preparations. Examples of a salt that the compounds described herein form with a base include the following: their salts with inorganic bases, such as sodium, potassium, magnesium, calcium, and aluminum salts; their salts with organic bases, such as methylamine, ethylamine, and ethanolamine; their salts with basic amino acids, such as lysine and ornithine; and an ammonium salt. The salts may be acid addition salts, which are particularly represented by the addition salts of the following acids: mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid and ethanesulfonic acid; acidic amino acids such as aspartic acid and glutamic acid.

The term "pharmaceutical composition" refers to a mixture of a compound described herein with other chemical components (collectively referred to herein as "excipients"), such as carriers, stabilizers, diluents, dispersing agents, suspending agents, and/or thickening agents. The pharmaceutical composition facilitates the administration of the compound to the body. There are numerous routes of administration of the compound in the art, including, but not limited to, rectal, oral, intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

The term subject refers to an animal, including, but not limited to, a primate (e.g., a human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms subject and patient are used interchangeably herein to refer to, for example, a mammalian subject such as a human. The terms treat, treating, and treatment, in the context of treating a disease or disorder, include alleviating or eliminating the disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or slowing the progression, spread, or worsening of the disease, disorder, or condition, or one or more symptoms thereof. The term treatment of a malignant tumor refers to one or more of the following actions: (1) inhibiting, to some extent, tumor growth, including (i) slowing and (ii) completely stopping growth; (2) decreasing the number of tumor cells; (3) maintaining tumor size; (4) decreasing tumor size; (5) inhibition, including (i) reduction, (ii) slowing, or (iii) complete prevention of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing, or (iii) complete prevention of metastasis; (7) enhancement of the anti-tumor immune response, which may result in (i) maintenance of tumor size, (ii) reduction in tumor size, (iii) slowing of tumor growth, (iv) reduction, slowing, or prevention of invasion; and/or (8) alleviation, to some extent, of the severity or number of one or more symptoms associated with the disorder.

The term halogen denotes fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). The term alkyl denotes a hydrocarbon chain, which may be straight or branched, containing the stated number of carbon atoms. For example, _C1-10 denotes that the group may contain from 1 to 10 (inclusive) carbon atoms. Non-limiting examples include methyl, ethyl, iso-propyl, tert-butyl, n-hexyl. The term haloalkyl denotes alkyl in which one or more hydrogen atoms are/are replaced by an independently selected halogen. The term alkoxy denotes an -O-alkyl radical (e.g., _-OCH3 ). The term alkylene denotes a branched or unbranched divalent alkyl (e.g., -CH=-).

The term alkenyl denotes a hydrocarbon chain, which may be straight or branched, containing one or more carbon-carbon double bonds. The alkenyl moiety contains the stated number of carbon atoms. For example, C2.6 denotes that the group may contain from 2 to 6 (inclusive) carbon atoms in it. The term alkynyl denotes a hydrocarbon chain, which may be straight or branched, containing one or more carbon-carbon triple bonds. The alkynyl moiety contains the stated number of carbon atoms. For example, C2-6 denotes that the group may contain from 2 to 6 (inclusive) carbon atoms in it. The term aromatic generally refers to a ring that includes a cyclic group of resonance-stabilized 4n+2 pi electrons, where n is an integer (e.g., 1 or 2). Aromatic moieties include aryl and heteroaryl groups. The term non-aromatic describes any moiety that does not meet the definition of aromatic.

The term "aryl" denotes a 6-carbon monocyclic, 10-carbon bicyclic, or 14-carbon tricyclic aromatic ring system in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent, and where the ring containing the monocyclic radical is aromatic, and where at least one of the fused rings containing the bicyclic or tricyclic radical is aromatic, such as tetrahydronaphthyl. Examples of aryl groups also include phenyl, naphthyl, and the like.

The term "cycloalkyl" as used herein includes saturated cyclic hydrocarbon groups containing from 3 to 10 carbon atoms, preferably from 3 to 8 carbon atoms, and more preferably from 3 to 6 carbon atoms, wherein the cycloalkyl group may be optionally substituted. Preferred cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. The term "cycloalkylene" as used herein means divalent cycloalkyl.

The term heteroaryl means an aromatic 5-8-membered monocyclic, 8-12-membered bicyclic, or 11-14-membered tricyclic ring system containing 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, wherein said heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent, and wherein the ring containing the monocyclic radical is aromatic, and wherein at least one of the fused rings containing the bicyclic or tricyclic radical is aromatic (but need not be the ring that contains the heteroatom, e.g., tetrahydroisoquinolinyl). Examples of heteroaryl groups also include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term heterocyclyl denotes a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic

- 11 040307 or an 11-14 membered tricyclic ring system containing 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, wherein said heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, or 3 atoms of each ring may be substituted with a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like. The term heterocycloalkylene denotes divalent heterocyclyl.

Furthermore, the atoms constituting the compounds of the present embodiments are intended to include all isotopic forms of such atoms. Isotopes as used herein include those atoms that have the same atomic number but different mass numbers. By way of general example and without limitation, hydrogen isotopes include tritium and deuterium, and carbon isotopes include ^13C and ^14C . The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other distinguishing features and advantages of the invention will be apparent from the description and drawings, as well as from the claims.

Detailed description

The present disclosure provides chemical entities (e.g., a compound or pharmaceutically acceptable salt and/or hydrate and/or co-crystal and/or drug combination thereof) that modulate (e.g., agonize or partially agonize) NLRP3, which are useful, for example, for treating a condition, disease, or disorder in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity (e.g., a condition, disease, or disorder associated with an insufficient immune response) that contributes to the pathology and/or symptoms and/or progression of the condition, disease, or disorder (e.g., a malignancy) in a subject (e.g., a human). The present disclosure also relates to compositions, as well as other methods for using and preparing them.

Compounds of the invention

In one aspect, the compounds of formula I or a pharmaceutically acceptable salt thereof are

where W' represents H;

W is independently selected from: -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) _3-4 -OH, -NH-(CH ₂ ) _1-2 -CH(CH ₃ )OH, -

NH-(CH ₂ ) _1-2 -C(CH ₃ ) ₂ OH, -O-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrimidinyl),

-NH-(CH ₂ ) _1-2 -(pyridazinyl), -NH-(CH ₂ ) _1-2 -CF ₂ (pyridyl),

R ³ is independently pyrazolyl or thienyl; and

R ⁴ independently represents H or F.

In one aspect, the compounds of formula I or a pharmaceutically acceptable salt thereof are wherein W' is H;

W is independently selected from: -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) _3-4 -OH,

-NH-(CH ₂ ) _1-2 -CH(CH ₃ )OH,

-NH-(CH ₂ ) _1-2 -C(CH ₃ ) ₂ OH, -O-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrimidinyl),

-NH-(CH ₂ ) _1-2 -(pyridazinyl),

-NH-(СН2)1-2-CF2(pyridyl),

R ³

R ⁴ independently represents

independently represents H or F.

In one aspect, the compounds of formula I or a pharmaceutically acceptable salt thereof are

- 12 040307

where W' represents H;

W is independently selected from: -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) ₃ . ₄ -OH, -NH-(CH ₂ ) ₁ . ₂ -CH(CH ₃ )OH,

-NH-(CH2)i-2-C(CH3)2OH, -NH-(CH2)1-2-(pyrazolyl),

R ³ independently represents

R ⁴ independently represents H or F.

In another aspect, there are provided compounds of formula (I) or a pharmaceutically acceptable salt thereof, wherein W' is H;

W is independently selected from: -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) ₃ . ₄ -OH, -NH-(CH ₂ ) ₁ . ₂ -CH(CH ₃ )OH,

-NH-(CH2)i-2-C(CH3)2OH, -NH-(CH2)1-2-(pyrazolyl) and

R ³ is

R ⁴ is independently H or F. In another aspect, the compound is selected from:

- 13 040307 or a pharmaceutically acceptable salt thereof. In another aspect, the compound is selected from:

or a pharmaceutically acceptable salt thereof. In another aspect, the compound is selected from:

or a pharmaceutically acceptable salt thereof. In another aspect, the compound is selected from:

- 14 040307

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a compound selected from the examples provided, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a compound selected from any subset of compounds or an individual compound of the examples provided within the scope of any of the above aspects.

Pharmaceutical compositions and administration

According to some embodiments, a chemical compound (e.g., a compound that modulates (e.g., agonizes or partially agonizes) NLRP3, or a pharmaceutically acceptable salt and/or hydrate and/or co-crystal and/or drug combination thereof) is administered as a pharmaceutical composition that comprises the chemical compound and one or more pharmaceutically acceptable excipients and optionally one or more additional therapeutic agents, as described herein.

In some embodiments, a pharmaceutical composition is described comprising a compound of the present invention or a salt thereof and one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition is described comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. In some embodiments, a pharmaceutical composition is described comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

In some embodiments, the chemical compounds may be administered in combination with one or more conventional pharmaceutical excipients. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric delivery matrices, whey proteins such as human serum albumin, buffering agents such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethyleneglycol, carboxymethylcellulose sodium, polyacrylates, waxes, block copolymers of polyethylene and polyoxypropylene, and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives can also be used to improve the delivery of the compounds described herein. Dosage forms or compositions containing a chemical compound as described herein in the range of 0.005 to 100% in a ratio derived from a non-toxic excipient can be prepared. The provided compositions may contain from 0.001 to 100% of the chemical compound provided herein, in one embodiment from 0.1 to 95%, in another embodiment from 75 to 85%, in a further embodiment from 20 to 80%. Existing methods for preparing such dosage forms are known or will be obvious to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 22 ^nd Edition (Pharmaceutical Press, London, UK. 2012).

- 15 040307

Routes of administration and components of the composition

In some embodiments, the chemical compounds or pharmaceutical compositions thereof described herein may be administered to a subject in need thereof by any acceptable route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intraperitoneal, intraarterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinusal, intravertebral, intraarticular, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, subcutaneous, epidural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral, and vaginal. In some embodiments, the preferred route of administration is parenteral (e.g., intratumoral). In some embodiments, the preferred route of administration is systemic.

Compositions can be formulated for parenteral administration, such as injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectable preparations or as liquid solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions by adding liquid prior to injection can also be prepared, and the preparations can also be emulsified. The preparation of such formulations will be known to those skilled in the art in light of the present disclosure.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. It must also be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Protection against the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin, in the compositions. Sterile injectable solutions were prepared by incorporating the active compounds in the required amount in an appropriate solvent with various other components listed above, if necessary, followed by filtered sterilization. Typically, dispersions were prepared by incorporating various sterilized active components into a sterile vehicle containing the basic dispersion medium and the required other components listed above. For sterile powders, vacuum drying and freeze-drying techniques are preferred for obtaining sterile injectable solutions, resulting in a powder of the active ingredient plus any additional required component from its previously sterile-filtered solution.

Intratumoral injections have been discussed, for example, in Lammers, et al., Effect of Intratumoral Injection on the Biodistribution and the Therapeutic Potential of HPMA Copolymer-Based Drug Delivery Systems Neoplasia 10:788–795 (2006).

Pharmacologically acceptable excipients useful in a rectal composition in the form of a gel, cream, enema, or rectal suppository include, but are not limited to, any one or more of cocoa butter glycerides, synthetic polymers such as polyvinylpyrrolidone, PEG (such as PEG ointment), glycerin, glycinose gelatin, hydrogenated vegetable oils, poloxamers, mixtures of polyethylene glycols of various molecular weights and polyethylene glycol fatty acid esters, petrolatum, anhydrous lanolin, shark liver oil, sodium saccharinate, menthol, sweet almond oil, sorbitol, sodium benzoate, SBN anoxide, vanilla essential oil, aerosol, parabens in phenoxyethanol, sodium methyl para-hydroxybenzoate, sodium propyl para-hydroxybenzoate,

- 16 040307 diethylamine, carbomer, carbopol, methyl oxybenzoate, macrogol cetostearyl ether, cocoyl caprylocaprate, isopropyl alcohol, propylene glycol, liquid paraffin, xanthan gum, carboxymethabisulfite, sodium edetate, sodium benzoate, potassium metabisulfite, grapefruit seed extract, methylsulfonylmethane (MSM), lactic acid, glycine, vitamins such as vitamin A and E, and potassium acetate.

In some embodiments, suppositories can be prepared by mixing the chemical compounds described herein with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository wax, which are solid at ambient temperature and liquid at body temperature and thus melt in the rectum and release the active compound. According to other embodiments, the composition for rectal administration is in the form of an enema. In other embodiments, the compounds described herein or their pharmaceutical composition are suitable for topical delivery to the digestive or gastrointestinal tract by oral administration (e.g., solid or liquid dosage forms).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the chemical compound is mixed with one or more pharmaceutically acceptable excipients, such as sodium citrate or dicalcium phosphate, and/or: a) fillers or diluents, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) dissolution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. When capsules, tablets, and pills are used, the dosage form may also contain buffering agents. Solid compositions of this type can also be used as fillers in filled soft and hard gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.

According to one embodiment, the compositions will be in a unit dosage form such as a pill or tablet, and thus the composition may comprise, together with a chemical compound provided herein, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate, or the like; and a binder such as starch, acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives, or the like. In another solid dosage form, a powder, granules, solution, or suspension (e.g., in propylene carbonate, vegetable oils, PEG, poloxamer 124, or triglycerides) is encapsulated in a capsule (gelatin or cellulose-based capsule). Also contemplated are unit dosage forms in which one or more chemical compounds provided herein or additional active agents are physically separated; For example, capsules with granules (or tablets within a capsule) of each drug; bilayer tablets; dual-chamber gel capsules, and the like. Also available for oral use are enteric-coated or delayed-release dosage forms.

Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives, which are particularly useful for preventing the growth or attack of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. In some embodiments, the excipients are sterile and generally free of unwanted substances. These compositions can be sterilized using conventional, well-known sterilization techniques. For various excipients in oral dosage forms, such as tablets and capsules, sterility is not required. In most cases, the USP/NF standard is sufficient.

According to some embodiments, solid oral dosage forms may further include one or more components that chemically and/or structurally provide the composition for delivering the chemical compound to the stomach or lower GI (gastrointestinal tract); for example, to the ascending colon and/or transverse colon and/or distal colon and/or small intestine. Illustrative formulation techniques are described, for example, in Filipski, K.J., et al., Current Topics in Medicinal Chemistry, 2013, 13, 776-802. Examples include upper GI targeting techniques, such as Accordion Pill (Intec Pharma), floating capsules, and mucoadhesive materials.

Other examples include lower GI targeting techniques. Several enteric/pH-sensitive coatings and excipients are available to target different sites of the GI tract. These materials are typically polymers that are designed to dissolve or degrade in specific pH ranges selected based on the GI site for the desired drug release. These materials also function to protect acid-labile drugs from gastric juices or to limit exposure in cases where the active ingredient may irritate the upper GI tract (e.g., hydroxypropyl methylcellulose phthalate series, Coateric (polyvinyl acetate phthalate), cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, Eudragit series (methacrylic acid-methyl methacrylate copolymers), and Marcoat). Other approaches include dosage forms that respond to local flora in the GI tract, a capsule for pressure-controlled delivery to the colon, and Pulsincap.

Ophthalmic compositions may include, without limitation, one or more of any of the following: viscogens (e.g., carboxymethylcellulose, glycerin, polyvinylpyrrolidone, polyethyleneglycol); stabilizers (e.g., Pluronic (triblock copolymers), cyclodextrins); preservatives (e.g., benzalkonium chloride, ETDA, SofZia (boric acid, propylene glycol, sorbitol, and zinc chloride; Alcon Laboratories, Inc.), Purite (stabilized oxychlor complex; Allergan, Inc.)).

Compositions for topical use may include ointments and creams. Ointments are semi-solid preparations that typically contain petrolatum or other petroleum derivatives as a base. Creams containing the selected active agent are typically viscous liquid or semi-solid emulsions, often either oil-in-water or water-in-oil. Cream bases are typically water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also sometimes called the internal phase, typically contains petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase often, although not necessarily, exceeds the oil phase in volume and typically contains a humectant. The emulsifier in a cream formulation is typically a nonionic, anionic, cationic, or amphoteric surfactant. As with other carriers or vehicles, the ointment base must be inert, stable, non-irritating and hypoallergenic.

According to any of the above embodiments, the pharmaceutical compositions described herein may include one or more of the following: lipids, multilamellar vesicles having crosslinks between lipid bilayers, biodegradable nanoparticles or microparticles based on a copolymer of poly-D,L-lactic acid and glycolic acid [PLGA] or based on polyanhydride, and nanoporous particles onto which lipid bilayers are deposited.

Dosage

The dosage may vary depending on the patient's needs, the severity of the condition being treated, and the specific compound used. A medical professional can determine the appropriate dosage for a specific situation. The total daily dose may be divided and administered in portions throughout the day or by continuous delivery. In some embodiments, the compounds described herein are administered at a dose of from about 0.001 to about 500 mg/kg (e.g., from about 0.001 to about 200 mg/kg; from about 0.01 to about 200 mg/kg; from about 0.01 to about 150 mg/kg; from about 0.01 to about 100 mg/kg; from about 0.01 to about 50 mg/kg; from about 0.01 to about 10 mg/kg; from about 0.01 to about 5 mg/kg; from about 0.01 to about 1 mg/kg; from about 0.01 to about 0.5 mg/kg; from about 0.01 to about 0.1 mg/kg; from about 0.1 to about 200 mg/kg; from about 0.1 to about 150 mg/kg; from about 0.1 to about 100 mg/kg; from about 0.1 to about 50 mg/kg; from about 0.1 to about 10 mg/kg; from about 0.1 to about 5 mg/kg; from about 0.1 to about 1 mg/kg; from about 0.1 to about 0.5 mg/kg).

Treatment regimens

The above doses may be administered daily (e.g., as a single dose or as two or more divided doses) or non-daily (e.g., every other day, every two days, every three days, once a week, twice a week, once every two weeks, once a month).

According to some embodiments, the period of administration of the compound described herein is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. According to a further embodiment, the period during which the administration was stopped is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. According to an embodiment, the therapeutic compound is administered to the subject during a period of time followed by a separate period of time. According to another embodiment, the therapeutic compound is administered during a first period and a second period that followed the first period, with the administration stopped during the second period, which was followed by a third period in which the administration was started

- 18 040307 administration of a therapeutic compound, and then a fourth period following the third period in which the administration was stopped. In an aspect of the present invention, the period of administration of the therapeutic compound, followed by the period in which the administration was stopped, is repeated for a definite or indefinite period of time. According to a further embodiment, the administration period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. According to a further embodiment, the period during which the administration was stopped was 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more.

Treatment methods

According to some embodiments, methods of treating a subject having a condition, disease, or disorder are provided, wherein enhancing NLRP3 signaling can correct a deficiency in innate immune activity (e.g., a condition, disease, or disorder associated with an insufficient immune response) that contributes to the pathology and/or symptoms and/or progression of the condition, disease, or disorder (e.g., a cancer).

Indications

According to any of the methods described herein, the subject may have a malignant tumor. In some examples of any of the methods described herein, the mammal has been identified as having a malignant tumor or diagnosed with a malignant tumor.

Non-limiting examples of malignant tumor include acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendiceal cancer, teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, colon cancer, colorectal cancer, craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, eye cancer, fallopian cancer tubal cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, cardiac cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, oral and lip cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestinal cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer. In some embodiments, non-limiting examples of cancer include breast cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, and prostate cancer.

Methods for diagnosing a subject as having a malignant tumor or identifying a mammal as having a malignant tumor are well known in the art. For example, a medical professional (e.g., a physician, physician assistant, or paramedical professional) may be able to diagnose a malignant tumor in a mammal by observing the mammal for one or more symptoms of a malignant tumor. Non-limiting examples of symptoms of a malignant tumor include weakness, a lump or palpable thickening under the skin, a change in body weight, jaundice, darkening or redness of the skin, non-healing ulcers, changes in existing moles, changes in bodily functions, persistent coughing or difficulty breathing, difficulty swallowing, hoarseness, persistent stomach upset or discomfort after eating, persistent muscle or

- 19 040307 joint pain of unknown etiology, persistent fever or night sweats of unknown etiology, and bleeding or hematoma of unknown etiology. Methods for diagnosing a subject with a malignant tumor or identifying a subject as having a malignant tumor may additionally include performing one or more diagnostic tests (e.g., performing one or more diagnostic tests on a biopsy or blood sample).

According to some examples of any of the methods described herein, the subject may be a subject having a cancer, a subject diagnosed with having a cancer, or a subject identified as having a cancer that was refractory to prior treatment for the cancer. Diagnostic tests for diagnosing a subject as having a cancer or for identifying a mammal as having a cancer are known in the art. According to some embodiments, methods are provided for treating a subject having a condition, disease, or disorder in which enhancing NLRP3 signaling can correct a deficiency in innate immune activity (e.g., a condition, disease, or disorder associated with an insufficient immune response) that contributes to the pathology and/or symptoms and/or progression of the condition, disease, or disorder (e.g., a cancer).

In some embodiments of the present invention, a method of treating a malignant tumor is provided, wherein the malignant tumor may be any type of malignant tumor that does not elicit an optimal response of the innate immune system.

The innate immune system refers to the portion of the immune system that responds to threats to the body, such as infections or cancer, in a non-antigen-specific manner and stimulates the adaptive, antigen-specific immune system. In general, complete removal of a harmful factor and long-term protection (=immunity) require the activity of the adaptive, antigen-specific immune system, which, in turn, depends on stimulation by the innate immune system. In some embodiments, the present invention provides a method of treatment in a case where the cancer type is selected based on resistance to T-cell immune checkpoint inhibition, either independently of the cancer type and based on a failure to respond to previous T-cell checkpoint inhibitor therapy, or based on a cancer type that is typically resistant to T-cell checkpoint inhibitor therapy, such as hormone receptor-positive breast cancer, allite stable colorectal cancer, pancreatic cancer, and prostate cancer. In some other embodiments, the present invention provides a method of treating cancer comprising an NLPR3 agonist of the present invention for the treatment of non-inflammatory tumors with low CD8+ T cell infiltration to enhance tumor immunogenicity and stimulate inflammatory responses. For example, the combination may be used to treat a solid tumor based on biopsy results demonstrating low CD8+ T cell infiltration or low expression of genes produced by CD8+ T cells.

Resistance to T-cell checkpoint inhibition refers to cancer progression during therapy or lack of response within 6 months of therapy according to response evaluation criteria for the relevant cancer type, such as RECIST1.1 for most solid tumors.

T cell infiltration refers to the percentage of T cells among all nucleated cells, determined by immunohistochemistry in tumor biopsy specimens. CD8+ T cell infiltration refers to the percentage of CD8+ cells among all nucleated cells, determined by immunohistochemistry in tumor biopsy specimens. In addition to immunohistochemistry for quantifying CD8+ T cells in biopsy specimens, gene expression induced by interferon-γ-producing CD8+ T cells can be measured by mRNA quantification using, for example, next-generation sequencing to obtain information about CD8+ T cell infiltration. Levels of low and high CD8+ T cell infiltration are developed by various groups using immunohistochemical mRNA quantification techniques and take into account the spectrum of CD8+ T cell infiltration in malignancies as well as in specific cancers.

According to any of the methods described herein, a subject may have an infectious disease. According to some examples of any of the methods described herein, the subject has been identified as having an infectious disease or has been diagnosed with an infectious disease. For example, the infectious disease may be caused by a bacterium, virus, fungus, parasite, or mycobacterium.

Non-limiting examples of infectious disease include: Acinobacter infection, actinomycosis, African sleeping sickness, acquired immunodeficiency syndrome, amebiasis, anaplasmosis, anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, atrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidiasis, Baylisascaris infection, BK virus infection, black piedra, Blastocystis hominis infection, blastomycosis, Libyan hemorrhagic fever, botulism, Brazilian hemorrhagic fever, brucellosis, bubonic plague, Burkholderi infection, Buruli ulcer, Calicivirus infection, Campylobacteriosis, Candidiasis, Cat scratch disease, Cellulitis, Chagas disease, Chancroid, Chickenpox, Chikungunya, Chlamydia infection, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Viral upper respiratory tract infection, Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous wandering larva syndrome, Cysticercosis, Cytomegalovirus infection, Dengue fever, Desmodesmus infection, dientamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis, Enterococcus infection, Enterovirus infection, epidemic typhus, erythema infectiosum, exanthema succedaneum, fasciolopsiasis, fascioliasis, fatal familial insomnia, filariasis, Clostridium myonecrosis food poisoning, free-living ameba infection, Fusobacterium infection, gas gangrene, geotrichosis, Gerstmann-Sträussler-Scheinker syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale, group A streptococcal infection, streptococcal infection group B, Haemophilus influenzae infection, hand, foot, and mouth disease, hantavirus pulmonary syndrome, heartland virus disease, Heliobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex infection, histoplasmosis, hookworm, bocavirus infection, human ehrlichiosis, human granulocytic anaplasmosis, human metapneumovirus infection, monoritary human ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, hymenolepiasis, Epstein-Barr infectious mononucleosis, influenza, isosporiasis, Kawasaki disease, keratitis, Kingella kingae infection, kuru, Lassa fever, Legionnaires' disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, Middle East respiratory syndrome, melioidosis, meningitis, meningococcal disease, metagonimiasis, microsporidiosis, molluscum contagiosum, monkeypox, mumps, murine typhus, mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis, variant Creutzfeldt-Jakob disease, nocardiosis, onchocerciasis, paracoccidioidomycosis, paragonimiasis, pasteurellosis, pediculosis capitis, pediculosis pubis, pelvic inflammatory disease, whooping cough, Plague, pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapsing fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsialpox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, severe acute respiratory syndrome, scabies, schistosomiasis, septicemia, shigellosis, herpes zoster, sporotrichosis, staphylococcal food poisoning, staph infection, strongyloidiasis, subacute sclerosing panencephalitis, Syphilis, taeniasis, tetanus, tinea barbae, trichophytosis profunda, tinea corporis, tinea cruris, tinea mani, lichen nigricans, tinea pedis, tinea pedis, tinea versicolor, toxocariasis, trachoma, toxoplasmosis, trichinosis, trichomoniasis, trichuriasis, tuberculosis, tularemia, typhoid fever, Ureaplasma urealyticum infection, valley fever, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white piedra, Yersinia psuedotuberculosis infection, yersiniosis, yellow fever, and zygomycosis.

Methods for diagnosing the presence of an infectious disease in a subject or identifying a subject as having an infectious disease are well known in the art. For example, a healthcare professional (e.g., a physician, physician assistant, or paramedical professional) may diagnose an infectious disease in a subject by observing the subject for one or more symptoms of an infectious disease. Non-limiting examples of symptoms of an infectious disease include fever, diarrhea, fatigue, and muscle pain. Methods for diagnosing the presence of an infectious disease in a mammal or identifying a subject as having an infectious disease may further include performing one or more diagnostic tests (e.g., performing one or more diagnostic tests on a biopsy or blood sample). Diagnostic tests for diagnosing the presence of an infectious disease in a subject or for identifying a subject as having an infectious disease are known in the art.

Combination therapy

This disclosure contemplates both monotherapy and combination therapy regimens. In some embodiments, the methods described herein may further comprise administering one or more additional therapies (e.g., one or more additional therapeutic agents and/or one or more therapeutic regimens) in combination with the administration of the compounds described herein.

According to some embodiments, the methods described herein may further comprise administering one or more additional types of anti-cancer therapies.

One or more additional types of anti-cancer therapies may include, but are not limited to, surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine, Oncophage, Provenge), gene therapy, and combinations thereof. Immunotherapy includes, but is not limited to, adoptive cell therapy, stem cell and/or dendritic cell harvesting, blood transfusions, lavages, and/or other treatment modalities, including, but not limited to, tumor freezing.

In some embodiments, the one or more additional types of anti-cancer therapies is chemotherapy, which may involve the administration of one or more additional chemotherapeutic agents.

In some embodiments, the additional type of anticancer therapy (chemotherapeutic agent) includes an immunomodulatory moiety, such as an immune checkpoint inhibitor. According to some such embodiments, the immune checkpoint inhibitor targets an immune checkpoint receptor selected from CTLA-4, PD-1, PD-L1, PD-1 - PD-L1, PD-1 - PD-L2, T-cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), galectin 9 - TIM3, phosphatidylserine - TIM3, lymphocyte activation gene protein 3 (LAG3), MHC class II - LAG3, 4-1BB-4-1BB ligand, 0X40-0X40 ligand, GITR, GITR ligand-GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA, HVEM-CD160, HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80-PDL-1, PDL2-CD80, CD244, CD48CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2, butyrophilins, including BTNL2, the Siglec family, representatives of the TIGIT and PVR families, KIR, ILT and LIR, NKG2D and NKG2A, MICA and MICB, CD244, CD28, CD86-CD28, CD86-CTLA, CD80-CD28, phosphatidylserine, TIM3, phosphatidylserine - TIM3, SIRPA-CD47, VEGF, neurophilin, CD160, CD30, and CD155 (e.g., CTLA-4, or PD1, or PD-L1) and other immunomodulatory agents such as interleukin-2 (IL-2), indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFe), CD39, CD73, adenosine-C039-C073, and CXCR4-CXCL12. See, e.g., Postow, M. J. Clin. Oncol. 33, 1 (2015).

In some embodiments, the immune checkpoint inhibitor targets an immune checkpoint receptor selected from CTLA-4, PD-1, PD-L1, PD-1-PD-L1, and PD-1-PDL2.

In some embodiments, the immune checkpoint inhibitor is selected from nivolumab (also known as OPDIVO; previously designated 5C4, BMS-936558, MDX-1106, or ONO-4538), pembrolizumab (also known as KEYTRUDA, lambrolizumab, and MK-3475. See WO 2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; AMP-514; see WO 2012/145493), cemiplimab (REGN-2810) (Regeneron; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015/35606 and US Patent No. 2015/0079109), INCSHR1210 (SHR-1210; Jiangsu Hengrui Medicine; see WO 2015/085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (ANB011; Tesaro Biopharmaceutical; see WO 2014/179664), GLS-010 (WBP3055; Wuxi/Harbin Gloria Pharmaceuticals; see Si-Yang Liu et al., J. Hematol. Oncol. 70:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGD013 (Macrogenics); IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, WO 2017/133540); BMS-936559 (previously designated 12A4 or MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013/173223), MPDL3280A (also known as RG7446, atezolizumab, and TECENTRIQ; U.S. Patent No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (IMFINZI; MEDI-4736; AstraZeneca; see WO 2011/066389), avelumab (Pfizer; MSB-0010718C; BAVENCIO; see WO 2013/079174), STI-1014 (Sorrento; see WO 2013/181634), CX-072 (Cytomx; see WO 2016/149201), KN035 (3D Med/Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see e.g. WO 2017/034916), CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)); urelumab, PF-05082566, MEDI6469, TRX518, varlilumab, CP-870893, BMS-986016, MGA271, lirilumab, IPH2201, emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, bavituximab, CC-90002, bevacizumab, MNRP1685A, ipilimumab (YERVOY; U.S. Patent No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO 2016/196237), and tremelimumab (formerly ticilimumab, CP-675,206; AstraZeneca; see, e.g., WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133–39 (2007)).

In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, JS001, BGB-A317, INCSHR1210, TSR-042, GLS-010, STI-1110, MGD013, IBI308, BMS-936559, atezolizumab, durvalumab, avelumab, STI-1014, CX-072, KN035, LY3300054, CK-301, urelumab, PF-05082566, MEDI6469, TRX518, varlilumab, BMS-986016, ipilimumab, AGEN1884 and tremelimumab. In some of these embodiments, the immune checkpoint inhibitor is selected from urelumab, PF-05082566, MEDI6469, TRX518, varlilumab, CP-870893, pembrolizumab (PD1), nivolumab (PD1), atezolizumab (formerly MPDL3280A) (PDL1), MEDI4736 (PD-L1), avelumab (PD-L1), PDR001 (PD1), BMS-986016, MGA271, lirilumab, IPH2201,

- 22 040307 emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, bavituximab, CC-90002, bevacizumab and MNRP1685A.

In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab, and avelumab.

In some embodiments, the immune checkpoint inhibitor is selected from nivolumab and ipilimumab.

According to some embodiments, the additional anti-cancer agent (chemotherapeutic agent) is a STING agonist. For example, a STING agonist may include cyclic dinucleotides such as cAMP, cGMP, and cGAMP, as well as modified cyclic dinucleotides that include one or more of the following modification features: a 2'-O/3'-O linkage, a phosphorothioate linkage, an adenine and/or guanine analog, a 2'-OH modification (e.g., -OCH ₃ or a substitution, e.g., -F or N3). See, for example, WO 2014/189805.

In some embodiments, the additional chemotherapeutic agent is an alkylating agent. Alkylating agents are so named for their ability to alkylate a variety of nucleophilic functional groups under conditions present in cells, including, but not limited to, cancer cells. In another embodiment, the alkylating agent includes, but is not limited to, cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, and/or oxaliplatin. In an embodiment, alkylating agents can function by disrupting cellular function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules, or they can function by modifying cellular DNA. In a further embodiment, the alkylating agent is synthetic, semisynthetic, or a derivative. In some embodiments, the additional chemotherapeutic agent is an antimetabolite. Antimetabolites disguise themselves as purines or pyrimidines, the building blocks of DNA, and generally prevent the incorporation of these substances into DNA during S phase (the cell cycle), halting normal development and division. Antimetabolites can also affect RNA synthesis. In one embodiment, the antimetabolite includes, but is not limited to, azathioprine and/or mercaptopurine. In another embodiment, the antimetabolite is synthetic, semisynthetic, or a derivative.

According to some embodiments, the additional chemotherapeutic agent is a plant alkaloid and/or terpenoid. Such alkaloids are derived from plants and block cell division primarily by preventing microtubule function. In an embodiment, the plant alkaloid and/or terpenoid is a vinca alkaloid, podophyllotoxin, and/or taxane. Vinca alkaloids generally bind to specific sites in tubulin, inhibiting the assembly of tubulin into microtubules, typically during the M phase of the cell cycle. In an embodiment, the vinca alkaloid is obtained from, but is not limited to, the Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea). In an embodiment, the vinca alkaloid includes, but is not limited to, vincristine, vinblastine, vinorelbine, and/or vindesine. In an embodiment, the taxane includes, but is not limited to, taxol, paclitaxel, and/or docetaxel. In a further embodiment, the plant alkaloid or terpenoid is synthetic, semi-synthetic, or a derivative. In a further embodiment, the podophyllotoxin is, but is not limited to, etoposide and/or teniposide. In an embodiment, the taxane is, but is not limited to, docetaxel and/or ortataxel. In an embodiment, the cancer therapeutic agent is a topoisomerase. Topoisomerases are essential enzymes that maintain DNA topology. Inhibition of type I or type II topoisomerase interferes with both transcription and DNA replication by disrupting proper DNA supercoiling. In a further embodiment, the topoisomerase inhibitor is, but is not limited to, a type I topoisomerase inhibitor or a type II topoisomerase inhibitor. In an embodiment, the type I topoisomerase inhibitor is, but is not limited to, camptothecin. According to another embodiment, camptothecin is, but is not limited to, exatecan, irinotecan, lurtothecan, topotecan, BNP 1350, CKD 602, DB 67 (AR67), and/or ST 1481. According to an embodiment, the type II topoisomerase inhibitor is, but is not limited to, epipodophyllotoxin. According to a further embodiment, the epipodophyllotoxin is, but is not limited to, amsacrine, etoposide, etoposide phosphate, and/or teniposide. According to a further embodiment, the topoisomerase is synthetic, semi-synthetic, or a derivative, including naturally occurring substances such as, but not limited to, epipodophyllotoxins naturally occurring in the root of Podophyllum peltatum.

According to some embodiments, the additional chemotherapeutic agent is a stilbenoid. According to a further embodiment, the stilbenoid includes, but is not limited to, resveratrol, piceatannol, pinosylvin, pterostilbene, alpha-viniferin, ampelopsin A, ampelopsin E, diptoindonesin C, diptoindonesin F, epsilon-viniferin, flexuosol A, gnetin H, hemsleyanol D, hopeaphenol, trans-diptoindonesin B, astringin, piceid, and diptoindonesin A. According to a further embodiment, the stilbenoid is synthetic, semi-synthetic, or a derivative.

According to some embodiments, the additional chemotherapeutic agent is a cytotoxic antibiotic. In an embodiment, the cytotoxic antibiotic is, but is not limited to, actinomycin, anthracenedione, anthracycline, thalidomide, dichloroacetic acid, nicotinic acid, 2-deoxyglucose, and/or clofazimine. In an embodiment, actinomycin is, but is not limited to, actinomycin D, bacitracin, colistin (polymyxin E), and/or polymyxin B. In another embodiment, the anthracycline is, but is not limited to, mitoxantrone and/or pixantrone. In a further embodiment, the anthracycline is, but is not limited to, bleomycin, doxorubicin (adriamycin), daunorubicin (daunomycin), epirubicin, idarubicin, mitomycin, plicamycin, and/or valrubicin. According to a further embodiment, the cytotoxic antibiotic is synthetic, semi-synthetic or a derivative.

According to some embodiments, the additional chemotherapeutic agent is selected from endostatin, angiogenin, angiostatin, chemokines, angioarrestin, angiostatin (plasminogen fragment), anti-angiogenic factors derived from basement membrane collagen (tamstatin, canstatin, or arrestin), anti-angiogenic antithrombin III, cartilage-derived inhibitor (CDI) signal transduction inhibitors, complement fragment CD59, fibrinonectin fragment, gro-beta, heparinases, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon-inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease, plasminogen activator inhibitor, platelet factor 4 (PF4), 16-kD fragment of prolactin, proliferin-related protein (PRP), various retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growth factor-beta (TGF-β), vasculostatin, vasostatin (fragment of calreticulin), etc. According to some embodiments, the additional chemotherapeutic agent is selected from abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, bleomycin, N,N-dimethyl-L-valyl-Lvalyl-N-methyl-L-valyl-L-prolyl-1-L-proline-tert-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'-deoxy-8'-norvine-kaleukoblastin, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine, dolastatin, doxorubicin (Adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea taxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, onapristone, paclitaxel, prednemustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate and vinflunine.

According to some embodiments, the additional chemotherapeutic agent is platinum, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, etoposide and teniposide, paclitaxel, docetaxel, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, 5-fluorouracil, leucovorin, methotrexate, gemcitabine, taxane, leucovorin, mitomycin C, tegafur/uracil, idarubicin, fludarabine, mitoxantrone, ifosfamide and doxorubicin. Additional agents include mTOR (mammalian target of rapamycin) inhibitors, including but not limited to rapamycin, everolimus, temsirolimus, and deforolimus.

In other embodiments, the additional chemotherapeutic agent may be selected from those mentioned in U.S. Pat. No. 7,927,613.

According to another embodiment, the methods may further comprise administering one or both of (i) one or more antifungal agents (e.g., selected from the group of bifonazole, butoconazole, clotrimazole, econazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, eposiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravusconazole, terconazole, voriconazole, abafungin, amorolfine, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, 5-fluorocytosine, griseofulvin, haloprogin, tolnaflate, undecylenic acid and balsam of Peru) and (ii) one or more antibiotics (e.g. selected from the group of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalosin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditorene, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, cefto- 24 040307 biprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, torezolid, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, amoxicillin, calvulanate, ampicillin, subbactam, piperacillin, tazobactam, ticarcillin, clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver Sulfadiazine, Sulfadimethoxine, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfonamidochrysoidine, Demeclocycline, Minocycline, Oxytetracycline, Tetracycline, Clophasemine, Dapsone, Dapreomycin, Cycloserine, Ethambutol, Ethionamide, Isiniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic Acid, Metronidazole, mupirocin, platensimicin, quinupristin, dalopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim and teixobactin).

In some embodiments, the second therapeutic agent or regimen is administered to the subject prior to exposure to the chemical compound or prior to administration of the chemical compound (e.g., about 1 hour, or about 6 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 1 week, or about 1 month).

In other embodiments, the second therapeutic agent or regimen is administered to the subject at approximately the same time as exposure to the chemical compound or administration of the chemical compound. For example, the second therapeutic agent or regimen and the chemical compound are provided to the subject simultaneously in the same dosage form. Alternatively, the second therapeutic agent or regimen and the chemical compound are provided to the subject concurrently in separate dosage forms.

According to still other embodiments, the second therapeutic agent or regimen is administered to the subject after exposure to the chemical compound or after administration of the chemical compound (e.g., after about 1 hour, or after about 6 hours, or after about 12 hours, or after about 24 hours, or after about 48 hours, or after about 1 week, or after about 1 month).

Patient selection

In some embodiments, the methods described herein further include the step of identifying a subject (e.g., a patient) in need of such treatment (e.g., by biopsy, endoscopy, or other conventional method known in the art). In some embodiments, the NLRP3 protein can serve as a biomarker for certain types of cancer.

In some embodiments, the chemical compounds, methods, and compositions described herein may be used for certain groups of treatment-resistant patients (e.g., patients resistant to checkpoint inhibitors).

In some embodiments, the compounds of the present invention can be used in therapy. In some embodiments, the present invention relates to the combined preparation of a compound of the present invention or a pharmaceutically acceptable salt thereof and additional therapeutic agent(s) for simultaneous, separate, or sequential use in therapy.

In some embodiments, a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, can be used as a medicament. In some embodiments, the compounds of the invention can be used to manufacture a medicament for the treatment of cancer. In some embodiments, the compounds of the present invention can be used to manufacture a medicament that modulates NLRP3 activity. In some embodiments, modulation involves agonizing NLRP3.

Methods of obtaining

As will be appreciated by one of ordinary skill in the art, methods for synthesizing compounds of the formulas herein will be readily apparent to those skilled in the art. For example, the compounds described herein can be synthesized, for example, using one or more of the methods described herein and/or using the methods described, for example, in U.S. Patent No. 2015/0056224. Transformations in synthetic chemistry and protecting group methodologies (protection and deprotection) used to synthesize the compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and R. G. M. Wuts, Protective Groups in Organic

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Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and their subsequent editions. The starting materials used in preparing the compounds of the invention are known, prepared by known methods, or are commercially available. One skilled in the art will also appreciate that the conditions and reagents described herein may be interchangeable with alternative equivalents recognized in the art. For example, in many reactions, triethylamine can be interchanged with other bases, such as non-nucleophilic bases (e.g., diisopropylamine, 1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine, or tetrabutylphosphazene).

One skilled in the art will appreciate the variety of analytical techniques that can be used to characterize the compounds described herein, including, for example, 1H NMR, heteronuclear NMR, mass spectrometry, liquid chromatography, and infrared spectroscopy. The above list represents a subset of the characterization methods available to those skilled in the art and is not intended to be limiting.

To further illustrate the above, the following non-limiting exemplary synthetic schemes are included. Variations of these examples within the scope of the claims are within the skill of one skilled in the art and are considered to be within the scope of the invention as described and claimed herein. The reader will appreciate that a skilled artisan, having knowledge of this disclosure, and a person skilled in the art will be able to prepare and use this invention without exhaustive examples.

The following abbreviations have the following meanings:

ACN = acetonitrile

Ac ₂ O = acetic anhydride

AcOH = acetic acid

BnOH = benzyl alcohol

CDCl ₃ = chloroform-d

CD ₃ OD = methanol-d

CH ₂ Cl ₂ = dichloromethane

CH ₃ ReO ₃ = methyltrioxorenium conc. = concentrated

Cs2CO3 = cesium carbonate

CuI = copper(I) iodide d = doublet DCM = dichloromethane DCE = 1,2-dichloroethane DIAD = diisopropyl azodicarboxylate DIPEA = N,N-diisopropylethylamine DMF = N,N-dimethylformamide DMSO = dimethyl sulfoxide ES = electrospray ionization _Et2O = diethyl ether EtOAc = ethyl acetate EtOH = ethanol eq = equivalents g = grams h = hours

HCl = hydrogen chloride (usually as a solution)

_H2O = water

H ₂ O ₂ = hydrogen peroxide

HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HCl = hydrogen chloride or hydrochloric acid

HPLC = high-performance liquid chromatography

I2 = iodine

K ₂ CO ₃ = potassium carbonate

K ₂ HPO ₄ = potassium phosphate, dibasic

KI = potassium iodide l = liter LC-MS = gas chromatography-mass spectrometry LiBH ₄ = lithium borohydride m = multiplet

- 26 040307 m/z = mass-to-charge ratio

M = molar m-CPBA = meta-chloroperoxybenzoic acid mg = milligram(s)

MeOH = methanol

MHz = megahertz ml = milliliter(s) mmol = millimole(s) NaH = sodium hydride NaHCO ₃ = sodium hydrogen carbonate Na2CO ₃ = sodium carbonate NaOH = sodium hydroxide Na ₂ SO ₄ = sodium sulfate

NEt ₃ and TEA = trimethylamine

NH ₄ OH and NH ₃ H ₂ O = ammonium hydroxide

NH ₄ HCO ₃ = ammonium hydrogen carbonate nm = nanometer

PBr ₃ = phosphorus tribromide

PdCl ₂ (PPh ₃ ) ₂ = bis(triphenylphosphine)palladium (II) dichloride

Pd(dppf)Cl ₂ = 1,1'-bis(diphenylphosphino)ferrocene

Pd(dppf)Cl2DCM = 1,1'-bis(diphenylphosphino)ferrocene-dichloromethane complex

Pd(OH)2 = palladium hydroxide

PMB = para-methoxybenzyl

POCl3 = phosphorus oxychloride ppm = parts per million

Pt = platinum

Pt/C = platinum on carbon supports

RP = reversed-phase s = singlet t = triplet

T3P = 1-propanephosphonic anhydride t-BuOK = potassium tert-butoxide

TFA = trifluoroacetic acid

TLC = thin layer chromatography

TsCl and TosCl = para-toluenesulfonyl chloride °C = degrees Celsius μm = micrometer μmol = micromole(s)

General methods for compounds of the invention

The compounds of the present invention can be prepared by a number of methods well known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereof, as understood by those skilled in the art. Preferred methods include, but are not limited to, the methods described below. The compounds of the present invention can be prepared using the reactions and procedures described in this section. The reactions are carried out in solvents appropriate to the reagents and materials used and suitable for the transformations being carried out. Furthermore, in describing the synthetic methods described below, it should be understood that all suggested reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, experimental duration, and assay procedures, are selected as standard conditions for this reaction, which should be readily recognizable to one skilled in the art. It is clear to a person skilled in organic synthesis that the functionality present in the various parts of a molecule must be compatible with the proposed reagents and reactions.

Such limitations on the substituents compatible with the reaction conditions will be apparent to those skilled in the art, necessitating the use of alternative procedures. This may sometimes require a decision to change the order of synthetic steps or to select one specific process scheme over another to obtain the target compound of the invention. It should also be recognized that another important consideration when planning any synthetic route in this area is the judicious choice of the protecting group used to protect the reactive functional groups present in the compounds described in this invention. An authoritative source describing many alternatives for the trained practitioner is Greene and Wuts (Protective Groups In Organic Synthesis, Third Edition, Wiley and Sons, 1999).

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Compounds of formula (I) can be prepared by the methods shown in the following schemes. As shown therein, the final product is a compound having the same structural formula as formula (I). It should be understood that any compound of formula (I) can be prepared using the schemes by appropriately selecting reactants with appropriate substitution. Solvents, temperatures, pressures, and other reaction conditions can be readily selected by one skilled in the art. The starting materials are commercially available or can be readily prepared by one skilled in the art. The components of the compounds are as defined herein or elsewhere in this description. The synthesis of compounds of formula (I) can be accomplished using the methods shown in Schemes 1 and 2.

Scheme 1

Step 1: The first step of Scheme 1 begins with a suitably functionalized quinolinol (i). If desired, the R ⁷ , R ^{8 ,} and R ¹⁰ groups can be the R ³ , R ^{4 ,} and W' groups found in the final product. Alternatively, one or more of these groups can be groups that can be modified at a later stage of the synthesis, such as bromine. This quinolinol can be purchased commercially or can be synthesized by methods known to one skilled in the art. In Step 1, the alcohol group of compound (i) can be converted to a halogen group or an ester sulfonate, such as chlorine, bromine, or triflate. If the target group Z is chlorine, this conversion can be accomplished by treating compound (i) with a reagent such as phosphoryl chloride in a solvent such as toluene. Alternatively, if the target group Z is bromine, this transformation can be accomplished by treating compound (i) with a reagent such as phosphorus tribromide in a solvent such as DMF. Alternatively, if the target group Z is a triflate, this transformation can be accomplished by treating compound (i) with a reagent such as trifluoromethanesulfonyl chloride, a reagent such as 4-dimethylaminopyridine, and a base such as Hunig's base in a solvent such as dichloromethane.

Step 2: In step 2 of Scheme 1, compound (ii) is converted to the N-oxide (iii) by treatment with an appropriate oxidizing agent such as meta-chloroperoxybenzoic acid in a solvent such as DCM.

Step 3: In step 3 of Scheme 1, compound (iii) is converted to amine (iv) by treatment with an appropriate activating reagent such as tosyl chloride and an ammonia source such as ammonium chloride and triethylamine in an appropriate solvent such as DCM.

Step 4: In Step 4 of Scheme 1, the halogen Z of compound (iv) is converted to the R ⁹ group of compound (v). The R ⁹ group may be a W group required in the final compound; alternatively, it may be a group that can be converted to a W group at a later stage of the synthesis. One skilled in the art will appreciate that the method for performing this transformation will depend on the nature of the R ⁹ and Z groups. For example, if Z is chlorine and the desired R ⁹ group is an amine, this transformation can be accomplished by heating compound (iv) to a suitable temperature, such as 120°C, with a suitable amine and a base, such as Hunig's base, in a solvent such as DMSO. Alternatively, if Z is chlorine and the target group R ⁹ is an ether, this transformation can be accomplished by heating compound (iv) to a suitable temperature, such as 100°C, with a suitable alcohol and a base, such as potassium tert-butoxide, in a solvent, such as NMP. Alternatively, if Z is bromine and the target group R ⁹ is an alkyne, this transformation can be accomplished by heating compound (iv) to a suitable temperature, such as 70°C, with a suitable alkyne, copper(I) iodide, a suitable base, such as Hunig's base, and a suitable palladium source, such as tetrakis(triphenylphosphine)palladium(0), in a suitable solvent, such as THF. Alternatively, if Z is triflate and the target group R ⁹ is an optionally substituted alkyl group, this step can be accomplished by treating compound (iv) with a suitable alkyl boronic acid or ester, a catalyst such as PdCl2(dppf)-DCM complex, and a base such as cesium carbonate in a solvent such as dioxane.

Steps 5 through 8 of Scheme 1 consist of a series of optional functional group manipulations to convert substituents ^R7 , ^R8 , ^R9, and ^R10 in intermediate (v) to substituents ^R3 , ^R4 , W, and W', which are the desired substituents in the final compound (ix). One skilled in the art will appreciate that some or all of these steps may be optional depending on the groups present in compounds (v) and (ix). One skilled in the art will also appreciate that for some substrates, these steps may be performed in an alternative order.

Step 5: Step 5 of Scheme 1 is an optional step or series of steps for converting the R ⁷ group in intermediate (v) to the R ³ group found in molecule (vi). For example, if R ⁷ is bromine and the desired R ³ group is an aromatic or heteroaromatic group, this transformation can be accomplished by reacting intermediate (v) with an optionally protected aromatic or heteroaromatic boronic acid or boronic ester, a catalyst such as PdCl2(dppf)-DCM complex, and a base such as tripotassium phosphate in a mixture of solvents such as dioxane and water. If the installed group contains a protecting group, an additional optional step can be carried out to remove that protecting group under appropriate conditions, if desired. For example, if the installed group was a pyrazole with a tetrahydropyran protecting group, the tetrahydropyran could be removed by reaction with an acid such as trifluoroacetic acid in a solvent such as dichloromethane. Alternatively, if ^R7 is bromine and the target group ^R3 is an aromatic or heteroaromatic group, this transformation could be accomplished by reacting intermediate (v) first with a compound such as PdCl2(dppf)-DCM bis(pinacolato)diborone complex, a reagent such as potassium acetate, and a catalyst such as PdCl2(dppf)-DCM complex in a solvent such as dioxane, followed by reacting the resulting boronic ester with a suitable aryl or heteroaryl halide, a base such as sodium carbonate, and a catalyst such as tetrakis(triphenylphosphine)palladium(0) in an appropriate mixture of solvents such as dioxane and water. Alternatively, if R ⁷ is bromine and the target group R ³ is a heterocycle linked through a nitrogen atom, this step can be accomplished by reacting intermediate (v) with the appropriate heterocycle in the presence of a copper source such as copper(I) iodide, a base such as sodium carbonate, and a ligand such as N,N'dimethylethane-1,2-diamine in a suitable solvent such as DMSO.

Step 6: Step 6 of Scheme 1 is an optional step or series of steps for converting the R ⁹ group in intermediate (vi) to the W group found in molecule (vii). For example, if the R ⁹ group contains a Boc-protected amine and the target W group contains an amide, this transformation can be accomplished by first removing the Boc group with a suitable acid-solvent combination, such as hydrochloric acid and dioxane, and then forming the target amide by reaction with a suitable carboxylic acid, a coupling agent such as T3P, and a base such as triethylamine in a solvent such as DMF. Alternatively, if the R ⁹ group contains an unsaturated group such as an alkyne and the target W group is fully saturated, this transformation can be accomplished by reaction with hydrogen and a suitable catalyst, such as palladium on a carbon support.

Step 7: Step 7 of Scheme 1 is an optional step or series of steps to convert the R ⁸ group in intermediate (vii) to the R ⁴ group found in molecule (viii).

Step 8: Step 8 of Scheme 1 is an optional step or series of steps for converting the R ¹⁰ group in intermediate (vii) to the W' group located in molecule (ix). For example, if the R ¹⁰ group contains an alcohol protected by a benzyl ether and the target W' group is the corresponding alcohol, this conversion can be accomplished by reaction with a suitable acid, such as hydrochloric acid. If the R ¹⁰ group contains an alcohol and the target W' group contains an amine at the same location, this conversion can be accomplished by first reacting intermediate (vii) with reagents such as thionyl chloride in a solvent such as dichloromethane, then by reacting the resulting chloride with an amine such as ethylamine, sodium iodide, and a base such as potassium carbonate in a solvent such as acetonitrile.

One skilled in the art will appreciate that some of these steps may be performed in an alternative order, depending on the target groups in the final molecule (ix). For example, for some molecules, the transformation of the R ⁷ group to R ³ described in step 5 may be performed before the transformation of the Z group to the R ⁹ group described in step 4.

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Scheme 2

XIV XV

As an alternative to the route described in Scheme 1, some compounds of formula (I) can be prepared by the route described in Scheme 2.

Step 1: Step 1 of Scheme 2 begins with an appropriately functionalized aminobenzaldehyde (x) and an appropriately functionalized nitrile (xi). If desired, the ^R7 , ^R8, and ^R10 groups can be the ^R3 , ^R4 , and W' groups found in the final product. Alternatively, one or more of these groups can be groups that can be modified at a later stage of the synthesis, such as bromine. These compounds can be purchased commercially or can be synthesized by methods known to one skilled in the art. Step 1 of Scheme X involves the reaction of (x) and (xi) in the presence of a suitable combination of base and solvent, such as potassium tert-butoxide in DMSO or sodium hydroxide in ethanol, to form aminoquinoline (xii). Steps 2-4 of Scheme 2 consist of a series of optional functional group manipulations to transform substituents ^R7 , ^R8, and ^R10 in intermediate (xii) to substituents ^R3 , ^R4 , and W' required in the final compound (xv). One skilled in the art will appreciate that some or all of these steps may be optional depending on the groups present in compounds (v) and (x). One skilled in the art will also appreciate that for some substrates, these steps may be performed in an alternative order.

Step 2: Step 2 of Scheme 2 is an optional step or series of steps for converting the R ⁷ group in intermediate (xii) to the R ³ group found in molecule (xiii). For example, if R ⁷ is bromine and the target R ³ group is an aromatic or heteroaromatic group, this conversion can be accomplished by reacting intermediate (xii) with an optionally protected aromatic or heteroaromatic boronic acid or boronic ester, a catalyst such as PdCl ₂ (dppf)-DCM complex, and a base such as tripotassium phosphate in a mixture of solvents such as dioxane and water. If the group being installed contains a protecting group, an additional optional step can be performed to remove that protecting group under appropriate conditions, if desired. For example, if the group installed was a pyrazole with a tetrahydropyran protecting group, the tetrahydropyran could be removed by reaction with an acid such as trifluoroacetic acid in a solvent such as dichloromethane.

Step 3: Step 3 of Scheme 2 is an optional step or series of steps to convert the R ⁸ group in intermediate (xiii) to the R ⁴ group found in molecule (xiv).

Step 4: Step 4 of Scheme 2 is an optional step or series of steps for converting the R ¹⁰ group in intermediate (xiv) to the W' group found in molecule (xv). For example, if the R ¹⁰ group contains an alcohol protected by a benzyl ether and the target W' group is the corresponding alcohol, this conversion can be accomplished by reaction with a suitable acid, such as hydrochloric acid. If the R ¹⁰ group contains an alcohol and the target W' group is an amine, this conversion can be accomplished by first reacting intermediate (xiv) with reagents such as thionyl chloride in a solvent such as dichloromethane, and then reacting the resulting chloride with an amine such as ethylamine, sodium iodide, and a base such as potassium carbonate in a solvent such as acetonitrile.

Chemical shifts are reported in parts per million (ppm) downfield from internal tetramethylsilane (TMS) or from the TMS position determined by the deuterated NMR solvent. Apparent multiplicities are reported as singlet-s, doublet-d, triplet-t, quartet-q, or multiplet-m. Peaks that exhibit broadening are further designated as br. Integrations are approximate. It should be noted that integration intensities, peak shapes, chemical shifts, and coupling constants may depend on solvent, concentration, temperature, pH, and other factors. Furthermore, peaks that overlap or change with water or solvent peaks in the NMR spectrum may not provide reliable integration intensities. In some cases, NMR spectra are acquired using water peak suppression, which may result in overlapping peaks not being visible or having altered shapes and/or integration.

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Example 1. Synthesis of compound 101

Step 1: Synthesis of 7-(benzyloxy)heptanenitrile

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of BnOH (1.6 g, 14.80 mmol, 1.00 equiv.) in N,N-dimethylformamide (60 mL). This was followed by the addition of sodium hydride (1.02 g, 29.75 mmol, 2.00 equiv.) in several portions at 0 °C. The resulting solution was stirred for 30 min at 0 °C in an ice/water bath. To this solution, 7-bromoheptanenitrile (2.8 g, 14.73 mmol, 1.00 equiv.) was added in portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 16 h at room temperature. The reaction was then quenched by the addition of 500 mL of water. The resulting solution was extracted with ethyl acetate (2 x 500 mL), and the combined organic layers were concentrated under vacuum. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (50:1). 1.87 g (58%) of 7-(benzyloxy)heptanenitrile was obtained as a yellow solid.

Step 2: Synthesis of 3-[5-(benzyloxy)pentyl]-7-bromoquinolin-2-amine

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 7-(benzyloxy)heptanenitrile (3.05 g, 14.04 mmol, 1.00 equiv.) in N,N-dimethylformamide (50 mL). This was followed by the addition of t-BuOK (4.73 g, 42.15 mmol, 3.00 equiv.) in several portions at 0 °C. The resulting solution was stirred for 15 min at 0 °C in an ice/water bath. To this solution was added 2-amino-4-bromobenzaldehyde (2.8 g, 14.00 mmol, 1.00 equiv.) in several portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. The reaction was then quenched by adding 200 mL of water. The resulting solution was extracted with ethyl acetate (2 x 300 mL) and the combined organic layers were concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (40:1). This gave 1.87 g (33%) of 3-[5-(benzyloxy)pentyl]-7-bromoquinolin-2-amine as a yellow solid. LC-MS: (ES, m/z): [M+H]+=399.1.

Step 3: Synthesis of 3-[5-(benzyloxy)pentyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (compound 106)

A solution of 3-[5-(benzyloxy)pentyl]-7-bromoquinolin-2-amine (850 mg, 2.13 mmol, 1.00 equiv) in dioxane/H2O (10:1) (15 mL) was placed in a sealed 30 mL tube, purged _and maintained under an inert atmosphere of nitrogen. To the solution were added 3-(tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazole (828.6 mg, 4.27 mmol, 2.00 equiv), _Cs2CO3 (2.78 g, 8.53 mmol, 4.00 equiv), and _PdCl2 (dppf)DCM adduct (349 mg, 0.43 mmol, 0.20 equiv). The resulting solution was stirred for 16 h at 90°C in an oil bath. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column with dichloromethane/methanol (30:1). This gave 559 mg (68%) of 3-[5-(benzyloxy)pentyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine as a yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 387.2.

Step 4: Synthesis of 5-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]pentan-1-ol (compound 101)

A 50 mL round-bottomed flask was charged with a solution of 3-[5-(benzyloxy)pentyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (350 mg, 0.91 mmol, 1.00 equiv.) in concentrated hydrogen chloride (8 mL). The resulting solution was stirred for 1 h at 50 °C in an oil bath. The resulting mixture was concentrated under vacuum. The crude product was purified by preparative HPLC under the following conditions (HPLC-10): column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; mobile phase: water (10 mmol/L NH ₄ HCO ₃ ) and MeCN (27.0% MeCN to 60.0% over 8 min); detector: UV 254/210 nm. As a result, 59.8 mg (22%) of 5-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3yl]pentan-1-ol was obtained as a white solid. LC-MS: (ES, m/z): [M+H] ⁺ = 297.2. H NMR: 1H NMR (300 MHz, CD3OD-d ₄ ) δ 7.85 (br s, 1H), 7.73 (s, 1H), 7.65-7.62 (m, 3H), 6.71 (d, J=2.1 Hz, 1H), 3.55 (t, J= 6.3 Hz, 2H), 2.63 (t, J=7.5 Hz, 2H), 1.78-1.67 (m, 2H), 1.64-1.53 (m, 2H), 1.51-1.46 (m, 2H).

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Example 2: Preparation of 3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]propan-1-ol (compound 103)

Step 1: Synthesis of 5-(benzyloxy)pentanenitrile

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of phenylmethanol (3.02 g, 27.93 mmol, 1.00 equiv.) in N,N-dimethylformamide (50 mL). This was followed by the addition of sodium hydride (1.92 g, 56.00 mmol, 2.00 equiv., 70%) in several portions at 0 °C. The resulting solution was stirred for 30 min at 0 °C in an ice/water bath. To the solution was added 5-bromopentanenitrile (4.5 g, 27.77 mmol, 1.00 equiv.) in portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. The reaction was then quenched by the addition of 200 mL of water. The resulting solution was extracted with ethyl acetate (2 x 300 mL), and the combined organic layers were concentrated under vacuum. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (50:1). This yielded 700 mg (13%) of 5-(benzyloxy)pentanenitrile as a yellow oil.

Step 2: Synthesis of 3-[3-(benzyloxy)propyl]-7-bromoquinolin-2-amine

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 5-(benzyloxy)pentanenitrile (2.05 g, 10.83 mmol, 1.00 equiv.) in N,N-dimethylformamide (50 mL). This was followed by the addition of t-BuOK (3.641 g, 32.45 mmol, 3.00 equiv.) in several portions at 0 °C. The resulting solution was stirred for 15 min at 0 °C in an ice/water bath. To the solution was added 2-amino-4-bromobenzaldehyde (2.15 g, 10.75 mmol, 1.00 equiv.) in several portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. The reaction was then quenched by adding 200 mL of water. The resulting solution was extracted with ethyl acetate (2 x 300 mL) and the combined organic layers were concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (30:1). This gave 1.3 g (32%) of 3-[3(benzyloxy)propyl]-7-bromoquinolin-2-amine as a yellow oil. LC-MS: (ES, m/z): [M+H]+=371.3.

Step 3: Synthesis of 3-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (compound 104)

A solution of 3-[3-(benzyloxy)propyl]-7-bromoquinolin-2-amine (360 mg, 0.97 mmol, 1.00 equiv) in dioxane/H ₂ O (10:1) (8 mL) was placed in a sealed 15 mL tube, purged and maintained under an inert atmosphere of nitrogen. To the solution were added 3-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (377.6 mg, 1.95 mmol, 2.00 equiv), Cs ₂ CO ₃ (1.26 g, 3.87 mmol, 4.00 equiv), and the adduct PdCl ₂ (dppf) DCM (159 mg, 0.19 mmol, 0.20 equiv). The resulting solution was stirred for 16 h at 90°C in an oil bath. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane/methanol (25:1). This gave 249 mg (72%) of 3-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine as a yellow solid. LC-MS: (ES, m/z): [M+H]+=359.2.

Step 4. Synthesis of 3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]propan-1-ol (compound 103)

A 50 mL round-bottomed flask was charged with a solution of 3-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (400 mg, 1.12 mmol, 1.00 equiv.) in concentrated hydrogen chloride (10 mL). The resulting solution was stirred for 40 min at 50°C in an oil bath. The resulting mixture was concentrated under vacuum. The crude product was purified by preparative HPLC under the following conditions (HPLC-10): column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; mobile phase: water (10 mmol/L NH4HCO3) and MeCN (6.0% MeCN to 55.0% over 8 min); detector: UV 254/210 nm. This yielded 41.4 mg (14%) of 3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3yl]propan-1-ol as a yellow solid. LC-MS: (ES, m/z): [M+H]+=269.2. 1H NMR (300 MHz, DMSO-d ₆ ) δ 12.86 (s, 1H), 7.82-7.79 (m, 2H), 7.66-7.59 (m, 3H), 6.74 (s, 1H), 6.21 (br s, 2H), 4.52 (t, J= 5.1 Hz, 1H), 3.49-3.43 (m, 2H), 2.57 (t, J= 7.5 Hz, 2H), 1.77-1.72 (m, 2H).

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Example 3: Preparation of 4-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]butan-1-ol (compound 102)

Step 1: Synthesis of 6-(benzyloxy)hexanenitrile

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of BnOH (3.22 g, 29.78 mmol, 1.00 equiv.) in N,N-dimethylformamide (80 mL). This was followed by the addition of sodium hydride (2.05 g, 59.79 mmol, 2.00 equiv.) in several portions at 0 °C. The resulting solution was stirred for 30 min at 0 °C in an ice/water bath. To this solution was added 6-bromohexanenitrile (5.2 g, 29.54 mmol, 1.00 equiv.) in portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. The reaction was then quenched by the addition of 500 mL of water. The resulting solution was extracted with ethyl acetate (3 x 500 mL), and the combined organic layers were concentrated under vacuum. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (60:1). This yielded 1.06 g (18%) of 6-(benzyloxy)hexanenitrile as a yellow oil.

Step 2. Synthesis of 3-[4-(benzyloxy)butyl]-7-bromoquinolin-2-amine

A 250 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 6-(benzyloxy)hexanenitrile (2.3 g, 11.31 mmol, 1.00 equiv.) in N,N-dimethylformamide (50 mL). This was followed by the addition of t-BuOK (3.81 g, 33.95 mmol, 3.00 equiv.) in several portions at 0 °C. The resulting solution was stirred for 15 min at 0 °C in an ice/water bath. To this solution was added 2-amino-4-bromobenzaldehyde (2.25 g, 11.25 mmol, 1.00 equiv.) in several portions at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. The reaction was then quenched by adding 200 mL of water. The resulting solution was extracted with ethyl acetate (2 x 300 mL) and the combined organic layers were concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (40:1). This gave 1.8 g (41%) of 3-[4-(benzyloxy)butyl]-7-bromoquinolin-2-amine as a yellow oil. LC-MS: (ES, m/z): [M+H]+=385.1.

Step 3: Synthesis of 3-[4-(benzyloxy)butyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (compound 105)

A solution of 3-[4-(benzyloxy)butyl]-7-bromoquinolin-2-amine (1.37 g, 3.56 mmol, 1.00 equiv) in dioxane/H ₂ O (10:1) (15 mL) was placed in a sealed 30 mL tube, purged and maintained under an inert atmosphere of nitrogen. To the solution were added 3-(tetramethyl-1,3,2-dioxaborolan-2-yl)1H-pyrazole (1.38 g, 7.11 mmol, 2.00 equiv), Cs ₂ CO ₃ (4.64 g, 14.24 mmol, 4.00 equiv), and the adduct PdCl ₂ (dppf)Cl ₂ DCM (583 mg, 0.71 mmol, 0.20 equiv). The resulting solution was stirred for 16 h at 90°C in an oil bath. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane/methanol (35:1). This gave 1 g (76%) of 3-[4-(benzyloxy)butyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine as a yellow solid. LCMS: (ES, m/z): [M+H]+=373.2.

Step 4: Synthesis of 4-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]butan-1-ol (compound 102)

A 100 mL round-bottomed flask was charged with a solution of 3-[4-(benzyloxy)butyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (400 mg, 1.07 mmol, 1.00 equiv.) in concentrated hydrogen chloride (10 mL). The resulting solution was stirred for 40 min at 50 °C in an oil bath. The pH of the solution was adjusted to 8 with NH4OH. The resulting mixture was concentrated in vacuo. The crude product was purified by preparative HPLC under the following conditions (HPLC10): column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; mobile phase: water (10 mmol/L NH4HCO3) and MeCN (18.0% MeCN to 40.0% over 9 min); Detector: UV 254/210 nm. The result was 64.7 mg (21%) of 4-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]butan-1-ol as a white solid. LC-MS: (ES, m/z): [M+H]+=283.2. 1H NMR (300 MHz, DMSO-d6) δ 12.89 (br s, 1H), 7.82 (s, 1H), 7.687.58 (m, 4H), 6.74 (s, 1H), 6.24 (s, 2H), 4.38 (t, J= 5.1 Hz, 1H), 3.45-3.50 (m, 2H), 2.54 (t, J= 7.5 Hz, 2H), 1.671.45 (m, 4H).

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Example 4: Preparation of 3-[4-amino-7-(1H-pyrazol-3-yl)-[1,3]oxazolo[4,5-c]quinolin-2-yl]propan-1-ol (compound 107)

Step 1: Synthesis of 7-(1H-pyrazol-3-yl)quinolin-4-ol

A 500 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 7-bromoquinolin-4-ol (11.2 g, 49.99 mmol, 1.00 equiv.) in dioxane (250 mL) and water (50 mL). Sodium carbonate (15.9 g, 150.01 mmol, 3.00 equiv.), 3-(tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazole (19.4 g, 99.98 mmol, 2.00 equiv.), and Pd( _PPh3 ) ₄ (5 g, 4.33 mmol, 0.10 equiv.) were added to the solution. The resulting solution was stirred for 16 h at 90°C in an oil bath. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column with DCM/MeOH (0-10%). This gave 8.44 g (76%) of 7-(1H-pyrazol-3-yl)quinolin-4-ol as a light yellow solid. LC-MS: (ES, m/z): [M+H]+=212.2.

Step 2: Synthesis of 4-bromo-7-(1H-pyrazol-3-yl)quinoline

A 500 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 7-(1H-pyrazol-3-yl)quinolin-4-ol (8.44 g, 39.96 mmol, 1.00 equiv.) in N,N-dimethylformamide (200 mL). This was followed by the dropwise addition of PBr ₃ (21.6 g, 79.80 mmol, 2.00 equiv.) with stirring at 0 °C. The resulting solution was stirred for 30 min at room temperature. The reaction was then quenched by the addition of ice water. The pH of the solution was adjusted to 10 with sodium hydroxide. The resulting solution was extracted with ethyl acetate (3 x 500 mL) and the organic layers were combined. The solution was washed with brine (3 x 200 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (0-80%). This gave 5.3 g (48%) of 4-bromo7-(1H-pyrazol-3-yl)quinoline as a light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 275.1.

Step 3: Synthesis of 4-[3-(benzyloxy)prop-1-yn-1-yl]-7-(1H-pyrazol-3-yl)quinolone

A solution of 4-bromo-7-(1H-pyrazol-3-yl)quinoline (548 mg, 2.00 mmol, 1.00 equiv) in tetrahydrofuran (20 mL) was placed in a 30 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen. Hunig's base (1.29 g, 10.00 mmol, 5.00 equiv), [(prop-2-yn-1-yloxy)methyl]benzene (584 mg, 3.99 mmol, 2.00 equiv), CuI (7.4 mg, 0.04 mmol, 0.20 equiv), and Pd( _PPh3 ) ₄ (231 mg, 0.20 mmol, 0.10 equiv) were added to the solution. The resulting solution was stirred for 16 h at 70°C in an oil bath. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (0-70%). This gave 500 mg (74%) of 4-[3-(benzyloxy)prop-1-yn-1-yl]-7-(1H-pyrazol-3-yl)quinoline as a light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 340.4.

Step 4: Synthesis of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinoline

A 100 mL round-bottomed flask was charged with a solution of 4-[3-(benzyloxy)prop-1-yn-1-yl]-7-(1H-pyrazol3-yl)quinoline (420 mg, 1.24 mmol, 1.00 equiv.) in methanol (20 mL). Palladium on carbon was added to the solution.

- 34 040307 hydrogen support (210 mg). The resulting solution was degassed and backfilled with hydrogen. The solution was then stirred for 16 h at room temperature. The solids were filtered off. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (0-80%). This gave 343 mg (81%) of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinoline as a light yellow solid. LC-MS: (ES, m/z): [M+H]+=344.4.

Step 5: Synthesis of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate

A 50 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinoline (343 mg, 1.00 mmol, 1.00 equiv.) in dichloromethane (10 mL). To the solution was added m-CPBA (344 mg, 1.99 mmol, 2.00 equiv.). The resulting solution was stirred for 5 h at room temperature. The reaction was then quenched by adding 10 mL _of aqueous _Na2S2O4 . The resulting solution was extracted with DCM:MeOH (10: ₁ , 3x10 mL) and the organic layers were combined. The solution was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column with dichloromethane/methanol (0-5%) to afford 240 mg (67%) of 4-[3-(benzyloxy)propyl]-7(1H-pyrazol-3-yl)quinolin-1-ium-1-olate as a light yellow solid. LC-MS: (ES, m/z): [M+H]+=360.4.

Step 6: Synthesis of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (compound 108)

A 50 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate (240 mg, 0.67 mmol, 1.00 equiv.) in dichloromethane (6 mL). To the solution were added NH4OH (3 mL) and TsCl (176 mg, 2.00 equiv.). The resulting solution was stirred for 16 h at room temperature. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column with dichloromethane/methanol (0-10%). As a result, 220 mg (92%) of 4-[3-(benzyloxy)propyl]7-(1H-pyrazol-3-yl)quinolin-2-amine was obtained as a light yellow solid. LC-MS: (ES, m/z): [M+H]+=359.4.

Step 7: Synthesis of 3-[4-amino-7-(1H-pyrazol-3-yl)-[1,3]oxazolo[4,5-c]quinolin-2-yl]propan-1-ol (compound 107)

A 25 mL round-bottomed flask was charged with a solution of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (180 mg, 0.50 mmol, 1.00 equiv.) in concentrated hydrogen chloride (5 mL). The resulting solution was stirred for 5 h at room temperature. The resulting mixture was concentrated under vacuum. The pH of the solution was adjusted to 10 with NH4OH. The resulting solution was extracted with DCM:MeOH (10:1, 5x10 mL) and the organic layers were combined. The solution was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by preparative HPLC under the following conditions (HPLC-10): column XBridge ShieldRP18 OBD, 19x250 mm, 10 μm; Mobile phase: water (10 mmol/L NH4HO3) and MeCN (5.0% MeCN to 50.0% over 7 min); detector: UV 254/210 nm. The reaction yielded 45 mg (29%) of 3-[4-amino-7-(1H-pyrazol-3-yl)-[1,3]oxazolo[4,5-c]quinolin-2-yl]propan-1-ol as a white solid. LC-MS: (ES, m/z): [M+H]+=269.3. H-NMR: (DMSO-d6, 300 MHz, ppm): 5 13.38-12.91 (m, 1H), 7.847.81 (m, 2H), 7.67-7.64 (m, 2H), 6.78 (s, 1H), 6.58 (s, 1H), 6.30 (br s, 2H), 4.60 (t, J=5.4 Hz, 2H), 3.54-3.49 (m, 2H), 2.91 (t, J=7.8 Hz, 2H), 1.84-1.75 (m, 2H).

Example 5: Preparation of N-[3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]propyl]-N-ethylacetamide (compound 112)

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Step 1: Synthesis of 3-(3-chloropropyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine

A 100 mL round-bottomed flask was charged with a solution of 3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3yl]propan-1-ol (380 mg, 1.42 mmol, 1.00 equiv.) in dichloromethane (30 mL). SO ₂ Cl ₂ (15 mL) was added to the solution. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. This gave 574 mg (crude) of 3-(3-chloropropyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine as a yellow solid. LC-MS: (ES, m/z): [M+H]+=287.1.

Step 2: Synthesis of 3-[3-(ethylamino)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (compound 110)

A 100 mL round-bottomed flask was charged with a solution of 3-(3-chloropropyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine (574 mg, 2.00 mmol, 1.00 equiv.) in MeCN (20 mL). To the solution were added ethanamine (453 mg, 6.83 mmol, 5.00 equiv., 68%), potassium carbonate (554 mg, 4.01 mmol, 2.00 equiv.), and NaI (301 mg, 2.01 mmol, 1.00 equiv.). The resulting solution was stirred for 2 days at 70 °C in an oil bath. The resulting mixture was concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane/methanol (40:1). As a result, 754 mg (crude substance) of 3-[3(ethylamino)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine was obtained as a yellow solid. LC-MS: (ES, m/z): [M+H]+=296.2.

Step 3: Synthesis of N-[3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]propyl]-N-ethylacetamide (compound 112)

A 100 mL round-bottomed flask was charged with a solution of 3-[3-(ethylamino)propyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine (400 mg, 1.35 mmol, 1.00 equiv.) and triethylamine (411 mg, 4.06 mmol, 3.00 equiv.) in dichloromethane (20 mL). Acetic anhydride (211 mg, 2.07 mmol, 1.50 equiv.) was added to the solution. The resulting solution was stirred for 3 h at room temperature. The resulting mixture was concentrated in vacuo. The crude product was purified by preparative HPLC using the following conditions: column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; Mobile phase: water (10 mmol/L NH ₄ HCO ₃ ) and MeCN (17.0% MeCN to 55.0% over 8 min); detector: UV 254/210 nm. This gave 30.9 mg (7%) of N-[3-[2-amino-7-(1H-pyrazol-3-yl)quinolin-3-yl]propyl]N-ethylacetamide as a white solid. H-NMR: ^1H NMR (300 MHz, DMSO-d ₆ ) δ 13.38-12.90 (m, 1H), 7.87-7.63 (m, 5H), 6.79 (s, 1H), 6.45-6.28 (m, 2H), 2.60-2.55 (m, 6H), 2.00 (d, J= 5.1 Hz, 3H), 1.871.82 (m, 2H), 1.13 (t, J= 6.9 Hz, 2H), 1.03 (t, J=6.9 Hz, 2H). LC-MS: (ES, m/z): [M+H]+=338.2.

Example II-1. Synthesis of 3-substituted quinolines

PdCI ₂ (dppf)»DCM

K3PO4, dioxane/water

KOtBu, DMSO, then) DCM, TFA

Compound 1113

Step 1. Preparation of 2-amino-4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)benzaldehyde

2-Amino-4-bromobenzaldehyde (250 mg, 1.250 mmol), 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5 _- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (521 mg, 1.875 mmol), and _PdCl2 (dppf) _-CH2Cl2 adduct (102 mg, 0.125 mmol) were placed in a vial. The vial was placed under vacuum and backfilled with nitrogen. Tripotassium phosphate (2 M aqueous) (1875 µL, 3.75 mmol) and dioxane (6249 µL) were added, nitrogen was bubbled through the solution, the vial was capped, and the reaction mixture was heated to 100°C overnight. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were dried over sodium sulfate and concentrated. The residue was purified using ISCO (24 g column; hexane/EtOAc; gradient from 0 to 50%) to give 2-amino-4-(1-(tetrahydro-2H-pyran-2-yl)-1Hpyrazol-5-yl)benzaldehyde (207 mg, 0.763 mmol, 61.0% yield).

Step 2. Preparation of 3-(methoxymethyl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 113)

To a solution of 3-methoxypropanenitrile (12.55 mg, 0.147 mmol) and 2-amino-4-(1-(tetrahydro-2H-pyran2-yl)-1H-pyrazol-5-yl)benzaldehyde (20 mg, 0.074 mmol) in DMSO (400 µL) was added KOtBu (16.54 mg, 0.147 mmol). The reaction mixture was heated to 60 °C. After 5 h, the reaction was complete according to LC-MS. The reaction mixture was cooled, diluted with water and extracted twice with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.4 mL DCM and 0.4 mL TFA. After 2 h, the reaction was complete according to LC-MS. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 7% B, 7-47% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified using

- 36 040307 using preparative LC-MS under the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 3-(methoxymethyl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (3.2 mg, 16%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.33 (s, 1H), 8.06 (s, 1H), 7.98-7.88 (m, 2H), 7.82 (s, 1H), 6.87 (d, J=1.5 Hz, 1H), 4.50 (s, 2H), 3.36 (s, 3H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.75-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.99 min. M/Z = 255.2.

Unless otherwise stated, the same LC-MS analytical conditions were applied to the characterized compounds.

Compounds 114-122 were prepared according to synthetic methods similar to those described for compound 113, from the appropriate starting materials.

Connection No. Structure LC-MS [M+H] ⁺ RT (min) Dyamr (500 MHz, DMSO-d ₆ ) 114 Qc \_/ I o 336.2 0.96 5 7.84 (s, 1H), 7.71 (br s, 2H), 7.65 - 7.55 (m, 2H), 6.76 (s, 1H), 2.61 -2.54 (m, 2H), 2.45 2.26 (m, 6H), 1.82 - 1.70 (m, 2H), 1.57 - 1.45 (m, 4H), 1.38 (br s, 2H) 115 nh ₂ n ί T ΝγΜ Μ 310.1 1.44 5 7.88 (s, 1H), 7.81 (s, 1H), 7.72 (br s, 1H), 7.68 - 7.60 (m, 2H), 6.78 (s, 1H), 6.53 (br s, lH), 3.60 (br s, 2H), 2.40 (br s, 4H), 4 protons from morpholine are not visible in NMR. 116 \ / τ ' ί № 295.0 1.12 5 7.84 (s, 1H), 7.76 (s, 1H), 7.74 - 7.65 (in, 2H), 7.60 (br d, 7=7.9 Hz, 1H), 6.77 (s, 1H), 6.32 (brs, 1H), 4.03 -3.89 (m, 2H), 2.91 (brt, 7=11.3 Hz, 1H), 1.81 (brd, 7=13.1 Hz, 2H), 1.71 - 1.58 (m, 2H), one methylene of the THP ring is not visible, probably due to overlap with the suppressed water peak. 117 Ρ ΖΣ cm > > CHZ τζ / 324.2 1.24 5 7.87 (s, 1H), 7.79 (s, 1H), 7.72 - 7.59 (w, ZN), 6.75 (d, 7=1.9 Hz, 1H), 6.42 (brs, 1H), 3.91 (dt,7=12.4, 6.2 Hz, 1H), 3.81 -3.77 (w, 1H), 3.77-3.70 (rn, 1H), 3.64 - 3.57 (m, 1H), 2.59 - 2.56 (w, 1H), 1.95 - 1.73 (rn, 4H), 1.55-1.41 (m, 2H) 118 νη ₂ СН ₃ νΤ 351.3 0.93 δ 8.38 (s, 1H), 8.11 (s, 1H), 7.98 - 7.89 (m, 2H), 7.86 (s, 1H), 6.87 (d, 7=1.5 Hz, 1H), 4.02 -3.86 (m, 1H), 3.26 (brs, 1H), 2.80 - 2.70 (m, 6H), 2.06 (brd, 7=10.4 Hz, 2H), 1.83 1.63 (m, 2H), several protons from the piperidine ring are not visible, probably due to obscuration by water/DMSO.

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119 Forehead H iY 308.2 0.99 δ 8.28 (s, 1H), 8.08 (s, 1H), 8.00 -7.90 (m, 2H), 7.84 (br s, 1H), 6.87 (d, 7=1.5 Hz, 1H), 4.43 (s, 2H), 3.49 -3.37 (m, 2H) (overlapped by the suppressed water peak), 2.38 (brt, 7=8.1 Hz, 2H), 2.07 1.92 (m, 2H) 120 I o ^z vK IZ 'g 280.9 1.14 δ 7.86 (s, 1H), 7.79 (s, 1H), 7.74 - 7.68 (m, 1H), 7.67 - 7.57 (m, 2H), 6.78 (s, 1H), 6.20 (br s, 1H), 0.94 (br s, 2H), 0.72 (br s, 2H). Methylene, located next to the alcohol, is not visible, possibly due to overlap with the suppressed water peak. 121 z Q- y _z \_/ T ----( M Q 324.2 0.96 δ 8.30 (brs, 1H), 8.11 (brs, 1H), 7.98-7.81 (m, 3H), 6.86 (s, 1H), 4.16-3.66 (m, 2H), 3.11 (brs, 1H). The peaks of the morpholino-ethyl chain are broadened and have low integration. 122 z \ ' ^Z=L AH o o I 337.06 0.99 δ 7.84 (s, 1H), 7.76 - 7.68 (m, 2H), 7.65 - 7.57 (m, 2H), 6.76 (d, 1=1.8Hz, 1H), 2.78-2.69 (m, 2H), 2.60 (brt, J=7.0 Hz, 2H), 2.47 - 2.25 (m, 4H),2.17 (s, 3H)

Step 2A. Alternative route to quinoline: preparation of 3-(1H-imidazol-5-yl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 123)

To a solution of 2-(1H-imidazol-5-yl)acetonitrile (11.84 mg, 0.111 mmol) and 2-amino-4-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)benzaldehyde (20 mg, 0.074 mmol) in EtOH (369 μL) was added sodium hydroxide (1 M in EtOH) (14.74 μL, 0.015 mmol). The reaction mixture was heated to 70 °C. After 1 h, 75 μL of 1 M sodium hydroxide in EtOH was added and heating was continued overnight. LCMS indicated that the reaction was complete. The reaction mixture was cooled and concentrated. The residue was dissolved in 0.4 mL of DCM and 0.4 mL of TFA was added. After 45 min, LC-MS showed the reaction to be complete. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then a 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation to afford 3-(1H-imidazol-5-yl)-7-(1H-pyrazol-5yl)quinolin-2-amine (6.4 mg, 31%). ¹ H NMR (500 MHz, DMSO-d6) δ 8.27 (s, 1H), 7.86 (d, J=8.3 Hz, 2H), 7.81 (s, 1H), 7.73-7.59 (m, 3H), 6.77 (d, J=1.8 Hz, 1H). LC RT: 0.86 min. M/Z=277.2.

Compounds 124-127 were prepared according to the synthetic methods described for compound 123 from the appropriate starting materials.

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Connection No. Structure LC-MS [m+ng] RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 124 nh ₂ o ΐ JI n n m 331.1 1.19 δ 8.90 (s, 1H), 8.56 (s, 1H), 8.34 (brd,7=4.5 Hz, 1H), 8.15 (brd, 7=7.7 Hz, 1H), 7.91 (s, 1H), 7.84 - 7.79 (m, 1H), 7.75 (brs, 1H), 7.42 (dd, 7=8.1, 4.7 Hz, 1H), 6.93 - 6.75 (m, 2H) 125 m 318.1 1.10 δ 8.48 (s, 1H), 8.01 - 7.95 (m, 1H), 7.91 (brd, 7=4.8 Hz, 2H), 7.80 (d, 7=8.3 Hz, 1H), 7.73 (s, 1H), 7.68 (brd, 7=8.4 Hz, 1H), 7.48 (br d, 7=7.5 Hz, 2H),6.81 (d, 7=1.9 Hz, 1H), 4.67 (br s, 2H) 126 NH ₂ HN-N 1A ^ ^сн з ^N ^|| N H ij Μ 292.1 0.95 δ 8.77 (brs, 1H), 7.96 - 7.52 (m, 5H), 6.84 (brs, 1H), 2.48 (brs, 3H) 127 N M 326.9 1.36 6 8.33 (s, 1H), 8.23-8.14 (m, 2H), 7.98 (br d, 7=7.6 Hz, ZN), 7.87 (brs, 1H), 7.73 (d, 7=8.5 Hz, 1H), 7.50 (brd, 7=8.5 Hz, 1H), 6.89 (s, 1H)

Example P-2: Synthesis of 4-amino-substituted quinolines

CI

DMSO, /Pr ₂ NEt, then DCM, TFA n

Step 1. Obtaining 7-bromo-4-chloroquinoline

To a suspension of 7-bromoquinolin-4-ol (2.5 g, 11.16 mmol) in toluene (20 mL) was added POCl ₃ (2.080 mL, 22.32 mmol). The reaction mixture was heated to 100 °C. After 1.5 h, the reaction mixture was cooled, and then ice was added. The reaction mixture was stirred vigorously for approximately 30 min, then water was added. The reaction was extracted twice with DCM. The organic layers were washed with saturated aqueous NaHCO ₃ and brine, then dried over sodium sulfate and concentrated. LC-MS showed that some product remained in the original aqueous layer. The aqueous layer was stirred, and saturated aqueous NaHCO ₃ was carefully added. The precipitated solid was filtered, washed with water, and dried. The organic layer and filtered solid were combined and dried under high vacuum to give 7-bromo-4-chloroquinoline (2.46 g, 10.14 mmol, 91% yield). Ή NMR (400 MHz, chloroform-d ) δ 8.80 (d, J = 4.7 Hz, 1H), 8.33 (d, J = 1.9 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.75 (dd, J = 9.0, 2.0 Hz, 1H), 7.52 (d, J = 4.8 Hz, 1H).

Step 2. Obtaining 7-bromo-4-chloroquinoline 1-oxide

To a solution of 7-bromo-4-chloroquinoline (2.0 g, 8.25 mmol) in DCM (55.0 mL) was added mCPBA (6.10 g, 24.74 mmol). The reaction mixture was stirred overnight, then quenched with saturated sodium thiosulfate solution. The reaction mixture was stirred for 0.5 h, then saturated aqueous sodium bicarbonate solution was added. The reaction was extracted twice with DCM. The organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 7-bromo-4-chloroquinoline 1-oxide (2.16 g, 8.36 mmol, quantitative yield). 1H NMR (400 MHz, chloroform-d) δ

8.99 (d, J=1.9 Hz, 1H), 8.43 (d, J=6.6 Hz, 1H), 8.10 (d, J=9.0 Hz, 1H), 7.86 (dd, J=9.0, 2.0 Hz, 1H), 7.40 (d,

J=6.6 Hz, 1H).

Step 3. Obtaining 7-bromo-4-chloroquinoline-2-amine

In one round-bottomed flask, 7-bromo-4-chloroquinoline 1-oxide (9400 mg, 36.4 mmol) was suspended in DCM (150 mL). Ts-Cl (7626 mg, 40.0 mmol) was added. This mixture was stirred for one hour. In a second round-bottomed flask, ammonium chloride (9725 mg, 182 mmol) (dried in an oven at 110°C overnight) was suspended in DCM (150 mL). Triethylamine (25.3 mL, 182 mmol) was added, and the mixture was stirred for 0.5 h, then the contents of the first round-bottomed flask were added to the second. The reaction mixture was stirred overnight, then filtered and concentrated. The residue was dissolved in 100 mL of hot DCM. The solution was cooled to room temperature, and the solid was filtered. The filter cake was washed with 100 mL of -20°C DCM. The filter cake was suspended in water (50 mL) and filtered. The solid was the desired product 7-bromo-4-chloroquinolin-2-amine. The DCM filtrate was evaporated, suspended in water (100 mL), and filtered. The filter cake was washed with 100 mL of -20°C DCM to obtain additional product. The combined solids were dried under high vacuum to afford 7-bromo-4-chloroquinolin-2-amine (6.52 g, 69.6%). 1H NMR (400 MHz, DMSO-d ₆ ) δ 7.79 (d, J=8.7 Hz, 1H), 7.65 (d, J=1.9 Hz, 1H), 7.39 (dd, J=8.8, 2.0 Hz, 1H), 6.98 (s, 1H), 6.88 (s, 2H).

Step 4. Preparation of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine

To each of two 40 mL vials were placed (1-(tetrahydro-2H-pyran-2-yl)1H-pyrazol-5-yl)boronic acid (0.714 g, 3.64 mmol), 7-bromo-4-chloroquinolin-2-amine (0.750 g, 2.91 mmol), and the PdCl ₂ (dppf)-DCM adduct (0.238 g, 0.291 mmol). The vials were evacuated three times and backfilled with nitrogen. Dioxane (14.56 mL) and tripotassium phosphate (2 M aqueous) (4.37 mL, 8.74 mmol) were added to each vial, nitrogen was bubbled through the solution, and the reaction mixture was heated to 100°C overnight. The contents of the vials were cooled, diluted with EtOAc and water, and combined. The reaction mixture was extracted three times with EtOAc, and then the organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified using ISCO (80 g column; DCM/MeOH; gradient from 0 to 10%) to afford 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline-2-amine (1.14 g, 59.5% yield). 1H NMR (400 MHz, chloroform-d) δ 8.11 (d, J=8.6 Hz, 1H), 7.82 (d, J=1.4 Hz, 1H), 7.65 (d, J=1.5 Hz, 1H), 7.52 (dd, J=8.5, 1.7 Hz, 1H), 6.90 (s, 1H), 6.45 (d, J=1.8 Hz, 1H), 5.38-5.26 (m, 1H), 4.90 (br s, 1H), 4.22-4.09 (m, 2H), 3.65 (td, J=11.7, 2.3 Hz, 1H), 2.68-2.51 (m, 1H), 2.14-1.51 (m, 5H).

Step 5: Preparation of K4-((1H-pyrazol-3-yl)methyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (compound 128)

To a solution of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) and (1H-pyrazol-3-yl)methanamine (59.1 mg, 0.608 mmol) in DMSO (0.5 mL) was added Hunig's base (0.032 mL, 0.182 mmol). The reaction mixture was heated to 120 °C overnight. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were concentrated, then dissolved in 0.4 mL DCM and 0.4 mL TFA. After 1 h, the reaction was complete according to LC-MS. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 3 min hold at 0% B, 0-32% B over 25 min, then 5 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give N4-((1H-pyrazol-3yl)methyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (4.6 mg, 24.7%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.00 (br d, J=8.2 Hz, 1H), 7.75 (s, 1H), 7.72 (br s, 1H), 7.58 (br s, 1H), 7.54 (br d, J=7.9 Hz, 1H), 7.43 (br s, 1H), 6.76 (s, 1H), 6.62-6.41 (m, 1H), 6.20 (s, 1H), 5.76 (s, 1H), 4.42 (br d, J=5.2 Hz, 2H). LC RT: 0.99 min. M/Z= 306.18.

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Step 5b: Method of application of amine salts.

Preparation of N4-(1-(6-methoxypyridin-2-yl)ethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 2 TFA (compound 129)

To a solution of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) and 1-(6-methoxypyridin-2-yl)ethan-1-amine, HCl (115 mg, 0.608 mmol) in DMSO (0.5 mL) was added Hunig's base (0.159 mL, 0.912 mmol). The reaction mixture was heated to 100 °C overnight. Then the reaction temperature was raised to 120 °C for 5.5 h. The reaction mixture was cooled, diluted with water and extracted three times with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.4 mL DCM and 0.4 mL TFA. After approximately 1 h, the reaction was complete as determined by LC-MS. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LCMS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 9% B, 9-46% B over 23 min, then 6 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give N4-(1-(6-methoxypyridin-2-yl)ethyl)-7(1H-pyrazol-5-yl)quinoline-2,4-diamine, 2 TFA (4.4 mg, 12%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.51 (br d, J=8.5 Hz, 1H), 8.18 (br d, J=6.4 Hz, 1H), 8.01-7.81 (m, 3H), 7.68 (brt, J=7.6 Hz, 1H), 7.58 (br s, 2H), 6.96 (br d, J=7.3 Hz, 1H), 6.86 (br s, 1H), 6.71 (br d, J=7.9 Hz, 1H), 5.63 (s, 1H), 4.70 (br t, J=6.6 Hz, 1H), 3.88 (s, 3H), 1.68 (br d, J=6.7 Hz, 3H). LC/MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 1.46 min. M/Z = 361.31.

Compounds 130-166, compounds 222-290, and compound 351 were prepared according to the synthetic methods described for compound 129 from the appropriate starting materials.

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Compound No. Structure LC-MS [M+H]+ RT (min) Ή NMR (500 MHz, DMSO-de) 130 Z Z \_/ X o o X so 309.2 1.21 δ 7.81 (s, 1H), 7.76 - 7.67 (m, 2H), 7.58 (br d, 7=8.2 Hz, 1H), 6.75 (s, 1H), 6.31 (brs, 1H), 6.24 (s, 1H), 3.08 (brs, 4H), 2.60 (br s, 4H), 2.29 (s, 3H) 131 nh ₂ л n igo Ό 296.3 1.12 δ 8.11 (brs, 1H), 8.02 (brs, 1H), 7.96 - 7.90 (m, 1H), 7.89 - 7.80 (m, 2H), 6.81 (d, 7=2.0 Hz, 2H), 6.34 (s, 2H), 3.88 (m, 4H), 3.36 - 3.29 (m, 2H). Two protons from the morpholine ring are not visible, likely due to water occlusion/suppression. 132 nh ₂ on l JL L yHG i V 311.9 1.05 δ 7.89 (brd, 7=8.5 Hz, 1H), 7.76-7.66 (br, 2H), 7.51 (br s, 1H), 6.81 (br d, 7=4.0 Hz, 1H), 6.75 (s, 1H), 6.32-6.11 (m, 1H), 5.70 (s, 1H), 3.25 (brs, 1H), 1.80 (brt, 7=7.5 Hz, 2H), 1.20 (s, 6H). NMR is missing one proton from the side chain, probably due to overlap with the suppressed water peak. 133 nh ₂ ⁿ OH m 284.3 0.69 δ 8.04 (brd, 7=8.6 Hz, 1H), 7.80 (brs, 1H), 7.72 (br s, 1H), 7.67 - 7.56 (br, 1H), 6.76 (s, 1H), 5.80 (s, 1H), 4.04 -3.95 (br, 1H), 3.89 (s, 2H), 1.18 (d, 7=6.1 Hz, 3H) 134 ζ Xvz \=/ Ή ^z \_/ X ΙΖ oJ ¹ ъ X 283.9 1.09 δ 8.26 (d, 7=8.7 Hz, 1H), 8.00-7.91 (br, 2H), 7.88 - 7.78 (br, 2H), 7.47 (br s, 2H), 6.83 (d, 7=2.1 Hz, 1H), 5.89 (s, 1H), 4.05 -3.98 (br, 1H), 3.89 (s, 2H), 1.18 (d, 7=6.2 Hz, 3H) 135 nh ₂ 10 Μ 317.0 1.06 δ 8.56 (brd, 7=4.3 Hz, 1H), 8.07 (brd, 7=8.5 Hz, 1H), 7.81 - 7.68 (br, 4H), 7.65 - 7.56 (br, 1H), 7.33 (br d, 7=7.6 Hz, 1H), 7.31-7.24 (br, 1H), 6.78 (s, 1H), 5.59 (s, 1H), 4.55 (brd, 7=5.5 Hz, 2H)

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136 nh ₂ H cn ₃ V 284.3 0.99 δ 8.02 (brd, 7=8.5 Hz, 1H), 7.84 (s, 1H), 7.74 (brs, 1H), 7.61 (br d, 7=4.9 Hz, 1H), 6.78 (s, 1H), 6.59 (brd, 7=12.2 Hz, 1H), 6.20 (s, 1H), 3.74 (brs, 2H), 2.91 (s, 3H). One methylene from the side chain is not visible, likely due to overlap with the suppressed water peak. 137 νη ₂ l 1 n Gu Γ ν Υ 1 1 323.9 1.13 δ 8.15 (brd, 7=8.5 Hz, 1H), 7.91 - 7.68 (br, 4H), 6.83 (s, 1H), 5.80 (s, 1H), 3.86 (brd, 7=10.7 Hz, 2H), 3.68 - 3.53 (br, 2H), 3.28 (br t, 7= 11.6 Hz, 2H), 1.98 (brs, 1H), 1.66 (brd, 7=12.2 Hz, 2H), 1.33 - 1.19 (br, 2H) 138 ζ \ ^4ζτ γΑ— ^ζ \=< Vz \_/ I ) ' Μ ΙΖ U ο-ζ ? 'ο X 325.1 1.08 δ 8.16 (brd, 7=8.5 Hz, 1H), 8.01 - 7.74 (br, 4H), 7.49 - 7.29 (br, 1H), 6.84 (brs, 1H), 5.82 (s, 1H), 3.36 (brs, 2H), 2.98 (s, 3H), 2.85 (s, 3H), 2.76 (brt, 7=6.9 Hz, 2H) 139 CO Τ Ο \ ΖΙ 7 ΖΙ τ / ⁼ \ >=\ ^ζ V? ΙΖ ζ 311.2 1.03 δ 7.91 (brd, 7=8.5 Hz, 2H), 7.78 -7.66 (br, 2H), 7.51 (brd, 7=7.6 Hz, 1H), 6.85 (brs, 1H), 6.75 (s, 1H), 6.36 (br s, 1H), 5.73 (s, 1H), 2.60 (brd, 7=4.3 Hz, 3H). The side-chain methylenes are not visible in NMR, likely due to overlap with the suppressed water peak. 140 with ^Ο ΖΙ σι ,-----{ X ^ζ ν /=\ ^ζ Λ? χζ 'Ζ. 332.2 0.87 δ 8.25 - 8.09 (m, 2H), 7.99 (br s, 1H), 7.88 (brs, 2H), 7.75 (br s, 2H), 6.86 (br s, 1H), 5.87 (s, 1H), 3.76 (br d, 7=5.5 Hz, 2H), 3.56 (brt, 7=6.3 Hz, 2H), 3.10 (s, 3H) 141 νη ₂ Un ^Η И jQl %'cCH ₃ V 296.2 1.06 δ 8.01 (brd,7=8.5 Hz, 1H), 7.77 (s, 1H), 7.73 (br s, 1H), 7.56 (brd, 7=7.9 Hz, 1H), 6.76 (s, 1H), 6.60 (brd, 7=17.4 Hz, 1H), 6.45 (br s, 1H), 5.82 (s, 1H), 3.13 (br d, 7=5.2 Hz, 2H), 1.22 (s, 6H) 142 X O ό CM / ( t / \ ^z 4 /=\ ^z A_/ χζ &'Ζ. 296.0 0.75 δ 8.28 (brd,7=8.5 Hz, 1H), 7.97 (br s, 1H), 7.88 (br s, 1H), 7.79 (brd,7=8.5 Hz, 1H), 7.53 (br s, 2H), 6.83 (br s, 1H), 5.82 (s, 1H), 4.46 (br s, 1H), 4.02 3.94 (m, 1H), 3.94 -3.84 (m, 1H), 3.77 - 3.67 (m, 1H), 3.54

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(br d, 7=10.7 Hz, 1H), 2.121.93 (m, 2H) 143 z \ ' ^Н1 Xa—z \=( 4—z )=/ σ ό I 296.3 1.05 5 8.06 (brd,7=8.5 Hz, 1H), 7.81 (brs, 1H), 7.75 (brs, 1H), 7.55 (brd,7=3.1 Hz, 1H), 6.76 (s, 1H), 6.48 (brd,7=5.5 Hz, 1H), 5.83 (s, 1H), 4.41 (brs, 1H), 3.85 (br dd,7=10.1,4.0 Hz, 1H), 3.75 (brd,7=7.6 Hz, 1H), 2.12- 1.99 (m, 1H), 1.97-1.91 (m, 1H). Two protons from the pyrrolidine ring are not visible, probably due to overlap with the suppressed water peak. 144 z \=< Vz \_/ I ) ' m IZ O I 298.1 1.07 5 8.23 (brd,7=8.2 Hz, 1H), 8.12 (brs, 1H), 7.96 (br s, 1H), 7.89-7.78 (sh, 2H), 7.62 (br s, 2H), 6.85 (brs, lH),5.80(s, 1H), 3.47 (brd,7=4.9 Hz, 1H), 3.28 (brd,7=10.4 Hz, 1H), 1.76-1.67 (m, 2H), 1.60-1.46 (m, 2H). Two protons from the side chain are not visible, probably due to poor integration or overlap with the suppressed water peak. 145 z / ^- V- z \=/ V ^- z \_/ I 2 · yu P O I 310.2 1.07 5 8.14 - 7.97 (m, 2H), 7.91 - 7.80 (br, 3H), 6.83 (brs, 1H), 3.87 -3.77 (m, 1H), 3.55 (brd, 7=8.5 Hz, 1H), 3.13 (brt, 7=11.4 Hz, 1H), 2.98 -2.87 (br, 1H), 1.96 (brs, 2H), 1.67 (brd, 7=8.5 Hz, 2H). One proton from piperidine is missing, probably due to overlap with the suppressed water peak or poor integration. 146 o /=4 У ZI CM ? \ /=\ ^г V0 ΞZ 'z 373.2 1.21 5 8.76 (brs, 1H), 8.12 (brd, 7=7.3 Hz, 1H), 7.96-7.71 (sh, 7H), 7.57-7.41 (m, 5H), 6.83 (brs, 1H), 5.85 (s, 1H), 3.61 (br d, 7=5.2 Hz, 2H), 3.48 (brd, 7=4.3 Hz, 1H). One proton from the side chain is missing, likely due to overlap with the suppressed water peak or poor integration.

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147 nh ₂ OH 8%» 296.3 1.07 δ 8.04 (br d, 7=8.5 Hz, 1H), 7.79 (brs, 1H), 7.77 -7.69 (m, 1H), 7.61 (brs, 1H), 6.78 (br s, 1H), 5.79 (s, 1H), 2.99 (s, 1H), 0.66 (br s, 2H), 0.61 (br s, 2H). One proton from the side chain is not visible, possibly due to overlap with the suppressed water peak or poor integration. 148 nh ₂ fS 317.3 0.92 δ 8.50 (br d, 7=4.6 Hz, 2H), 8.04 (br d, 7=8.5 Hz, 1H), 7.79 -7.65 (m, ZN), 7.59 (br d, 7=6.7 Hz, 1H), 7.35 (br d, 7=4.6 Hz, 2H), 6.78 (s, 1H), 4.51 (brd, 7=5.5 Hz, 2H) 149 nh ₂ 1Λ ^NN '' ^N lAJ ^h and ⁿ A 318.3 1.01 δ 9.20 (brs, 1H), 8.88 (brs, 1H), 8.29 (br d, 7=8.2 Hz, 1H), 7.98 - 7.79 (m, 4H), 7.74 - 7.64 (m, 4H), 6.86 (brs, 1H), 5.75 (s, 1H), 4.87 (brd, 7=5.8 Hz, 2H) 150 Z ₄ z \=< Ά z \_/ I 2 ^f GO IZ d 317.3 1.11 δ 12.50 (br s, 1H), 8.78 (br s, 1H), 8.66 (br s, 1H), 8.53 (br s, 1H), 8.26 (br d, 7=8.5 Hz, 1H), 7.94 (s, 1H), 7.90 - 7.79 (m, 3H), 7.65 (br s, 2H), 7.50 - 7.43 (m, 1H), 6.86 (s, 1H), 5.74 (s, 1H), 4.62 (br d, 7=5.2 Hz, 2H) 151 nh ₂ ^N ii ^Ν<ί> Ίι nJU ^h 331.2 1.06 δ 12.50 (br s, 1H), 8.62 (brd, 7=4.9 Hz, 1H), 8.20 (brs, 1H), 8.15 (brd,7=8.5 Hz, 1H), 7.99 - 7.90 (m, 2H), 7.86 - 7.79 (m, 2H), 7.69 (br s, 2H), 7.54 (br d, 7=7.6 Hz, 1H), 7.48 - 7.40 (m, 1H), 6.84 (s, 1H), 5.89 (s, 1H), 3.71 (brd, 7=6.1 Hz, 1H), 3.23 (brt, 7=7.0 Hz, 1H). Two protons are missing from the side chain, either due to overlap with the suppressed water peak or due to poor integration. 152 nh ₂ 1 Ii M / N kAJ ^hn A 320.3 0.77 δ 8.68 - 8.53 (m, 1H), 8.11 - 7.98 (br, 2H), 7.90 (br, s, 1H), 7.85 -7.76 (m, 2H), 7.56 (br, s, 2H), 7.39 (br, s, 1H), 6.85 (s, 1H), 5.78 (s, 1H), 4.43 (br, s, 2H), 2.94 - 2.83 (m, 1H). One proton from the side chain is missing, likely due to poor integration or overlap with the suppressed water peak.

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153 nh ₂ A 1 N%J ^H OH 300.3 0.64 δ 7.92 (brd, 7=8.2 Hz, 1H), 7.73 (br s, 2H), 7.52 (br s, 1H), 6.76 (s, 1H), 6.65 (br s, 1H), 6.24-6.10 (m, 1H), 5.74 (s, 1H), 3.85 - 3.77 (m, 1H), 3.33 3.26 (m, 1H), 3.14-3.05 (m, 2H). One proton from the side chain is not visible, likely due to overlap with the water peak. 154 nh ₂ TRO n. II 1 VJ he 347.1 1.06 δ 8.64 (brs, 1H), 8.49 (brs, 1H), 8.20 (br d, 7=7.6 Hz, 2H), 7.96-7.79 (m, 4H), 7.61 (br s, 2H), 7.46-7.34 (m, 1H), 6.85 (s, 1H), 5.92 (s, 1H), 4.99 (br s, 1H). The methylene side chain is not visible in the NMR spectrum, likely due to overlap with the suppressed water peak. 155 <N / ( ^z 4 >=\ IZ 'z 347.0 1.05 δ 8.55 (br d, 7=4.0 Hz, 1H), 8.00 (brd, 7=8.5 Hz, 1H), 7.877.80 (m, 1H), 7.79 (br s, 1H), 7.74 (br s, 1H), 7.60 (br d, 7=7.6 Hz, 2H), 7.34 - 7.25 (m, 1H), 7.11 (brs, 1H), 6.79 (s, 2H), 5.83 (s, 1H), 4.98 (br dd, 7=8.1,3.5 Hz, 1H), 3.68-3.58 (m, 1H), 3.49 -3.30 (m, 1H) 156 z \ ' ^ZT Xa—z W Vz )=/- ¹ IZ 4 O I 337.2 0.92 δ 8.74 (brs, 1H), 8.27 (brd, 7=8.5 Hz, 1H), 7.97 (brs, 1H), 7.87 (brs, 2H), 7.62 (brs, 2H), 7.33 (s, 1H), 6.86 (brs, 1H), 5.82 (s, 1H), 4.59 (brd,7=5.5 Hz, 2H), 2.65 (s, 3H) 157 [Rae] nh ₂ 324.0 0.96 δ 8.35 (br d, 7=7.6 Hz, 1H), 8.01-7.90 (m, 1H), 7.88 - 7.74 (m, 2H), 7.57 (br s, 2H), 7.27 (br d, 7=6.7 Hz, 1H), 6.85 (br s, 1H), 5.87 (s, 1H), 4.99 (brd, 7=3.4 Hz, 1H), 4.05 (brs, 1H), 3.55 - 3.33 (m, 1H), 1.96-1.29 (m, 8H) 158 Z \=< Vz \_/ I / o I co 337.3 0.74 δ 8.21 (brd,7=5.2 Hz, 1H), 8.08 (brd, 7=7.9 Hz, 1H),791 - 7.72 (m, 2H), 7.68 - 7.54 (m, 1H), 6.78 (br s, 1H), 6.75-6.55 (m, 1H), 5.84 (brs, 1H), 4.34 (brs, 1H), 3.89 (brs, 1H), 3.79 -3.57(m, 1H), 2.18 (brd, 7=6.1 Hz, 1H), 1.95 (brd, 7=5.5 Hz, 1H), 1.82 (s, 3H). Two protons from the pyrrolidine ring are not visible due to overlap with the suppressed water peak or poor integration.

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159 nh ₂ Δ n η jOj й^. V 306.1 0.66 δ 8.81 (brs, 1H), 8.56 (brs, 1H), 8.22 (brd,7=8.2 Hz, 1H), 7.96 (brs, 1H), 7.91 -7.82 (m, 2H), 7.75 (br s, 2H), 7.55 (br s, 1H), 6.86 (s, 1H), 5.83 (s, 1H), 4.60 (br d,7=4.3 Hz, 1H). One proton from methylene is not visible, likely due to overlap with the suppressed water peak or poor integration. 160 νη ₂ n Dr N^X^J ^Η OH m 324.1 0.97 δ 8.33 (brd, 7=8.5 Hz, 1H), 7.98 - 7.90 (br, 1H), 7.90 - 7.75 (br, 2H), 7.65 (br d, 7=7.6 Hz, 1H), 7.49 (br s, 2H), 6.85 (br s, 1H), 5.91 (s, 1H), 4.97 (brd, 7=4.9 Hz, 1H), 3.60 (brd, 7=4.6 Hz, 1H), 3.46-3.22 (m, 2H), 2.03 - 1.92 (m, 2H), 1.71 (brd, 7=5.2 Hz, 2H), 1.43 1.19 (m, 4H) 161 Z ₄ \ ' ^ΖΞ ζ \=/ V- Z u ' м IZ ^: 1з 367.2 1.20 6 8.75 (brd,7=4.3 Hz, 1H), 8.29 -8.22 (m, 2H), 8.01 (br t, 7=7.6 Hz, 1H), 7.95 (br s, 1H), 7.88 - 7.79 (m, 2H), 7.75 (d, 7=7.9 Hz, 1H), 7.69 (br s, 1H), 7.63 - 7.57 (m, 1H), 6.83 (d, 7=1.8 Hz, 1H), 6.07 (s, 1H), 4.27 (td, 7=14.9, 6.1 Hz, 2H) 162 /\X\/° ZI cm /------( /=\ ΞΕΖ 'ζ 324.3 1.11 δ 8.23 (brd,7=8.2 Hz, 1H), 7.92 - 7.69 (m, 3H), 7.40 - 7.04 (m, 2H), 6.82 (brs, 1H), 5.87 (s, 1H), 3.65 - 3.53 (m, 1H), 3.31 -3.19 (m, 1H), 1.96 (brs, 2H), 1.70 (brs, 3H), 1.41 - 1.18 (m, 6H) 163 νη ₂ Ν=\ Μ 321.3 0.93 δ 8.49 (s, 1H), 8.03 - 7.89 (m, 2H), 7.82 (brs, 1H), 7.76 (br s, 1H), 7.66 (br d, 7=7.0 Hz, 1H), 7.43 (brs, 1H), 6.98 - 6.83 (m, 1H), 6.80 (s, 1H), 5.75 (s, 1H), 4.50 (brt, 7=5.6 Hz, 2H), 3.68 (br d, 7=4.9 Hz, 1H). One proton from the side chain is not visible, likely due to overlap with the suppressed water peak or poor integration. 164 ζ \ ^ζτ χ ^- ^— ^ζ \=Ζ y-ζ \_/ I 2 —' No. « 343.2 1.20 δ 8.43 (br d, 7=4.0 Hz, 1H), 7.95 (br s, 1H), 7.90 (br d, 7=8.5 Hz, 1H), 7.80 (brs, 2H), 7.64 (br d, 7=7.6 Hz, 1H), 7.27 (br dd, 7=7.5, 4.7 Hz, 1H), 6.82 (s, 1H), 6.38 (s, 1H), 4.49 (br s,

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2H), 3.21 (br s, 2H), 3.16 (s, 2H) 165 nh ₂ nA ? ^Нз nAjJ A V 284.0 0.94 5 8.29 (brd,7=8.5 Hz, 1H), 7.90 (br s, 1H), 7.80 (br d, 7=7.1 Hz, 2H), 7.40 (br s, 1H), 7.30 (brs, 1H), 6.82 (d, 7=1.9 Hz, 1H), 5.90 (s, 1H), 3.763.67 (m, 1H), 3.65 - 3.50 (m, 2H), 1.27 (d, 7=6.5 Hz, ZN) 166 nh ₂ 1Л H fj] G ³ ch, M 254.4 1.02 5 8.05 (brd, J=8.5 Hz, 1H), 7.95 (brs, 1H), 7.87 -7.65 (m, 4H), 6.83 (s, 1H), 6.04 (s, 1H), 3.15 (s, 6H) 222 z o ZI cm >----( x / \ ^z 4 >=\ ^z -JJ ΙΖ _χ A 'z 292.9 0.79 5 8.23 (brd,7=8.2 Hz, 1H), 8.12-8.05 (m, 1H), 7.96 (brs, 1H), 7.91 -7.81 (m, 1H), 7.57 (br dd, 7=5.3, 4.1 Hz, 2H), 6.91 -6.83 (m, 1H), 5.81 (s, 1H), 3.44 -3.33 (m, 2H), 2.65 (brt, 7=6.9 Hz, 2H), 2.03 - 1.94 (m, 2H) 223 z П-z \=/ V- z \_/ X y ^f ΓΌ IZ ° 5 and / Ο-ω— о A* “ ω 360.3 0.84 5 8.21 (brd,7=8.2 Hz, 1H), 8.02 - 7.95 (m, 1H), 7.93 - 7.84 (m, 3H), 7.83 - 7.69 (m, 2H), 6.86 (s, 1H), 6.05 (s, 1H), 3.71 (br d, 7=6.4 Hz, 2H), 3.05 (s, 3H), 1.40 (s, 6H) 224 ^T1 ( ZI ^x CM 7 ( X /—\ ⁿ V /=\ XZ ₄ Λ z 321.0 0.66 5 8.24 -8.11 (m, 2H), 7.94 (br s, 1H), 7.83 (brd,7=9.2 Hz, 2H), 7.72 (br s, 1H), 6.84 (d, 7=2.1 Hz, 1H), 5.87 (s, 1H), 3.72-3.61 (m, 2H), 3.15-3.06 (m, 2H) 225 nh ₂ 320.1 0.71 5 8.86 (s, 1H), 8.22-8.10 (m, 2H), 7.95 (br s, 1H), 7.83 (br d, 7=8.5 Hz, 2H), 7.78 (br s, 2H), 7.46 (s, 1H), 6.84 (d, 7=2.2 Hz, 1H), 5.89 (s, 1H), 3.66-3.56 (m, 2H), 3.06 (t, 7=6.8 Hz, 2H) 226 Z _x \ ^ZT z \=( z \_/ X /--- ^rw IZ d I 307.1 0.71 5 8.74 -8.61 (m, 1H), 8.21 (br d, 7=8.2 Hz, 1H), 7.99 - 7.90 (m, 1H), 7.89 - 7.77 (m, 3H), 7.65 (br s, 2H), 6.85 (br s, 1H), 5.86 (s, 1H),4.63 (brd,7=5.2 Hz, 2H)

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227 nh ₂ Λ n G II n ' ^NH 307.0 0.92 6 8.73 (brs, 1H), 8.55 (brs, 1H), 8.25 (br d, 7=7.6 Hz, 1H), 8.03 -7.92 (m, 1H), 7.87 (brs, 2H), 7.73 - 7.57 (m, 2H), 6.86 (brs, 1H), 5.88 (brs, 1H), 4.76 - 4.50 (m, 2H) 228 A ZI CM ' ( I /\ ^z 4 )=\ ^g vj z 306.1 0.76 6 7.98 (d, 7=8.9 Hz, 1H), 7.76 (s, 1H), 7.72 (br s, 1H), 7.60 (br s, 2H), 7.56 (br d, 7=7.6 Hz, 1H), 7.44 - 7.34 (m, 1H), 6.78 (d, 7=1.8 Hz, 1H), 6.69-6.51 (m, 1H), 5.79 (s, 1H),4.31 (br d, 7=5.5 Hz, 2H) 229 nh ₂ 11 ⁿ rr G NV CH ₃ 320.1 1.00 6 8.62 (brs, 1H), 8.23 (d, 7=8.8 Hz, 1H), 7.98 - 7.92 (m, 1H), 7.84 (br d, 7=8.2 Hz, 2H), 7.63 (d, 7=1.8 Hz, 1H), 7.58 (brd, 7=1.6 Hz, 1H), 6.85 (d, 7=1.9 Hz, 1H), 6.21 (d, 7=2.2 Hz, 1H), 5.86 (s, 1H), 4.46 (d, 7=6.0 Hz, 2H), 3.80 (s, 3H) 230 nh ₂ A and xj «№> M 307.2 0.98 6 12.74 (s, 1H), 8.64 (brt, 7=5.0 Hz, 1H), 8.37 (s, 1H), 8.22 (d, 7=8.5 Hz, 1H), 7.96 (s, 1H), 7.88 - 7.75 (m, 4H), 7.23 (s, 1H), 6.85 (d, 7=2.1 Hz, 1H), 5.92 (s, 1H), 4.64 (brd, 7=5.2 Hz, 2H) 231 Ζ _χ \ ^ZI \A— ^z \=Z 1- z )=/^ ^z __/ I 335.3 0.97 6 8.55 (s, 1H), 8.28-8.18 (m, 1H), 8.11 - 8.04 (m, 1H), 8.00 (s, 1H), 7.95 (brt, 7=5.1 Hz, 1H), 7.85 (br d, 7=8.7 Hz, 2H), 7.63 (brd, 7=1.3 Hz, 2H), 6.86 (d, 7=1.9 Hz, 1H), 5.76 (s, 1H), 4.34 (t, 7=6.9 Hz, 2H), 3.29 (q, 7=6.1 Hz, 1H), 2.28-2.18 (m, 2H). One proton is not visible in NMR, probably due to overlap with the suppressed peak of water. 232 Q ⁵ ZI CM /-----( X / \ ² 4 /=\ ^z Sj IZ A 310.3 1.08 6 12.46 (br s, 1H), 8.31 (br d, 7=8.8 Hz, 1H), 7.93 (br d, 7=4.0 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.67 - 7.57 (t, 2H), 7.42 (br d, 7=6.4 Hz, 1H), 6.85 (d, 7=2.3 Hz, 1H), 5.90 (s, 1H), 4.31 4.23 (t, 1H), 3.77 -3.67 (t, 1H), 2.09- 1.98 (t, 1H), 1.95 1.76 (t, 3H), 1.72- 1.64 (t, 1H), 1.60- 1.49 (t, 1H)

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233 Г ² Z°” Ϊ Jl .□ йАГ ^й м 296.0 0.74 5 8.07 (d, 7=8.8 Hz, 1H), 7.81 7.69 (m, 2H), 7.57 (br d, 7=7.6 Hz, 1H), 7.09 - 6.94 (m, 1H), 6.78 (d, 7=1.8 Hz, 1H), 5.51 (s, 1H), 4.36 (brt,7=6.5 Hz, 1H), 4.04-3.91 (m, 1H), 2.43 - 2.32 (m, 2H), 2.30 - 2.20 (m, 2H) 234 Z Xvz \=/ Vz \_/ I /------' ¹⁴³ IZ 296.0 0.73 5 8.05 (d, 7=8.5 Hz, 1H), 7.77 7.73 (m, 1H), 7.73 - 7.68 (m, 1H), 7.54 (brd, 7=8.2 Hz, 1H), 7.03 - 6.93 (m, 1H), 6.77 (d, 7=1.8 Hz, 1H), 6.57 - 6.43 (m, 1H), 5.61 (s, 1H), 3.99-3.88 (m, 1H), 2.80-2.71 (m, 2H), 1.96 - 1.87 (m, 2H). One proton is not visible in NMR, probably due to overlap with the suppressed peak of water. 235 nh ₂ La 310.3 1.13 5 8.31 (brd,7=7.9 Hz, 2H), 7.93 (br s, 1H), 7.84 (br d, 7=8.5 Hz, 2H), 7.57 (br s, 2H), 7.41 (br d, 7=6.7 Hz, 1H), 6.85 (d, 7=1.8 Hz, 1H), 5.90 (s, 1H), 4.28 (brs, 1H), 3.78 -3.67 (m, 1H), 2.09-1.98 (m, 1H), 1.95 1.76 (m, 3H), 1.75 - 1.63 (m, 1H), 1.61-1.48 (m, 1H) 236 z _x Xvz Х=/ Ά z )=Л IZ Ζ— ^Ζ 'Z 335.0 1.00 5 8.20 - 8.13 (m, 1H), 8.11 (br d, 7=8.8 Hz, 1H), 8.00 - 7.92 (m, 1H), 7.90 - 7.78 (m, 3H), 7.72 - 7.62 (m, 2H), 6.85 (d, 7=1.9 Hz, 1H), 5.81 (s, 1H), 4.64 (t, 7=6.1 Hz, 2H), 3.803.73 (m, 2H), 2.21 (s, 3H) 237 νη ₂ 1^1 ⁿ N XJ Of/) nAV ,, An Ν _χ Τ H ₃ C 320.0 0.95 5 8.01 (brd,7=8.5 Hz, 1H), 7.80 -7.75 (m, 1H), 7.73 (brs, 1H), 7.54 (brd,7=8.2 Hz, 1H), 7.45 (s, 1H), 7.30-7.16 (m, 1H), 6.77 (d, 7=1.9 Hz, 1H), 5.78 (s, 2H), 4.26 (brd,7=4.8 Hz, lH), 2.19(s, 3H) 238 ζ _χ ΥΥ— ζ \=Ζ Χ—ζ \_/ I /--- ^{, Μ} ^ζ _/ I Λ 345.3 1.14 5 12.46 (s, 1H), 8.72 - 8.65 (m, 1H), 8.60 (brd,7=5.0 Hz, 1H), 8.21 (d, 7=8.6 Hz, 1H), 8.178.07 (m, 2H), 7.93 (d, 7=1.4 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.73 7.58 (m, 3H), 6.85 (d, 7=2.2 Hz, 1H), 5.80 (s, 1H), 3.39-3.28 (m, 2H), 2.87 - 2.80 (m, 2H), 2.10-1.98 (m, 2H)

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239 nh ₂ s <T % 351.0 1.09 δ 12.42 (s, 1H), 8.83 (s, 1H), 8.21 (brt, δ=5.6 Hz, 1H), 8.15 (d, δ=8.7 Hz, 1H), 7.93 (s, 1H), 7.87-7.81 (m, 2H), 7.69 - 7.59 (m, 1H), 6.85 (d, δ=2.3 Hz, 1H), 5.82 (s, 1H), 3.21-3.13 (m, 2H), 2.30 (s, 3H). Two protons are not visible in NMR, likely due to overlap with the suppressed water peak. 240 XZ Д r ZI cm > I xz\ ^z 4 >=\ ΧΖ _χ A z 306.0 0.94 δ 8.73 (br t, 7=5.6 Hz, 1H), 8.42 -8.29 (m, 1H), 8.21 (d, 7=8.7 Hz, 1H), 7.98 (s, 1H), 7.94 7.82 (m, 4H), 7.58 (s, 2H), 6.87 (d, 7=2.3 Hz, 1H), 5.70 (s, 1H), 4.89 (d, 7=5.5 Hz, 2H) 241 z^z X / Z—' ^~C/=\ \ ) XZ _s A 'z 334.0 0.99 δ 8.72 (brs, 1H), 8.21 (brd, 7=8.6 Hz, 1H), 8.08-8.01 (m, 1H), 7.98-7.91 (m, 1H), 7.85 (br d, 7=8.4 Hz, 2H), 7.70 - 7.60 (m, 3H), 7.47 (s, 1H), 6.86 (d, 7=1.9 Hz, 1H), 5.77 (s, 1H), 4.27 (brt, 7=6.9 Hz, 2H), 3.35 3.25 (m, 1H), 2.23 (quin, 7=6.9 Hz, 2H). One proton is not visible in the NMR, probably due to overlap with the suppressed water peak. 242 g ZT - ¹ - CM » ( T /\ ^Ζ Λ >=\ ^z V/ xz _x A z 320.3 1.00 δ 8.69 - 8.59 (m, 1H), 8.27 (br d, 7=8.1 Hz, 1H), 8.01-7.93 (m, 1H), 7.86 (br d, 7=7.6 Hz, 2H), 7.77-7.61 (m, 2H), 7.35 (s, 1H), 6.86 (s, 1H), 6.27 (s, 1H), 5.82 (s, 1H),4.61 (brd, 7=5.3 Hz, 2H), 3.87 (s, 3H) 243 nh ₂ H K/VN ^N U ⁴ 320.1 0.87 δ 8.61 (br d, 7=4.6 Hz, 1H), 8.52 (brs, 1H), 8.23 (brd, 7=8.5 Hz, 1H), 7.97 (br s, 1H), 7.87 (br d, 7=8.9 Hz, 2H), 7.79 (br d, 7=1.3 Hz, 2H), 7.45 (br s, 1H), 6.86 (s, 1H), 5.85 (s, 1H), 4.55 (br d, 7=4.4 Hz, 2H), 3.76 (s, 3H) 244 nh ₂ A. H £J йА> M 323.2 0.88 δ 9.03 (s, 1H), 8.78 (brs, 1H), 8.18 (brd,7=8.4 Hz, 1H), 7.94 (brd, 7=9.5 Hz, 2H), 7.90 - 7.80 (m, 2H), 7.75 - 7.60 (m, 1H), 6.86 (brs, 1H), 5.88 (brs, 1H), 4.81 (brd, 7=4.5 Hz, 2H)

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245 z z \=Z 1 I f m zz o I 310.0 0.89 5 8.05 (brd,7=8.4 Hz, 1H), 7.81 -7.76 (m, 1H), 7.75 - 7.70 (m, 1H), 7.63 - 7.54 (m, 1H), 6.96 -6.84 (m, 1H), 6.80 (brs, 1H), 5.72 (s, 1H), 4.23-4.13 (m, 1H), 2.35 - 2.24 (m, 1H), 2.08 - 1.96 (m, 1H), 1.81 (brs, 1H), 1.81 - 1.70 (m, 1H), 1.701.57 (m, 2H). One proton is not visible in NMR, probably due to overlap with the suppressed water peak. 246 z \ ^zx Kz ^=4 1 2 · m zz o I 310.0 0.89 5 8.32 (brd,7=8.4 Hz, 1H), 7.97 -7.89 (m, 1H), 7.87 - 7.79 (m, 2H), 7.76 (brd, 7=6.3 Hz, 1H), 7.68 - 7.54 (m, 2H), 6.85 (s, 1H), 5.85 (s, 1H), 4.35 -4.24 (m, 1H), 4.12-4.03 (m, 1H), 2.30- 2.18 (m, 1H), 2.10-2.00 (m, 1H), 1.99 - 1.82 (m, 2H), 1.74 - 1.61 (m, 1H), 1.61 - 1.51(m, 1H) 247 z \ ^zx z \=Z 4—z \_/ T Z------' ¹⁴³ zz o T 310.1 0.83 5 8.02 (d, 7=8.5 Hz, 1H), 7.78 7.75 (t, 1H), 7.74 - 7.68 (t, 1H), 7.60 - 7.50 (t, 1H), 6.81 6.72 (t, 2H), 5.72 (s, 1H), 4.22 -4.14 (t, 1H), 3.86 -3.78 (t, 1H), 2.30 (dt, 7=13.8, 7.0 Hz, 1H), 2.07- 1.96 (t, 1H), 1.841.71 (t, 2H), 1.70- 1.56 (t, 2H) 248 z \ ^zz: /L-Z \=Z 4—z \_/ T Z------' ¹⁴³ zz T 310.2 0.89 5 8.13 (d, 7=8.5 Hz, 1H), 7.79 (s, 1H), 7.76-7.71 (t, 1H), 7.67 (brd,7=7.9 Hz, 1H), 7.17 (br dd, 7=7.9, 5.2 Hz, 1H), 6.82 (d, 7=1.8 Hz, 1H), 5.78 (s, 1H), 4.32 -4.23 (t, 1H), 4.05 (brd, 7=6.4 Hz, 1H), 2.27 - 2.20 (t, 1H), 2.05- 1.91 (t, 2H), 1.881.82 (t, 1H), 1.64- 1.49 (t, 2H) 249 nh ₂ /u 307.2 0.86 5 8.05 (s, 1H), 7.97 (d, 7=8.5 Hz, 1H), 7.75 (s, 1H), 7.71 (br s, 1H), 7.55 (brd,7=7.3 Hz, 2H), 7.18 (s, 1H), 6.77 (d, 7=1.8 Hz, 1H), 6.31 (brs, 1H), 5.74 (s, 1H), 4.56 (brd,7=5.5 Hz, 2H) 250 nh ₂ s///? V 324.1 0.94 5 8.00 (d, 7=8.9 Hz, 1H), 7.77 (s, 1H), 7.73 (br s, 1H), 7.59 7.51 (t, 1H), 6.76 (d, 7=1.8 Hz, 1H), 6.71 -6.56 (t, 1H), 6.16 (brd,7=3.7 Hz, 1H), 5.77 (s, 1H), 4.02 (br s, 1H), 1.85 - 1.65 (t, 4H), 1.64- 1.47 (t, 2H),

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1.44 - 1.30 (m, 2H). One proton is not visible in NMR, likely due to overlap with the suppressed water peak. 251 z \ ' ^ZI k=Z V- z \_/ X /----' ^yu XZ O( ) I \__/ 324.1 1.13 δ 8.35 (brd, 7=8.5 Hz, 1H), 7.94 (brs, 1H), 7.89 -7.79 (w, 2H), 7.71 (br s, 2H), 7.27 (br d, 7=7.0 Hz, 1H), 6.84 (s, 1H), 5.89 (s, 1H), 4.05 (brs, 1H), 3.56 -3.47 (m, 1H), 1.95-1.47 (m, 6H), 1.42 - 1.30 (m, 2H) 252 A ZI - ¹ - CM !-----1 X /=\ IZ &'z 320.2 0.82 6 8.87 (s, 1H), 8.51 (brt,7=4.9 Hz, 1H), 8.23 (d, 7=8.5 Hz, 1H), 7.96 (br s, 1H), 7.89-7.75 (m, 4H), 7.60 (s, 1H), 6.86 (d, 7=2.4 Hz, 1H), 5.91 (s, 1H), 4.66 (br d, 7=4.6 Hz, 2H), 3.86 (s, 3H) 253 co X o ZI I ( T. ^z 4 >=\ ^z 'Y XZ 'z 240.2 0.72 δ 8.10 (d, 7=8.8 Hz, 1H), 8.067.98 (m, 1H), 7.95 - 7.74 (m, 3H), 7.48 - 7.30 (m, 1H), 6.83 (s, 1H), 5.70 (s, 1H), 2.91 (d, 7=4.6 Hz, 3H) 254 Z _s \ ' ^ZI /Vz \=Z У-z \_/ X /---' ^M IZ 0-0 \ x \ / ω '----( О I 314.1 0.73 δ 7.97 (d, 7=8.5 Hz, 1H), 7.80 7.75 (m, 1H), 7.73 (br s, 1H), 7.59 -7.52 (m, 1H), 6.82 (brd, 7=2.1 Hz, 1H), 6.77 (d, 7=1.8 Hz, 1H), 6.62 - 6.47 (m, 1H), 5.74 (s, 1H), 4.01 -3.90 (m, 1H), 3.38 (brd,7=5.2 Hz, 1H), 3.29 (s, 3H), 3.28-3.21 (m, 1H), 3.18-3.10 (m, 1H). One proton is not visible in NMR, probably due to overlap with the suppressed water peak. 255 z \ ^ZI Xvz ^=/ V- z \_/ I ) ¹ yu X IZ co \ °' ) 284.0 0.89 δ 8.26-8.21 (m, 1H), 8.13 (brt, 7=5.3 Hz, 1H), 7.94 (br s, 1H), 7.84 (br d, 7=7.3 Hz, 2H), 7.58 (brs, 1H), 6.85 (d, 7=2.1 Hz, 1H), 5.85 (s, 1H), 3.62 (t, 7=5.5 Hz, 2H). Five protons are not visible in the NMR spectrum, likely due to overlap with the suppressed water peak. 256 z 'zx ΛΧ _z \=Z У“ ^H \_/ X ) ^f ho d ^! 316.2 1.10 δ 8.11 -8.05 (m, 1H), 7.78 (s, 1H), 7.74 (br s, 1H), 7.67 (br s, 1H), 7.59 (brd,7=8.5 Hz, 1H), 7.41-7.33 (m, 5H), 7.31 - 7.12 (m, 1H), 6.78 (d, 7=1.8 Hz, 1H), 6.49 (br s, 2H), 5.62 (s, 1H), 4.49 (br d, 7=5.9 Hz, 2H)

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257 \ ' ^z=c \=/ Z \_/ X ° хЧГ 409.2 1.10 δ 8.83 - 8.75 (m, 1H), 8.29 (br d, 7=8.4 Hz, 1H), 7.96 (br s, 1H), 7.90 (brd,7=8.1 Hz, 2H), 7.59 (br s, 2H), 7.47 -7.26 (m, 2H), 7.26-7.16 (m, 1H), 7.12 (br d, 7=7.4 Hz, 2H), 6.88 (s, 1H), 5.68 (s, 1H), 4.57 (brd, 7=5.6 Hz, 2H), 2.96 (s, 3H) 258 nh ₂ A h Hf M 11 NH o=s=o 1 409.1 0.92 6 8.13-8.03 (m, 1H), 7.79 (s, 2H), 7.77 - 7.70 (m, 1H), 7.70 7.49 (m, 1H), 7.35 (d, 7=8.4 Hz, 2H), 7.18 (d, 7=8.5 Hz, 2H), 6.81 (d, 7=1.8 Hz, 1H), 6.776.63 (m, 1H), 5.64 (s, 1H), 4.44 (br d, 7=5.8 Hz, 2H), 2.96 (s, 3H) 259 nh ₂ A 1 H fi[ L ^H cd O\ 346.1 1.17 δ 8.83 - 8.60 (m, 1H), 8.26 (br d,7=8.5 Hz, 1H), 7.85 (brs, 2H), 7.50 (br s, 1H), 7.27 (t, 7=8.1 Hz, 1H), 6.95 - 6.90 (m, 2H), 6.89 - 6.80 (m, 2H), 5.69 (s, 1H), 4.52 (brd, 7=5.8 Hz, 2H), 3.72 (s, 3H) 260 z _s \ ' ^{ZI z} \=< Ήz \_/ X /---' ^yu /—Z __/ X ^m VH 334.1 1.19 δ 8.90 - 8.75 (m, 1H), 8.33 8.24 (m, 1H), 8.00 - 7.95 (m, 1H), 7.89 (br d, 7=8.9 Hz, 2H), 7.61 (br s, 2H), 7.53 -7.35 (m, 2H), 7.27-7.17 (m, 2H), 7.12 (brt, 7=9.0 Hz, 1H), 6.88 (s, 1H), 5.70 (s, 1H), 4.60 (brd, 7=5.8 Hz, 2H) 261 OJ ' ( T /\ ^Z A >=\ ^ZH U xz z 'z 320.1 1.06 δ 8.21 (brd, 7=6.7 Hz, 2H), 7.85 (br d, 7=2.0 Hz, 2H), 7.68 (brs, 2H), 7.57 (s, 1H), 6.90-6.85 (m, 1H), 6.20 (s, 1H), 5.88 (s, 1H), 3.O1 (brt, 7=7.5 Hz, 2H). One methylene is not visible, likely due to overlap with the suppressed water peak. 262 Z ₄ \ ' ^ζχ \=Z Ήz \_/ X 320.3 1.03 δ 8.13 (brd, 7=7.2 Hz, 1H), 8.01-7.90 (br, 1H), 7.85 (brd, 7=9.3 Hz, 1H), 7.80 - 7.70 (br, 1H), 7.70 - 7.59 (br, 1H), 7.59 7.40 (br, 1H), 6.86 (d, 7=2.1 Hz, 1H), 6.23 (t, 7=1.6 Hz, 1H), 5.76 (s, 1H), 3.59 -3.49 (br, 4H)

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263 nh ₂ N XO* ¹ M 320.1 0.90 δ 8.24 (brd,7=8.9 Hz, 1H), 8.03 (s, 1H), 7.87 (brs, 2H), 7.83 (brd,7=8.5 Hz, 1H), 7.77 (br s, 1H), 6.85 (br s, 1H), 6.35 -6.24 (m, 1H), 6.21 (s, 1H), 4.62 (br s, 2H), 3.01 (br s, 3H) 264 νη ₂ Δ η Μ] 1 ¹ %% Μ 331.0 0.96 δ 8.52 - 8.48 (m, 1H), 8.48 8.46 (m, 1H), 7.85 - 7.79 (m, 2H), 7.79 - 7.66 (m, 2H), 7.66 7.50 (m, 1H), 7.40 (dd, 7=7.6, 4.6 Hz, 1H), 6.76 (d, 7=2.1Hz, 1H), 6.77-6.71 (m, 1H), 6.26 (br s, 1H), 6.24 - 6.20 (m, 1H), 4.42 (s, 2H), 2.76 (s, 3H) 265 νη ₂ Π ζ-χ ^Η ίΥι / \^Ο Μ 310.2 1.08 δ 7.80 (d, J=1.2 Hz, 1H), 7.76 (s, 1H), 7.71 (br s, 1H), 7.56 (br d, J=8.2 Hz, 1H), 6.76 (d, J=2.1 Hz, 1H), 6.30-6.19 (m, 2H), 3.88-3.76 (m, 4H), 3.45-3.34 (m, 2H), 2.06 (quin, J=5.6 Hz, 2H). One proton from the side chain is missing in the NMR, likely due to overlap with the suppressed water peak. 266 Η ΖΙ MF t ( ΠΣ / \ ^Ζ Α /=\ ^ζ VA ΙΖ Λ 'ζ 326.2 0.88 δ 8.08 - 7.98 (m, 1H), 7.83 7.71 (br, 2H), 7.57 (br d, J=8.4 Hz, 1H), 7.13-7.00 (m, 1H), 6.84 -6.76 (m, 1H), 6.73 - 6.58 (m, 1H), 5.75 (s, 1H), 3.85 (br d, J=9.6 Hz, 2H), 3.81 -3.74 (m, 1H), 3.72 -3.55 (m, 2H), 3.41-3.29 (m, 1H), 3.26 - 3.20 (m, 1H). One proton from the side chain is missing in the NMR, probably due to overlap with the suppressed water peak. 267 \ '' ^Z - ^:L ζ \=< Α ζ ΞΖ ΙΖ^__/О 325.1 0.58 δ 8.32 - 8.20 (m, 1H), 8.17 (br t, J=5.5 Hz, 1H), 7.97 (brs, 1H), 7.86 (br d, J=8.2 Hz, 2H), 7.78 (br s, 2H), 6.86 (d, 1=1.2 Hz, 1H), 5.91 (s, 1H), 4.02 (br dd, J=12.1, 3.2 Hz, 2H), 3.80-3.69 (m, 1H), 3.46 -3.33 (m, 1H), 3.29-3.13 (m, 1H), 3.10-3.00 (m, 1H), 2.94 - 2.89 (m, 1H). The NMR is missing one proton from the side chain, probably due to overlap with the suppressed water peak.

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268 nh ₂ ϊΛ Η s-drA V 325.1 0.68 δ 8.20 (d, J=8.9 Hz, 1H), 7.98 7.92 (m, 1H), 7.91 -7.82 (m, 2H), 7.78 - 7.69 (m, 1H), 6.87 (d, J=1.8 Hz, 1H), 5.90 (s, 1H), 4.03 -3.93 (m, 1H), 3.86-3.78 (m, 1H), 3.16-3.07 (m, 1H), 3.03 -2.95 (m, 1H). A number of protons from the side chain are missing in the NMR, probably due to overlap with the suppressed peak of water. 269 νη ₂ ϊΛ Η ν 325.1 0.92 δ 8.18 (d, J=8.8 Hz, 1H), 8.138.06 (m, 1H), 7.97 (s, 1H), 7.92 - 7.87 (m, 1H), 7.85 (br s, 1H), 7.79 (br s, 2H), 6.87 (d, J=2.1 Hz, 1H), 5.92 (s, 1H), 4.06 (br dd, J=11.9, 2.1 Hz, 1H), 3.973.89 (m, 1H), 3.78 - 3.53 (m, 2H), 3.52-3.41 (m, 1H), 3.34 (brd, J=13.1 Hz, 1H),3.163.07 (m, 1H), 2.97-2.86 (m, 1H) 270 And ² II? ^Нз Ϊ Η II СН ₃ к Ju ^й J 381.1 1.21 δ 13.27-13.10 (m, 1H), 12.6912.60 (m, 1H), 8.34 (brd, J=5.0 Hz, 1H), 8.30 (brd, J=8.5 Hz, 1H), 7.96 (br s, 1H), 7.89-7.83 (m, 2H), 7.76 - 7.75 (m, 1H), 7.81-7.73 (m, 1H), 6.86 (d, 1=1.7Hz, 1H), 5.59 (s, 1H), 4.40 -4.32 (m, 1H), 4.29 - 4.22 (m, 2H), 3.99 (br dd, 1=8.4,4.8 Hz, 2H), 1.40 (s, 9H). One extra proton, probably due to the TFA salt. 271 nh ₂ l L n Ml ^N A ^F m 301.9 1.10 δ 13.25 - 13.12 (m, 1H), 8.01 7.95 (m, 1H), 7.90 - 7.87 (m, 1H), 7.86 - 7.77 (m, 4H), 6.84 (d, 1=1.9Hz, 1H), 5.65 (s, 1H), 4.96 (brt, 1=11.8Hz, 4H) 272 nh ₂ aA Ao M '' he 310.2 1.02 ^a δ 8.02 - 7.96 (m, 1H), 7.81 (s, 1H), 7.78 - 7.72 (m, 1H), 7.63 7.55 (m, 1H), 6.77 (d, 1=1.7Hz, 1H), 6.67 - 6.50 (m, 2H), 6.08 6.00 (m, 1H), 4.88 -4.77 (m, 1H), 4.09 - 4.02 (m, 1H), 3.88 3.80 (m, 2H), 2.21-2.13 (m, 1H), 2.03 - 1.92 (m, 2H), 1.90 (s, 2H), 1.79- 1.70 (m, 1H)

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273 z \ ^n:s Κζ V- z \_/ X IZ^ 280.2 1.17 ^a 5 8.16-8.11 (m, 1H), 7.82 7.79 (m, 1H), 7.78 - 7.73 (m, 1H), 7.68 - 7.62 (m, 1H), 7.46 7.37 (m, 1H), 6.99 - 6.83 (m, 1H), 6.80 (d, 1=1.7Hz, 1H), 5.66 -5.63 (m, 1H), 4.01-3.95 (m, 1H), 2.45 -2.38 (m, 2H), 2.15-2.06 (m, 2H), 1.88 (s, 2H), 1.84- 1.75 (m, 2H) 274 nh ₂ ,^ΐΧ ^0Η 324.1 0.96 5 7.85 - 7.81 (m, 1H), 7.75 7.73 (m, 1H), 7.72 (s, 1H), 7.63 -7.55 (m, 1H), 6.76 (d, J=1.7 Hz, 1H), 6.43 - 6.33 (m, 2H), 6.23 (s, 1H), 4.65 - 4.52 (m, 1H), 2.69 (brt, 1=10.3Hz, 1H), 1.98-1.91 (m, 1H), 1.90 (s, 1H), 1.84-1.74 (m, 3H), 1.21 1.12 (m, 1H). Several protons from the piperidine ring are not visible, probably due to overlap with water/DMSO. 275 nh ₂ NA r-0 AA 296.1 0.97 5 8.17 - 8.09 (m, 1H), 7.827.80 (m, 1H), 7.78 - 7.72 (w, 1H), 7.62 - 7.53 (m, 1H), 6.81 6.74 (m, 1H), 5.79 -5.69 (w, 1H), 4.16-4.05 (w, 1H), 4.003.95 (m, 2H), 3.94 -3.85 (w, 2H), 2.34 -2.21 (w, 1H), 2.19 2.03 (m, 1H) 276 nh ₂ nA rQ йАА 296.4 0.96 5 8.27 - 8.22 (m, 1H), 7.90 7.87 (m, 1H), 7.87 - 7.85 (w, 1H), 7.80 - 7.77 (m, 1H), 6.92 6.81 (m, 1H), 5.93 -5.86 (w, 1H), 4.39 - 4.29 (m, 1H), 4.11 4.03 (m, 2H), 3.99 -3.88 (w, 2H), 2.51 -2.40 (m, 1H), 2.22 2.13 (w, 1H) 277 nh ₂ nA rQ AA <j 296.2 0.98 5 8.39 - 8.29 (m, 1H), 7.98 7.91 (m, 1H), 7.89 - 7.74 (w, 2H), 6.93 - 6.82 (m, 1H), 5.875.78 (m, 1H), 4.25 -4.14 (w, 1H), 4.01 -3.88 (m, 2H), 3.883.74 (m, 2H), 2.35 - 2.25 (t, 1H), 2.20 - 2.04 (t, 1H) 278 nh ₂ n^h A ⁰ «jA ^M 310.2 0.87 5 8.40 - 8.25 (t, 2H), 8.02 7.93 (t, 1H), 7.88-7.81 (t, 1H), 6.91 -6.80 (t, 1H), 6.01 5.90 (t, 1H), 3.79 -3.64 (t, 1H), 3.57 - 3.36 (t, 2H), 2.93 2.91 (t, 2H), 2.00- 1.90 (t, 2H), 1.81 - 1.64 (t, 2H)

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279 nh ₂ ήΰ and JU ^й Μ 310.4 0.75 5 8.29 - 8.15 (m, 1H), 7.94 7.85 (m, 2H), 7.82 - 7.76 (m, 1H), 7.39-7.31 (m, 1H), 6.87 6.82 (m, 1H), 4.14-4.05 (m, 1H), 3.64 - 3.54 (m, 2H), 3.51 3.41 (m, 2H), 1.99-1.71 (m, 4H) 280 νη ₂ Ал mJU ^й μ 282.0 0.75 5 8.13 - 8.03 (m, 1H), 7.80 7.74 (m, 1H), 7.63 - 7.54 (m, 1H), 7.39 - 7.28 (m, 1H), 6.84 6.75 (m, 1H), 6.26 - 6.09 (m, 1H), 5.48-5.41 (m, 1H), 4.96 4.87 (m, 2H), 4.71 -4.58 (m, 2H) 281 νη ₂ ό η nJU ^й ν 308.0 1.32 5 8.40 - 8.24 (m, 1H), 7.98 7.92 (m, 1H), 7.89 - 7.80 (m, 1H), 7.76 - 7.65 (m, 1H), 6.93 6.81 (m, 1H), 5.94 -5.83 (m, 1H), 3.03 -2.85 (m, 3H), 2.06 1.95 (m, 2H), 1.87- 1.76 (m, 2H), 1.51 - 1.29 (m, 4H) 282 . ΰ-θ kJU ^η μ 323.9 1.02 5 8.37 - 8.25 (m, 1H), 7.93 7.78 (m, 1H), 7.74 - 7.67 (m, 1H), 7.66 - 7.58 (m, 1H), 7.43 7.32 (m, 1H), 7.30-7.21 (m, 1H), 5.92 - 5.84 (m, 1H), 2.99 2.84 (m, 1H), 2.52-2.51 (m, 1H), 2.41 -2.32 (m, 1H), 2.121.86 (m, 1H), 1.61-1.43 (m, 1H), 1.37- 1.22 (m, 1H), 1.191.12 (m, 2H) 283 νη ₂ and JU Μ 324.0 1.02 5 8.35 - 8.23 (m, 1H), 7.78 7.65 (m, 1H), 7.73 - 7.65 (m, 1H), 7.63 - 7.55 (m, 1H), 7.39 7.32 (m, 1H), 7.30 - 7.20 (m, 1H), 5.91 -5.83 (m, 1H), 3.473.27 (m, 1H), 2.58 -2.54 (m, 1H), 2.54 - 2.47 (m, 1H), 2.41 2.33 (m, 1H), 2.13 - 1.90 (m, 1H), 1.60- 1.42 (m, 1H), 1.381.21 (m, 1H), 1.17-1.11 (m, 2H) 284 nh ₂ Ух H ГТ _Г ” ^ΰ ^ά 0.99 331.2 δ 8.59 (brs, 1H), 8.50 (brd, 7=3.1 Hz, 1H), 8.24-8.12 (m, 2H), 7.94 (s, 1H), 7.88 - 7.80 (m, 3H), 7.71 -7.60 (m, 2H), 7.45 (dd, 7=7.6, 5.2 Hz, 1H), 6.85 (d, 7=2.1Hz, 1H), 5.89 (s, 1H), 3.62 - 3.38 (m, 2H), 3.04 (brt, 7=7.5 Hz, 2H)

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285 nh ₂ cl hVn hnU 309.2 0.86 δ 13.22-13.17 (m, 1H), 8.20 8.12 (m, 1H), 8.11-8.02 (m, 1H), 8.01-7.92 (m, 1H), 7.91 7.82 (m, 2H), 7.80 - 7.64 (m, 2H), 6.86 (d, 7=1.9 Hz, 1H), 5.93-5.81 (m, 1H), 3.92 - 3.83 (m, 2H), 3.31 - 3.25 (m, 1H), 3.25 -3.18 (m, 1H), 3.17 - 3.14 (m, 1H), 2.21 -2.11 (m, 1H), 2.04- 1.90 (m, 2H), 1.75 - 1.67 (m, 1H) 286 nh ₂ rS v ^h HN' ⁿ 309.2 0.61 δ 7.82 - 7.76 (m, 2H), 7.72 (br s, 1H), 7.55 (br d, J=7.9 Hz, 1H), 6.76 (d, J=1.5 Hz, 1H), 6.23 (s, 1H), 6.19 (s, 1H), 3.44-3.32 (m, 2H), 3.08 -2.95 (m, 4H), 1.94 (brd, J=4.6 Hz, 2H). The two diazepine protons are not observed, likely due to overlap with the suppressed H2O peak. 287 nh ₂ Λ ,a _v U ^й ^ ^он Μ 300.3 0.72 δ 8.11 (brd,7=8.5 Hz, 1H), 7.90 -7.82 (br, 1H), 7.80 - 7.66 (br, 2H), 7.62 - 7.45 (m, 1H), 7.24 - 7.03 (br, 1H), 6.82 (s, 1H), 5.81 (s, 1H), 3.86-3.79 (br, 1H), 3.66 -3.53 (br, 1H), 3.49 -3.32 (br, 2H), 3.25-3.16 (m, 1H) 288 νη ₂ аШ hJJ ^й м 310.1 1.13 δ 8.34 - 8.26 (t, 1H), 7.96 7.90 (t, 1H), 7.88-7.81 (t, 2H), 7.62 - 7.58 (t, 1H), 6.90 6.77 (t, 1H), 6.04-5.92 (t, 1H), 4.02 - 3.90 (t, 1H), 3.863.78 (t, 1H), 3.67 -3.63 (t, 1H), 3.46-3.25 (t, 2H), 2.162.03 (t, 1H), 1.83 - 1.71 (t, 2H), 1.71 - 1.57 (t, 1H) 289 νη ₂ уА О _Β jJ ^s Μ 310.2 1.05 δ 8.39 - 8.23 (t, 1H), 8.01 7.92 (t, 1H), 7.93 - 7.79 (t, 2H), 7.63 - 7.59 (t, 1H), 6.90 6.82 (t, 1H), 6.03 -5.92 (t, 1H), 4.03 - 3.93 (t, 1H), 3.88 3.77 (t, 1H), 3.46 -3.33 (t, 2H), 2.20 - 2.02 (t, 1H), 1.861.72 (t, 2H), 1.72- 1.55 (t, 1H) 290 νη ₂ Μ 342.4 0.95 δ 8.17-8.16 (t, 1H), 8.168.14 (t, 1H), 8.11-8.09 (t, 1H), 7.89 - 7.85 (t, 2H), 7.80 7.78 (t, 1H), 3.86 -3.76 (t, 1H), 3.74-3.61 (t, 1H), 2.38 2.27 (t, 1H), 2.11 - 1.86 (t, 4H), 1.67- 1.32 (t, 4H) 291 Ζ ₄ \ ' ^Ζ=Ε tky _z \_/ I ζ-------' ^{143 τζ} . / U 340.4 0.90 δ 8.43 - 8.33 (t, 1H), 8.00 7.93 (t, 1H), 7.93 - 7.89 (t, 1H), 7.88 - 7.82 (t, 2H), 6.89 6.81 (t, 1H), 5.94 -5.85 (t, 1H), 4.20 -4.10 (t, 2H), 4.10 4.03 (t, 1H), 4.00 - 3.92 (t, 1H), 3.88 - 3.80 (t, 1H), 3.80 3.64 (t, 2H), 1.20-1.12 (t, 3H) 351 νη ₂ η ju m Jj 310.3 0.84 δ 8.32 - 8.19 (t, 1H), 8.17 8.05 (t, 1H), 7.99-7.91 (t, 1H), 7.62-7.41 (t, 1H), 6.926.80 (t, 1H), 5.91-5.81 (t, 1H), 3.86 - 3.77 (t, 1H), 3.763.71 (t, 1H), 3.71 -3.63 (t, 1H), 3.59 -3.44 (t, 1H), 3.323.23 (t, 2H), 2.75 -2.61 (t, 1H), 2.14- 1.96 (t, 1H), 1.741.58 (t, 1H)

^a LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase

A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with

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0.1% trifluoroacetic acid; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm).

Example II-3: Synthesis of 4-(1H-imidazol-1-yl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 167)

To a solution of 1H-imidazole (45.6 mg, 0.669 mmol) and 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1Hpyrazol-5-yl)quinolin-2-amine (22 mg, 0.067 mmol) in NMP (446 µL) was added potassium tert-butoxide (18.77 mg, 0.167 mmol). The reaction mixture was heated to 100°C overnight. The reaction mixture was diluted with water and extracted twice with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.4 mL DCM and 0.4 mL TFA. After 1 h, the reaction mixture was concentrated and azeotroped with DCM. The reaction was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 2% B, 2-42% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give (1.8 mg, 9.5%). Ή NMR (500 MHz, DMSO-d ₆ ) δ 8.50-8.28 (m, 1H), 8.10 (br s, 1H), 7.95-7.75 (m, 3H), 7.57 (br d, J=8.2 Hz, 1H), 7.40 (br s, 1H), 7.00-6.80 (m, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase A: 5:95. acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.75-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.89 min. M/Z=277.4.

Example II-4: Synthesis of 4-Substituted Quinolines from an Unprotected Intermediate

Step 1: Preparation of 4-chloro-7-(1H-pyrazol-5-yl)quinolin-2-amine

4-Chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (100 mg, 0.304 mmol) was dissolved in DCM (1.5 mL) and TFA (1.5 mL). After 1 h, the reaction was complete according to LC-MS. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in a small amount of DCM, and then saturated sodium bicarbonate solution was added. The precipitated solid was filtered, washed with water, and dried. The solid was suspended in saturated sodium bicarbonate solution, stirred for 15 min, then filtered and washed twice with water to give 4-chloro-7-(1H-pyrazol-5-yl)quinolin-2-amine (66 mg, 0.270 mmol, 89% yield). Ή NMR (400 MHz, DMSO4) δ 7.93-7.86 (m, 2H), 7.84-7.72 (m, 2H), 6.91 (s, 1H), 6.84 (d, J=2.1 Hz, 1H), 6.64 (br s, 2H).

Step 2: Preparation of N4-(3-(dimethylamino)propyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 2 TFA (compound 168)

To N1,N1-dimethylpropane-1,3-diamine (104 mg, 1.022 mmol) in DMA (681 µL) was added Hunig's base (53.5 µL, 0.307 mmol). The reaction mixture was heated to 120 °C overnight, then the reaction mixture was heated to 150 °C for an additional 24 h. The reaction mixture was cooled, quenched with AcOH, diluted with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 µm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 3 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford N4-(3-(dimethylamino)propyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 2 TFA (12.2 mg, 22.1%). Ή NMR (500 MHz, DMSO-d6) δ 8.21 (br d, J=8.5 Hz, 1H), 8.09 (br s, 1H), 7.95 (br s, 1H), 7.85 (br d, J=7.9 Hz, 2H), 7.78 (br s, 2H), 6.85 (s, 1H), 5.85 (s, 1H), 3.44-3.33 (m, 1H), 3.23-3.13 (m, 2H), 2.80 (s, 6H), 2.11-1.98 (m, 2H). Note: one proton is missing from the side chain methylene, likely due to overlap with the suppressed water peak. Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 µm particles; mobile phase

A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.75 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC

RT: 0.90 min. M/Z= 311.1.

Compounds 169-171 were prepared according to the synthetic methods described for compound 168 from the appropriate starting materials.

Connection No. Structure LC-MS [M+H] ⁺ RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 169 nh ₂ A ΑΑ _Ν ^° .nAU ^h hn T 270.0 1.11 δ 8.03 (brd,7=8.5 Hz, 1H), 7.80 (s, 1H), 7.74 (brs, 1H), 7.66 (brd, 7=7.6 Hz, 1H), 7.28 (brs, 1H), 6.83 (s, 1H),5.76 (s, 1H), 3.67 (brt, 7=5.8 Hz, 2H), 3.36 -3.27 (m, 2H) 170 co 0 ZI CN , ( T /\ >=\ A z 325.1 1.05 δ 8.17 (brd, 7=8.5 Hz, 1H), 8.07-8.01 (m, 1H), 7.97 (br s, 1H), 7.90 - 7.73 (m, 3H), 6.83 (d, 7=1.9 Hz, 1H), 5.95 (s, 1H), 3.71 (brd, 7=5.2 Hz, 2H), 1.29 - 1.19 (m, 6H). Several protons from the amine side chain are missing, likely due to overlap with the suppressed water peak. 171 nh ₂ Al .«AJ ⁿ V 284.0 0.81 δ 7.97 (brd, 7=8.5 Hz, 1H), 7.77 (s, 1H), 7.73 (br s, 1H), 7.55 (br d, 7=7.9 Hz, 1H), 6.96 (brs, 1H), 6.77 (s, 1H), 5.71 (s, 1H), 3.55 (brt, 7=6.1 Hz, 1H), 3.25 (brd, 7=5.8 Hz, 2H), 1.85 - 1.78 (m, 2H). One proton from the side chain alcohol is missing, likely due to overlap with the suppressed water peak.

Example 5: Synthesis of a 4-ether-substituted quinoline from an unprotected intermediate

N-ι

N-

KOfBu, DMSO

Cl

HE

Step 1: Synthesis of 4-((2-amino-7-(1H-pyrαzol-5-yl)quinolin-4-yl)oxy)butan-1-ol, TFA (compound 172)

To a solution of 4-chloro-7-(1H-pyrazol-5-yl)quinolin-2-amine (22 mg, 0.090 mmol) and butane-1,4-diol (81 mg, 0.899 mmol) in DMSO (599 µL) was added potassium tert-butoxide (20.18 mg, 0.180 mmol). The reaction mixture was heated to 100 °C overnight, then potassium tert-butoxide (10.09 mg, 0.90 mmol) was added and the reaction mixture was heated to 120 °C. After 8 h, the reaction mixture was cooled, quenched with AcOH, diluted with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 µm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient: 0 min hold at 0% B, 0-46% B over 25 min, then 6 min hold at 100% B; Flow rate: 20 mL/min; Column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford 4-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)butan-1-ol, TFA (5.2 mg, 14%). 1H NMR (500 MHz, DMSO-d6) δ 8.32-8.13 (m, 1H), 8.07-7.96 (m, 2H), 7.93-7.81 (m, 2H), 6.84 (s, 1H), 6.36 (s, 1H), 4.29 (br t, J=6.3 Hz, 2H), 3.55-3.34 (m, 2H), 1.98-1.86 (m, 2H), 1.73-1.60 (m, 2H). LC RT: 1.04 min. M/Z=299.31.

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Example II-6: Synthesis of a 4-amino-substituted quinoline with an N-linked pyrazole

Step 1: Preparation of 4-chloro-7-(1H-pyrazol-1-yl)quinolin-2-amine

7-Bromo-4-chloroquinolin-2-amine (250 mg, 0.971 mmol), 1H-pyrazole (132 mg, 1.942 mmol), copper(I) iodide (370 mg, 1.942 mmol), and sodium carbonate (412 mg, 3.88 mmol) were placed in a vial under pressure. The vial was placed under vacuum three times and backfilled with nitrogen. DMSO (9708 µL) was added, and nitrogen was bubbled through the solution. N,N'-dimethylethane-1,2-diamine (257 mg, 2.91 mmol) was added, and the reaction mixture was heated to 120°C. After 4 h, the reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were washed with a solution of half water/half saturated ammonium hydroxide, dried over sodium sulfate, and concentrated. The residue was dissolved in DCM/MeOH and absorbed onto silica gel. The residue was purified using ISCO (24 g column; DCM/MeOH; gradient from 0 to 10%) to give 4-chloro-7-(1H-pyrazol-1-yl)quinolin-2-amine (144 mg, 0.589 mmol, 60.6% yield). ^1H NMR (400 MHz, DMSO4) δ 8.67 (d, J=2.5 Hz, 1H), 7.96 (d, J=8.9 Hz, 1H), 7.90 (d, J=2.0 Hz, 1H), 7.85-7.79 (m, 2H), 6.93 (s, 1H), 6.77 (s, 2H), 6.61-6.57 (m, 1H).

Step 2: Preparation of 3-((2-amino-7-(1H-pyrazol-1-yl)quinolin-4-yl)amino)propan-1-ol (compound 173)

To a solution of 4-chloro-7-(1H-pyrazol-1-yl)quinolin-2-amine (20 mg, 0.082 mmol) and 3-aminopropan-1-ol (61.4 mg, 0.817 mmol) in DMSO (0.5 mL) was added Hunig's base (0.043 mL, 0.245 mmol). The reaction mixture was heated to 120 °C overnight. LC-MS showed that the reaction was complete. The reaction mixture was cooled, diluted with MeOH and a small amount of AcOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 3 min hold at 0% B, 0-38% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford 3-((2-amino-7-(1H-pyrazol-1-yl)quinolin-4-yl)amino)n-ropan-1-ol (14.2 mg, 61.3%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.55 (br s, 1H), 8.06 (br d, J=8.9 Hz, 1H), 7.75 (br d, J=14.0 Hz, 2H), 7.61 (br d, J=7.9 Hz, 1H), 7.03 (br s, 1H), 6.68 (br s, 1H), 6.56 (br s, 1H), 5.72 (s, 1H), 3.52 (br s, 2H), 3.30-3.17 (m, 2H), 1.84-1.77 (m, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 µm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 1.08 min. M/Z=283.96.

Compounds 174-176, compounds 292-309 were prepared according to the synthetic methods described for compound 173, from the appropriate starting materials.

Connection No. Structure LC-MS [M+H]+ RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 174 t o ZI 7 ZI CN , ( t / \ ^z Ч /=\ Μ 310.9 1.08 δ 8.55 (brs, 1H), 8.00 (brd, 7=8.9 Hz, 1H), 7.89 (brs, 1H), 7.75 (br d, 7=17.4 Hz, 2H), 7.59 (br d, 7=7.9 Hz, 1H), 6.98 (br s, 1H), 6.55 (br s, 1H), 6.49 (brs, 1H), 5.74 (s, 1H), 3.55 3.35 (m, 2H), 2.59 (brd, 7=4.3 Hz, 3H). Two protons from the side chain are not visible, likely due to overlap with the suppressed water peak or poor integration. 175 A V- ζ \__/ ζ: ΙΖ d 317.0 1.19 δ 8.60 (brs, 1H), 8.53 (brs, 1H), 8.45 (brd,7=3.1 Hz, 1H), 8.09 (brd, 7=8.9 Hz, 1H), 7.76 (brs, 2H), 7.71 (s, 1H), 7.67 (brd,7=4.6 Hz, 1H), 7.61 (brd, 7=9.2 Hz, 1H), 7.41 - 7.32 (m, 1H), 6.56 (br s, 1H), 6.25-6.16

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(m, 1H), 5.65 (s, 1H), 4.49 (br d, J=5.2 Hz, 2H) 176 nh ₂ УаУ ^oh £Г ^^с^ ^{снз 3} Vn 297.9 1.11 5 8.63 (brs, 1H), 8.37 (brd, 7=8.9 Hz, 1H), 7.97 (s, 1H), 7.91-7.79 (m, 3H), 7.60 (br s, 2H), 6.64 (br s, 1H), 5.98 (s, 1H), 3.26 (br d, 7=5.5 Hz, 2H), 1.24- 1.16 (m, 6H) 292 G ^A—z IZ li 306.1 0.82 5 8.55 (s, 1H), 8.07 (d, 7=8.8 Hz, 1H), 7.76 (d, 7=1.5 Hz, 1H), 7.72 (d, 7=2.3 Hz, 1H), 7.61-7.54 (m, 2H), 7.49 - 7.40 (m, 1H), 6.61 -6.52 (m, 1H), 6.35 (brd,7=2.1 Hz, 1H), 6.21 (d, 7=2.1 Hz, 1H), 5.78 (s, 1H), 4.42 (brd, 7=5.6 Hz, 2H) 293 NH ₂ - ' 331.2 1.2 5 8.67 - 8.54 (m, 2H), 8.28 8.16 (m, 2H), 8.03 - 7.94 (m, 2H), 7.91 -7.81 (m, 2H), 7.79 7.66 (m, 1H), 7.56 (brd, J=7.6 Hz, 1H), 7.50 - 7.42 (m, 1H), 6.64 (d, 1=1.8Hz, 1H), 5.88 (s, 1H), 3.68 (br s, 2H), 3.23 (brt, J=7.0 Hz, 2H) 294 G ^~A—z ^=/ V- z \_/ X xz ά x^ 307.0 0.78 5 8.59 (d, 7=2.1 Hz, 1H), 8.30 (br s, 1H), 8.14 (d, 7=8.8 Hz, 1H), 7.79 (s, 3H), 7.68 (dd, 7=8.9,2.1 Hz, 1H), 6.67 - 6.48 (m, 2H), 5.75 (s, 1H), 4.54 (br d, 7=5.8 Hz, 2H) 295 nh ₂ A n N. JU &H u 310.1 0.91 5 8.57 (d, J=2.1 Hz, 1H), 8.07 (d, J=8.8 Hz, 1H), 7.78 (d, 1=1.2Hz, 1H), 7.76 (d, J=2.1 Hz, 1H), 7.63 (dd, J=8.9,2.1 Hz, 1H), 6.58 (brd, >1.8 Hz, 2H), 6.41 -6.35 (m, 1H), 5.82 (s, 1H), 4.26 (brd, >2.4 Hz, 1H), 3.69-3.61 (m, 1H), 2.11 1.98 (m, 1H), 1.87 - 1.64 (m, 4H), 1.61 - 1.50 (m, 1H) 296 G <^A—z ^=/ z \_/ X z-------' ¹⁴³ IZ O X 270.2 0.93 ^a 5 8.57 (d, 7=2.4 Hz, 1H), 8.11 (d, 7=9.2 Hz, 1H), 7.78 (dd, 7=6.7, 1.5 Hz, 2H), 7.66 (dd, 7=8.9, 2.1 Hz, 1H), 7.14 (brs, 1H), 6.88 (br s, 1H), 6.58 (s, 1H), 5.77 (s, 1H), 3.78-3.61 (m, 1H), 3.31 (q, 7=5.6 Hz, 2H). One of the ethylene proton signals is minimized, probably due to overlap with the suppressed water peak.

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297 С? Ч— З \=/ А ^н \_/ I >' N) IZ L ω А 337.1 1.14 δ 8.56 (d, 7=2.4 Hz, 1H), 8.04 (d, 7=8.9 Hz, 1H), 7.84 - 7.72 (m, 2H), 7.64 (dd, 7=8.9, 2.1 Hz, 1H), 7.59 (d, 7=3.1 Hz, 1H), 7.33 -7.17 (m, 1H), 6.70 (br s, 1H), 6.57 (d, 7=1.5 Hz, 1H), 5.82 (s, 1H), 3.71 -3.54 (m, 1H), 3.40 (t, 7=7.0 Hz, 1H). The two ethylene proton signals are minimized, probably due to overlap with the suppressed water peak. 298 O %—ζ Α ^ζ \_/ I y ' th ΙΖ A C- ^ΖΞΕ 306.1 0.74 δ 8.57 (d, 7=2.1 Hz, 1H), 8.13 (d, 7=8.8 Hz, 1H), 7.85 - 7.76 (t, 2H), 7.68 (br dd, 7=8.9, 2.1 Hz, 2H), 6.96 (s, 2H), 6.77 (br s, 1H), 6.58 (d, 7=1.5 Hz, 1H), 5.75 (s, 1H), 4.48 (br d, 7=5.5 Hz, 2H) 299 nh ₂ 367.2 1.26 Ή NMR (500 MHz, DMSO-de) δ 8.76 (br d, 7=4.3 Hz, 1H), 8.58 (d, 7=2.4 Hz, 1H), 8.13 (d, 7=9.2 Hz, 1H), 8.00 (td, 7=7.7, 1.7 Hz, 1H), 7.80 (dd, 7=7.6, 1.8 Hz, 2H), 7.75 (d, 7=7.9 Hz, 1H), 7.69 (dd, 7=8.9, 1.8 Hz, 1H), 7.64 - 7.52 (m, 2H), 6.71 (br s, 1H), 6.60 - 6.52 (m, 1H), 5.97 (s, 1H), 4.18 (td, 7=14.7, 6.3 Hz, 2H) 300 νη ₂ 317.2 0.96 6 8.62 - 8.52 (m, 2H), 8.13 (d, 7=8.8 Hz, 1H), 7.81 -7.72 (m, 3H), 7.70 - 7.59 (m, 2H), 7.34 (d, 7=7.9 Hz, 1H), 7.32 - 7.26 (m, 1H), 6.57 (d, 7=1.8 Hz, 1H), 6.16 (brs, 2H), 5.60 (s, 1H), 4.55 (d, 7=5.8 Hz, 2H) 301 G <Хч—ζ \=/ Α ^ζ )=/-? ΙΖ II co co 312.1 1.24 δ 8.57 (d, 7=2.1 Hz, 1H), 8.05 (d, 7=9.2 Hz, 1H), 7.79 (s, 2H), 7.65 (dd, 7=8.9, 1.5 Hz, 1H), 7.22 (br d, 7=4.6 Hz, 1H), 6.77 (brs, 1H), 6.58 (s, 1H),5.73 (s, 1H), 3.34 -3.22 (m, 2H), 1.841.73 (m, 2H), 1.20 (s, 6H) 302 A zz ZI X / ⁼ \ ^H \\ /=\ ^z Ay Z—, =0 320.2 1.04 δ 8.62 - 8.54 (m, 1H), 8.04 (d, 1=9.2Hz, 1H), 7.81 (d, J=1.8 Hz, 1H), 7.79 (d, 1=1.2Hz, 1H), 7.75 (d, 1=1.8Hz, 1H), 7.69 (dd, J=9.0, 2.0 Hz, 1H), 7.49 (d, 1=1.2Hz, 1H), 7.34 (br s, 1H), 6.78 - 6.65 (m, 2H), 6.58 (d, 1=1.8Hz, 1H), 6.26-6.21 (m, 1H), 5.76 (s, lH),4.43(t, J=6.4 Hz, 2H), 3.63 (q, J=6.0 Hz, 2H)

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303 nh ₂ 337.1 1.01 5 8.54 (d, 1=2.4Hz, 1H), 8.13 8.08 (m, 1H), 7.76 (d, 1=1.2Hz, 1H), 7.73 (d, J=2.1 Hz, 1H), 7.61 (brdd, 1=8.9,2.1 Hz, 2H), 7.21 (s, 1H), 6.62 - 6.48 (m, 2H), 6.41 - 6.37 (m, 1H), 5.72 (s, 1H), 4.48 (brd, J=5.8 Hz, 2H), 2.63 (s, 3H) 304 νη ₂ An GP Η * Ν. Αχ J OH 310.1 0.93 5 8.62 - 8.52 (m, 1H), 8.06 (d, J=9.2 Hz, 1H), 7.77 (s, 1H), 7.74 (d, 1=1.8Hz, 1H), 7.62 (dd, J=8.7, 2.0 Hz, 1H), 6.63 6.58 (m, 1H), 6.56 (d, 1=1.8Hz, 1H), 6.44 - 6.35 (m, 1H), 5.81 (s, 1H), 4.24 (brd, J=2.4 Hz, 1H), 2.07 - 1.98 (m, 1H), 1.85 1.80 (m, 1H), 1.79 - 1.71 (m, 2H), 1.70- 1.62 (m, 1H), 1.601.50 (m, 2H). Several protons from the cyclopentyl ring are not visible, likely due to overlap with water/DMSO. 305 νη ₂ ' ^CHs 332.1 0.99 5 8.62 - 8.56 (b, 1H), 8.04 (d, J=8.8 Hz, 1H), 7.80 (d, J=2.1 Hz, 1H), 7.79 (d, J=1.2 Hz, 1H), 7.68 (dd, J=9.0, 1.7 Hz, 1H), 7.34 - 7.24 (b, 1H), 6.65 - 6.53 (b, 3H), 5.80 (s, 1H), 3.74-3.62 (b, 2H), 3.58 -3.47 (b, 1H), 3.08 (s, 3H). The proton from the ethyl chain is not visible, probably due to overlap with water/DMSO. 306 νη ₂ 311.1 0.77 5 8.60-8.52 (br, 1H), 8.14 (brt, I=5.2 Hz, 1H), 7.99 (d, J=8.5 Hz, 1H), 7.77 (d, J=1.2 Hz, 1H), 7.75 (d, I=2.1 Hz, 1H), 7.62 (dd, J=9.0, 2.0 Hz, 1H), 7.05 (br s, 1H), 6.60-6.51 (rn, 2H), 5.73 (s, 1H), 1.85 (s, 3H). Several protons from the ethyl chain are not visible, likely due to overlap with water/DMSO. 307 νη ₂ Λ ⁿ „ LJ η ¢// ^Ν 306.1 0.97 5 8.94-8.90 (rn, 1H), 8.65 (d, J=2.4 Hz, 1H), 8.62 - 8.57 (br, 1H), 8.30 (brd, J=9.2 Hz, 1H), 8.01 (d, J=1.8 Hz, 1H), 7.97 7.91 (in, 1H), 7.90-7.81 (br, 3H), 7.61 -7.57(t, 1H), 6.67 6.60 (t, 1H), 5.82 (s, 1H), 4.63 (br d, J=5.2 Hz, 2H) 308 νη ₂ nA ν^\ 321.1 0.74 5 8.57 (d, 7=2.1 Hz, 1H), 8.50 (s, 1H), 8.01 (d, 7=10.0 Hz, 2H), 7.79 (dd, 7=3.5, 1.7 Hz, 2H), 7.66 (dd, 7=8.9, 1.8 Hz, 1H), 7.27 (br s, 1H), 6.64 -6.56 (t, 2H), 5.76 (s, 1H), 4.49 (t, 7=6.0 Hz, 2H), 3.76 -3.55 (t, 2H) 309 νη ₂ Αυ Vn 318.1 1.11 5 9.17 - 9.10 (t, 1H), 8.54 (br d, 7=2.5 Hz, 1H), 8.12 (d, 7=9.2 Hz, 1H), 8.00 - 7.89 (t, 1H), 7.77 (d, 7=1.2 Hz, 1H), 7.74 (d, 7=1.9 Hz, 1H), 7.69 - 7.66 (t, 1H), 7.65 (d, 7=4.7 Hz, 1H), 7.64-7.61 (t, 1H), 6.59 -6.54 (t, 1H), 6.48 - 6.34 (t, 1H), 5.64 (s, 1H), 4.77 (brd, 7=5.6 Hz, 2H)

^a LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase

A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with

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0.1% trifluoroacetic acid; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm).

Example II-7: Synthesis of a 4-ether-substituted quinoline with an N-linked pyrazole

Step 1: Preparation of N-(2-((2-amino-7-(1H-pyrazol-1-yl)quinolin-4-yl)oxy)ethyl)acetamide, TFA (compound 177)

Potassium tert-butoxide (22.93 mg, 0.204 mmol) was added to a solution of N-(2-hydroxyethyl)acetamide (63.2 mg, 0.613 mmol) and 4-chloro-7-(1H-pyrazol-1yl)quinolin-2-amine (20 mg, 0.082 mmol) in NMP (0.5 mL). The reaction mixture was heated to 100°C overnight. The temperature was then raised to 120°C and the reaction mixture was heated overnight. Potassium tert-butoxide (11.5 mg, 0.102 mmol) was added and the reaction mixture was heated for an additional 6 h. The reaction mixture was cooled, diluted with MeOH and a small amount of AcOH, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 1% B, 1-41% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The substance was further purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 4 min hold at 0% B, 0-32% B for 25 min, then 6 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give N-(2-((2-amino-7-(1H-pyrazol-1-yl)quinolin-4-yl)oxy)ethyl)acetamide, TFA (2.8 mg, 8.1%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.59 (br s, 1H), 8.23 (br s, 1H), 8.01 (br d, J=8.8 Hz, 1H), 7.79 (br d, J=8.9 Hz, 2H), 7.67 (br d, J=8.5 Hz, 1H), 6.57 (br s, 1H), 6.18 (s, 1H), 4.12 (br t, J=4.7 Hz, 2H), 3.56 (br d, J=5.5 Hz, 1H), 1.86 (s, 3H). One proton from the side chain is not visible due to poor integration or overlap with the suppressed water peak. LC-MS conditions: column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0-100% B over 3 min, then 0.75 min hold at 100% B; flow rate: 1.0 mL/min; detection: UV at 220 nm. LC RT: 1.09 min. M/Z=312.1.

Example II-8: Preparation of a 4-substituted quinoline with a C-linked pyrazole

Step 1: Preparation of 2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)ethan-1-ol (compound 178)

To a solution of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (80 mg, 0.243 mmol) and ethane-1,2-diol (151 mg, 2.433 mmol) in NMP (1622 µL) was added potassium tert-butoxide (54.6 mg, 0.487 mmol). The reaction mixture was heated to 100°C overnight. LCMS showed that the reaction was complete. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were concentrated. The residue was dissolved in 1 mL DCM and 1 mL TFA. After 2 h, LCMS showed that the reaction was complete, and some trifluoroacetoacetic ester had been formed. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in 1 mL of MeOH and potassium carbonate (67.3 mg, 0.487 mmol) was added. After 1.5 h, LC-MS showed that the trifluoroacetoacetic ester was completely hydrolyzed. The reaction mixture was concentrated. The residue was purified using ISCO (24 g column; DCM/MeOH; gradient 0 to 20%) to give 2-((2-amino

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7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)ethan-1-ol (28 mg, 41.3% yield). 1H NMR (400 MHz, methanol-d ₄ ) δ 8.23 (d, J=8.5 Hz, 1H), 8.00-7.88 (m, 2H), 7.78 (br s, 1H), 6.85 (d, J=2.2 Hz, 1H), 6.37 (s, 1H), 4.37 (t, J=4.5 Hz, 2H), 4.09-4.01 (m, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.75-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.71 min. M/Z = 271.24.

Compounds 179-201, compounds 310-312 were prepared according to the synthetic methods described for compound 178, from the appropriate starting materials.___________________

Connection No. Structure LC-MS [m+n] ⁺ RT (min) Ή NMR (500 MHz, DMSO-cU) 179 nh ₂ M 285.4 0.93 δ 8.05 - 7.96 (m, 2H), 7.89 (br d, 7 8.8 Hz, 1H), 7.81 (br s, 1H), 6.83 (d, 7 = 2.2 Hz, 1H), 6.39 (s, 1H), 4.35 (t, 7 = 6.2 Hz, 2H), 3.66 (brt, 7=6.1Hz, 2H), 2.05 (quin, 7=6.2Hz, 2H) 180 o*° o o I /=\ ^ζ Λ /=\ ² V? iz _x 416.2 1.25 δ 7.87 (d, 7=8.4 Hz, 1H), 7.83 (s, 1H), 7.71 (brs, 1H), 7.64 (br s, 1H), 6.75 (s, 1H), 6.23 (s, 1H), 4.17 (t, 7=6.3 Hz, 2H), 3.04 (br d, 7=4.9 Hz, 4H), 2.96 -2.87 (m, 4H), 2.57 (brt,7=7.2 Hz, 2H), 1.88 (quin, 7=6.8 Hz, 2H), 1.66 (quin, 7=7.2 Hz, 2H)

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181 nh ₂ 352.1 1.11 5 7.94 (brd, 7=8.5 Hz, 1H), 7.90 (brs, 1H), 7.83 - 7.68 (m, 2H), 6.81 (s, 1H), 6.23 (s, 1H), 4.17 (brt, 7=5.6 Hz, 2H), 2.24 2.15 (m, 2H), 2.12-2.02 (m, 2H), 1.95 - 1.84 (m, 2H). The two methylenes are not visible in the NMR, likely due to overlap with the suppressed water peak. 182 z _x \ ' ^Hz: ζ \=Z Vz \_/ I О о I с 269.0 1.59 δ 8.40 - 8.19 (br, 1H), 8.06 - 7.97 (br, 2H), 7.93 - 7.83 (br, 2H), 6.84 (s, 1H), 6.37 (s, 1H), 4.23 (brt, 7=6.3 Hz, 2H), 1.96 - 1.84 (br, 2H), 1.07 (t, 7=7.3 Hz, 3H) 183 ζ \ ^ΖΞΕ \=Ζ χ— ^ζ ο <4 ο 338.1 1.13 δ 8.11 - 8.01 (br, 2H), 7.93 (br d, 7=8.4 Hz, 1H), 7.83 (br s, 1H), 6.83 (d, 7=2.2 Hz, 1H), 6.33 (s, 1H), 5.14 (s, 2H), 3.51 (brt, 7=6.7 Hz, 2H), 3.38 (brt, 7=6.9 Hz, 2H), 2.00 - 1.90 (m, 2H), 1.86 - 1.75 (m, 2H) 184 νη ₂ nA a ⁿ ' ^CH3 7\Α ___1 η Ν-Αζ να 381.0 1.21 5 7.84 (d, 7=8.4 Hz, 1H), 7.78 (s, 1H), 7.69 (s, 1H), 7.56 (brd, 7=7.8 Hz, 1H), 6.73 (d, 7=1.9 Hz, 1H), 6.19 (s, 1H), 4.13(t, 7=6.4 Hz, 2H), 2.42 - 2.26 (m, 8H), 2.13 (s, 2H), 1.87-1.81 (m, 2H), 1.69 - 1.59 (m, 2H). One methylene from the side chain is not visible in NMR, probably due to overlap with the suppressed water peak. 185 Ζ ₄ ' ^ζτ ΑΛ—Ζ \ Αχ 2 ' yu ο ο < ο τ 299.1 1.30 5 7.85 (d, 7=8.4 Hz, 1H), 7.80 (s, 1H), 7.70 (s, 1H), 7.58 (brd, 7=8.6 Hz, 1H), 6.74 (d, 7=1.9 Hz, 1H), 6.22 (s, 1H), 4.29 4.19 (br, 2H), 3.88 - 3.79 (br, 2H), 3.58 (q, 7=7.0 Hz, 2H), 1.15 (t, 7=7.0 Hz, 3H) 186 ζ \=/ Ά ^ζ \_/ τ 2 ' Μ ο 0 311.3 1.05 5 8.10 -7.98 (w, 2H), 7.92 (br d, 7=8.2 Hz, 1H), 7.86 (br s, 1H), 6.85 (s, 1H), 6.47 (s, 1H), 4.98 -4.86 (m, 1H), 3.97 - 3.88 (m, 2H), 3.59 (brt,7=8.5 Hz, 1H), 3.42-3.31 (m, 1H),2.11 (br d, 7=9.8 Hz, 2H), 1.82 (br d, 7=8.5 Hz, 2H)

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187 nh ₂ sJr ³ T ₀ Μ 325.2 1.30 5 8.02 (br d, 7=8.4 Hz, 2H), 7.92 - 7.77 (m, 2H), 6.82 (d, 7=1.9 Hz, 1H), 6.40 (s, 1H), 4.15 (d, 7=6.2 Hz, 2H), 3.93 (br dd, 7=11.2, 3.0 Hz, 2H), 3.41 (brt, 7=10.9 Hz, 1H), 2.21 (br s, 1H), 1.77 (brd, 7=11.1 Hz, 2H), 1.47 (qd, 7=12.2, 4.4 Hz, 2H). One proton is missing in the THP side chain, probably due to overlap with the suppressed water peak. 188 ζ \ ' ^Z=L dd- Ζ \ У? 2 ' th ¢/ ZI °h o I ω 312.3 0.97 5 8.10 (brs, 1H), 8.04 (d, 7=8.5 Hz, 1H), 7.93 (br s, 1H), 7.78 (brs, 2H), 6.83 -6.77 (m, 1H), 6.30 (s, 1H), 4.22 (brt, 7=5.0 Hz, 2H), 3.59 (q, 7=5.3 Hz, 2H), 1.86 (s, ZN) 189 νη ₂ |0 ⁿ m Ί _ζ ΝγΜ ν 318.0 1.12 5 8.62 (br d, 7=4.3 Hz, 1H), 7.95 (brd, 7=8.2 Hz, 1H), 7.92 -7.86 (m, 1H), 7.81 (s, 1H), 7.73 (br s, 1H), 7.65 - 7.56 (w, 2H), 7.42 - 7.36 (m, 1H), 6.78 (s, 1H), 6.31 (brs, 2H), 6.26 (s, 1H), 5.33 (s, 2H) 190 ζ УУ— ζ \=< Az \__/ ZC <е d 318.1 1.06 5 8.77 (s, 1H), 8.60 (brd,7=4.3 Hz, 1H), 7.98 (brd,7=7.6 Hz, 1H), 7.85 (brd,7=8.5 Hz, 1H), 7.80 (s, 1H), 7.72 (brs, 1H), 7.57 (br d, 7=8.2 Hz, 1H), 7.51 - 7.45 (w, 1H), 6.77 (s, 1H), 6.32 (br d, 7=7.9 Hz, 3H), 5.31 (s, 2H) 191 a ⁿ 'nh УУм/ Ν Ν Η ₂ Οχ Ό, Όη 325.3 0.99 5 7.84-7.75 (m, 2H), 7.71 (br s, 1H), 7.55 (brd, 7=8.2 Hz, 1H), 6.75 (s, 1H), 6.22 (br d, 7=9.2 Hz, 2H), 4.45 (brd, 7=3.7 Hz, 1H), 3.62 (brs, 1H), 2.17-2.05 (m, 2H), 1.89 (brs, 2H), 1.65 - 1.53 (m, 2H), 1.45 1.30 (w, 2H) 192 νη ₂ he LU Η MJ ⁰ ΝχΑζ Μ 311.0 1.02 5 8.33 - 8.12 (m, 1H), 8.03 (br d, 7=7.9 Hz, 2H), 7.96 - 7.80 (m, 2H), 6.85 (s, 1H), 6.34 (s, 1H), 4.18 (s, 2H), 0.63 (br d, 7=11.0 Hz, 4H). One methylene from the side chain is missing, probably due to overlap with the suppressed water peak.

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193 nh ₂ och ₃ Uh U n gg ⁰ M 299.4 1.10 6 8.02 (br d, 7=8.2 Hz, 2H), 7.98 - 7.80 (m, 2H), 6.85 (s, 1H), 6.37 (s, 1H), 4.32 (brt, 7=6.0 Hz, 2H), 3.61 -3.40 (m, 2H) (overlapped by the suppressed peak of water), 3.27 (s, 2H), 2.19- 2.06 (m, 2H) 194 nh ₂ A Jl ? ^Nz n ^N ^J/^ ^BUT 329.3 0.82 6 8.00 (br d, 7=8.2 Hz, 2H), 7.88 (br d, 7=9.8 Hz, 2H), 6.84 (brs, 1H), 6.38 (s, 1H), 4.06 (br s, 2H), 3.24 (brs, 2H), 1.00 (s, 3H). One methylene from the side chain is missing in the NMR, likely due to overlap with the suppressed water peak. 195 nh ₂ on _M A A ^cNz A 1 J ^SNz n P ⁰ mAA m 313.2 1.22 6 7.86-7.77 (w, 2H), 7.72 (br s, 1H), 7.57 (br d, 7=7.6 Hz, 1H), 6.77 (s, 1H), 6.30 (br s, 2H), 6.21 (s, 1H), 4.23 (brt, 7=6.7 Hz, 2H), 1.99 (brt, 7=6.7 Hz, 2H), 1.22 (s, 6H) 196 On νη ₂ ν a\ / n rr° nAV M 335.3 1.07 6 7.87 (brd, 7=8.5 Hz, 1H), 7.81-7.68 (m, 3H), 7.58 (brd, 7=7.0 Hz, 1H), 7.46 (s, 1H), 6.77 (s, 1H), 6.25 (brd, 7=11.0 Hz, 3H), 6.11 (s, 1H), 4.38 (brt, 7=6.6 Hz, 2H), 4.05 (brt, 7=5.6 Hz, 2H), 2.36 (brt, 7=6.1 Hz, 2H) 197 \ ' ^z:c \ Fl pvA ^=/ z \_/ I 2 ' yu a ο-Λό ΞΕ 313.2 0.79 6 8.09 (brd,7=8.5 Hz, 1H), 7.99-7.69 (w, ZN), 6.82 (br s, 1H), 6.34 (br s, 1H), 4.97 (br s, 1H), 4.58 -4.44 (m, 1H), 4.16 (brdd, 7=9.5, 5.5 Hz, 1H), 4.03 -3.86 (m, 2H), 3.79-3.64 (m, 2H) 198 X o CN > ( T /—\ >=\ ^Z A IZ Λ Z 339.2 0.82 6 7.84 (brd, 7=8.2 Hz, 1H), 7.78 (s, 1H), 7.75 - 7.50 (m, 2H), 6.76 (s, 1H), 6.35 (s, 1H), 6.24 (brs, 2H), 6.18 (s, 1H), 4.18 (br s, 2H), 3.60 -3.38 (m, 4H) (overlapped by the suppressed peak of water), 3.24 (brt, 7=7.8 Hz, 2H) 199 nh ₂ A °4 and UA N-AV M 310.0 1.06 6 8.19 (brs, 1H), 7.84-7.77 (m, 2H), 7.72 (br d, 7=4.9 Hz, 1H), 7.57 (brd, 7=7.6 Hz, 1H), 6.77 (s, 1H), 6.34 (s, 1H), 6.28 (br s, 2H), 5.02 (brt, 7=7.5 Hz, 1H), 2.74 - 2.62 (m, 1H), 2.19-2.06 (m, 1H). Two protons are not visible, possibly due to

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overlaps with suppressed water peak or poor integration. 200 NH ₂ Ϊ JI ? ³ */7- · · 326.0 0.79 δ 8.02 - 7.65 (m, 5H), 6.80 (br s, 1H), 6.24 (s, 1H), 4.18 (br s, 2H), 2.00 (br t, 7=6.3 Hz, 2H), 1.81 (s, 3H). Two side-chain protons are not visible, possibly due to overlap with the suppressed water peak. 201 Z /Vz )=/> o Z-Z V 321.3 1.13 δ 8.08 (s, 1H), 8.01-7.90 (m, 2H), 7.84 - 7.62 (m, ZN), 6.81 (br d, 7=15.9 Hz, 2H), 6.67 (br s, 1H), 3.71-3.55 (m, 2H), 2.69 (brt, 7=6.7 Hz, 2H) 310 X o o t / ⁼ \ ² Ч >=\ ^ζ V? iz _s 313.3 1.05 δ 7.89 (d, 7=8.5 Hz, 1H), 7.83 (s, 1H), 7.75 (br s, 1H), 7.70 7.54 (m, 1H), 6.79 (d, 7=1.8 Hz, 2H), 6.53 (br s, 2H), 6.22 (s, 1H), 3.87 (s, 2H), 3.39 (br s, 1H), 1.03 (s, 6H). One methylene is not visible, possibly due to overlap with the suppressed water peak. 311 νη ₂ N^J H ₃ C 322.0 1.01 δ 8.02-7.91 (m, ZH), 7.87 7.72 (m, 2H), 6.82 (s, 1H), 6.46 (s, 1H), 5.57 (brs, 2H), 4.11 (s, 3H) 312 nh ₂ A N ₄ J H ₃ C 322.2 1.00 δ 8.05 - 7.98 (m, 3H), 7.92 7.83 (m, 2H), 6.85 (d, 7=2.1 Hz, 1H), 6.54 (s, 1H), 5.65 (s, 2H), 3.97 (s, 3H)

Example II-9: Synthesis of 2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)propane-1,3-diol

Step 1: Preparation of 2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)propane-1,3-diol (compound 202)

To a solution of 2-phenyl-1,3-dioxan-5-ol (110 mg, 0.608 mmol) and 4-chloro-7-(1-(tetrahydro-2H-pyran-2yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) in NMP (406 µL) was added potassium tert-butoxide (17.06 mg, 0.152 mmol). The reaction mixture was heated to 100°C. After 5 h, the reaction mixture was cooled, diluted with water, and extracted three times with EtOAC. The organic layers were concentrated. The residue was dissolved in 0.8 mL MeOH, and 0.2 mL concentrated HCl was added. After 4 h, 0.2 mL HCl was added. After an additional 4 h, the reaction mixture was heated to 50°C overnight. The reaction mixture was concentrated and azeotroped with MeOH. The residue was dissolved in MeOH, neutralized _with solid _K2CO3 , filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 2-((2-amino-7-(1Hpyrazol-5-yl)quinolin-4-yl)oxy)propane-1,3-diol (4.1 mg, 28.1%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.94 (br d, J=8.2 Hz, 1H), 7.81 (s, 1H), 7.74 (br s, 1H), 7.60 (br d, J=7.6 Hz, 1H), 6.78 (s, 1H), 6.52 (br s, 1H), 6.30 (s, 1H), 4.49-4.40 (m, 1H), 3.81-3.61 (m, 4H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B:

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95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.56 min. M/Z=301.0.

Example II-10: Synthesis of 4-diaminoethane-substituted quinolines

Step 1: Preparation of tert-butyl (2-((2-amino-7-bromoquinolin-4-yl)amino)ethyl)carbamate

To a solution of 7-bromo-4-chloroquinolin-2-amine (200 mg, 0.777 mmol) and tert-butyl (2-aminoethyl)carbamate (622 mg, 3.88 mmol) in DMSO (3883 µL) was added Hunig's base (407 µL, 2.330 mmol). The reaction mixture was heated to 120°C overnight. The reaction mixture was diluted with water and extracted three times with EtOAc. The organic layers were dried over sodium sulfate and concentrated. The residue was purified using ISCO (24 g column; DCM/MeOH; gradient from 0 to 20%). The material was triturated with DCM/hexanes to give tert-butyl (2-((2-amino-7-bromoquinolin-4-yl)amino)ethyl)carbamate (170 mg, 57.4% yield) containing a small amount of residual tert-butyl (2-aminoethyl)carbamate. This material was used in the next step without further purification. 1H NMR (400 MHz, chloroform-d) δ 7.79 (d, J=2.0 Hz, 1H), 7.54 (d, J=8.7 Hz, 1H), 7.32-7.28 (m, 1H), 6.71 (br d, J=1.2 Hz, 1H), 6.125.87 (m, 2H), 5.53 (s, 1H), 5.14-5.04 (m, 1H), 3.63-3.53 (m, 2H), 3.36-3.26 (m, 2H), 1.48 (s, 9H).

Step 2: Preparation of tert-butyl (2-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5yl)quinolin-4-yl)amino)ethyl)carbamate

1-(Tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (171 mg, 0.613 mmol), tert-butyl (2-((2-amino-7-bromoquinolin-4-yl)amino)ethyl)carbamate (187 mg, 0.490 mmol), and _PdCl2 (dppf)-DCM adduct (40.1 mg, 0.049 mmol) were placed in a vial under positive pressure. The vial was placed under vacuum three times and backfilled with nitrogen. Dioxane (3270 µL) and tripotassium phosphate (2 M aqueous) (736 µL, 1.471 mmol) were added, and nitrogen was bubbled through the solution. The vial was capped and heated to 100°C overnight. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified using ISCO (12 g column; DCM/MeOH; gradient 0 to 20%) to afford tert-butyl (2-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)carbamate (160 mg, 0.354 mmol, 72.1% yield). 1H NMR (400 MHz, chloroform-d) δ 7.77 (d, J=8.5 Hz, 1H), 7.72 (s, 1H), 7.60 (d, J=1.5 Hz, 1H), 7.34 (br d, J=8.4 Hz, 1H), 6.85 (br s, 1H), 6.68-6.43 (m, 1H), 6.40 (d, J=1.7 Hz, 1H), 5.63 (s, 1H), 5.57 (br t, J=5.8 Hz, 1H), 5.25 (dd, J=10.2, 1.9 Hz, 1H), 4.13 (br dd, J=9.4, 1.6 Hz, 1H), 3.66-3.57 (m, 1H), 3.52 (br d, J=4.4 Hz, 2H), 3.28 (br d, J=3.3 Hz, 2H), 2.63-2.47 (m, 1H), 2.01 (br s, 1H), 1.86 (br d, J=12.7 Hz, 1H), 1.80-1.66 (m, 1H), 1.62-1.49 (m, 2H), 1.48-1.44 (m, 9H).

Step 3: Preparation of N4-(2-aminoethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine

M HCl in dioxane (3535 µL) was added to tert-butyl (2-((2-amino-7-(1-(tetrahydro-2H-pyran-2yl)-1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)carbamate (160 mg, 0.354 mmol). After 2 h, the reaction mixture was concentrated and azeotroped twice with DCM. 217 mg of material was obtained and carried on to the next reaction, assuming 50% purity. 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.20 (br d, J=8.5 Hz, 1H), 8.06-7.95 (m, 2H), 7.87 (br d, J=8.5 Hz, 4H), 6.86 (br s, 1H), 5.87 (s, 1H), 3.55 (br d, J=4.6 Hz, 2H), 3.21 (br d, J=5.2 Hz, 1H). One proton from the side chain is not visible, probably due to overlap with the suppressed water peak.

Step 4: Preparation of N-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-2-methylthiazole-4-carboxamide, TFA (compound 203)

N4-(2-Aminoethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (30 mg, 0.056 mmol) (assumed 50% HCl by weight) and 2-methylthiazole-4-carboxylic acid (16.01 mg, 0.112 mmol) were dissolved in DMF (0.4 mL). Triethylamine (0.078 mL, 0.559 mmol) and T3P (50% in DMF) (49.8 mg, 0.078 mmol) were added. After 2 h, the reaction was quenched with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm,

- 72 040307 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give N-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-2-methylthiazole-4-carboxamide, TFA (15.1 mg, 52.5%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.69 (br s, 1H), 8.16 (br d, J=8.5 Hz, 1H), 8.11 -8.04 (m, 2H), 7.92 (br s, 1H), 7.87-7.77 (m, 2H), 7.59 (br s, 2H), 6.85 (s, 1H), 5.85 (s, 1H), 3.67 -3.35 (m, 4H), 2.68 (s, 3H). LC RT: 1.07 min. M/Z =394.34.

Compound 204 was prepared according to the synthetic methods described for compound 203 from the appropriate starting materials.

^1H NMR (500 MHz, DMSO-d6) δ 9.22-9.06 (m, 1H), 8.65 (br d, J=4.3 Hz, 1H), 8.11-8.04 (m, 1H), 8.037.93 (m, 2H), 7.77 (s, 1H), 7.72 (br s, 1H), 7.63-7.51 (m, 2H), 7.03 (br s, 1H), 6.77 (s, 1H), 6.51 (br s, 1H), 5.78 (s, 1H), 3.67 (br d, J=6.1 Hz, 1H), 3.51-3.33 (m, 1H). Two protons from the side chain are not visible, likely due to poor integration or overlap with the suppressed water peak. LC RT: 1.06 min. M/Z = 374.3.

Example II-11: Preparation of methyl (2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)carbamate (compound 205)

To a suspension of N4-(2-aminoethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (30 mg, 0.056 mmol) (assumed 50% HCl by weight) in DMF (0.4 mL) was added methyl chloroformate (6.49 µL, 0.084 mmol). After 1.5 h, 4 µL of methyl chloroformate was added. After 40 min, the reaction was quenched with MeOH. K ₂ CO ₃ was added and the reaction mixture was stirred overnight. The reaction was quenched with AcOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 µm particles; Mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; Flow rate: 20 mL/min; Column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-60% B over 40 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford methyl (2-((2-amino-7(1H-pyrαzol-5-yl)quinolin-4-yl)amino)ethyl)carbomethylate (3.6 mg, 19.3%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.96 (br d, J=1.9 Hz, 1H), 7.86-7.71 (m, 2H), 7.64 (br d, J=6.7 Hz, 1H), 7.33-7.17 (m, 1H), 6.83 (br s, 1H), 5.83-5.69 (m, 1H), 3.55-3.46 (m, 2H), 3.37-3.22 (m, 3H). Two protons from the side chain are not visible, probably due to overlap with the suppressed peak of water. LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.89 min. M/Z = 327.12.

Example II-12: Preparation of N-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)methanesulfonamide, TFA (compound 206)

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To a suspension of N4-(2-aminoethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (30 mg, 0.056 mmol) (assumed 50% HCl by weight) in DMF (0.4 mL) was added MsCl (6.53 μL, 0.084 mmol). After 1.5 h, 3.5 μL of MsCl was added. After 40 min, the reaction was quenched with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford N-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)methanesulfonamide, TFA (5.7 mg, 21.5%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.17 (br d, J=8.5 Hz, 1H), 8.04 (br s, 1H), 7.93 (br s, 1H), 7.85 (br d, J=9.2 Hz, 2H), 7.62 (br s, 2H), 7.29 (br t, J=5.8 Hz, 1H), 6.86 (s, 1H), 3.43 (br d, J=5.5 Hz, 2H), 3.28 (br d, J=5.8 Hz, 2H), 2.93 (s, 3H). Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 µm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.85 min. M/Z = 347.27.

Example II-13: Preparation of 1-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-3-ethylurea, TFA (compound 207)

To a suspension of N4-(2-aminoethyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine (30 mg, 0.056 mmol) (assumed 50% HCl by weight) in DMF (0.4 mL) was added ethyl isocyanate (6.64 µL, 0.084 mmol). After 40 min, the reaction was quenched with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 µm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid. Gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation to yield 1-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-3-ethylurea (7.3, 38.5%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.36 (br s, 1H), 8.43-8.31 (m, 1H), 8.12 (br d, J=8.5 Hz, 1H), 7.94 (s, 1H), 7.88-7.78 (m, 2H), 7.63 (br s, 2H), 6.86 (s, 1H), 5.79 (s, 1H), 3.44-3.25 (m, 2H), 3.04 (br d, J=6.7 Hz, 2H), 0.99 (t, J=7.2 Hz, 3H). One methylene from the side chain is missing, probably due to overlap with the suppressed water peak. LC RT: 1.07 min. M/Z= 339.94.

Example II-14: Preparation of 3-(2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-1,1-dimethylurea (compound 208)

CH ₃

To a suspension of N4-(2-aminoethyl)-7-(1H-nirazol-5-yl)quinoline-2,4-diamine (30 mg, 0.056 mmol) (assuming 50% HCl by weight) in DMF (0.4 mL) was added dimethylcarbamoyl chloride (7.71 μL, 0.084 mmol). After 1.5 h, dimethylcarbamoyl chloride (7.71 μL, 0.084 mmol) was added. After additional

- 74 040307 Dimethylcarbamoyl chloride (7.71 µL, 0.084 mmol) was added over an additional 1.5 h and the reaction mixture was stirred overnight. Triethylamine (0.078 mL, 0.559 mmol) was added. After 2 h, the reaction was quenched with MeOH, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: column XBridge C18, 200 mm × 19 mm, 5 µm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid. Gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; Flow rate: 20 mL/min; Column temperature: 25°C. Fraction collection was triggered by UV signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate. Gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation to afford 3-(2-((2-amino7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)ethyl)-1,1-dimethylurea (8.7 mg, 45.9%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.91 (br d, J=8.5 Hz, 1H), 7.82-7.66 (m, 2H), 7.60 (br d, J=7.6 Hz, 1H), 7.34 (br s, 1H), 6.80 (s, 2H), 6.69-6.57 (m, 1H), 5.73 (s, 1H), 3.36 (br d, J=5.5 Hz, 2H), 3.26-3.16 (m, 2H), 2.80 (s, 6H). LC RT: 0.96 min. M/Z= 340.22.

Example II-15: Synthesis of 4-diaminopropane-substituted quinolines

Step 1: Preparation of tert-butyl (3-((2-amino-7-bromoquinolin-4-yl)amino)propyl)carbamate

To a solution of 7-bromo-4-chloroquinolin-2-amine (520 mg, 2.019 mmol) and tert-butyl (3-aminopropyl)carbamate (1759 mg, 10.10 mmol) in DMSO (5 mL) was added Hunig's base (1.058 mL, 6.06 mmol). The reaction mixture was heated to 120°C overnight. The reaction mixture was partitioned between DCM and water. The organic layer was dried over sodium sulfate and evaporated. The residue was purified by ISCO (40 g column; DCM/EtOAc; gradient 0 to 100%) to give tert-butyl (3-((2-amino-7-bromoquinolin-4-yl)amino)propyl)carbamate (478 mg, 1.20 mmol, 60% yield). 1H NMR (400 MHz, DMSOd ₆ ) δ 7.86 (d, J=8.8 Hz, 1H), 7.43 (d, J=2.0 Hz, 1H), 7.15 (dd, J=8.7, 2.0 Hz, 1H), 6.89 (brt, J=5.3 Hz, 1H), 6.72 (brt, J=5.0 Hz, 1H), 6.12 (s, 2H), 5.71 (s, 1H), 3.21-3.11 (m, 2H), 3.10-2.97 (m, 2H), 1.85-1.70 (m, 2H), 1.38 (s, 9H).

Step 2: Preparation of tert-butyl (3-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5yl)quinolin-4-yl)amino)propyl)carbamate

A two-phase solution of 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-1H-pyrazole (420 mg, 1.512 mmol), tert-butyl (3-((2-amino-7-bromoquinolin-4-yl)amino)propyl)carbamate (478 mg, 1.209 mmol), _PdCl2 (dppf)-DCM adduct (99 mg, 0.121 mmol), and tripotassium phosphate (2 M aqueous) (1.814 mL, 3.63 mmol) in dioxane (10 mL) was heated to 110°C overnight. The reaction mixture was diluted with 100 mL DCM, dried over sodium sulfate, and evaporated under reduced pressure. The residue was purified by ISCO (40 g column; DCM/MeOH; gradient 0 to 25%) to give tert-butyl (3-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)carbamate (400 mg, 0.85 mmol, 71% yield). LC-MS conditions: column: Aquity UPLC BEH C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase A: 100% water with 0.05% TFA; mobile phase B: 100% acetonitrile with 0.05% TFA; gradient: 2% B to 98% B over 1 min, then 0.50 min hold at 100% B; Flow rate: 0.8 ml/min. LC RT: 0.77 min. M/Z=467.

Step 3: Preparation of N4-(3-aminopropyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl

To a solution of tert-butyl (3-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline-4

- 75 040307 (yl)amino)propyl)carbamate (400 mg, 0.857 mmol) in dioxane (10 mL) was added HCl (4 M in dioxane) (4 mL, 16.00 mmol). After 2 h, the reaction mixture was evaporated under high vacuum to give N4(3-aminopropyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl (336 mg, 0.85 mmol, 100% yield). LC-MS conditions: column: Aquity UPLC BEH C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase A: 100% water with 0.05% TFA; mobile phase B: 100% acetonitrile with 0.05% TFA; Gradient: 2% B to 98% B over 1 min, followed by a 0.50-min hold at 100% B; Flow rate: 0.8 mL/min. LC RT: 0.45 min. M/Z=283.

Step 4: Preparation of N-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)-1-methyl-1-imidazole-2-carboxamide, 2 TFA (compound 209)

To a solution of N4-(3-aminopropyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl (30 mg, 0.077 mmol), 1-methyl-1H-imidazole-2-carboxylic acid (19.32 mg, 0.153 mmol), and triethylamine (0.213 mL, 1.532 mmol) in DMF (1 mL) was added T3P (50% in DMF) (97 mg, 0.153 mmol). After stirring the reaction at room temperature overnight, the reaction mixture was concentrated under high vacuum. The reaction mixture was diluted with 1 mL of 1:1 DMF:acetic acid, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 5 min hold at 0% B, 0-33% B for 25 min, then 5 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals.

Fractions containing the desired product were combined and dried by centrifugal evaporation to give N-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)-1-methyl-1-imidazole-2-carboxamide as a bis-trifluoroacetate salt (7.1 mg, 15%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.44 (br s, 1H), 8.22 (br d, J=8.7 Hz, 1H), 8.02 (br s, 1H), 7.93 (br s, 1H), 7.90-7.76 (m, 3H), 7.55 (br s, 1H), 7.30 (s, 1H), 6.99 (s, 1H), 6.87-6.80 (m, 1H), 5.85 (s, 1H), 3.94 (s, 3H), 2.01-1.93 (m, 2H). Four protons from the side chain are missing, probably due to overlap with the suppressed water peak. LC RT: 1.01 min. M/Z=391.1.

Compounds 210-212 were prepared according to the synthetic methods described for compound 209 from the appropriate starting materials.

Compound No. Structure LC-MS [M+H] ⁺ RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 210 nh ₂ xa k H ₃ cA A ³ S CH ₃ 422.1 1.21 δ 8.40-8.29 (m, 1H), 8.00 (br d, 7=8.9 Hz, 1H), 7.81 (br s, 1H), 7.74 (brs, 1H), 7.58 (brd, 7=7.9 Hz, 1H), 7.15-7.03 (m, 1H), 6.92-6.79 (m, 1H), 6.76 (s, 1H), 5.72 (s, 1H), 3.25 (brd, 7=5.8 Hz, 1H), 2.68 (s, 3H), 2.59 (s, 3H), 1.96-1.83 (m, 2H). Three protons from the side chain are not visible, possibly due to overlap with the suppressed water peak. 211 nh ₂ ⁿ id O gA 388.1 1.02 δ 8.77 (brs, 1H), 8.74-8.69 (m, 2H), 8.24 (brd, 7=8.6 Hz, 1H), 8.02 (br s, 1H), 7.99-7.91 (m, 2H), 7.87-7.79 (m, 2H), 7.79-7.72 (m, 2H), 7.72-7.52 (m, 1H), 6.83 (d, 7=1.9 Hz, 1H), 5.86 (s, lH), 2.02 (brt, 7=6.7 Hz, 2H). Four protons from the side chain are not visible, possibly due to overlap with the suppressed water peak. 212 nh ₂ A ^H If O A □ ° 388.1 0.95 δ 8.99 (s, 1H), 8.75 - 8.63 (m, 2H), 8.26 - 8.14 (m, 2H), 8.01 (br s, 1H), 7.93 (brd, 7=11.6 Hz, 1H), 7.84 7.74 (m, 2H), 7.50 (br dd, 7=7.8, 4.9 Hz, 2H), 6.82 (d, 7=1.9 Hz, 1H), 5.85 (s, 1H), 2.00 (quin, 7=6.7 Hz, 2H). Four protons from the side chain are not visible, possibly due to overlap with the suppressed water peak.

- 76 040307

Example II-16: Synthesis of N-(3-((2-amino-7-(III-pyrazol-5-yl)quinolin-4-yl)amino)propyl)acetamide, 2

TFA (compound 213)

To a solution of 4-(3-aminopropyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl (30 mg, 0.077 mmol), and triethylamine (0.213 mL, 1.532 mmol) in THF (1 mL) was added acetyl chloride (0.016 mL, 0.230 mmol). After stirring the reaction at room temperature overnight, the reaction mixture was concentrated under high vacuum. The reaction mixture was diluted with 1:1 DMF:acetic acid, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 3 min hold at 0% B, 0-33% B over 23 min, then 5 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford N-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)acetamide as bis-trifluoroacetate salt (17.5 mg, 52%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.01-7.89 (m, 2H), 7.74 (br s, 2H), 7.55-7.44 (m, 1H), 6.80 (br s, 1H), 6.75 (br s, 1H), 6.30 (br s, 1H), 5.70 (s, 1H), 3.23-3.11 (m, 2H), 1.84-1.73 (m, 5H). Two protons from the side chain are not visible, probably due to overlap with the suppressed peak of water. LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min. RT: 0.91 min. M/Z=325.1.

Example II-17: Synthesis of 3-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)-1,1-dimethylurea, 2 TFA (compound 214)

To a solution of 4-(3-aminopropyl)-7-(1H-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl (30 mg, 0.077 mmol), and triethylamine (0.213 mL, 1.532 mmol) in DMF (1 mL) was added dimethylcarbamoyl chloride (0.021 mL, 0.230 mmol). After stirring the reaction at room temperature overnight, the reaction mixture was concentrated under high vacuum. The reaction mixture was diluted with 1:1 DMF:acetic acid, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B over 25 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford 3-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)-1,1-dimethylurea as bis-trifluoroacetate salt (7.8 mg, 18%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.00 (br d, J=8.2 Hz, 1H), 7.78 (br s, 1H), 7.76-7.67 (m, 1H), 7.58 (br s, 1H), 7.06 (br s, 1H), 6.77 (br s, 2H), 6.35 (br s, 1H), 5.70 (br s, 1H), 3.20 (br s, 2H), 3.18-3.06 (m, 2H), 2.54 (brd, J=1.5 Hz, 6H), 1.80 (br s, 2H). LC/MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min. LC RT: 0.96 min. M/Z=354.1.

Example II-18: Preparation of N-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propyl)methanesulfonamide (compound 215)

- 77 040307

Compound 215

To a solution of 4-(3-aminopropyl)-7-(N-pyrazol-5-yl)quinoline-2,4-diamine, 3 HCl (30 mg, 0.077 mmol), and triethylamine (0.213 mL, 1.532 mmol) in DMF (1 mL) was added Ms-Cl (0.018 mL, 0.230 mmol). After stirring the reaction at room temperature overnight, the reaction mixture was concentrated under high vacuum. The reaction mixture was diluted with 1:1 DMF:acetic acid, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B over 24 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford X-(3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4yl)amino)propyl)methanesulfonamide as bis-trifluoroacetate salt (7.2 mg, 15%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.04-7.91 (m, 1H), 7.75 (br s, 2H), 7.54 (br s, 1H), 7.12-7.02 (m, 1H), 6.93-6.66 (m, 2H), 6.55-6.19 (m, 1H), 5.72 (s, 1H), 3.32-3.19 (m, 1H), 3.19-3.04 (m, 2H), 2.95-2.86 (m, 3H), 1.89 (s, 2H). One proton from the side chain is not visible, probably due to overlap with the suppressed water peak. LCMS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min. LC RT: 0.89 min. M/Z=361.1.

Example II-19: Synthesis of 3-(2-amino-7-(1H-pyrazol-1-yl)quinolin-4-yl)propan-1-ol (compound 216)

Step 1. 7-Bromoquinolin-4-yl trifluoromethanesulfonate

In a 1000 mL round-bottomed flask, 7-bromoquinolin-4-ol (22.4 g, 99.98 mmol, 1 equiv), Hunig's base (3.8 g, 299.93 mmol, 3 equiv), and DMAP (1.2 g, 10.00 mmol, 0.100 equiv) were placed in DMF (1000 mL, 6.84 mmol, 0.068 equiv), then trifluoromethanesulfonyl chloride (25.3 g, 150 mmol, 1.502 equiv) was added. The resulting solution was stirred for 16 h at room temperature in an ice/water bath. The resulting solution was diluted with 1.5 L of ethyl acetate. The resulting mixture was washed with 2 x 500 mL of water and 2 x 500 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated. The resulting mixture was concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5). This gave 20 g (56.18%) of 7-bromoquinolin-4-yl trifluoromethanesulfonate as a yellow solid. LC-MS: (ES, m/z): [M+H]+=355.9.

Step 2. 4-[3-(Benzyloxy)prop-1-yn-1-yl]-7-bromoquinoline

In a 1000 mL round-bottomed flask, which was purged and maintained under an inert atmosphere of nitrogen, 7-bromoquinolin-4-yl trifluoromethanesulfonate (10 g, 28.08 mmol, 1 eq.) in THF (280 mL) was added, followed by Hunig's base (10.9 g, 84.24 mmol, 3 eq.), CuI (1.1 g, 5.62 mmol, 0.2 eq.), [(prop-2-yn-1-yloxy)methyl]benzene (6.2 g, 42.12 mmol, 1.5 eq.), Pd(PPh ₃ ) ₄ (3.2 g, 0.1 eq.). The resulting solution was stirred for 16 h at 70 °C in an oil bath. The resulting mixture was concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5) to give 6 g (60.66%) of 4-[3-(benzyloxy)prop-1-yn-1-yl]-7-bromoquinoline in

- 78 040307 as a yellow solid. LC-MS: (ES, m/z): [M+H]+=352.0.

Step 3. 4-[3-(Benzyloxy)propyl]-7-bromoquinoline

A 500 mL round-bottomed flask was charged with 4-[3-(benzyloxy)prop-1-yn-1-yl]-7-bromoquinoline (6 g, 17.03 mmol, 1 equiv) and PtO ₂ (0.56 g, 2.64 mmol, 0.155 equiv) in acetonitrile (170 mL). The resulting solution was stirred for 16 h at room temperature under H ₂ . The solids were filtered off. The filtrate was concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:5). This gave 4.8 g (79.09%) of 4-[3-(benzyloxy)propyl]7-bromoquinoline as a yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 356.1.

Step 4. 4-[3-(Benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinoline

A 100 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with 4-[3-(benzyloxy)propyl]-7-bromoquinoline (1 g, 2.81 mmol, 1 equiv), 1H-pyrazole (955.5 mg, 14.03 mmol, 5 equiv), K3PO4 (1.2 g, 5.61 mmol, 2 equiv), CuI (106.9 mg, 0.56 mmol, 0.2 equiv), and (1S,2S)cyclohexane-1,2-diamine (32.1 mg, 0.28 mmol, 0.1 equiv) in dioxane (28 mL, 0.32 mmol, 0.113 equiv). The resulting solution was stirred for 5 days at 100°C in an oil bath. The solids were filtered off.

The resulting mixture was concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:1). This gave 370 mg (38.38%) of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinoline as a yellow solid. LC-MS: (ES, m/z): [M+H]+=344.2.

Step 5. 4-[3-(Benzyloxy)propyl]-7-( 1 H-pyrazol-1 -yl)quinolin-1 -ium-1 -olate

In a 50 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinoline (300 mg, 0.87 mmol, 1 equiv) was added in DCM (10 mL), followed by m-CPBA (226.1 mg, 1.31 mmol, 1.5 equiv).

The resulting solution was stirred for 12 h at room temperature. The reaction was then quenched by adding 10 mL _{of Na2S2O4} . The pH of the solution was adjusted to 10 _{with NaHCO3} . The resulting solution was extracted with 3x10 mL of dichloromethane. The resulting mixture was washed with 1x10 mL of brine. The resulting mixture was concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). This gave 200 mg (63.70%) of 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinolin-1-ium-1-olate as a yellow solid. LC-MS: (ES, m/z): [M+H]+=360.2.

Step 6. 4-[3-(Benzyloxy)propyl]-7-( 1 H-pyrazol-1-yl)quinolin-2-amine

In a 50 mL round-bottomed flask, 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinolin-1-ium-1-olate (150 mg, 0.42 mmol, 1 eq.) was added to DCM (2 mL, 0.02 mmol) and NH4OH (1 mL), followed by the addition of TsCl (159.1 mg, 0.83 mmol, 2 eq.). The resulting solution was stirred for 20 min at room temperature. The resulting mixture was concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). As a result, 100 mg (66.85%) of 4-[3-(benzyloxy)propyl]-7(1H-pyrazol-1-yl)quinolin-2-amine was obtained as a yellow solid. LC-MS: (ES, m/z): [M+H]+=359.2.

Step 7. 3-[2-Amino-7-(1H-pyrazol-1-yl)quinolin-4-yl]propan-1-ol (compound 216)

A 850 mL round-bottomed flask, purged and maintained under nitrogen, was charged with 4-[3-(benzyloxy)propyl]-7-(1H-pyrazol-1-yl)quinolin-2-amine (100 mg, 0.28 mmol, 1 equiv) in concentrated HCl (6 mL, 0.16 mmol, 0.590 equiv). The resulting solution was stirred for 2 h at 40 °C. The pH of the solution was adjusted to 10 with ammonium hydroxide. The resulting mixture was concentrated. The crude product was purified by preparative HPLC and flash chromatography using the following conditions: column Flash, C18 spherical, 20-35 μm, 100 Å, 20 g; Mobile phase: water (10 mmol/L NH4HCO3) and MeCN (15% phase B to 75% over 9 min); detector: UV 254/210 nm. This yielded 46 mg (61.45%) of 3-[2-amino-7-(1Hpyrazol-1-yl)quinolin-4-yl]propan-1-ol as a white solid. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.62 (d, J=2.5 Hz, 1H), 7.92 (d, J=8.9 Hz, 1H), 7.84 (d, J=2.2 Hz, 1H), 7.78 (d, J=1.7 Hz, 1H), 7.70 (dd, J=8.9, 2.3 Hz, 1H), 6.64-6.51 (m, 2H), 6.48 (s, 2H), 4.60 (t, J=5.2 Hz, 1H), 3.53 (q, J=6.0 Hz, 2H), 2.97-2.89 (m, 2H), 1.86-1.75 (m, 2H). LC Methods: Column: Kinetex EVO, 3.0 mm x 50 mm, 2.6 μm particles; Mobile Phase A: Water with 0.03% NH4OH; Mobile Phase B: Acetonitrile; Temperature: 40°C; Gradient: 10% B to 95% B over 1.9 min, then a 0.6-min hold at 95% B; Flow Rate: 1.2 mL/min. LC, Retention Time: 0.981 min. LC-MS: (ES, m/z): [M+H] ⁺ = 269.1.

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Example II-20: Synthesis of N-(2-(2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)ethyl)acetamide (compound

217)

Step 1. Benzyl N-[2-(7-bromoquinolin-4-yl)ethyl]carbamate

To _a 500 mL 3-necked round-bottomed flask, purged and maintained under nitrogen, were placed 7-bromoquinolin-4-yl trifluoromethanesulfonate (7.5 g, 21.06 mmol, 1 equiv), _Cs2CO3 (20585.8 mg, 63.18 mmol, 3.0 equiv), (2-[[(benzyloxy)carbonyl]amino]ethyl)boronic acid (9394.4 mg, 42.12 mmol, 2 equiv), and Pd(dppf)Cl2 (1541.0 mg, 2.11 mmol, 0.1 equiv) in toluene (200 mL) and H2O (50 mL). The resulting solution was stirred for 16 h at 70°C. The resulting mixture was cooled to room temperature and concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:1). This gave 3.5 g (43.14%) of benzyl N-[2-(7-bromoquinolin-4-yl)ethyl]carbamate as a white solid. LC-MS: [M+H]+=385.0.

Step 2. Benzyl N-[2-[7-(1H-pyrazol-3-yl)quinolin-4-yl]ethyl]carbamate

To a 500 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, were placed benzyl N-[2-(7-bromoquinolin-4-yl)ethyl]carbamate (2 g, 5.19 mmol, 1 equiv), 3-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1007.3 mg, 5.19 mmol, 1.0 equiv), Pd( _PPh3 ) ₄ (599.9 mg, 0.52 mmol, 0.1 equiv), and _Na2CO3 (1100.5 mg, 10.38 mmol, 2.0 equiv) in dioxane (80 mL, 944.33 mmol, 181.903 equiv) and _H2O (20 _mL , 1110.17 mmol, 213.848 equiv). The resulting solution was stirred for 16 h at room temperature on an oil bath. The resulting mixture was cooled to room temperature and concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). This gave 1.8 g (93.10%) of benzyl N-[2-[7-(1H-pyrazol-3-yl)quinolin-4yl]ethyl]carbamate as an off-white solid. LC-MS: [M+H] ⁺ = 373.2.

Step 3. 4-(2-[[(Benzyloxy)carbonyl]amino]ethyl)-7-(1 H-pyrazol-3 -yl)quinolin-1 -ium-1 -olate

In a 100 mL round-bottomed flask, benzyl N-[2-[7-(1H-pyrazol-3-yl)quinolin-4yl]ethyl]carbamate (1.7 g, 4.56 mmol, 1 equiv) was placed in DCM (50 mL, 0.59 mmol, 0.129 equiv), followed by the addition of mCPBA (1575.4 mg, 9.13 mmol, 2.0 equiv). The resulting solution was stirred for 16 h at room temperature. The reaction was then quenched by the addition of 50 mL of saturated aqueous Na ₂ S ₂ O ₃ solution. The resulting solution was extracted with 3 x 50 mL dichloromethane, and the combined organic layers were concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). As a result, 1.4 g (78.96%) of 4-(2-[[(benzyloxy)carbonyl]amino]ethyl)-7-(1H-pyrazol-3-yl)quinol yne-1-ium-1-olate was obtained as a solid. LC-MS: [M+H] ⁺ = 389.2.

Step 4. Benzyl N-[2-[2-amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]ethyl]carbamate

A 25 mL round-bottomed flask was charged with 4-(2-[[(benzyloxy)carbonyl]amino]ethyl)-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate (100 mg, 0.26 mmol, 1 equiv) in DCM (4 mL) and NH4OH (2 mL). TsCl (97.8 mg, 0.51 mmol, 2.0 equiv) was added. The resulting solution was stirred for 2 h at room temperature. The reaction was then quenched by adding 10 mL MeOH. The resulting

- 80 040307 The resulting mixture was concentrated. The crude product was purified by preparative HPLC using the following conditions: column XBridge ShieldRP18 OBD, 19x250 mm, 10 μm; mobile phase: water (10 mmol/L NH ₄ HCO ₃ ) and MeCN (phase B from 30% to 47% over 10 min); detector: UV 254/210 nm. This gave 39.4 mg (39.50%) of benzyl N-[2-[2-amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]ethyl]carbamate as a white solid. 1H NMR: (300 MHz, DMSO-d ₆ ) δ 7.87-7.63 (m, 5H), 7.46-7.27 (m, 5H), 6.77 (s, 1H), 6.57 (s, 1H), 6.31 (s, 2H), 5.02 (s, 2H), 3.29 (m, 2H), 3.03-2.98 (m, 2H). LC Methods: Column: Kinetex EVO, 3.0 mm x 50 mm, particles 2.6 μm; mobile phase A: water with 0.03% NH4OH; mobile phase B: acetonitrile; temperature: 40°C; gradient: 10% B to 95% B over 1.9 min, then 0.60 min hold at 95% B; Flow rate: 1.2 ml/min. LC, retention time: 1.291 min. LCMS: [M+H]+=388.2.

Step 5. 4-(2-Aminoethyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine

In a 25 mL round-bottomed flask, benzyl N-[2-[2-amino-7-(1H-pyrazol-3-yl)quinolin-4yl]ethyl]carbamate (50 mg, 0.13 mmol, 1 equiv) in MeOH (3 mL, 0.09 mmol, 0.725 equiv) was added, followed by the addition of Pd/C (15 mg, 0.14 mmol, 1.092 equiv). The resulting solution was stirred for 16 h at room temperature under N2. The solids were filtered. The resulting mixture was concentrated. The crude product was purified by preparative HPLC using the following conditions: column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; Mobile phase: water (10 mmol/L NH4HCO3) and MeCN (10% phase B to 20% over 6 min); detector: UV 254/210 nm. This yielded 14 mg (42.83%) of 4-(2-aminoethyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine as a white solid. LC Methods: Column: Kinetex EVO, 3.0 mm x 50 mm, particles 2.6 μm; mobile phase A: water with 0.03% NH3H2O; mobile phase B: acetonitrile; temperature: 40°C; gradient: 10% B to 40% B over 2.49 min, 40% B to 95% B over 0.9 min, then a 0.75 min hold at 95% B. Flow rate: 1.2 mL/min. LC, retention time: 1.338 min. LC-MS: [M+H] ⁺ = 254.1. H-NMR: (400 MHz, DMSC-d6) δ 7.89-7.84 (m, 2H), 7.72 (s, 1H), 7.62 (d, J = 8 Hz, 1H), 6.84-6.78 (m, 1H), 6.58 (s, 1H), 6.33 (d, J = 4 Hz, 1H), 3.99-3.37 (m, 1H), 3.02-2.87 (m, 3H), 1.72-1.64 (m, 2H), 1.58-1.52 (m, 2H).

Step 6. N-[2-[2-amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]ethyl]acetamide (compound 217)

In a 25 mL round-bottomed flask, 4-(2-aminoethyl)-7-(1H-pyrazol-3-yl)quinolin-2-amine (50 mg, 0.20 mmol, 1 equiv) was placed in DCM (3 mL, 0.04 mmol, 0.179 equiv), then (CH ₃ CO) ₂ O (20.2 mg, 0.20 mmol, 1.0 equiv) and Hunig's base (76.5 mg, 0.59 mmol, 3.0 equiv) were added. The resulting solution was stirred for 2 h at room temperature. The reaction was then quenched by adding 5 mL MeOH. The resulting mixture was concentrated. The crude product was purified by preparative HPLC using the following conditions: column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; Mobile phase: water (10 mmol/L NH4HCO3) and MeCN (12% phase B to 35% over 6 min); detector: UV 254/210 nm. This yielded 12.7 mg (21.78%) of N-[2-[2-amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]ethyl]acetamide as a solid. H NMR: (300 MHz, DMSO-d ₆ ) δ 8.06-8.02 (m, 1H), 7.90-7.86 (m, 2H), 7.74-7.65 (m, 2H), 6.80 (s, 1H), 6.58 (s, 1H), 6.34 (s, 2H), 3.38-3.32 (m, 2H), 3.03-2.98 (m, 2H), 1.82 (s, 3H). LC Methods: Column: Kinetex EVO, 3.0 mm x 50 mm, particles 2.6 μm; Mobile phase A: water with 0.03% NH ₄ OH; Mobile phase B: acetonitrile; Temperature: 40°C; Gradient: 10% B to 95% B over 1.9 min, then 0.15 min hold at 95% B; Flow rate: 1.2 mL/min. LC, retention time: 0.851 min. LC-MS: [M+H] ⁺ = 296.1.

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218)

Preparation of 4-(2-amino-7-(1 H-pyrazol-5-yl)quinolin-4-yl)butan-1-ol (compound

Step 1. 4-[7-(1H-pyrazol-3-yl)quinolin-4-yl]but-3-yn-1-ol

A solution of 4-bromo-7-(1H-pyrazol-3-yl)quinoline (1.2 g, 4.38 mmol, 1.00 equiv.) in tetrahydrofuran (20 mL) was placed in a hermetically sealed 40 mL tube. Pd(PPh ₃ ) ₄ (500 mg, 0.43 mmol, 0.10 equiv.), CuI (166 mg, 0.87 mmol, 0.20 equiv.), DIPEA (1.7 g, 13.15 mmol, 3.00 equiv.), and but-3-yn-1-ol (620 mg, 8.85 mmol, 2.00 equiv.) were added. The resulting solution was stirred for 16 h at 70 °C in an oil bath. The resulting mixture was cooled to room temperature and concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane/methanol (0-10%) to afford 500 mg (43%) of 4-[7-(1H-pyrazol-3-yl)quinolin-4-yl]but-3-yn-1-ol as a crude brown solid. LC-MS: (ES, m/z): [M+H]+=264.1.

Step 2. 4-[4-[(tert-Butyldimethylsilyl)oxy]but-1-yn-1-yl]-7-(1 H-pyrazol-3-yl)quinoline

A 250 mL round-bottomed flask was charged with a solution of 4-[7-(1H-pyrazol-3-yl)quinolin-4-yl]but-3-yn-1ol (500 mg, 1.90 mmol, 1.00 equiv.) in dichloromethane (100 mL), TBSCl (1.45 g, 4.00 equiv.), and triethylamine (780 mg, 7.71 mmol, 4.00 equiv.). The resulting solution was stirred for 2 days at 40 °C in an oil bath. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column with ethyl acetate:petroleum ether (1:0). As a result, 650 mg (91%) of 4-[4-[(tert-butyldimethylsilyl)oxy]but-1-yn-1-yl]-7-(1H-pyrazol-3-yl)quinoline was obtained as a light brown solid. LC-MS: (ES, m/z): [M+H] ⁺ = 378.2.

Step 3. 4-[4-[(tert-Butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinoline

A 100 mL round-bottomed flask was charged with a solution of 4-[4-[(tert-butyldimethylsilyl)oxy]but-1-yn-1-yl]-7-(1H-pyrazol-3-yl)quinoline (550 mg, 1.46 mmol, 1.00 equiv.) in CH3OH (30 mL). Palladium on carbon (200 mg) was added. The resulting solution was stirred for 16 h at room temperature under _H2 . The solids were filtered. The organic mixture was concentrated, and the residue was applied to a silica gel column with dichloromethane/methanol (10:1). This gave 350 mg (63%) of 4-[4-[(tert-butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinoline as a light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 382.2.

Step 4. 4-[4-[(tert-Butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate

A 50 mL round-bottomed flask was charged with a solution of 4-[4-[(tert-butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinoline (300 mg, 0.79 mmol, 1.00 equiv.) in dichloromethane (20 mL) and m-CPBA (270 mg, 1.56 mmol, 2.00 equiv.) was added. The resulting solution was stirred for 5 h at room temperature. The reaction was then quenched by adding Na ₂ S ₂ O ₃ . The resulting mixture was washed with 50 mL of sodium bicarbonate. The resulting aqueous solution was extracted

3x50 mL dichloromethane. The organic layers were combined and concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). This gave 320 mg (102%) of 4-[4[(tert-butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate as a white solid. LC-MS: (ES, m/z): [M+H] ⁺ = 398.2.

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Step 5. 4-[4-[(tert-Butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine

A 50 mL round-bottomed flask was charged with a solution of 4-[4-[(tert-butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinolin-1-ium-1-olate (270 mg, 0.68 mmol, 1.00 equiv.) in dichloromethane (15 mL) and NH4OH (5 mL). TsCl (260 mg, 2.00 equiv.) was added. The resulting solution was stirred for 2 h at room temperature. The resulting mixture was concentrated in vacuo. This gave 240 mg (89%) of 4-[4-[(tert-butyldimethylsilyl)oxy]butyl]-7-(1H-pyrazol-3-yl)quinolin-2-amine as a light yellow solid. LC-MS: (ES, m/z): [M+H]+=397.2.

Step 6. 4-[2-Amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]butan-1-ol (compound 218)

A 50 mL round-bottomed flask was charged with a solution of 4-[4-[(tert-butyldimethylsilyl)oxy]butyl]-7-(1Hpyrazol-3-yl)quinolin-2-amine (200 mg, 0.50 mmol, 1.00 equiv.) in hydrogen chloride in methanol (10 mL). The resulting solution was stirred for 4 h at room temperature. The pH of the solution was adjusted to 10 with NH4OH. The resulting mixture was concentrated under vacuum. The crude product was purified by preparative HPLC using the following conditions: column XBridge Shield RP18 OBD, 19x250 mm, 10 μm; mobile phase: water (10 mmol/L NH4HCO3) and MeCN (10.0% MeCN to 70.0% over 7 min); detector: UV 254/210 nm. As a result, 101 mg (71%) of 4[2-amino-7-(1H-pyrazol-3-yl)quinolin-4-yl]butan-1-ol was obtained as a white solid. 1H-NMR: (300 MHz, DMSO-d ₆ ) δ 13.38-12.90 (d, J= 8.4 Hz, 1H), 7.85-7.55 (m, 4H), 6.78 (s, 1H), 6.59 (d, J=7.2 Hz, 1H), 6.35-6.27 (d, J=24 Hz, 2H), 4.42-4.39 (m, 1H), 3.48-3.42 (m, 2H), 2.90-2.85 (m, 2H), 1.72-1.64 (m, 2H), 1.581.52 (m, 2H). LC methods: Column: Waters Xbridge shield RP18, 4.6 mm x 50 mm, 3.5 µm particles; Mobile phase A: water with 0.03% NH ₄ OH; mobile phase B: acetonitrile; temperature: 40°C; gradient: 10% B to 70% B over 2.4 min, 70% B to 95% B over 0.7 min, then 0.98 min hold at 95% B; flow rate: 1.5 mL/min. LC, retention time: 1.837 min. LC-MS: (ES, m/z): [M+H] ⁺ = 283.

Example II-22: Preparation of 3-(2-amino-7-(isothiazol-3-yl)quinolin-4-yl)propan-1-ol (compound 219)

He

Step 1. [4-[3-(Benzyloxy)propyl]quinolin-7-yl]boronic acid

A 100 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with 4-[3-(benzyloxy)propyl]-7-bromoquinoline (1 g, 2.81 mmol, 1 equiv), KOAc (551.0 mg, 5.61 mmol, 2 equiv), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1425.6 mg, 5.61 mmol, 2 equiv), and Pd(dppf) _Cl2 (205.4 mg, 0.28 mmol, 0.1 equiv) in dioxane (20 mL). The resulting solution was stirred for 16 h at 90°C in an oil bath. The solids were filtered off. The resulting mixture was concentrated. This yielded 904 mg of [4-[3-(benzyloxy)propyl]quinolin-7-yl]boronic acid as a light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 322.2.

Step 2. 4-[3-(Benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolone

A 50 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with [4- _{[3-(benzyloxy)propyl]quinolin-7-yl]boronic acid (904 mg, 2.81 mmol, 1 equiv), Na2CO3 (} 596.6 mg, 5.63 mmol, 2 equiv), 3-bromo-1,2-thiazole (923.3 mg, 5.63 mmol, 2 equiv), and Pd( _PPh3 ) ₄ (325.2 mg, 0.28 mmol, 0.1 equiv) in dioxane (20 mL) and _H2O (5 mL). The resulting solution was stirred for 4 h at 80°C in an oil bath. The resulting mixture was cooled to room temperature and concentrated. The residue was applied to a silica gel column with ethyl acetate/petroleum ether (1:1) to give 500 mg (49.28%) of 4-[3-(benzyloxy)propyl]7-(1,2-thiazol-3-yl)quinoline as a light brown solid. LC-MS: (ES, m/z): [M+H] ⁺ = 361.1.

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Step 3. 4-[3-(Benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-1-ium-1-olate

In a 25 mL 3-necked round-bottomed flask, which was purged and maintained under an inert atmosphere of nitrogen, 4-[3-(benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinoline (450 mg, 1.25 mmol, 1 equiv) in DCM (12 mL) was added and m-CPBA (430.9 mg, 2.50 mmol, 2 equiv) was added. The resulting solution was stirred for 5 h at room temperature. The reaction was then quenched by the addition of Na ₂ S ₂ O ₄ . The pH of the solution was adjusted to 10 with NaHCO ₃ . The resulting solution was extracted with 3 x 20 mL ethyl acetate and concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (20:1). As a result, 130 mg (27.66%) of 4-[3-(benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-1-ium-1-olate was obtained as a light yellow solid. LCMS: (ES, m/z): [M+H]+=377.1.

Step 4. 4-[3-(Benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-2-amine

A 25 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with 4-[3-(benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-1-ium-1-olate (130 mg, 0.35 mmol, 1 eq.) in DCM (3 mL) and NH4OH (1.5 mL, 38.52 mmol). TsCl (131.7 mg, 0.69 mmol, 2 eq.) was added. The resulting solution was stirred for 2 h at room temperature. The resulting mixture was concentrated. The residue was applied to a silica gel column with dichloromethane/methanol (10:1). As a result, 120 mg (92.55%) of 4-[3-(benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-2-amine was obtained as a light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ = 376.1.

Step 5. 3-(2-Amino-7-(isothiazol-3-yl)quinolin-4-yl)propan-1-ol (compound 219)

In a 25 mL round-bottomed flask, 4-[3-(benzyloxy)propyl]-7-(1,2-thiazol-3-yl)quinolin-2-amine was placed in hydrogen chloride in MeOH (4 M). The resulting solution was stirred for 16 h at room temperature. The pH of the solution was adjusted to 10 with NH4OH. The resulting mixture was concentrated. The crude product was purified by preparative HPLC under the following conditions (Preparative HPLC-018): column: XBridge Prep OBD C18, 19x250 mm, 5 μm; mobile phase: water (10 mmol/L NH4HCO ₃ ) and MeCN (10% phase B to 50% over 7 min); detector: UV. As a result, 26 mg (41.91%) of 3-[2-amino-7-(1,2-thiazol-3-yl)quinolin-4-yl]propan-1-ol was obtained as a white solid. 1H NMR: (400 MHz, DMSO-d ₆ , ppm): δ 9.19 (d, J=4.4 Hz, 1H) 8.08-8.05 (m, 2H), 7.92-7.84 (m, 2H), 6.65 (s, 1H), 6.41 (s, 2H), 4.63-4.60 (m, 1H), 3.55-3.50 (m, 2H), 2.95-2.92 (m, 2H), 1.83-1.79 (m, 2H). LC Methods: Column: Kinetex EVO 3.0 mm x 50 mm, 2.6 μm particles; Mobile Phase A: Water with 0.03% NH4OH; Mobile Phase B: Acetonitrile; Temperature: 40°C; Gradient: 10% B to 95% B over 2 min, then 0.60 min hold at 95% B; Flow Rate: 1.2 mL/min. LC, Retention Time: 1.073 min. LC-MS: (ES, m/z): [M+H] ⁺ = 286.

Method for connection 109:

Compound 109 was prepared following a method similar to that presented for compound 110.

M/Z=295.6.

Compound 111 was prepared following a method similar to that presented for compound 112. 1H NMR (400 MHz, Methanol-L) δ 7.95-7.84 (m, 1H), 7.80 (d, J=6.1 Hz, 1H), 7.73-7.58 (m, 3H), 6.75 (br s, 1H), 3.47-3.34 (m, 4H), 2.71 (br d, J=6.4 Hz, 2H), 2.10 (d, J=9.1 Hz, 3H), 1.83-1.62 (m, 4H), 1.26-1.01 (m, 3H). LC-MS conditions: column: BEH C18 2.1x50 mm, particles 1.7 μm; Mobile phase A: water with 0.05% trifluoroacetic acid; mobile phase B: acetonitrile with 0.05% trifluoroacetic acid; temperature: 25°C; gradient: 2% B to 98% B over 1.6 min, then 0.4 min hold at 98% B; flow rate: 0.8 mL/min; detection: MS and UV (220 nm). LC RT: 0.62 min. M/Z=352.1.

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Example III-1: Synthesis of (S)-3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)propane-1,2-diol

Step 1. Preparation of (S)-3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)propane-1,2-diol (compound 220)

To a solution of (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (60.3 mg, 0.456 mmol) and 4-chloro-7-(1(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) in NMP (406 µL) was added potassium tert-butoxide (17.06 mg, 0.152 mmol). The reaction mixture was heated to 100°C overnight. The reaction mixture was diluted with water and extracted three times with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.4 mL DCM and 0.4 mL TFA. After 1 h, the reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and subjected to SCP purification by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B over 24 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give (7(S)-3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)propane-1,2-diol (7.6 mg, 41%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.10 (br d, J=8.2 Hz, 1H), 8.04 (br s, 1H), 7.94-7.80 (m, 2H), 6.86 (s, 1H), 6.39 (s, 1H), 4.30 (br dd, J=9.5, 3.4 Hz, 1H), 4.17 (br dd, J=9.6, 6.3 Hz, 1H), 3.98 (br s, 1H), 3.56 (br s, 1H), 3.34 (br s, 1H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.57 min. M/Z = 301.0.

Compound 221 was prepared according to the synthetic methods described for compound 220 from the appropriate starting materials.

NH ₂

OH ¹ H NMR (400 MHz, MeOH-d ₄ ) δ 8.18 (d, J=8.5 Hz, 1H), 7.93 (s, 1H), 7.86 (br d, J=8.3 Hz, 1H), 7.75 (d, J=1.7 Hz, 1H), 6.83 (d, J=2.0 Hz, 1H), 6.36 (s, 1H), 4.42-4.33 (m, 1H), 4.32-4.24 (m, 1H), 4.20-4.10 (m, 1H), 3.76 (d, J=5.6 Hz, 2H). LC RT: 0.84 min. M/Z=301.0.

Example III-2: Preparation of 4-((1H-pyrazol-5-yl)methoxy)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 313)

CZo

II /Ν-ΝΗ

Ν-Ν^Γ ^Η0 ► [I ΖΙ Λ

II I Ύ KOtBu, NMP;

then DCM,TFA CT

0' Connection 313

4-((1H-pyrazol-5-yl)methoxy)-7-(1H-pyrazol-5-yl)quinolin-2-amine was prepared from (1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)methanol (55.4 mg, 0.304 mmol) and 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) in the same manner as described in Example II-8. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.97-7.61 (m, 5H), 6.80 (s, 1H), 6.54-6.39 (m, 2H), 5.29 (br s, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50 min hold at 100% B; flow rate: 1 mL/min. Detection: MS and UV (220 nm). LC RT: 1.16 min. M/Z= 307.3.

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Example III-3: Synthesis of 7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 314) n ₂ , Pd-C N-NH

NH ₂ THF/MeOH;

----Ϊ I T

TFA, DCM

Compound 314

4-Chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-N-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) was dissolved in MeOH (0.75 mL) and THF (0.25 mL). The flask was briefly evacuated twice and backfilled with nitrogen. Pd-C (10% on carbon support, 50% humidity, 3 mg, 0.028 mmol) was added, then the flask was attached to a hydrogen balloon, briefly evacuated, backfilled with hydrogen, and stirred overnight. The reaction mixture was diluted with MeOH and filtered through a syringe filter. The filtrate was concentrated. The residue was dissolved in 0.5 mL DCM and 0.5 mL TFA. After 1 h, the reaction mixture was concentrated and azeotroped with DCM.

The residue was dissolved in DMF, filtered through a syringe filter, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 2% B, 2-42% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 7-(1H-pyrazol-5-yl)quinolin-2-amine (5.3 mg, 41%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 8.9 Hz, 1H), 7.83 (s, 1H), 7.71 (br s, 1H), 7.67-7.58 (m, 2H), 6.78 (d, J = 2.1 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H), 6.38 (s, 1H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.91 min. M/Z = 211.2.

Example III-4: Synthesis of 2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propane-1,3-diol (compound 315)

To a solution of 2,2-dimethyl-1,3-dioxan-5-amine (39.9 mg, 0.304 mmol) and 4-chloro-7-(1-(tetrahydro-2Hpyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol) in DMSO (406 μL) was added 2,2-dimethyl-1,3-dioxan-5-amine (39.9 mg, 0.304 mmol). The reaction mixture was heated to 120°C. After 16 h, additional 2,2-dimethyl-1,3-dioxan-5-amine (39.9 mg, 0.304 mmol) and Hunig's base (31.9 μL, 0.182 mmol) were added. After an additional 24 h, the reaction mixture was cooled, diluted with water, and extracted three times with EtOAC. The organic layers were concentrated. The residue was dissolved in 0.5 mL MeOH, and 0.3 mL concentrated HCl was added. After 4 h, the reaction mixture was concentrated and azeotroped with MeOH. The residue was dissolved in MeOH, neutralized _with solid _K2CO3 , filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate. Gradient: 2-min hold at 0% B, 0-45% B over 20 min, then 4-min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford 2-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propane-1,3-diol (5.9 mg, 31%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.99 (br d, J=8.5 Hz, 1H), 7.93 (s, 1H), 7.78 (br s, 1H), 7.76-7.70 (m, 1H), 7.62-7.55 (m, 1H), 6.79 (s, 1H), 5.76 (s, 1H), 3.86-3.78 (m, 1H), 3.57-3.49 (m, 1H), 3.45 (br t, J=5.3 Hz, 1H), 3.37-3.28 (m, 1H), 3.15-3.08 (m, 1H). Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.61 min. M/Z=300.1.

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Example III-5: Synthesis of 7-(1H-pyrazol-5-yl)-N4-(pyrimidin-2-ylmethyl)quinoline-2,4-diamine (compound 316)

A vial was charged with first-generation Brettphos precatalyst (4.86 mg, 6.08 μmol), 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (20 mg, 0.061 mmol), and sodium tert-butoxide (14.61 mg, 0.152 mmol). The vial was placed under vacuum and backfilled with nitrogen twice. Dioxane (0.5 mL) and pyrimidin-2-ylmethanamine (13.28 mg, 0.122 mmol) were added, nitrogen was bubbled through the solution, and the reaction mixture was heated to 100°C overnight. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.5 mL DCM and 0.5 mL TFA. After 1 h, the reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-30% B over 25 min, then a 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the target product were pooled and dried by centrifugal evaporation to yield 7-(1H-pyrαzol-5-yl)-N4-(pyrimidin-2-ylmethyl)quinoline-2,4-diamine (4.9 mg, 25%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.79 (d, J=4.9 Hz, 2H), 8.10 (d, J=8.5 Hz, 1H), 8.05-7.92 (m, 1H), 7.81 (br s, 1H), 7.787.72 (m, 1H), 7.70-7.64 (m, 1H), 7.42 (t, J=4.9 Hz, 1H), 6.81 (d, J=1.8 Hz, 1H), 5.61 (s, 1H), 4.68 (br d, J=5.8 Hz, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 µm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 1.01 min. M/Z = 318.3,

Compound 317 was prepared according to the synthetic methods described for compound 316 from the appropriate starting materials.

^1H NMR (500 MHz, DMSO-d ₆ ) δ 9.09 (s, 1H), 8.81 (s, 2H), 8.04 (br d, J=8.7 Hz, 1H), 7.79 (s, 1H), 7.777.71 (m, 1H), 7.68-7.60 (m, 1H), 6.82-6.77 (m, 1H), 5.68 (s, 1H), 4.53 (br d, J=3.4 Hz, 2H). LC RT: 0.90 min. M/Z= 318.2.

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Example Ш-6: Synthesis of 6-substituted 4-aminoquinolines

Connection|318

Step 1. Obtaining 5-(((3-bromo-4-fluorophenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione

A solution of 3-bromo-4-fluoroaniline (1.9 g, 10.00 mmol) and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (2.234 g, 12.00 mmol) in dioxane (5 mL) was heated to 120°C for 20 min. The reaction mixture was then cooled to room temperature and diluted with 50 mL of diethyl ether. The solid was filtered and dried to give 5-(((3-bromo-4-fluorophenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (2.77 g, 8.0 mmol, 80%). ^1H NMR (400 MHz, DMSO-d6) δ 11.23 (br d, J=14.4 Hz, 1H), 8.53 (d, J=14.5 Hz, 1H), 8.06 (dd, J=6.0, 2.8 Hz, 1H), 7.69-7.61 (m, 1H), 7.44 (t, 7=8.7 Hz, 1H), 1.73-1.63 (m, 6H).

Step 2. Obtaining 7-bromo-6-fluoroquinolin-4-ol

A solution of 5-(((3-bromo-4-fluorophenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (2.77 g, 8.05 mmol) in phenyl ether (7 mL) was heated to 240°C for 10 min. The reaction mixture was then cooled to room temperature and diluted with 50 mL of diethyl ether. The solid was filtered and dried to give a 1:1 mixture of 7-bromo-6-fluoroquinolin-4-ol and 5-bromo-6-fluoroquinolin-4-ol (0.982 g, 4.0 mmol, 50%).

Step 3. Obtaining 7-bromo-4-chloro-6-fluoroquinoline

To a 1:1 suspension of 7-bromo-6-fluoroquinolin-4-ol and 5-bromo-6-fluoroquinolin-4-ol (982 mg, 4.06 mmol) in toluene (7 mL) was added _POCl3 (0.756 mL, 8.11 mmol). The reaction mixture was then heated to 100°C for 1 h. The cooled reaction mixture was poured onto ice and then partitioned between DCM and saturated sodium carbonate solution. The organic layer was dried over sodium sulfate and concentrated. The residue was purified with ISCO (80 g column; hexanes/ethyl acetate; gradient from 0 to 100%) to afford 7-bromo-4-chloro-6-fluoroquinoline (203 mg, 0.8 mmol, 19%). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.88 (d, J=4.7 Hz, 1H), 8.54 (d, J=6.8 Hz, 1H), 8.09 (d, J=9.5 Hz, 1H), 7.88 (d, J=4.8 Hz, 1H).

Step 4. Obtaining 7-bromo-4-chloro-6-fluoroquinoline 1-oxide

To a solution of 7-bromo-4-chloro-6-fluoroquinoline (0.203 g, 0.779 mmol) in DCM (10.0 mL) was added mCPBA (0.576 g, 2.34 mmol). The reaction mixture was stirred overnight, then quenched with saturated sodium thiosulfate solution. The reaction mixture was stirred for 0.5 h, then saturated aqueous sodium bicarbonate solution was added. The reaction was extracted twice with DCM. The organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 7-bromo-4-chloro-6-fluoroquinoline 1-oxide (0.215 g, 0.779 mmol, quantitative yield). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.87 (d, J=6.7 Hz, 1H), 8.60 (d, J=6.6 Hz, 1H), 8.13 (d, J=9.2 Hz, 1H), 7.80 (d, J=6.6 Hz, 1H).

Step 5: Preparation of 7-bromo-4-chloro-6-fluoroquinolin-2-amine

In a round-bottomed flask, 7-bromo-4-chloro-6-fluoroquinoline 1-oxide (240 mg, 0.868 mmol) was suspended in DCM (8 mL). TsCl (182 mg, 0.955 mmol) was added. The mixture was stirred for 1 h.

- 88 040307 Ammonium chloride (232 mg, 4.34 mmol) (dried in an oven at 110°C overnight) was suspended in DCM (4 mL) in a second round-bottomed flask. Triethylamine (0.605 mL, 4.34 mmol) was added and the mixture was stirred for 0.5 h, then the contents of the first round-bottomed flask were added to the second. The reaction mixture was stirred overnight, then filtered and concentrated. The residue was purified with ISCO (24 g column; hexanes/ethyl acetate; gradient 0 to 100%) to give 7-bromo-4-chloro-6-fluoroquinolin-2-amine (128 mg, 0.47 mmol, 54%).

Step 6: Preparation of 4-chloro-6-fluoro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline-2-amine

1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.174 g, 0.626 mmol), 7-bromo-4-chloroquinolin-2-amine (0.115 g, 0.417 mmol) and PdCl ₂ (dppf)-DCM adduct (0.034 g, 0.042 mmol) were placed in a vial under elevated pressure.

The vial was placed under vacuum three times and backfilled with nitrogen. Dioxane (10 mL) and tripotassium phosphate (2 M aqueous) (0.63 mL, 1.25 mmol) were added, nitrogen was bubbled through the solution, and the reaction mixture was heated to 100°C overnight. The reaction mixture was cooled to room temperature, diluted with 50 mL ethyl acetate, dried over sodium sulfate, and concentrated. The residue was purified by ISCO (12 g column; hexanes/ethyl acetate; gradient 0 to 100%) to give 4-chloro-6fluoro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (0.113 g, 0.22 mmol, 52% yield).

Step 7: Preparation of 3-((2-amino-6-fluoro-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propan-1-ol (compound 318)

To a solution of 4-chloro-6-fluoro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (60 mg, 0.173 mmol) and 3-aminopropan-1-ol (39 mg, 0.52 mmol) in DMSO (0.5 mL) was added Hunig's base (0.3 mL, 1.7 mmol). The reaction mixture was heated to 120 °C overnight. The reaction mixture was cooled, diluted with water and extracted three times with DCM. The organic layers were concentrated, dissolved in 5 mL DCM and 4 N HCl in dioxane (1.038 mL, 4.15 mmol) was added. After 20 min, the reaction was complete according to LC-MS. The reaction mixture was concentrated. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LCMS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation. The material was further purified by preparative LCMS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 3-((2-amino-6-fluoro-7-(1H-pyrazol-5yl)quinolin-4-yl)amino)propan-1-ol (3.7 mg, 7.1%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.19-8.13 (m, 1H), 8.11 (br s, 1H), 7.98-7.92 (m, 1H), 7.88 (br s, 1H), 7.58 (br s, 1H), 6.77 (br s, 1H), 5.83 (s, 1H), 3.55 (br d, J=4.0 Hz, 2H), 3.45-3.25 (m, 2H), 1.84 (quin, J=6.6 Hz, 2H). One methylene from the side chain is not visible, probably due to overlap with the suppressed water peak. LC RT: 0.80 min. M/Z=302.1.

Compound 319 and compound 320 were prepared according to the synthetic methods described for compound 318 from the appropriate starting materials.

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Compound No. Structure LC-MS [M+H] ⁺ RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 319 nh ₂ iiUZJ ⁿ G Υ τ Cd _СНз 298.1 0.81 δ 7.95 - 7.90 (m, 1H), 7.76 (br s, 1H), 7.57 (s, 1H), 7.24 - 7.05 (m, 2H), 6.56 (s, 1H), 5.72 (s, 1H), 3.29 (brd, 7=6.1 Hz, 2H), 2.49 (s, 3H), 1.87- 1.79 (m, 2H). One methylene is not visible, possibly due to overlap with the suppressed water peak. 320 ζ zx ^o— \ V ^{- z} / \={ \\_ _z \_/ X /-----' ¹⁴³ xz o ^z X 314.1 0.83 δ 8.31 - 8.00 (m, 2H), 7.92 - 7.68 (m, 2H), 7.63 - 7.38 (br, 2H), 6.87 (br s, 1H), 5.84 (s, 1H), 3.98 (s, 3H), 3.57 (t, 7=6.1 Hz, 1H), 1.91 - 1.82 (m, 2H). Three protons are not visible, possibly due to overlap with the suppressed water peak.

Example 321. Preparation of (S)-7-(1H-pyrazol-3-yl)-N4-(pyrrolidin-3-yl)quinoline-2,4-diamine (compound 321)

To a solution of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (100 mg, 0.304 mmol) in NMP (1 mL) were added tert-butyl (3S)-3-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1Hpyrazol-5-yl)quinolin-4-yl)amino)pyrrolidine-1-carboxylate and Hunig's base (0.531 mL, 3.04 mmol). The resulting mixture was heated to 150 °C overnight. The reaction mixture was cooled, diluted with water, and extracted three times with EtOAc. The organic layers were concentrated. The residue was dissolved in 0.4 mL DCM and 0.4 mL TFA. After 1 h, LC-MS showed complete deprotection of THP. The reaction mixture was concentrated and azeotroped with DCM. The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative reversed-phase HPLC using the following conditions: column: Luna 30 x 100 mm; 5 μm particles; mobile phase A: 10:9 methanol:water with 0.1% trifluoroacetic acid; mobile phase B: 90:10 methanol:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-100% B for 10 min, then a 2 min hold at 100% B; flow rate: 40 mL/min. column temperature: 25°C, to give (S)-7-(1H-pyrazol-3-yl)-N4-(pyrrolidin-3-yl)quinoline-2,4-diamine (55 mg, 0.187 mmol, 61.4% yield). 1H NMR (400 MHz, methanol^) δ 8.34-8.27 (m, 1H), 7.98-7.91 (m, 1H), 7.90-7.84 (m, 1H), 7.80-7.77 (m, 1H), 6.94-6.83 (m, 1H), 6.02-5.89 (m, 1H), 3.75-3.64 (m, 2H), 3.35-3.30 (m, 1H), 2.44-2.28 (m, 2H), 2.13-1.98 (m, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.58 min. M/Z=295.0.

Example III-8: Preparation of 7-(1H-pyrazol-3-yl)-N4-(2-(thiophen-2-yl)ethyl)quinoline-2,4-diamine (compound 322)

SA/d/ ^NH Cs ₂ CO ₃ , dioxane

Ж_IJ Connection 322

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Step 1. Obtaining N-(7-bromo-4-chloroquinolin-2-yl)benzamide

A solution of 7-bromo-4-chloroquinoline 1-oxide (2.326 g, 9 mmol) in dichloroethane (22.50 mL) was treated with benzoyl isocyanate (2.94 g, 18.00 mmol), and the resulting mixture was heated to reflux for 16 h. The reaction mixture was cooled to room temperature, applied to a silica gel column, and eluted with 5-25% EtOAc-hexane. (The column and mobile phase were kept warm with a heat gun to prevent vigorous precipitation of the product.) Concentration of the appropriate fractions gave N-(7-bromo-4-chloroquinolin-2-yl)benzamide (2.8 g, 7.74 mmol, 86% yield) as an off-white solid, mp 142-143°C. LC-MS: Waters Acquity SDS using the following method: linear gradient from 2 to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18, 2.1 mm × 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 1.10 min. M/Z= 363.0.

Step 2. Obtaining 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline

A solution of 2-(thiophen-2-yl)ethan-1-amine (132 mg, 1.037 mmol) and N-(7-bromo-4-chloroquinolin-2-yl)benzamide (75 mg, 0.207 mmol) in DMSO (518 µL) was heated at 120°C for 4 h, then cooled to room temperature. The reaction was then purified by preparative reversed-phase HPLC (MeOH-water gradient, 0.1% TFA in both mobile phases). Concentration of the appropriate fractions afforded 7-bromo-N-4-(2-(thiophen-2-yl)ethyl)quinoline-2,4-diamine, TFA (39 mg, 41% yield) as a colorless glass. LC-MS method: Waters Acquity SDS using the following method: linear gradient from 2 to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18 2.1 mm x 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.75 min. M/Z = 350.2.

Step 3. Preparation of 7-(1H-pyrazol-3-yl)-N4-(2-(thiophen-2-yl)ethyl)quinoline-2,4-diamine (compound 351)

A mixture of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (29.4 mg, 0.151 mmol), 7-bromoK4-(2-(thiophen-2-yl)ethyl)quinoline-2,4-diamine, TFA (35 mg, 0.076 mmol), PdCNdppfj-CIbCF adduct (6.18 mg, 7.57 μmol), and cesium carbonate (99 mg, 0.303 mmol) in nitrogen-saturated dioxane (757 μL) was placed under nitrogen atmosphere and heated at 95°C for 2 h. The reaction mixture was cooled and stirred at room temperature. The reaction was quenched with 50% aqueous HOAc, diluted with 2 mL DMF, and purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:ammonium acetate; gradient: 0 min hold at 12% B, 12-52% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 7-(1H-pyrazol3-yl)-N4-(2-(thiophen-2-yl)ethyl)quinoline-2,4-diamine (13.6 mg, 54% yield). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.03 (d, J=8.9 Hz, 1H), 7.83 (br. s, 1H), 7.73-7.78 (m, 1H), 7.63-7.68 (m, 1H), 7.41-7.48 (m, 1H), 7.34 (d, J=4.6 Hz, 1H), 6.96-7.00 (m, 3H), 6.84 (d, J=1.8 Hz, 1H), 5.80 (s, 1H), 3.47-3.53 (m, 2H), 3.20 (t, J=7.0 Hz, integration distorted by water suppression). LC RT: 1.36 min. M/Z= 336.1.

Compounds 323-327 were prepared according to the synthetic methods described for compound 321 from the appropriate starting materials.

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Compound No. Structure LC-MS [M+H] ⁺ RT (min) Ή NMR (500 MHz, DMSO-cL) 323 nh ₂ «АА ^н <Г 295.3 0.45 δ 8.29 - 8.18 (m, 1H), 7.94 - 7.93 (m, 1H), 7.92 - 7.90 (m, 1H), 7.83 7.77 (m, 1H), 6.91 - 6.84 (m, 1H), 5.98 -5.90 (m, 1H), 4.62-4.51 (m, 1H), 3.81 -3.70 (m, 2H), 3.66 3.53 (m, 2H), 2.60 - 2.48 (m, 2H) 324 nh ₂ A O yDH ⁿ V 309.2 0.82 δ 8.25 -8.17(m, 1H), 8.08 - 8.00 (m, 1H), 7.93 - 7.88 (m, 2H), 6.89 6.79 (m, 1H), 6.41 - 6.29 (m, 1H), 3.77 - 3.66 (m, 1H), 3.61 - 3.49 (m, 1H), 3.00 -2.87 (m, 1H), 2.171.94 (m, 3H), 1.86 - 1.73 (m, 1H), 1.74 - 1.58 (m, 2H) 325 UUD \_/ t /-------' ¹⁴³ IZ I 295.4 0.55 δ 8.17-8.12 (m, 1H), 7.91 - 7.88 (m, 1H), 7.88 - 7.85 (m, 1H), 7.80 7.78 (m, 1H), 6.90 - 6.81 (m, 1H), 5.95 -5.86 (m, 1H), 4.18-4.08 (m, 2H), 3.83 -3.70 (m, 2H), 3.67 3.62 (m, 1H), 3.39 - 3.35 (m, 2H) 326 nh ₂ Ух Ат νΆΑ й JU ^н А V 323.2 0.65 δ 8.02 - 7.94 (m, 2H), 7.91 - 7.87 (m, 1H), 7.81 - 7.77 (m, 1H), 7.65 7.59 (m, 1H), 7.52 - 7.46 (m, 1H), 4.12-3.81 (m, 1H), 3.09 - 2.66 (m, 2H), 2.18 - 1.85 (m, 2H), 1.821.60 (m, 2H), 1.44 - 1.20 (m, 4H) 327 nh ₂ Ukh n Ag S A ⁿ A/ m 309.2 0.45 δ 8.20-8.10 (m, 1H), 7.95 - 7.86 (m, 2H), 7.82 - 7.77 (m, 1H), 6.88 6.83 (m, 1H), 5.94 - 5.87 (m, 1H), 3.64 - 3.54 (m, 2H), 3.50 - 3.42 (m, 2H), 3.24 -3.08 (m, 2H), 2.99 2.65 (m, 2H), 1.89 - 1.76 (m, 1H)

Example III-9: Synthesis of 4-alkoxy-substituted quinolines via the Mitsunobu reaction

Step 1. Obtaining 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline

A stirred suspension of 2-(thiophen-3-yl)ethan-1-ol (256 mg, 2.000 mmol), triphenylphosphine (367 mg, 1.400 mmol), and 7-bromoquinolin-4-ol (224 mg, 1 mmol) in THF (5000 µL) was heated to reflux, then cooled to room temperature. This suspension was treated with DIAD (272 µL, 1.400 mmol) for 1-2 min. The resulting mixture was stirred for 1 h at room temperature and then purified by flash chromatography (25-50% EtOAc-hexane). Concentration of appropriate fractions afforded a light amber oil. The oil was treated with ~5 mL hexanes and stirred. A small amount of EtOAc was added, and the mixture was stirred. A small amount of dichloromethane was added and the mixture was stirred and heated. A solution was obtained which was stirred while cooling. The product precipitated on the glass and the mixture was then evaporated to dryness to give 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline (270 mg, 0.81 mmol, 81% yield) as an off-white solid, mp 92-95°C. LC-MS: Waters Acquity SDS using the following procedure: linear gradient from 2 to 98% solvent B over

- 92 040307 for 1.00 min; UV visualization at 220 or 254 nm; column: VEN C18 2.1 mm x 50 mm; particles

1.7 µm (heated to 50°C); flow rate: 0.8 ml/min; mobile phase A: 100% water,

0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.76 min. M/Z = 336.1.

Step 2. Preparation of 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline 1-oxide

A solution of 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline (250 mg, 0.748 mmol) in chloroform (3740 µL) was treated with m-CPBA (516 mg, 2.99 mmol). The reaction mixture was stirred for 1 h at room temperature, then poured into EtOAc-hexane (to give an organic phase with a density <1). A precipitate formed, which was redissolved by adding additional EtOAc and EtOH and warmed. This mixture was shaken with 5% aqueous sodium thiosulfate. Saturated aqueous sodium bicarbonate was added, and when effervescence ceased, the mixture was gently shaken. The organic phase was washed (brine), dried, separated, and chromatographed on silica gel (5-10% MeOH-CH ₂ Cl ₂ ). Concentration of appropriate fractions gave 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline 1-oxide (140 mg, 0.40 mmol, 53% yield) as an amber glass. LC-MS: Waters Acquity SDS using the following procedure: linear gradient from 2% to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18 2.1 mm x 50 mm; 1.7 μm particles (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA. Mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.79 min. M/Z = 352.1.

Step 3. Preparation of 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinolin-2-amine

A solution of 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinoline 1-oxide (137 mg, 0.391 mmol) in chloroform (3 mL) was treated with 1 mL of concentrated aqueous ammonia and stirred rapidly for 3-4 min. Stirring was slowed, and the resulting mixture was treated with Ts-Cl (78 mg, 0.411 mmol) in 1 mL of chloroform via syringe (inside) over ~40 s. The reaction mixture was stirred for 20 min at room temperature, then applied to a silica gel column and eluted with 5-10% MeOH-CH ₂ Cl ₂ . Concentration of the appropriate fractions gave 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinolin-2-amine (113 mg, 0.324 mmol, 83% yield) as an oil that crystallized on standing to give a waxy brownish solid, mp 150-156°C. LC-MS: Waters Acquity SDS using the following procedure: linear gradient from 2 to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18 2.1 mm x 50 mm; 1.7 μm particles (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA. Mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.78 min. M/Z = 351.1.

Step 4. Preparation of 7-(1H-pyrazol-3-yl)-4-(2-(thiophen-3-yl)ethoxy)quinolin-2-amine (compound 329)

A suspension of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (16.67 mg, 0.086 mmol), 7-bromo-4-(2-(thiophen-3-yl)ethoxy)quinolin-2-amine (15 mg, 0.043 mmol), _PdCl2 (dppf) _-CH2Cl2 adduct (7.01 mg, 8.59 μmol), and cesium carbonate (42.0 mg, 0.129 mmol) in nitrogen-saturated dioxane (429 _μL ) was placed under nitrogen atmosphere and heated at 95°C for 2 h. The reaction mixture was cooled to room temperature, quenched with a few drops of 50% aqueous HOAc, filtered, and purified by preparative LC-MS at The chromatographs were run under the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 19% B, 19-59% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 7-(1H-pyrazol3-yl)-4-(2-(thiophen-3-yl)ethoxy)quinolin-2-amine (7.1 mg, 48% yield). 1H NMR (500 MHz, DMSO-d ₆ ) δ 7.82-7.88 (m, 2H), 7.76 (br. s, 1H), 7.65 (br. s, 1H), 7.50 (dd, J=4.8, 2.9 Hz, 1H), 7.39 (d, J=2.1 Hz, 1H), 7.18 (d, J=4.6 Hz, 1H), 6.80 (d, J=1.8 Hz, 1H), 6.64-6.81 (m, 2H), 6.25 (s, 1H), 4.35 (t, J=6.4 Hz, 2H), 3.21 (t, J=6.6 Hz, integration distorted by water suppression). LC RT: 1.49 min. M/Z=337.1.

Compound 330 and compound 331 were prepared according to the synthetic methods described for compound 329 from the appropriate starting materials.

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Connection No. Structure LC-MS [M+H] ⁺ RT (min) 'NMR (500 MHz, DMSO-d ₆ ) 331 nh ₂ fS A _/S ^U ⁰ \J 353.1 2.16 δ 7.85 (d, 7=8.3 Hz, 1H), 7.58-7.63 (m, 2H), 7.47-7.53 (m, 2H), 7.38 (d, 7=1.8 Hz, 1H), 7.15-7.20 (m, 2H), 6.74 (s, 2H), 6.25 (s, 1H), 4.34 (t, 7=6.4 Hz, 2H), 3.20 (t, 7=6.4 Hz, 2H). 331 U) o ^z \=/ Vz 353.1 2.12 δ 8.38 (br. s, 3H), 8.07-8.11 (m, 1H), 8.07-8.11 (m, 1H), 7.97 (d, 7=8.9 Hz, 1H), 7.81-7.86 (m, 1H), 7.73-7.77 (m, 1H), 7.59-7.63 (m, 1H), 7.50-7.53 (m, 1H), 7.40-7.43 (m, 1H), 7.17-7.20 (m, 1H), 4.44 (t, 7=6.4 Hz, 2H), 3.25 (t, 7=6.4 Hz, integration distorted by water suppression). The primary amine is combined into three because this sample is a TFA salt.

Example III-10: Synthesis of 4-alkoxy-substituted quinolines via Sn _Ar on 7-bromo-4-chloroquinoline-2-amine

Step 1. Obtaining (3-(((2-amino-7-bromoquinolin-4-yl)oxy)methyl)oxetan-3-yl)methanol

A stirred solution of oxetane-3,3-diyldimethanol (229 mg, 1.942 mmol) in DMSO (388 μL) was treated with KOtBu (582 μL, 0.582 mmol) in THF over 1–2 min. This solution was stirred for 5 min and then treated with 7-bromo-4-chloroquinolin-2-amine (50 mg, 0.194 mmol). The resulting solution was heated at 90°C for 20 min, then the temperature was raised to 95°C and stirring was continued for an additional 3 h. The reaction mixture was cooled to room temperature and pipetted into water (with stirring). A suspension was obtained with some material adhering to the glass. Stirring was continued for 40 min, after which the suspension was filtered, washed with water and air-dried to give (3-(((2-amino-7-bromoquinolin-4yl)oxy)methyl)oxetan-3-yl)methanol (52 mg, 0.153 mmol, 79% yield) as an amorphous brownish solid. LC-MS: Waters Acquity SDS using the following procedure: linear gradient from 2 to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18 2.1 mm x 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; Mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.57 min. M/Z = 341.1.

Step 2. Preparation of (3-(((2-amino-7-(thiophen-3-yl)quinolin-4-yl)oxy)methyl)oxetan-3-yl)methanol (compound 332)

A suspension of (3-(((2-amino-7-bromoquinolin-4-yl)oxy)methyl)oxetan-3-yl)methanol (20 mg, 0.059 mmol), thiophen-3-ylboronic acid (15.09 mg, 0.118 mmol), _PdCl2 (dppf) _-CH2Cl2 adduct (4.82 mg, 5.90 μmol) _, and cesium carbonate (57.6 mg, 0.177 mmol) in nitrogen-saturated dioxane (590 μL) was placed under nitrogen atmosphere and heated to 95°C. After 2 h, LC-MS indicated the reaction was complete. The reaction mixture was cooled and quenched with a few drops of 50% aqueous HOAc. When all solids had dissolved, the reaction mixture was diluted with 2 mL DMF and filtered. The crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 14% B, 14-54% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give (3-(((2-amino-7(thiophen-3-yl)quinolin-4-yl)oxy)methyl)oxetan-3-yl)methanol (11.6 mg, 55% yield). ¹ H NMR (500 MHz, DMSO-d6) δ 7.95-7.97 (m, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.70 (d, J=1.8 Hz, 1H), 7.65-7.68 (m, 1H), 7.61-7.64 (m, 1H), 7.50 (dd, J=8.6, 1.5 Hz, 1H), 6.34 (s, 2H), 6.25 (s, 1H), 4.55 (d, J=6.1 Hz, 2H), 4.48 (d, J=6.1 Hz, 2H), 4.28 (s, 2H), 3.82 (s, 2H). LC RT: 1.33 min. M/Z= 343.3.

Compound 333 was prepared according to the synthetic methods described for compound 332 from the appropriate starting materials.

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1H NMR (500 MHz, DMSO-d ₆ ) δ 7.84 (d, J=8.3 Hz, 1H), 7.81 (s, 1H), 7.74 (d, J=1.3 Hz, 1H), 7.59 (dd,

J=8.3, 1.0 Hz, 1H), 6.78 (s, 1H), 6.33 (s, 2H), 6.26 (s, 1H), 4.55 (d, J=5.6 Hz, 1H), 4.49 (d, J=5.1 Hz, 1H), 4.28 (s, 2H), 3.82 (s, 2H). LC RT: 0.93 min. M/Z= 326.9.

Example III-11: Synthesis of 4-alkoxy-substituted quinolines via SNA-on 7-bromo-4-chloroquinoline-2-amine

Step 1. Preparation of (1-(((2-Amino-7-bromoquinolin-4-yl)oxy)methyl)cyclobutyl)methanol (1-(((2-Amino-7-bromoquinolin-4-yl)oxy)methyl)cyclobutyl)methanol was prepared from cyclobutane-1,1-diyldimethanol and 7-bromo-4-chloroquinolin-2-amine in 89% yield using the method for the preparation of (3-(((2-amino-7-bromoquinolin-4-yl)oxy)methyl)oxetan-3-yl)methanol. LC-MS: Waters Acquity SDS using the following procedure: linear gradient 2 to 98% solvent B over 1.00 min; UV visualization at 220 or 254 nm; column: BEH C18 2.1 mm x 50 mm; 1.7 μm particles (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. LC RT: 0.82 min. M/Z = 339.2.

Step 2. Preparation of (1-(((2-amino-7-(thiophen-2-yl)quinolin-4-yl)oxy)methyl)cyclobutyl)methanol (compound 334)

A suspension of 4,4,5,5-tetramethyl-2-(thiophen-2-yl)-1,3,2-dioxaborolane (24.92 mg, 0.119 mmol), (1(((2-amino-7-bromoquinolin-4-yl)oxy)methyl)cyclobutyl)methanol (20 mg, 0.059 mmol), cesium carbonate (58.0 mg, 0.178 mmol), and _PdCl2 (dppf) _-CH2Cl2 adduct (4.84 mg, 5.93 μmol) in nitrogen-saturated dioxane (593 μL) was placed under nitrogen atmosphere and heated at 95°C for 1.5 h. The reaction mixture was then cooled to room temperature, quenched with 50% aqueous HOAc, and filtered. The crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 20% B, 20-60% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give (1-(((2-amino-7-(thiophen-2-yl)quinolin-4-yl)oxy)methyl)cyclobutyl)methanol (11.2 mg, 56% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.84 (d, J=8.4 Hz, 1H), 7.61 (d, J=1.6 Hz, 1H), 7.56-7.60 (m, 2H), 7.47 (dd, J=8.4, 1.8 Hz, 1H), 7.17 (dd, J=4.7, 3.9 Hz, 1H), 6.40 (s, 2H), 6.24 (s, 1H), 4.06 (s, 2H), 1.87-2.00 (m, 6H). One methylene is not visible, probably due to overlap with the suppressed water peak. LC RT: 1.79 min. M/Z=341.2.

Compounds 335-337 were prepared according to the synthetic methods described for compound 334 from the appropriate starting materials.

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Connection No. Structure LC-MS [M+H]+ RT (min) Ή NMR (500 MHz, DMSO-d ₆ ) 335 NH ₂ X JI . n L I <> 325.2 1.21 5 7.84 (d, 7=8.4 Hz, 1H), 7.81 (s, 1H), 7.74 (d, 7=0.7 Hz, 1H), 7.59 (dd, 7=8.5, 0.6 Hz, 1H), 6.78 (d, 7=2.0 Hz, 1H), 6.35 (s, 2H), 6.23 (s, 1H), 4.06 (s, 2H), 1.89-2.00 (m, 6H). One methylene group is not visible, probably due to water suppression. 336 nh ₂ ΪΧ 341.2 1.79 5 7.94-7.97 (m, 1H), 7.84 (d, 7=8.4 Hz, 1H), 7.70 (br. s, 1H), 7.65-7.68 (m, 1H), 7.61-7.64 (m, 1H), 7.53 (dd, 7=8.5, 1.5 Hz, 1H), 6.42 (br. s, 2H), 6.24 (s, 1H), 4.06 (s, 2H), 1.90-1.98 (m, 6H). One methylene group is not visible, probably due to water suppression. 337 nh ₂ 352.9 1.95 5 8.04 (br s, 1H), 8.00 (d, 7=8.2 Hz, 1H), 7.78 (br. s, 1H), 7.67-7.73 (br, 2H), 7.61-7.64 (br, 1H), 7.46 (br. s, 2H), 6.29 (s, 1H), 4.28 (t, 7=4.6 Hz, 2H), 2.08-2.16 (br, 2H). Two protons from the side chain are not visible, probably due to overlap with the DMSOde peak.

Example III-12: Synthesis of 4-amino-substituted quinolines using N-(7-bromo-4-chloroquinolin-2-yl)benzamide

Compound 338

Step 1. Obtaining I-(4-chloro-7-(thiophen-3-yl)quinolin-2-yl)benzamide

N-(7-bromo-4-chloroquinolin-2-yl)benzamide (164 mg, 0.454 mmol), thiophen-3-ylboronic acid (75 mg, 0.590 mmol), and potassium phosphate (337 mg, 1.587 mmol) were charged into a reaction vial. Dioxane (6.25 mL) and water (0.25 mL) were then added. The reaction was degassed for 10 min under a stream of nitrogen. Tetrakis(triphenylphosphine)palladium(0) (52.4 mg, 0.045 mmol) was then added, and the vial was sealed and heated to 90°C. After 3 h, the cooled reaction mixture was partitioned between water and ethyl acetate. The aqueous layer was extracted with additional ethyl acetate. The combined organic layers were then washed with brine. The product was dried over magnesium sulfate, filtered, and evaporated to yield the crude product. The product was purified on a 24 g silica gel column, eluting with 20-100% ethyl acetate in hexanes. Evaporation of the product containing fractions afforded N-(4-chloro-7-(thiophen-3-yl)quinolin-2-yl)benzamide (158 mg, 0.433 mmol, 95% yield). ¹ H NMR (400 MHz, DMSO-d6) δ 11.35 (br s, 1H), 8.53 (s, 1H), 8.20 (m, 1H), 8.17 (s, 2H), 8.12-8.05 (m, 3H), 7.79-7.71 (m, 2H), 7.64 (t, J=7.2 Hz, 1H), 7.55 (t, J=7.8 Hz, 2H).

Step 2. Preparation of (3-(((2-amino-7-(thiophen-3-yl)quinolin-4-yl)amino)methyl)oxetan-3-yl)methanol (compound 338)

K-(4-chloro-7-(thiophen-3-yl)quinolin-2-yl)benzamide (21.2 mg, 0.058

- 96 040307 mmol) in dimethyl sulfoxide (0.75 mL). (3-(Aminomethyl)oxetan-3-yl)methanol hydrochloride (44.6 mg, 0.291 mmol) and diisopropylethylamine (60.9 µL, 0.349 mmol) were added and the vial was purged with nitrogen. The reaction was then heated to 120°C and stirred overnight. The cooled reaction mixture was diluted with DMSO (1 mL) and purified by preparative LC using the following conditions: XBridge C18 column, 200 mm × 19 mm, 5 µm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 10% B, 10-50% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford (3-(((2-amino-7-(thiophen-3-yl)quinolin-4-yl)amino)methyl)oxetan-3-yl)methanol (7.0 mg, 0.021 mmol, 35%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.01 (br s, 1H), 7.87 (d, J=8.9 Hz, 1H), 7.71 (s, H), 7.71-7.68 (m, 1H), 7.60 (d, J=4.9 Hz, 1H), 7.57 (br d, J=8.9 Hz, 1H), 6.91 (br s, 2H), 5.49 (s, 1H), 4.11 (br s, 4H), 3.59 (s, 4H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 1.13 min. MZ=341.92.

Example III-13: Synthesis of 4-heteroaryl-substituted quinolines

Step 1. Preparation of 4,7-bis(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine

To a solution of 7-bromo-4-chloroquinolin-2-amine (431.2 mg, 1.67 mmol) in anhydrous dioxane (5 mL) and anhydrous DMF (5 mL) at room temperature under nitrogen atmosphere was added K ₃ PO ₄ (2 M aqueous solution, 2.51 mL, 5.02 mmol), followed by the addition of 1-(tetrahydro-2h-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h-pyrazole (605 mg, 2.18 mmol). The mixture was sparged with argon for approximately 30 min, then PdCl ₂ (dppf)-CH ₂ Cl ₂ (68.4 mg, 0.084 mmol) was added and the mixture was heated with stirring at 90°C. After 12 h, the reaction was allowed to cool to room temperature and then partitioned between DCM and water. The layers were separated, and the aqueous layer was extracted twice more with DCM. These organic extracts were combined with the initial organic layer and washed with brine, dried over anhydrous sodium sulfate, filtered through a pad of Celite, then concentrated under vacuum to afford a dark brown oil. The crude product was purified by silica gel chromatography (Isco CombiFlash; RediSep normal phase silica gel flash column (40 g); EtOAc in hexane; gradient 0-100%) to afford the title compound as an oil (87.5 mg; 11.8% yield). 1H NMR (400 MHz, DMSO-d ₆ ) δ 7.75-7.72 (m, 1H), 7.69 (d, J=1.4 Hz, 1H), 7.60 (d, J=1.6 Hz, 1H), 7.47 (dd, J=8.4, 1.7 Hz, 1H), 7.28 (dd, J=8.5, 1.3 Hz, 1H), 6.88-6.83 (m, 1H), 6.77 (s, 2H), 6.57-6.51 (m, 2H), 5.34-5.27 (m, 1H), 5.07 (br dd, J=7.0, 2.2 Hz, 1H), 3.92-3.83 (m, 1H), 3.63-3.53 (m, 1H), 2.49-2.27 (m, 4H), 1.92 (br d, J=12.4 Hz, 2H), 1.79 (br d, J=12.7 Hz, 2H), 1.62-1.42 (m, 6H). LC-MS conditions: linear gradient from 2 to 98% solvent B over 1.7 min; UV visualization at 220 nm; column: BEH C182.1 mm x 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. MS (ES): m/z=445 [M+H]+. T _r = 0.71 min.

Step 2. Preparation of 4,7-di(1H-pyrazol-5-yl)quinolin-2-amine, HCl (compound 339) To a solution of 4,7-bis(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (87.5 mg, 0.197 mmol) in MeOH (1 mL) at room temperature in a sealed reaction vial was added HCl (4 N in dioxane; 0.3 mL, 1.20 mmol). The vial was capped, and the resulting solution was stirred for 20 min, then concentrated in vacuo to give a brownish solid. The crude material was dissolved in MeOH, filtered through an Acrodisc 13 mm, 0.45 µm syringe filter, and then purified by reversed-phase preparative HPLC under the following conditions: Column: Phen

- 97 040307

Axia C18 30x100, 5 μm particles; mobile phase A: 90:10 H2O:MeOH with 0.1% trifluoroacetic acid; mobile phase B: 10:90 H2O:MeOH with 0.1% trifluoroacetic acid; run time = 20 min with a 10 min gradient from 20 to 100%; mobile phase B; flow rate = 40 mL/min. Fractions containing the desired product, as determined by LC-MS, were pooled and concentrated under vacuum to remove volatiles. The resulting residue was treated with HCl (4 N in dioxane; 0.3 mL, 1.20 mmol) and stirred at ambient temperature for 10 min, then concentrated in vacuo to give the title compound as a light yellow solid (38.4 mg; 61.8% yield) as the HCl salt. 1H NMR (DMSO-d ₆ ): δ 8.84-8.62 (m, 2H), 8.16 (d, J=1.4 Hz, 1H), 8.04 (d, J=2.3 Hz, 1H), 7.97 (dd, J=8.7, 1.6 Hz, 1H), 7.88 (d, J=2.2 Hz, 1H), 7.30 (s, 1H), 6.91-6.85 (m, 2H), 6.00-5.11 (m, 3H). The following method was used: Waters Acquity SDS, linear gradient from 2 to 98% solvent B over 1.7 min; UV visualization at 220 nm; column: BEH C18 2.1 mm x 50 mm; 1.7 μm particles (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. T _r = 0.55 min. MS (ES): m/z = 277 [M+H]+.

Example III-14: Synthesis of 4-aminoethyl-substituted quinolines

Step 1. Preparation of 7-bromo-N4-(2-(pyridazin-3-yl)ethyl)quinoline-2,4-diamine

To a homogeneous mixture of 7-bromo-4-chloroquinolin-2-amine (150 mg, 0.58 mmol) in NMP (2 mL) in a sealed reaction vial was added 2-pyridazin-3-ylethanamine (100 mg, 0.81 mmol) followed by DIPEA (0.42 mL, 2.40 mmol). The vial was capped and the mixture was stirred and heated at 120°C for 15 h. After cooling to room temperature, the reaction mixture was immediately purified by silica gel chromatography (Isco CombiFlash; RediSep normal phase silica gel flash column (24 g); MeOH in DCM; 0-20% gradient) to afford the title compound as a yellow residue (101.4 mg; 45.5% yield). LC-MS conditions: linear gradient from 2 to 98% solvent B over 1.7 min; UV visualization at 220 nm; column: BEH C18 2.1 mm x 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. T _r = 0.62 min. MS (ES): m/z = 344 [M+H] ⁺ .

Step 2. Preparation of 7-(1H-pyrazol-3-yl)-N4-(2-(pyridazin-3-yl)ethyl)quinoline-2,4-diamine (compound 340)

A mixture of 7-bromo-N4-(2-(pyridazin-3-yl)ethyl)quinoline-2,4-diamine (20 mg, 0.06 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)pyrazole (24.80 mg, 0.13 mmol) _{and Cs2CO3} (56.8 mg, 0.17 mmol) in dioxane (1.5 mL) and water (0.2 mL) in a sealed reaction vial was bubbled with argon for approximately 10 min, then _PdCl2 ( _dppf ) _-CH2Cl2 adduct (9.49 mg, 0.012 mmol) was added. The vial was sealed and the reaction mixture was heated at 90°C for 18 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo to remove volatiles, dissolved in DMF, and then purified by preparative HPLC/MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give the title compound (9.7 mg; 47.5% yield). 1H NMR (500 MHz, DMSO-d ₆ ) δ 9.11 (br d, J=3.1 Hz, 1H), 8.24-8.17 (m, 1H), 8.14 (br d, J=8.5 Hz, 1H), 7.97-7.88 (m, 1H), 7.87-7.79 (m, 2H), 7.70-7.57 (m, 3H), 6.85 (d, J=1.2 Hz, 1H), 5.90 (s, 1H), 3.81-3.72 (m, integration distorted by water suppression), 3.33 (br t, J=1.2 Hz, integration distorted by water suppression). Conditions

- 98 040307

LC-MS: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; Flow rate: 1 mL/min; Detection: MS and UV (220 nm). LC RT: 0.76 min.

M/Z= 332.12.

Compound 341: N4-(2-(pyridazin-3-yl)ethyl)-7-(thiophen-3-yl)quinoline-2,4-diamine

HN

Compound 341 (8.8 mg; 41.7% yield) was prepared following a method similar to the synthesis of compound 340, except that 4,4,5,5-tetramethyl-2-(thiophen-3-yl)-1,3,2-dioxaborolane (26.9 mg; 0.13 mmol) was used instead of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)pyrazole. 1H NMR (500 MHz, DMSO-d ₆ ) δ 9.17-9.04 (m, 1H), 8.25-8.18 (m, 1H), 8.17-8.11 (m, 1H), 8.09-8.03 (m, 1H), 7.79-7.70 (m, 3H), 7.68-7.59 (m, 4H), 5.90 (s, 1H), 3.77 (q, J=6.5 Hz, integration distorted by water suppression), 3.33 (t, J=7.2 Hz, integration distorted by water suppression). LC RT: 1.27 min. M/Z=348.08.

Compound 342: N4-(2-(pyridazin-3-yl)ethyl)-7-(thiophen-2-yl)quinoline-2,4-diamine

HN

Compound 342 (8.6 mg; 40.8% yield) was prepared following a method similar to the synthesis of compound 340, except that 4,4,5,5-tetramethyl-2-(thiophen-2-yl)-1,3,2-dioxaborolane (26.9 mg; 0.13 mmol) was used instead of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)pyrazole. 1H NMR (500 MHz, DMSO-d ₆ ) δ 9.19-8.98 (m, 1H), 8.19 (brt, J=5.2 Hz, 1H), 8.10 (d, J=8.5 Hz, 1H), 7.73-7.57 (m, 6H), 7.24-7.17 (m, 1H), 5.87 (s, 1H), 3.76-3.72 (m, integration distorted by water suppression), 3.31 (t, J=7.2 Hz, integration distorted by water suppression). LC RT: 1.23 min. M/Z= 348.10.

Example III-15: Preparation of 7-(1H-pyrazol-1-yl)-N4-(2-(pyridazin-3-yl)ethyl)quinoline-2,4-diamine (compound 343)

To a mixture of 7-bromo-N4-(2-(pyridazin-3-yl)ethyl)quinoline-2,4-diamine (20 mg, 0.06 mmol) in anhydrous DMSO (2.9 mL) in a sealed reaction vial were added 1H-pyrazole (7.91 mg, 0.12 mmol) and copper(I) iodide (22.13 mg, 0.12 mmol), followed by _Na2CO3 (24.63 _mg , 0.23 mmol). The homogeneous mixture was sparged with argon for approximately 5 min, then N',N'dimethylethylenediamine (0.02 mL, 0.19 mmol) was added. The vial was capped, and the reaction was heated at 120°C with stirring. After 18 h, the reaction mixture was cooled to room temperature, diluted with DMSO, then purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 5% B, 5-45% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give the title compound (7.5 mg; 37.2% yield). 1H NMR (500 MHz, DMSO-d ₆ ) δ 9.08 (br d, J=3.1 Hz, 1H), 8.51 (d, J=2.1 Hz, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.80-7.71 (m, 2H), 7.68-7.60 (m, 2H), 7.41-7.23

- 99 040307 (m, 1H), 6.74-6.50 (m, 2H), 5.84 (s, 1H), 3.77-3.59 (m, integration distorted by water suppression), 3.30 (br t, J=7.0 Hz, 2H). LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, particles 1.7 μm; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; Flow rate: 1 ml/min; detection: MS and UV (220 nm). LC RT: 0.83 min. M/Z= 332.12.

Example III-16: 7-(1 H-pyrazol-3-yl)-N4-(2-(tetrahydro-2 H-pyran-4-yl)ethyl)quinoline-2,4-diamine (compound 344)

Step 1. Preparation of 7-bromo-N4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinoline-2,4-diamine

To a homogeneous mixture of 7-bromo-4-chloroquinolin-2-amine (100 mg, 0.39 mmol) in NMP (0.5 mL) in a sealed reaction vial was added 4-(2-aminoethyl)tetrahydropyran hydrochloride (90 mg, 0.54 mmol), followed by DIPEA (0.35 mL, 2.00 mmol). The vial was capped, and the mixture was stirred and heated at 120°C for 20 h. After cooling to room temperature, the reaction was partitioned between EtOAc and brine. The layers were separated, and the aqueous layer was extracted again with EtOAc. The organic layers were combined, dried ( _Na2SO4 ), filtered, and concentrated in vacuo to give _a golden oil, which was immediately purified by silica gel chromatography (Isco CombiFlash; RediSep normal phase silica gel flash column (12 g); MeOH in DCM; gradient 0-20%) to give the title compound as an off-white solid (93.0 mg; 68.4% yield). ¹ H NMR (400 MHz, DMSO-d6) δ 8.20-8.09 (m, 1H), 7.93 (br s, 1H), 7.69 (d, J=2.0 Hz, 1H), 7.55-7.24 (m, 3H), 5.80 (s, 1H), 3.85 (dd, J=11.1, 3.5 Hz, 2H), 3.30-3.25 (m, 4H), 1.62 (br t, J=11.8 Hz, 5H), 1.33-1.13 (m, 2H). LC-MS conditions: linear gradient from 2 to 98% solvent B over 1.7 min; UV visualization at 220 nm; column: BEH C18 2.1 mm x 50 mm; 1.7 μm particles (heated to 50°C); flow rate: 0.8 mL/min; mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. T _r = 0.70 min. MS (ES): m/z = 350 [M+H]+.

Step 2. Preparation of 7-(1H-pyrazol-3-yl)-N4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinoline-2,4-diamine (compound 344)

A mixture of 7-bromo-X4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinoline-2,4-diamine (18 mg, _0.05 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)pyrazole (21.9 mg, 0.11 mmol) and _Cs2CO3 (50.2 mg, 0.15 mmol) in dioxane (1.5 mL) and water (0.2 mL) in a sealed reaction vial was bubbled with argon for approximately 10 min, then _PdCl2 (dppf) _-CH2Cl2 adduct ( _8.39 mg, 0.010 mmol) was added. The vial was sealed and the reaction mixture was heated at 90°C for 17 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo to remove volatiles, dissolved in DMF, and then purified by preparative HPLC/MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B for 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give the title compound (17.2 mg; 97% yield). ¹ H NMR (500 MHz, DMSO-d6) δ 8.22 (br d, J=8.5 Hz, 1H), 8.12-8.03 (m, 1H), 7.97-7.90 (m, 1H), 7.90-7.80 (m, 2H), 7.59 (br s, 2H), 6.85 (s, 1H), 5.79 (s, 1H), 3.85 (br dd, J=11.3, 3.1 Hz, 2H), 3.44-3.24 (m, integration distorted by water suppression), 1.70-1.43 (m, 6H), 1.31-1.18 (m, 2H). Some protons are not observed either because of overlap with the suppressed water peak or because of poor integration. LCMS conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; Flow rate: 1 mL/min; Detection: MS and UV (220 nm). LC RT: 1.12 min. M/Z=

338.26.

Compound 345: N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-7-(thiophen-3-yl)quinoline-2,4-diamine

Compound 345 (15.3 mg; 84% yield) was prepared following a method similar to the synthesis of compound 344, except that 4,4,5,5-tetramethyl-2-(thiophen-3-yl)-1,3,2-dioxaborolane (23.8 mg; 0.11 mmol) was used instead of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane)pyrazole. 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.26-8.19 (m, 1H), 8.14-8.05 (m, 2H), 7.81-7.77 (m, 1H), 7.76-7.71 (m, 2H), 7.65-7.52 (m, 3H), 5.80 (s, 1H), 3.85 (br dd, J=11.3, 3.4 Hz, 2H), 3.45-3.25 (m, integration distorted by water suppression), 1.68-1.58 (m, 5H), 1.30-1.18 (m, 2H). Protons may not be observed either due to overlap with the suppressed water peak or due to poor integration. LC RT: 1.62 min. M/Z= 354.06.

Example III-17: 7-(1H-pyrazol-1-yl)-N4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinoline-2,4-diamine (compound 346)

Br

ΝΗ·

Cu( _l )l, _Na2CO3

DMSO, 120°C

Compound 345

To a mixture of 7-bromo-N-(2-(tetrahydro-2H-pyran-4-yl)ethyl)quinoline-2,4-diamine (17.5 mg, 0.05 mmol) in anhydrous DMSO (2.5 mL) in a sealed reaction vial were added 1H-pyrazole (6.80 mg, 0.10 mmol) and copper(I) iodide (19.03 mg, 0.10 _mmol ), followed by _Na2CO3 (21.18 mg, 0.20 mmol). The homogeneous mixture was bubbled with argon for approximately 5 min, then N',N'dimethylethylenediamine (0.02 mL, 0.19 mmol) was added. The vial was capped, and the reaction was heated at 120°C with stirring. After 18 h, the reaction mixture was cooled to room temperature, diluted with DMSO, then purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 6% B, 6-46% B over 28 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give the title compound (13.3 mg; 76% yield). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.56 (br s, 1H), 8.18-8.03 (m, 1H), 7.83-7.70 (m, 2H), 7.60 (br dd, J=8.6, 2.0 Hz, 1H), 7.16-6.94 (m, 1H), 6.76-6.51 (m, 3H), 5.87-5.63 (m, 1H), 3.87-3.78 (m, 1H), 3.69-3.58 (m, 3H), 3.37-3.17 (m, 2H), 1.69-1.56 (m, 5H), 1.28-1.14 (m, 2H). Analytical LC-MS was used to determine final purity. Injection 1 conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then a 0.50 min hold at 100% B; Flow rate: 1 mL/min; Detection: MS and UV (220 nm). LC RT: 1.24 min. M/Z = 337.91.

Example III-18: 4-(1H-pyrazol-4-yl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 347)

- 101 040307

Step 1. Preparation of 4-(1H-pyrazol-4-yl)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine

To a mixture of 4-chloro-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (48.3 mg, 0.15 mmol) and 1-Boc-pyrazole-4-boronic acid pinacol ester (43.2 mg, 0.15 mmol) in anhydrous dioxane (3 mL) in a sealed reaction vial was added potassium phosphate (2 M aqueous; 0.22 mL; 0.44 mmol). The resulting mixture was sparged with argon for approximately 10 min, then Xphos Pd G2 (CAS: 1310584-14-5; 5.78 mg, 7.34 μmol) was added. The mixture was sparged with argon for approximately 2 min, then the vial was sealed and the mixture was stirred at 65°C. After 14 h, the reaction mixture was cooled to room temperature, then purified by silica gel chromatography (Isco CombiFlash; RediSep normal phase silica gel flash column (12 g); MeOH in DCM; gradient 0-20%) to give the title compound as a yellow residue (31.0 mg; 58.6% yield). The reaction mixture was purified by Waters Acquity SDS using the following procedure: linear gradient from 2 to 98% solvent B over 1.7 min; UV visualization at 220 nm; column: BEH C18 2.1 mm x 50 mm; particles 1.7 μm (heated to 50°C); flow rate: 0.8 mL/min. Mobile phase A: 100% water, 0.05% TFA; mobile phase B: 100% acetonitrile, 0.05% TFA. T _r = 0.66 min. MS (ES): m/z = 361 [M+H]+.

Step 2. Preparation of 4-(1H-pyrazol-4-yl)-7-(1H-pyrazol-5-yl)quinolin-2-amine (compound 347)

To a solution of 4-(1H-pyrazol-4-yl)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-2-amine (31.0 mg, 0.09 mmol) in anhydrous MeOH (1 mL) at room temperature under nitrogen atmosphere was added p-toluenesulfonic acid (4.2 mg, 0.024 mmol). The resulting mixture was stirred at ambient temperature for 1 h, then concentrated under vacuum to remove volatiles. The crude material was dissolved in DMF, filtered through an Acrodisc 13 mm, 0.45 μm syringe filter, and purified by reversed-phase preparative HPLC using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; Mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; gradient: 0 min hold at 2% B, 2-42% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were pooled and dried by centrifugal evaporation to afford the title compound (1.7 mg; 6.8%). 1H NMR (500 MHz, DMSO-d ₆ ) δ 8.11-7.94 (m, 2H), 7.92-7.84 (m, 2H), 7.78-7.70 (m, 1H), 7.68-7.58 (m, 1H), 6.79 (d, J=1.4 Hz, 1H), 6.72 (s, 1H), 6.38 (br s, 2H). Protons may not be observed due to either overlap with the suppressed water peak or poor integration. LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then a 0.50-min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.71 min. M/Z = 277.04.

Compound 348: 2-(4-(2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)-1H-pyrazol-1-yl)ethan-1-ol

Compound 348 (1.3 mg; 3.3% yield) was prepared following a method similar to the synthesis of compound 347, except that 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h-pyrazol-1-yl)ethanol (35.0 mg, 0.15 mmol) was used instead of 1-Boc-pyrazole-4-boronic acid pinacol ester. 1H NMR (500 MHz, DMSO-de) δ 8.29-8.16 (m, 1H), 8.04-7.92 (m, 2H), 7.90-7.83 (m, 1H), 7.82-7.55 (m, 2h), 6.88-6.68 (m, 2H), 4.27 (t, J=5.3 Hz, 2H), 3.88-3.74 (m, 2H). Protons may not be observed or

- 102 040307 due to overlap with the suppressed water peak or poor integration. LC RT: 0.80 min.

M/Z=321.07.

Example III-19: Preparation of (R)-1-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)-3-morpholinopropan-2-ol (compound 349)

HE

Step 1: Preparation of 7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-ol

7-Bromoquinolin-4-ol (1.5 g, 6.69 mmol) and 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazole (2.421 g, 8.70 mmol) were suspended in a 1:1 mixture of dioxane:DMF (33 mL). Nitrogen gas was bubbled through the reaction mixture for 5 min, then PdCl ₂ (dppf)-CH ₂ Cl ₂ adduct (0.273 g, 0.335 mmol) was added, followed by aqueous tripotassium phosphate (2 M, 10.04 mL, 20.08 mmol). Nitrogen gas was bubbled through the reaction mixture for another 5 min. The reaction was then heated under N2 for 16 h. After cooling to room temperature, the reaction mixture was partitioned between EtOAc and H2O. The organic layer was separated, and the aqueous phase was extracted with two additional portions of EtOAc. _The combined organic phases were dried over _Na2SO4 , filtered through Celite, and concentrated. The residue was triturated with _Et2O to give 7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-ol as a light brown solid (1.20 g). Ή NMR (400 MHz, DMSO-d6) δ 11.90 (br d, J=5.0 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.96 (dd, J=12, 6.0 Hz, 1H), 7.69 (d, J=1.2 Hz, 1H), 7.63 (d, J=1.6 Hz, 1H), 7.45 (dd, J=8.4, 1.5 Hz, 1H), 6.58 (d, J=1.8 Hz, 1H), 6.08 (d, J=7.3 Hz, 1H), 5.28 (dd, J=9.9, 2.0 Hz, 1H), 4.02 (br d, J=12.5 Hz, 1H), 3.62 (td, J=10.9, 3.3 Hz, 1H), 2.46-2.33 (m, 1H), 1.95 (br d, J=8.6 Hz, 1H), 1.80 (br d, J=13.0 Hz, 1H), 1.66-1.46 (m, 3H).

Step 2. Preparation of 4-(((R)-oxiran-2-yl)methoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5yl)quinoline

To a solution of 7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-ol (542 mg, 1.84 mmol) in THF (9 mL) were added (S)-oxiran-2-ylmethanol (0.244 mL, 3.67 mmol), triphenylphosphine (1267 mg, 3.67 mmol), and DIAD (0.714 mL, 3.67 mmol). The reaction mixture was stirred for 16 h at room temperature. Additional DIAD (0.200 mL, 1.03 mmol) was added. After 4 h, the reaction was complete as determined by LC-MS. The reaction mixture was concentrated and the residue was purified by column chromatography (40 g SiO ₂ , gradient elution from 0 to 30% CH ₂ Cl ₂ -acetone) to give 4-(((R)oxiran-2-yl)methoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline (392.7 mg, 61%). ^1H NMR (400 MHz, chloroform-d) δ 8.85-8.78 (m, 1H), 8.35 (d, J=8.6 Hz, 1H), 8.24-8.17 (m, 1H), 7.73-7.68 (m, 1H), 7.68-7.67 (m, 1H), 6.81 (d, J=5.3 Hz, 1H), 6.49 (d, J=1.8 Hz, 1H), 5.36-5.28 (m, 1H), 4.56 (ddd, J=11.l, 2.7, 0.9 Hz, 1H), 4.24-4.12 (m, 2H), 3.65 (td, J=11.7, 2.2 Hz, 1H), 3.54 (dq, J=6.0, 2.9 Hz, 1H), 3.06-3.00 (m,

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1H), 2.89 (dt, J=4.8, 2.4 Hz, 1H), 2.72-2.56 (m, 1H), 2.13-2.02 (m, 1H), 1.91 (br d, J=13.1 Hz, 1H), 1.84-1.72 (m, 1H), 1.40-1.24 (m, 2H).

Step 3. Preparation of 4-(((R)-oxiran-2-yl)methoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline 1-oxide mCPBA (511 mg, 2.22 mmol) was added to a solution of 4-(((R)-oxiran-2-yl)methoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline (390 mg, 1.11 mmol) in CH ₂ Cl ₂ (12.3 mL). The reaction mixture was stirred for 2 h, then quenched with saturated sodium thiosulfate solution. The biphasic mixture was stirred for 0.5 h, then saturated aqueous sodium bicarbonate solution was added. The reaction was extracted twice with CH ₂ Cl ₂ . The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give 4-(((R)-oxiran-2-yl)methoxy)7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline 1-oxide (408 mg, 1.11 mmol, quantitative yield). 1H NMR (400 MHz, chloroform-d) δ 9.04-8.95 (m, 1H), 8.50 (d, J=6.9 Hz, 1H), 8.35 (dd, J=8.7, 0.7 Hz, 1H), 7.87 (dt, J=8.6, 1.5 Hz, 1H), 7.67 (d, J=1.6 Hz, 1H), 6.74 (d, J=6.9 Hz, 1H), 6.58 (d, J=1.8 Hz, 1H), 5.35-5.30 (m, 1H), 4.68-4.54 (m, 1H), 4.26-4.19 (m, 1H), 4.15 (ddd, J=11.1, 6.2, 3.4 Hz, 1H), 3.84-3.73 (m, 1H), 3.56-3.49 (m, 1H), 3.04 (t, J=4.5 Hz, 1H), 2.90-2.85 (m, 1H), 2.70-2.57 (m, 1H), 2.12-2.04 (m, 1H), 1.92 (br d, J=12.4 Hz, 1H), 1.85-1.73 (m, 1H), 1.38-1.25 (m, 2H).

Step 4. Preparation of 4-((R)-2-hydroxy-3-morpholinopropoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)1H-pyrazol-5-yl)quinoline 1-oxide

4-(((R)-oxiran-2-yl)methoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline 1-oxide (59 mg, 0.16 mmol), ethanol (1.6 mL), and morpholine (16.8 mg, 0.193 mmol) were added to a 2-dram vial under pressure, and the reaction mixture was heated at 60°C. After 3 h, the reaction was complete according to LC-MS. The reaction mixture was concentrated, and the residue was dissolved in a small amount _{of CH2Cl2} followed by the addition of _Et2O , which resulted in the formation of a solid. The supernatant was decanted and the solid was washed with _Et2O . The solid was dried to give 4((R)-2-hydroxy-3-morpholinopropoxy)-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinoline 1 oxide (42.7 mg, 59%). ¹ H NMR (400 MHz, chloroform-d) δ 9.05-8.95 (m, 1H), 8.51 (d, J=6.8 Hz, 1H), 8.31 (d, J=8.6 Hz, 1H), 7.85 (dd, J=8.6, 1.1 Hz, 1H), 7.67 (d, J=1.7 Hz, 1H), 6.74 (d, J=6.9 Hz, 1H), 6.62-6.51 (m, 1H), 5.34-5.31 (m, 1H), 4.35-4.28 (m, 1H), 4.27 (s, 1H), 4.21 (dt, J=11.4, 2.0 Hz, 1H), 3.81-3.75 (m, 4H), 2.80-2.72 (m, 2H), 2.69-2.64 (m, 2H), 2.63-2.57 (m, 1H), 2.57-2.49 (m, 2H), 2.12-2.05 (m, 2H), 1.92 (br d, J=12.9 Hz, 2H), 1.78 (dt, J=7.9, 4.0 Hz, 2H), 1.59 (br dd, J=9.6, 3.3 Hz, 2H).

Step 5. Preparation of (2R)-1-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin4-yl)oxy)-3-morpholinopropan-2-ol

To a solution of 4-((R)-2-hydroxy-3-morpholinopropoxy)-7-(1-(tetrahydro-2H-pyran _- 2-yl)-1H-pyrazol5-yl)quinoline 1-oxide (42 mg, 0.092 mmol) in _CH2Cl2 was added ammonium hydroxide solution (30%) (0.9 mL, 14.3 mmol) followed by tosyl chloride (35.2 mg, 0.185 mmol). After 20 min, the reaction was complete according to LC-MS.

The reaction mixture was diluted with CH2Cl2 and water and extracted twice with CH2Cl2.

The combined organic layers were dried over sodium sulfate and concentrated to give crude (2R)-1-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4-yl)oxy)-3-morpholinopropan-2-ol (25 mg, 60%). LC RT: 0.56 min. M/Z=454.5.

Step 6: Preparation of (R)-1-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)-3-morpholinopropan-2-ol (compound 349)

To a solution of crude (2R)-1-((2-amino-7-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)quinolin-4yl)oxy)-3-morpholinopropan-2-ol (25 mg, 0.055 mmol) in CH ₂ Cl ₂ (0.7 mL) was added TFA (350 µL, 4.54 mmol) and the reaction mixture was stirred at room temperature. After 1 h, the reaction was complete according to LC-MS. The reaction mixture was concentrated and azeotroped with CH ₂ Cl ₂ (1x). The residue was dissolved in DMF, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 µm particles; Mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-40% B over 20 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to afford (R)-1-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)oxy)-3-morpholinopropan-2-ol (12.7 mg, 62%). 1H NMR (500 MHz, DMSO-d6) δ 8.16 (d, J=8.2 Hz, 1H), 7.99 (s, 1H), 7.90 (br d, J=7.9 Hz, 1H), 7.83 (br s, 1H), 6.87 (d, J=2.1 Hz, 1H), 6.36 (s, 1H), 4.50-4.42 (m, 1H), 4.29-4.20 (m, 2H), 3.32-3.20 (m, 2H), 2.94-2.87 (m, 1H). Some aliphatic protons are not visible in 1H NMR due to overlap with the water peak. LC RT: 0.96 min. M/Z= 370.22.

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Example Ш-20: Preparation of 3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)-1-morpholinopropan-1-one (compound 350)

Step 1: Preparation of tert-butyl 3-((2-amino-7-bromoquinolin-4-yl)amino)propanoate

Hunig's base (0.678 mL, 3.88 mmol) was added to a solution of 7-bromo-4-chloroquinolin-2-amine (100 mg, 0.388 mmol) and tert-butyl 3-aminopropanoate hydrochloride (353 mg, 1.942 mmol) in DMSO (1 mL). The reaction mixture was heated to 120°C overnight. The reaction mixture was cooled, evaporated, and dried under high vacuum. The residue was purified by ISCO (24 g column; hexanes/ethyl acetate; gradient 0 to 100% then 0 to 20% DCM/MeOH) to give tert-butyl 3-((2-amino-7-bromoquinolin-4-yl)amino)propanoate (140 mg, 0.38 mmol, 98%).

Step 2: Preparation of 3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propanoic acid

1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (133 mg, 0.478 mmol), tert-butyl 3-((2-amino-7-bromoquinolin-4yl)amino)propanoate (140 mg, 0.382 mmol), and PdCl ₂ (dppf)-CH ₂ Cl ₂ adduct (31.2 mg, 0.038 mmol) were placed in vials under elevated pressure. The vial was placed under vacuum three times and then backfilled with nitrogen. Dioxane (5 mL) and tripotassium phosphate (2 M aqueous) (0.573 mL, 1.147 mmol) were added, nitrogen was bubbled through the solution, and the reaction mixture was then heated to 100°C overnight. The reaction mixture was cooled to room temperature, diluted with 50 mL DCM, dried over sodium sulfate, and concentrated. The residue was purified using ISCO (24 g column; DCM/MeOH; gradient from 0 to 40%). After evaporation, the residue was dissolved in dioxane (5 mL). To this solution was added HCl (4 N dioxane) (3 mL, 12.00 mmol). After 16 h, the reaction mixture was concentrated under reduced pressure and dried under high vacuum.

Step 3: Preparation of 3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)-1-morpholinopropan-1one (compound 350)

A solution of 3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)propanoic acid (35 mg, 0.118 mmol), morpholine (0.021 mL, 0.235 mmol), TBTU (113 mg, 0.353 mmol), and TEA (0.164 mL, 1.177 mmol) in DMF (1 mL) was stirred at room temperature for 4 h. The reaction mixture was diluted with 0.5 mL DMF and 0.5 mL acetic acid, filtered through a syringe filter, and the crude material was purified by preparative LC-MS using the following conditions: XBridge C18 column, 200 mm x 19 mm, 5 μm particles; mobile phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; gradient: 0 min hold at 0% B, 0-25% B over 35 min, then 4 min hold at 100% B; flow rate: 20 mL/min; column temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containing the desired product were combined and dried by centrifugal evaporation to give 3-((2-amino-7-(1H-pyrazol-5-yl)quinolin-4-yl)amino)-1-morpholinopropan-1-one bis-trifluoroacetate (11.3 mg, 26%) ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.25-8.18 (m, 1H), 8.04 (br s, 1H), 7.96 (br s, 1H), 7.93-7.81 (m, 2H), 7.70-7.61 (m, 1H), 6.86 (s, 1H), 5.85 (s, 1H), 3.62-3.51 (m, 4H), 3.48 (br d, J=7.3 Hz, 2H), 2.80 (t, J=7.0 Hz, 2H). Three methylenes are not visible, probably due to overlap with the suppressed water peak. LC-MS conditions: column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; mobile phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; temperature: 50°C; gradient: 0% B to 100% B over 3 min, then 0.50 min hold at 100% B; flow rate: 1 mL/min; detection: MS and UV (220 nm). LC RT: 0.97 min. M/Z= 367.11.

Evaluation of biological activity

Measurement of Sh-Shch production in PMA-differentiated THP-1 cells

THP-1 cells were purchased from the American Type Culture Collection and passaged according to the supplier's instructions. Prior to experiments, cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS, penicillin (100 units/mL), and streptomycin.

- 105 040307 (100 μg/ml) and maintained in log phase until experimentation. Before the experiment, THP-1 cells were treated with PMA (phorbol 12-myristate 13-acetate) (10 μg/ml) for 24 h. On the day of the experiment, the medium was removed and the attached cells were trypsinized for 2 min, then the cells were collected, washed with PBS (phosphate-buffered saline), pelleted by centrifugation, resuspended in 2% heat-inactivated FBS with RPMI at a concentration of 1 x 10 ⁶ cells/ml and 100 μl were seeded into a 96-well plate. Compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium to achieve the desired concentration (e.g., 100, 30, 10, 3, 1, 0.3, or 0.1 μM). Cells were incubated with the compounds for 4 h. Cell-free supernatant was collected, and IL-1β production was assessed by ELISA. A vehicle-only control was analyzed simultaneously with each experiment. The final DMSO concentration was 1%. Compounds demonstrated a dose-dependent enhancement of IL-1β production in PMA-differentiated THP-1 cells.

Measurement of II-1β production in PMA-differentiated cells

TNR-1 (alternative method)

THP-1 cells were purchased from the American Type Culture Collection and passaged according to the supplier's instructions. Prior to experiments, cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS, penicillin (100 units/ml), streptomycin (100 μg/ml), HEPES (10 mM), and sodium pyruvate (1 mM) and maintained in log phase until experiments. Prior to experiments, THP-1 cells were treated with PMA (phorbol 12-myristate 13acetate) (20 μg/ml) overnight. On the day of the experiment, the medium was removed and adherent cells were trypsinized for 2 min. Cells were then collected, washed with PBS (phosphate-buffered saline), pelleted by centrifugation, and resuspended in 2% heat-inactivated FBS with RPMI at a concentration of 50,000 cells/well in a 384-well plate. Cell-free supernatant was collected, and II-1β production was assessed by ELISA. Compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium to achieve the desired concentration (e.g., 100, 30, 10, 3, 1, 0.3, or 0.1 μM). Cells were incubated with compounds for 2 h. A vehicle-only control was analyzed simultaneously with each experiment. The final DMSO concentration was 1%. The compounds demonstrated a dose-dependent enhancement of II-1β production in PMA-differentiated THP-1 cells.

Measurement of IL-1β production using the hTRF protocol (second alternative method)

Serial dilutions of compounds in DMSO were added to 384-well small-volume plates at 100 nL/well using an ECHO 550 acoustic dispenser (Labcyte) to achieve the target starting concentration of 10 μM in the assay. THP-1 cells in RPMI medium (Gibco, 11875) with 10% FBS at a density of ^1x106 cells/mL in a T175 flask were treated with a final concentration of phorbol 12-myristate 13-acetate (PMA) (Sigma, P1585) of 50 ng/mL overnight at 37°C under 5% CO2 for differentiation. Cells were harvested the following day after a good wash with dPBS using 0.5% trypsin. A cell solution was prepared at a concentration of 1 x ¹⁰⁶ cells/mL for 50,000 cells at 50 μL/well in RPMI medium with 2% FBS. Cells were seeded using a multichannel pipette with compound dilutions into 384-well Greiner black, transparent-bottomed, tissue culture-treated plates (781090). The plates were incubated at 37°C under 5% CO2 for 2 h.

After 2 h of incubation, the cell plates were centrifuged for 5 min at 1200 rpm. Using Felix (CyBio), 8 µl of the supernatant was transferred to 384-well white low-volume plates (Perkin Elmer, 6008230). The supernatant was analyzed using the human IL-1-beta hTRF kit (CISBIO, 62HIL1BPEG). To obtain an IL1Beta standard curve, the kit instructions were followed, and then the kit antibodies were diluted 1:40 instead of 1:20 as specified in the kit. After combining, the antibodies were added to all plates at 5 µl/well. The plates were sealed and incubated at 4°C overnight. The plates were then read on a Perkin Elmer EnVision at 665/615 nm using an hTRF laser. The compounds demonstrated a dose-dependent enhancement of IL-1e production.

Measurement of P-SH production in human whole blood

Serial dilutions of compounds in DMSO were added to 384-well low-volume plates at 100 nL/well using an ECHO 550 acoustic dispenser (Labcyte) to achieve a target starting concentration of 10 μM in the assay. Human venous whole blood from healthy donors was pretreated with LPS (Invivogen, Cat # tlrl-eblps) at 1 ng/mL for 4 h at 37°C in a 95%/5% CO2 humidified incubator. Primed blood was added to the compound in the plate and incubated for an additional 4 h at 37°C. IL-1-beta in supernatants was measured using the AlphLISA kit (Cat # AL220) according to the manufacturer's instructions. The compounds demonstrated a dose-dependent increase in IL-1β production. _EC50 was determined using primed but untreated blood as a baseline.

Measurement of IL-1e production using the hTRF protocol in a mouse model

Immortalized C57BL/6 murine macrophages were obtained from Ericke Latz, University of Bonn/University of Massachusetts, Worchester, MA. Cells were harvested using 0.05% trypsin and washed with PBS. Cells were seeded at 30,000 cells/well in 25 μl in DMEM (Gibco, 11965) supplemented with 2% FBS and incubated for 10 min at 37°C under 5% _CO2 . LPS-EB (Invivogen, tlrl-eblps) was added to a final concentration of 200 ng/ml at 5 μl/well, and cells were incubated for 2 h at 37°C under 5% CO2.

Cells were added with serial dilutions of compounds in DMSO in 384-well small volume plates at 60 nL/well using an ECHO 550 acoustic dispenser (Labcyte) to achieve a target starting concentration of 50 μM in the assay and incubated with compounds for an additional 2 h at 37°C under 5% CO2. After 2 h of incubation, cell plates were centrifuged for 5 min at 1200 rpm. Using Felix (CyBio), 8 μL of supernatant was transferred to 384-well white small volume plates (Perkin Elmer, 6008230). The human IL-1 beta hTRF kit (CISBIO, 62MIL1BPEH) was used for supernatant analysis. To generate a standard curve of IL-1 beta, the kit instructions were followed (the antibodies from the kit were diluted 1:40 instead of 1:20 as specified in the kit). After pooling, the antibodies were added to all plates at 5 µl/well. The plates were sealed and incubated at 4°C overnight. The plates were then read on a Perkin Elmer EnVision at 665/615 nm using an hTRF laser. Data were then converted to pg/ml IL-1 beta. The compounds demonstrated a dose-dependent increase in IL-1β production.

In vitro human TLR7 and TLR8 binding reporter assays

Logarithmically growing human HEK-Blue cells coexpressing the TLR7 or TLR8 gene and the NF-kB/AP1-inducible reporter gene SEAP (secreted embryonic alkaline phosphatase; Invivogen, San Diego, CA) were added to individual wells of a 384-well plate (15,000 cells/20 μl per well) and incubated for 24 h at 37°C, 5% _CO2 . The following day, test compounds or DMSO were dispensed into individual wells using acoustic fluid technology (100 nl per well), and the cells were then incubated for 18 h at 37°C, 5% _CO2 .

SEAP production in cells was measured using an Envision plate reader 30 min after the addition of freshly prepared Quanti-Blue reagent (prepared according to the manufacturer's instructions; Invivogen, San Diego, CA) to the HEK-Blue, TLR, and Nf-kBSEAP cellular reactions. All _EC50 (half-maximal effective concentration) values were determined using proprietary data analysis software. The normalized _EC50 value is the absolute value determined by setting Ymax to 100% using the RLU (relative light units) values of the reference standard from cells treated with 50 μM of the reference standard.

The table contains biological data for compounds that were analyzed using one or more of the above methods. Key to activity ranges: A = <1 µM; B = >1 µM, <20 µM; C = >20 µM, <100 µM; D = >100 µM; E <50% activity at 50 µM.

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Connection No. NLRP3 hILIB IC ₅ o (μM) TLR7 agonist, ECeo (μM) TLR8 agonist, ECeo (μM) 101 0.79 D D 102 1.38 D D 103 1.60 D D 104 0.69 D D 105 2.98 D D 106 3.82 D D 107 0.34 D D 108 1.15 109 4.33 D D BY 1.64 D D 111 2.46 D D 112 2.56 D D 113 11.90 D D 114 4.25 D D 115 3.04 D D 116 4.07 D D 117 5.80 E E 118 1.73 D D 119 3.07 D WITH 120 4.24 E E 121 3.03 E D 122 1.87 E E 123 3.01 E E 124 14.51 D D 125 1.98 E E 126 3.53 E E 127 1.15 D D 128 0.10 D D 129 2.29 D D 130 2.76 E D 131 0.85 D D 132 0.53 D D 133 0.30 D D 134 0.29 D D 135 0.12 D D 136 3.02 D D 137 0.55 D D 138 5.95 IN D 139 2.01 D D

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140 1.41 D D 141 0.63 D D 142 1.43 D D 143 5.81 D D 144 0.48 D D 145 2.55 D D 146 4.07 D D 147 0.99 D D 148 1.64 D D 149 0.14 D D 150 0.22 D D 151 0.31 D D 152 1.03 D D 153 1.11 D D 154 0.44 D D 155 0.42 D D 156 0.20 D D 157 0.73 D D 158 35.84 D D 159 0.52 D D 160 2.76 D D 161 0.15 D D 162 2.49 D D 163 0.16 D D 164 1.91 D D 165 0.47 D D 166 0.56 D D 167 8.33 D D 168 2.40 E D 169 1.37 D D 170 5.82 D D 171 0.38 E D 172 0.41 D D 173 0.28 D D 174 0.82 D D 175 0.97 D D 176 0.70 D D 177 3.07 D D

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178 0.70 D D 179 0.78 D D 180 9.72 D D 181 9.92 E D 182 1.14 D D 183 8.40 D D 184 7.27 E D 185 0.56 D D 186 1.08 D D 187 0.85 D D 188 6.31 D D 189 0.39 D D 190 0.66 D D 191 0.63 D D 192 0.63 D D 193 1.69 D D 194 2.23 D D 195 0.93 D D 196 1.11 D D 197 2.86 D D 198 11:37 D D 199 13.30 D D 200 1.89 D D 201 5.05 D D 202 1.89 D D 203 0.19 D D 204 0.64 D D 205 1.29 D D 206 0.84 D D 207 1.07 D D 208 1.55 D D 209 1.96 D D 210 20.75 D D 211 13.50 D D 212 9.27 D D 213 1.55 D D 214 4.61 D D 215 1.94 D D

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216 2.23 D D 217 14.35 D D 218 1.19 D D 219 2.75 D D 220 0.57 D D 221 0.52 D D 222 0.06 D D 223 1.35 D D 224 0.22 D D 225 0.13 D D 226 0.73 D D 227 0.33 D D 228 0.16 D D 229 0.09 D D 230 0.54 D D 231 1.27 D D 232 0.23 D D 233 0.09 D D 234 0.23 D D 235 0.86 D D 236 2.65 D D 237 2.57 D D 238 1.49 D D 239 4.83 D D 240 0.24 D D 241 2.25 D D 242 2.35 D D 243 1.02 D D 244 0.35 D D 245 1.34 D D 246 0.17 D D 247 0.48 D D 248 0.33 D D 249 0.05 D D 250 1.72 D D 251 0.37 D D 252 1.69 D D 253 0.27 D D

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254 0.21 D D 255 0.27 D D 256 0.22 D D 257 0.68 D D 258 1.84 D D 259 0.64 D D 260 0.32 D D 261 0.14 D D 262 0.12 D D 263 2.87 D D 264 2.89 D D 265 0.35 D D 266 0.39 D D 267 1.59 D D 268 0.97 D D 269 1.15 D D 270 7.37 D D 271 0.36 D D 272 6.22 D D 273 0.17 D D 274 1.98 D D 275 0.20 D D 276 0.52 D D 277 0.22 D D 278 1.31 D D 279 0.33 D D 280 1.57 D D 281 0.58 D D 282 5.66 D D 283 1.70 D D 284 0.38 D D 285 1.56 D D 286 11/13 D D 287 0.70 D D 288 0.39 D D 289 0.85 D D 290 1.00 D D 291 1.67 D D

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292 0.34 D D 293 0.87 D D 294 0.21 D D 295 0.31 D D 296 0.56 D D 297 0.57 D D 298 0.15 D D 299 0.65 D D 300 0.54 D D 301 1.01 D D 302 0.05 D D 303 0.14 D D 304 0.37 D D 305 0.64 D D 306 1.10 D D 307 0.11 D D 308 0.52 D D 309 0.42 D D 310 0.11 D D 311 3.13 D D 312 1.59 D D 313 0.11 D D 314 1.80 D D 315 1.52 D D 316 0.56 D D 317 0.69 D D 318 0.07 D D 319 25.7 D D 320 27.68 D D 321 11:59 D D 322 0.61 D D 323 5.72 D D 324 1.76 D D 325 23.16 D D 326 24.73 D D 327 4.66 D D 329 0.67 D D 330 24.59 D D

- 113 040307

331 14.62 D D 332 0.65 D D 333 1.31 D D 334 2.62 D D 335 0.63 D D 336 2.03 D D 337 9.17 D D 338 3.41 D D 339 0.48 D D 340 0.73 D D 341 0.62 D D 342 1.10 D D 343 0.61 D D 344 1.11 D D 345 1.77 D D 346 0.76 D E 347 1.18 D D 348 7.26 D D 349 5.80 D D 350 4.19 D D 351 0.17 D

CLAUSES OF THE INVENTION

Claims (29)

CLAIM 1. A compound characterized by formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof: where W' represents H; W is independently selected from -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) _3-4 -OH, -NH-(CH2)1-2-CH(CH3)OH, -NH -(CH2)i-2-C(CH3)2OH, -O-(CH2)1-2-(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrimidinyl), -NH-(CH ₂ ) _1-2 -(pyridazinyl), -NH-(CH ₂ ) _1-2 -CF ₂ (pyridyl), R ³ is independently pyrazolyl or thienyl; And R ⁴ is independently H or F. 2. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, where W' represents H; W is independently selected from -O-CH ₂ CH(OH)(CH ₂ OH), -NH-(CH ₂ ) _3-4 -OH, -NH-(CH2)1-2-CH(CH3)OH, -NH -(CH2)1-2-C(CH3)2OH, -O-(CH2)1-2-(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrazolyl), -NH-(CH ₂ ) _1-2 -(pyrimidinyl), -NH-(CH ₂ ) _1-2 -(pyridazinyl), -NH-(CH2)1-2-CF2(pyridyl), R ³ is independently R ⁴ is independently H or F. 3. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, where W' represents H; W is independently selected from: -O-CH2CH(OH)(CH2OH), -NH-(CH2)3-4-OH, -NH-(CH2)1-2-CH(CH3)OH, -NH-(CH2) i-2-C(CH3)2OH, -NH-(CH ₂ ) _1-2 -(pyrazolyl), - 114 040307 R ³ is independently R ⁴ is independently H or F. 4. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, where W' represents H; W is independently selected from: -O-CH ₂ CH(OH)(CH2OH), -NH-(CH ₂ ) _{3 -4} -OH, -NH-(CH2)i-2-CH(CH3)OH, -NH- (CH2)i-2-C(CH3)2OH, -NH-(CH ₂ ) ₁ . ₂ -(pyrazolyl) and R ³ is R ⁴ is independently H or F. 5. The connection according to claim 1, where the connection is selected from: or their pharmaceutically acceptable salt. 6. The connection according to claim 5, where the connection is selected from: - 115 040307 or their pharmaceutically acceptable salt. 7. The connection according to claim 5, where the connection is selected from: or their pharmaceutically acceptable salt. 8. The compound according to claim 1, where the compound is or a pharmaceutically acceptable salt thereof. 9. The compound according to claim 1, where the compound is or a pharmaceutically acceptable salt thereof. - 116 040307 11. The compound according to claim 1, where the compound is 12. A pharmaceutical composition containing a compound or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 to 11 and one or more pharmaceutically acceptable adjuvants. 13. The use of a compound or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 to 11 for the treatment of cancer. 14. The use of claim 13 wherein the cancer is selected from acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendix cancer, teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer , bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, colon cancer, cancer of the rectum and colon, craniopharyngioma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal intestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, lip and mouth cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, oral cancer, osteosarcoma, ovarian cancer, penis cancer, pharyngeal cancer, prostate cancer, cancer rectum, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, malignant tumor throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer. 15. Use according to claim 13 or 14, wherein the cancer is a refractory cancer. 16. The use of claim 13 wherein the cancer is selected from breast cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer and prostate cancer. 17. The use of claim 13 wherein the cancer is selected from hormone receptor positive breast cancer, microsatellite stable colon or rectal cancer, pancreatic cancer and prostate cancer. 18. Use according to any one of claims 13-17, wherein the compound is administered in combination with one or more additional cancer treatments. 19. The use of claim 18, wherein the one or more additional cancer treatments include surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy, or a combination thereof. 20. Use according to claim 19, wherein the adjunct cancer therapy comprises one or more agents selected from nivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, TSR-042, GLS-010, AM- 0001 STI-1110 AGEN2034 MGD013 IBI308 BMS-936559 Atezolizumab Durvalumab Avelumab 870893, BMS-986016, MGA271, lirilumab, IPH2201, emactuzumab, INCB024360, galunisertib, ulokuplumab, BKT140, bavituximab, CC-90002, bevacizumab, MNRP1685A, ipilimumab, MK-1308, AGEN-188umab4, and tremelimumab. 21. The use of claim 19 wherein the adjunctive cancer therapy comprises one or more of nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab, and avelumab. 22. The use of a pharmaceutical composition according to claim 12 for the treatment of cancer. 23. The use of claim 22 wherein the cancer is selected from acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendix cancer, teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer , malignant bone tumor, - 117 040307 malignant brain tumor, malignant breast tumor, bronchial tumor, carcinoid tumor, cardiac tumor, cervical malignant tumor, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasia, malignant tumor of the colon, malignant tumor of the rectum and colon, craniopharyngioma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head cancer, and neck cancer, heart cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, malignant tumor and larynx, chronic myelogenous leukemia, lip and mouth cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, oral cancer, osteosarcoma, ovarian cancer, penis cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer malignant tumor and malignant tumor of the vulva. 24. Use according to claim 22 or 23, wherein the cancer is a refractory cancer. 25. The use of claim 22 wherein the cancer is selected from breast cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer and prostate cancer. 26. The use of claim 22 wherein the cancer is selected from hormone receptor positive breast cancer, microsatellite stable colon or rectal cancer, pancreatic cancer and prostate cancer. 27. Use according to any one of claims 22-26, wherein the compound is administered in combination with one or more additional cancer treatments. 28. The use of claim 27, wherein the one or more additional cancer treatments include surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy, or a combination thereof. 29. Use according to claim 28, wherein the adjunctive cancer therapy comprises one or more agents selected from nivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, TSR-042, GLS-010, AM- 0001 STI-1110 AGEN2034 MGD013 IBI308 BMS-936559 870893, BMS-986016, MGA271, lirilumab, IPH2201, emactuzumab, INCB024360, galunisertib, ulokuplumab, VKT140, bavituximab, CC-90002, bevacizumab, MNRP1685A, ipilimumab, MK-1308, AGEN-1884, and treimumab. 30. The use of claim 28, wherein the adjunctive cancer therapy comprises one or more of nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab, and avelumab.