Haloallylamine sulfone derivative inhibitors of lysyl oxidase and uses thereof
阅读说明：本技术 赖氨酰氧化酶的卤代烯丙胺砜衍生抑制剂及其用途 (Haloallylamine sulfone derivative inhibitors of lysyl oxidase and uses thereof ) 是由 艾莉森·桃乐丝·芬得利 克雷格·伊旺·特纳 曼达·迪奥达 强纳森·史都华·甫特 沃尔夫冈·杰 于 2019-08-02 设计创作，主要内容包括：本发明涉及能够抑制某些胺氧化酶的新颖化合物。这些化合物可用于治疗人类受试者以及宠物和家畜的多种适应症,例如纤维化、癌症和/或血管生成。另外,本发明涉及含有这些化合物的药物组合物以及其各种用途。(The present invention relates to novel compounds capable of inhibiting certain amine oxidases. These compounds are useful for treating a variety of indications, such as fibrosis, cancer and/or angiogenesis, in human subjects as well as in companion animals and livestock. In addition, the invention relates to pharmaceutical compositions containing these compounds and to various uses thereof.)
1. A compound of formula I:
or a stereoisomer, pharmaceutically acceptable salt, polymorph form, solvate, hydrate or tautomeric form thereof; wherein:
a is aryl or heteroaryl;
each R1Independently selected from the group consisting of: X-R2Halogen, deuterium, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R4And R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substitutedIs substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R7Selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combined to form a composite with 0 to 1 additionA 5 to 7 membered ring with the heteroatom as a ring member;
and is
n is 0, 1,2, 3,4,5 or 6.
2. The compound of claim 1, wherein a is selected from the group consisting of: phenyl, naphthyl, pyridyl, quinolyl, benzothiazolyl, and indolyl.
3. The compound of claim 1 or 2, wherein a is selected from the group consisting of:
4. the compound of claim 1, wherein a is heteroaryl.
5. The compound of claim 1, wherein
A is selected from the group consisting of
R1Is methyl or isopropyl; and is
n is 0 or 1.
6. The compound of claim 1, wherein a isAnd n is 0.
7. The compound of claim 1, having formula Ia:
or a stereoisomer, pharmaceutically acceptable salt, polymorph form, solvate, hydrate or tautomeric form thereof; wherein:
each R1Independently selected from the group consisting of: X-R2Halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R7selected from the group consisting of: halogen, -OH, C1-6Alkyl, aryl, heteroaryl, and heteroaryl,O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having from 0 to 1 additional heteroatoms as ring members;
and is
n is 0, 1,2 or 3.
8. The compound of claim 7, wherein n is 0.
9. The compound of claim 7, wherein
Each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl and-S (O)2R6(ii) a Wherein each C1-6Alkyl optionally substituted with one or more halogens;
R6is C1-6An alkyl group;
and is
n is 1 or 2.
10. The compound of claim 1, selected from the group consisting of:
or a pharmaceutically acceptable salt or solvate thereof.
11. The compound of claim 1, selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof.
12. The compound of claim 1, selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof.
13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent.
14. A method of inhibiting amine oxidase activity of any of LOX, LOXL1, LOXL2, LOXL3, or LOXL4 in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1-12, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of claim 13.
15. A method of treating a disorder associated with any one of LOX, LOXL1, LOXL2, LOXL3, or LOXL4 protein, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-12, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of claim 13.
16. The method of claim 15, wherein the disorder is selected from the group consisting of: fibrosis, cancer and angiogenesis.
17. The method of claim 16, wherein, where the condition is fibrosis, the fibrosis is selected from the group consisting of: mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, crohn's disease, keloid, systemic sclerosis, joint fibrosis, dupuytren's contracture, adhesive joint capsulitis, pancreatic fibrosis, intestinal fibrosis, liver fibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis, fibrostenosis, cystic fibrosis, idiopathic lung fibrosis, radiation-induced fibrosis, peyronie's disease, and scleroderma, or is associated with: respiratory diseases, wound healing and repair abnormalities, scarring, hypertrophic scarring/keloids, post-operative scarring, cardiac arrest, and all conditions in which excessive or abnormal deposition of fibrous material is associated with disease, injury, implant, or surgery; the fibrosis is preferably selected from the group consisting of: bone marrow fibrosis, systemic sclerosis, liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis and radiation-induced fibrosis; and is
Wherein where the disorder is cancer, the cancer is selected from the group consisting of: lung cancer; breast cancer; large bowel cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer; hepatobiliary cancer; esophageal cancer; mesothelioma; non-hodgkin lymphoma; bladder cancer; uterine cancer; gliomas, glioblastoma, medulloblastoma and other brain tumors; myelofibrosis, renal cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer; carcinoids of the gastrointestinal tract, breast and other organs; signet ring cell carcinoma; a mesenchymal tumor comprising sarcoma, fibrosarcoma, hemangioma, angiomatosis, hemangiopericytoma, pseudohemangiomatoid interstitial hyperplasia, myofibroblasts, fibromatosis, inflammatory myofibroblastoma, lipoma, angiolipoma, granulocytoma, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, or leiomyosarcoma.
18. The method of any one of claims 15 to 17, further comprising administering a second therapeutic agent.
19. The method of claim 18, wherein the second therapeutic agent is selected from the group consisting of: anti-cancer agents, anti-inflammatory agents, anti-hypertensive agents, anti-fibrotic agents, anti-angiogenic agents, immunosuppressive agents, and metabolic agents.
20. Use of a compound of any one of claims 1 to 12, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for the treatment of a disorder associated with any one of LOX, LOXL1, LOXL2, LOXL3, or LOXL4 proteins.
Technical Field
The present invention relates to novel compounds capable of inhibiting certain amine oxidases. These compounds are useful for treating a variety of indications, such as fibrosis, cancer and/or angiogenesis in human subjects, as well as in companion animals and livestock. In addition, the invention relates to pharmaceutical compositions containing these compounds and to various uses thereof.
Background
The enzymes were the first family members described as Lysyl Oxidase (LOX) and LOX-like 1(LOXL1), LOXL2, LOXL3 and LOXL4 (J Cell Biochem 2003; 88: 660-. Lysyl oxidase isozymes are copper-dependent amine oxidases that initiate covalent crosslinking of collagen and elastin. The primary function of lysyl oxidase isozymes is to promote crosslinking of collagen and elastin by oxidative deamination of lysine and hydroxylysine amino acid side chains into aldehydes that react spontaneously with neighboring residues. The resulting cross-linked chains contribute to the stability of the extracellular matrix (ECM) and make it less susceptible to degradation by enzymes such as Matrix Metalloproteinases (MMPs). The activity of lysyl oxidase is essential to maintain the normal tensile and elastic properties of connective tissue of many organ systems of the human body.
Lysyl oxidase isozymes belong to a large group of amine oxidases, including flavin-dependent and copper-dependent oxidases, which are described by the nature of the catalytic cofactor. The flavin-dependent enzymes include monoamine oxidase-A (MAO-A), monoamine oxidase-B (MAO-B), polyamine oxidase and lysine demethylase (LSD1), and the copper-dependent enzymes include semicarbazide-sensitive amine oxidase (vascular adhesion protein-1, SSAO/VAP-1), retinal amine oxidase, diamine oxidase and lysyl oxidase isozymes. Copper-dependent amine oxidases have a second cofactor, which varies from enzyme to enzyme. In SSAO/VAP-1, it is an oxidized tyrosine residue (TPQ, oxidized to quinone), whereas in the lysyl oxidase isozyme, TPQ has been further processed (LTQ formation) by the addition of a proximal lysine residue (J. Cell. Biochem., 2003; 88: 660-.
Lysyl oxidase isozymes exhibit different in vivo expression patterns, indicating that a particular isozyme will have a particular biological effect. Catalytically active forms of LOX have been identified in the cytosolic and nuclear compartments and studies are underway to determine their role in these compartments. For example, LOX itself plays a major role in epithelial to mesenchymal transition (EMT), cell migration, adhesion, transformation, and gene regulation. Different patterns of LOX expression/activity have been associated with different pathological processes, including fibrotic diseases, Alzheimer's disease and other neurodegenerative processes, as well as tumor progression and metastasis (J.S.Pat.J. (Am J Surg) 2005; 189: 297-.
Direct replacement of dead or damaged cells with connective tissue after injury represents a survival mechanism that is conserved throughout evolution and appears to be most pronounced in humans, playing an important role after trauma, infection or disease has occurred. After chronic and/or repeated injury, scarring may develop, resulting in impaired function of some or all of the affected organs. Fibrosis can result from a variety of causes, such as chronic infection, chronic exposure to alcohol and other toxins, autoimmune and allergic reactions or surgery, radiation therapy, and chemotherapy. Thus, this pathological process can occur in almost any organ or tissue of the body, and is usually due to the simultaneous occurrence of inflammation, tissue destruction, and repair for weeks or months. In this case, fibrosis most often affects the lung, liver, skin, kidney and cardiovascular system.
For example, liver fibrosis can occur as a complication of hemochromatosis, wilson's disease, alcoholism, schistosomiasis, viral hepatitis, bile duct obstruction, exposure to toxins, and metabolic disorders. Liver fibrosis is characterized by the accumulation of extracellular matrix, which can be qualitatively distinguished from normal liver. This fibrosis can progress to cirrhosis, liver failure, cancer and eventually death (Pathology-Research and Practice 1994; 190: 910-.
Hypertension, hypertensive Heart disease, atherosclerosis, and myocardial infarction can lead to the accumulation of fibrotic tissue in the Heart and blood vessels, with the accumulation of extracellular matrix or fibrotic deposition leading to stiffening of the vascular system and stiffening of the Heart tissue itself (Am J physical Heart Circuit physical 2010; 299: H1-H9).
Pulmonary Arterial Hypertension (PAH) is a rare and rapidly fatal condition characterized by elevated pulmonary arterial pressure and caused by increased pulmonary vascular resistance. Despite heterogeneous disorders of various origins, it is increasingly recognized that PAH is associated with other diseases, such as connective tissue disease and scleroderma. Pathological features of PAH include vascular wall remodeling, excessive deposition and cross-linking with extracellular matrix (ECM). Lysyl oxidase dysregulation in the pulmonary vasculature of patients with Idiopathic Pulmonary Arterial Hypertension (IPAH) and leads to persistence of ECM components and inappropriate remodeling of collagen and elastin by cross-linking ("arteriosclerosis, thrombosis, and vascular biology 2014; 34: 1446-. The prognosis for patients with PAH is poor. Pharmacological targeting of lysyl oxidase may provide therapeutic intervention in situations where little or no current exists.
It has been demonstrated that there is a strong correlation between fibrosis and increased lysyl oxidase activity. For example, in experimental liver fibrosis in rats (Proc. Natl. Acad. Sci. USA) 1978; 75:2945-4The increase in enzyme activity was most pronounced in the rat model of induced liver fibrosis. In these studies, low levels of enzyme activity in healthy liver increased 15 to 30-fold in fibrotic liver.
In humans, there is also a significant correlation between lysyl oxidase activity measured in plasma and the progression of liver fibrosis. Lysyl oxidase activity levels are generally low in the serum of healthy subjects, but are significantly elevated in chronic active hepatitis, and even higher in liver cirrhosis. Thus, lysyl oxidase can serve as a marker for internal fibrosis.
Lysyl oxidase isozymes are highly regulated by hypoxia inducible factor 1 α (HIF-1 α) and TGF- β, the most prominent growth factors that cause fibrosis (Cell Biol 2009; 29: 4467-4483). Collagen cross-linking occurs in every type of fibrosis, so lysyl oxidase isozyme inhibitors can be used for idiopathic pulmonary fibrosis, scleroderma, kidney or liver fibrosis.
In normal wound healing, the formation of granulation tissue is a transient process that provides a scaffold for epithelial regeneration and repair. Subsequently, the tissue is remodeled and a vegetative normal scar is formed. However, after injury, humans cannot regenerate normal skin. In contrast, the repair (or healing) process results in scarring (scarring). Scars are not as beautiful as skin and functional. Scarring is a chronic problem, and excessive or hypertrophic scarring and its attendant aesthetic, functional, and psychological sequelae remain key challenges for the treatment of deep skin injuries and burns. A key factor in the poor appearance and flexibility of scars, particularly hypertrophic scars, is the change in collagen in the dermis. In scar tissue, collagen (mainly collagen I) is more densely packed and closely arranged in parallel bundles. In normal skin, collagen is not densely packed, but rather is a more "basket-woven" structure. These changes in the structure and amount of collagen are largely the basis for poor scar appearance and lead to loss of flexibility, discomfort and functional problems.
Dermal fibrosis or excessive scarring of the skin is the result of an excessive healing response characterized by disproportionate fibroblast proliferation and extracellular matrix (ECM) production in the dermis. Clinically, dermal fibrosis is manifested as areas of thickened, taut, and stiffened skin. The range of fibrotic skin diseases is wide, including but not limited to: hypertrophic scars, keloids, scleroderma (diffuse and limited subtype), scleredema (Buschke's disease), systemic amyloidosis, fatty skin sclerosis, presenile-like disorders, skin stiffness syndrome, dupuytren's contracture, nephrogenic fibrotic skin disease (NFD), mixed connective tissue disease, sclerosing mucoedema, graft-versus-host disease (GVHD), and eosinophilic fasciitis. Although each disorder has its own etiology and clinical features, it involves excessive collagen production and altered collagen remodeling. One possible mechanism of altered ECM remodeling is through covalent cross-linking. This directly suggests that the LOX enzyme family is involved in the pathogenesis of skin fibrosis (Laboratory research 2019; 99: 514-527). The expression of LOX and LOXL1-4 is elevated in scar fibroblasts compared to normal skin fibroblasts, with LOX and LOXL1 being the major isoforms found in skin tissue.
Studies involving two complementary in vitro skin-like models-human skin equivalents (hSEs) and self-assembled stromal tissue identified LOXL4 as a key isoform mediating the TGF-. beta.induced fibrotic phenotype (laboratory studies 2019; 99: 514-527).
The scarring process is a significant problem and challenge in the eye and surrounding structures. Ocular scars play a major role in primary disease (e.g., corneal and conjunctival scars) or treatment failure (e.g., postsurgical trabeculectomy) (U.S. version of the ophthalmic Surgery News (Ocular Surgery News), 2002, 1/10).
Glaucoma is a disease in which the optic nerve is damaged, resulting in progressive and irreversible loss of vision. Elevated intraocular pressure (IOP) is one of the major risk factors for the development and progression of glaucoma. Most treatments for glaucoma aim to lower intraocular pressure by reducing the formation of aqueous intraocular fluid or, as in the case of glaucoma filtration surgery, by increasing the outflow of intraocular fluid. Trabeculectomy, the current gold standard for IOP management, is a filtration procedure in which an eye hole is created in the anterior chamber below a partial thickness scleral flap to allow the outflow of intraocular water. Post-operative scarring is a major cause of treatment failure. The antimetabolites mitomycin c (mmc) and 5-fluorouracil (5-FU) are currently used in clinical practice to help limit the formation of postoperative ocular scar tissue. Although these agents have been shown to improve IOP outcomes from filtration surgery, they do so in a non-selective manner with significant side effects (ophthalmol 2002; 120:297 300). Safer and more targeted anti-fibrosis drugs are needed.
Gingival fibromatosis is a rare heterogeneous group of diseases that progress to a slowly progressive, localized or diffuse fibrous hyperplasia of the keratinized gingiva (gingival overgrowth or hyperplasia). In severe cases, too much tissue may cover the crown, leading to chewing, aesthetic, speech, functional and periodontal problems. Gingival overgrowth may be a spontaneous genetic disease, associated with inflammatory diseases of the oral cavity, or associated with other systemic diseases. However, most often due to side effects of systemic administration, such as the antiepileptic drugs phenytoin, the immunosuppressant cyclosporin A, and certain antihypertensive calcium channel blockers of dihydropyridines, especially nifedipine (crit rev oral biol 2004; 15: 165-. The pathological manifestations of gingival overgrowth include an excessive accumulation of extracellular matrix proteins, of which collagen I is the most predominant. A well-established concept of the mechanism of drug-induced gingival overgrowth is EMT, a mechanism in which the interaction of gingival cells with extracellular matrix is attenuated as epithelial cells transdifferentiate into fibroblastic, fibroblast-like cells (journal of physiology of the United states (AJP) 2010; 177: 208-218). Damaged epithelial, basement membrane and underlying stroma cause TGF-. beta.to stimulate lysyl oxidase activity and promote connective tissue fibrosis (laboratory research 1999; 79: 1655-1667).
The principle that lysyl oxidase isozyme blockers can continuously and strongly inhibit fibrosis is that the lack of cross-linking activity makes collagen susceptible to degradation by proteolytic enzymes (such as MMPs). Thus, any type of fibrosis should be reversed by using inhibitors of lysyl oxidase isozymes. Given the varying degree of involvement of all lysyl oxidase isozymes in fibrosis, inhibitors that exhibit sustained, strong inhibitory effects on all lysyl oxidase isozymes (i.e., inhibitors of pantox) should be most effective.
Rheumatoid Arthritis (RA) is a systemic autoimmune disorder characterized by chronic painful inflammation of the intima of the joints. However, in some people, the condition may develop into swelling and inflammation of the surrounding tissues and other body systems, including skin, eyes, lungs, heart and blood vessels. Rheumatoid arthritis is therefore a painful and debilitating disease that can result in a substantial loss of hand, wrist and foot function and mobility. Active rheumatoid arthritis originates in several joints, but may subsequently develop affecting multiple joints. Synovial hyperplasia, which involves infiltrating immune cells and resident Synovial Fibroblasts (SF), is a typical feature of RA. Rheumatoid Arthritis Synovial Fibroblasts (RASF) are the most common cell type in the affected area and are the leading cause of joint destruction. Activated RASF is able to migrate and is therefore involved in the spread of arthritis between joints. Cytokines from the infiltrated immune cells induce activation and proliferation of synovial fibroblasts. These activated SF in turn creates a pathogenic matrix to perpetuate chronic inflammation, ultimately leading to cartilage and bone destruction. By implanting RASF together with human cartilage in severely compromised mice, it has been demonstrated that activated RASF migrates in vivo, spreading the disease to the site of the implanted human cartilage. Furthermore, while RASF actively degrades cartilage, controls implanted with synovial fibroblasts from Osteoarthritis (OA) patients and skin fibroblasts from healthy donors are not (Nature medicine 2009; 15: 1414-. RASF differs in morphology and gene expression from non-activated healthy fibroblasts. RASF is characterized by resistance to apoptosis, expression of proto-oncogenes and lack of expression of tumor suppressor genes. RASF produces proinflammatory cytokines and chemokines that further attract immune cells to the synovium. In addition, the production of Matrix Metalloproteinase (MMP) enzymes facilitates the invasion and destruction of cartilage.
The type II collagen-induced arthritis (CIA) model is a common animal model of RA, as it well summarizes the characteristic immunological, pathological and arthritic manifestations observed in human RA. In CIA rats, high expression levels of LOX in synovium, synovial fluid and serum have been demonstrated. It was found that inhibition of LOX using β -aminopropionitrile (BAPN; a pan LOX inhibitor) can reduce inflammation, synovial hyperplasia, angiogenesis, and the expression of MMP-2 and MMP-9, indicating that LOX promotes synovial hyperplasia and angiogenesis in CIA rats. In addition, the knock-out of LOXL2 and antibodies to LOXL2 reduced collagen deposition, diffusion and RASF invasion (molecular medicine report (mol. Med. Rep.) 2017: 6736-6742).
Although there is no cure for RA, there are many treatments that can alleviate symptoms and alter the course of the disease. However, this treatment has significant side effects, partly associated with suppression of the immune system. Selective drugs against RASF would represent a more useful treatment for RA.
Osteoarthritis (OA) is a disease characterized by the degeneration of articular cartilage and underlying bone. OA is mainly caused by "wear" and can cause joint pain and stiffness. The most common affected joints are the joints of the fingers, knees, back and hips. Unlike other forms of arthritis (such as RA), osteoarthritis affects only the joints. In general, the joints on one side of the body are affected more than the joints on the other side. OA is a progressive and debilitating disease that can have a significant impact on work and normal daily activities.
Synovial fibrosis is a key factor in OA and is an imbalance in the expression of fibroblast proliferation and collagen synthesis and collagen degradation. This imbalance results in excessive deposition of collagen into the extracellular matrix (ECM) and results in thickening and stiffening of the synovium.
Genes encoding various lysyl oxidase family enzymes, including LOX, LOXL2, LOXL3, and LOXL4, have been shown to be highly expressed in mice with experimental OA and humans with advanced OA (Arthritis and rheumatism 2014; 66: 647-656).
Given that many members of the lysyl oxidase family of enzymes contribute differently to the development of rheumatoid arthritis and osteoarthritis, inhibitors of pantox may provide potentially more effective treatments.
BAPN is a widely used irreversible lysyl oxidase inhibitor based on a non-selective mechanism. BAPN has been used in animal studies (mainly rats, mice and hamsters) since the 60 s of the 20 th century and has effectively reduced various models (e.g., CCl)4Bleomycin, quartz, cancer) and collagen content in tissues (e.g., liver, lung, and dermis) (journal of cellular biochemistry (J Cell Biochem) 2003; 88:660-672). However, studies in human scleroderma patients have found poor tolerance to BAPN and have emphasized the need for safer alternatives (Clin. Pharmacol. Ther.) 1967: 593-.
Lysyl oxidase-catalyzed collagen crosslinking can proceed in two ways: lysine and hydroxylysine pathways. In the hydroxylysine pathway, immature divalent crosslinks including dehydrodihydroxylysine norleucine (deH-DHLNL) and dehydrohydroxylysine norleucine (deH-HLNL) are first formed, and then further developed (by independent reaction of lysyl oxidase) into mature trivalent crosslinks that molecularly form deoxypyridinoline (DPD) and Pyridinoline (PYD) between the three collagens. These mature and immature crosslinks can be measured by LC-MS/MS ("public science library, integration" (PLoS One) 2014; 9(11), e 112391).
Lysyl oxidase isozymes not only participate in the crosslinking of elastin and collagen during wound healing and fibrosis, but also regulate cellular motility and signal transduction. Its intracellular and intranuclear functions are involved in gene regulation and can lead to tumorigenesis and tumor progression (I.S. 2011; 19: 117-129). The down-and up-regulation of lysyl oxidase isozyme in tumor tissues and cancer cell lines has been described, suggesting a dual role of lysyl oxidase isozyme and LOX propeptide as a metastasis promoter gene and an anti-cancer gene.
In addition to its role in tissue remodeling, LOX isozymes play a key role in primary cancer and metastasis. Tumor growth is associated with reactive stroma, which is composed primarily of fibroblasts, called cancer-associated fibroblasts (CAF). Mice inoculated subcutaneously with a homogeneous mixture of tumor and CAF cells are known to have faster growth rates and higher incidence of metastases (Trends Mol Med.) (2013; 19(8): 447: 453)). CAF knockout models have shown a tumor promoting effect, however, this is a very abstract scenario compared to the patient's tumor microenvironment. CAF has been shown to have increased LOX expression compared to normal fibroblasts (models and mechanisms of disease (Dis Model Mech.) 2018; 11 (4)). The use of LOX inhibitors in a cancer setting can affect the tumor and stromal compartment, thereby helping to reduce tumor growth and metastasis.
Emerging evidence suggests a link between idiopathic pulmonary fibrosis and lung cancer, however, more research is needed. In both lung and liver mouse models, chemical or radiation-induced fibrosis leads to increases in alpha smooth muscle actin (a marker of fibroblasts), LOX expression, and metastatic tumor growth, which are reversed by LOX antibodies (Cancer research 2013; 73(6): 1721-1732).
To date, an increase in lysyl oxidase isozyme mRNA and/or protein has been observed in breast, central nervous system cancer cell lines, head and neck squamous cell carcinoma, esophageal, kidney, lung, prostate, clear cell renal and lung, ovarian, uterine, melanoma, and osteosarcoma patient samples from a cancer genomic map (TCGA). The gene expression data for TCGA patients of the LOX family are shown in table 1. The plus sign indicates mean gene expression above this data set.
TABLE 1
LOX family of TCGA patient gene expression data
A statistically significant clinical correlation between lysyl oxidase isozyme expression and tumor progression was observed in breast, head and neck squamous cell carcinoma, myelofibrosis, prostate, pancreatic, ovarian, and clear cell renal cell carcinoma. With regard to the role of lysyl oxidase isozymes in tumor progression, the most extensive studies in breast cancer have been performed using in vitro models of migration/invasion and mouse models of in vivo tumorigenesis and metastasis (Nature 2006; 440(7088): 1222-1226). Increased expression of lysyl oxidase isozyme was found in hypoxic patients and was associated with negative estrogen receptor status (ER-), decreased overall survival in ER patients not receiving adjuvant systemic therapy and lymph node negative patients, and shorter metastasis-free survival in ER patients and lymph node negative patients; in vivo models demonstrate that LOX inhibitors can have potential in breast cancer patients with bone metastasis by modulating RANKL-independent bone homeostasis 2015; 522(7554):106-110). Lysyl oxidase isozyme mRNA was shown to be up-regulated compared to primary cancer tissues compared to invasive and metastatic cell lines (MDA-MB-231 and Hs578T) and more aggressive breast cancer cell lines and distant metastatic tissues (cancer research 2002; 62(15):4478 and 4483).
The pathogenic process of primary myelofibrosis involves clonal myeloproliferation weighted by primary megakaryocytes and secondary tumor stromal reactions, including myelofibrosis, bone sclerosis, angiogenesis and extramedullary hematopoiesis. The bone marrow response includes excessive deposition of extracellular matrix proteins (such as fibrillar collagen), cytopenia, activation and recruitment of bone marrow fibroblasts, overproduction of cytokines and growth factors, and other changes leading to reduced hematopoietic capacity. Polycythemia vera or primary thrombocythemia may lead to secondary myelofibrosis. In myelofibrosis, disease progression is associated with an increase in the number of megakaryocytes overexpressing LOX. In the GATA1 low mouse myelofibrosis model, the pan-LOX inhibitor significantly reduced disease progression (including increased megakaryocyte numbers, fibrosis, and spleen size) ("J Biol Chem.) -2011; 286(31): 27630-.
In most tumor types, the primary treatment is surgical resection. The wound healing response is initiated surgically and may be associated with an increase in metastatic spread. Breast cancer models have shown that abdominal surgery increases lung metastasis. Furthermore, it proved to be caused by systematic LOX. Plasma collected from abdominal surgery mice (containing LOX) was injected into tumor-bearing mice, resulting in an increase in lung metastasis. Surgically-induced systemic LOX is blocked by BAPN, thereby reducing metastasis and increasing survival (Cell Rep 2017; 19(4): 774-784).
Tumor-associated endothelial cells have been shown to increase LOX expression in colon, breast and melanoma models, thereby stimulating angiogenesis and tumor growth (cancer research 2015; 73(2): 583-.
High collagen content is associated with high LOX gene expression, chemotherapy resistance and significantly reduced survival in patients with pancreatic, breast, lung, ovarian and colon cancers (Oncogene 2018; 37 (36)) 4921-4940, EMBO Mol Med 2015; 7(8)1063-1076, cancer target 2016; 7 (32100-32112). LOX inhibitors (both BAPN and LOX antibodies) and nursing chemotherapy were combined in a mouse model of proliferative tumors to reduce the tumor interstitial pressure that causes vasodilation (cancer target 2016; 7(22) 32100-32112). Increased vascular flow increases the concentration of chemotherapeutic agents at the site of the primary tumor, which results in lower metastatic burden and increased survival (cancer target 2016, 5, 31, 7(22) 32100-32112).
In squamous cell carcinoma of the head and neck, increased expression of the lysyl oxidase isozyme was found to be associated with CA-IX, a marker of hypoxia, and with decreased cancer-specific survival, decreased overall survival and decreased metastasis-free survival (cancer targets 2016; 7(31): 50781-50804). In oral squamous cell carcinoma, lysyl oxidase isoenzyme mRNA expression is up-regulated compared to normal mucosa.
Gene expression profiling of gliomas identified that the overexpressed lysyl oxidase isozyme was part of a molecular signature indicative of invasion and was associated with high grade tumors closely related to poor patient survival (public science library. complex. 2015 3/19; 10(3) e 0119781). Increased expression of lysyl oxidase isozyme protein in glioblastoma and astrocytoma tissues, as well as infiltrating U343 and U251 cultured astrocytoma cells.
In tissues, lysyl oxidase isozyme mRNA is upregulated in prostate cancer, correlated with Gleason score, and with high grade and short-term recurrence compared to benign prostatic hypertrophy (Oncol Rep 2008; 20: 1561-.
In clear cell RCC, smoking is associated with an allelic imbalance in chromosome 5q23.1 in which the LOX gene is located, and may involve duplication of the gene (Cancer Genet and cytogenetics 2005; 163 (1)) 7: 7-11.
SiHa cervical cancer cells exhibit increased in vitro invasiveness under hypoxic/anaerobic conditions; this phenomenon is inhibited by treatment with BAPN and LOX antisense oligonucleotides, LOX antibodies, LOX shRNA or extracellular copper chelators to inhibit the activity of the extracellular catalytically active lysyl oxidase (report in oncology 2013; 29(2), 541-548).
In a genetically engineered mouse model of ovarian cancer (ApoE knockout), connective tissue proliferative tumors with increased LOX gene expression were formed. BAPN treatment can significantly improve survival and reduce lung metastasis (J Exp Clin Cancer Res.) 2018; 37: 32. Some tumors from ovarian cancer patients have a single nucleotide polymorphism of the LOX gene G473A. Two independent studies have shown that individuals expressing the G473A polymorphism have an increased chance of developing ovarian cancer (J Int Med Res.) (2012; 40(3): 917-.
In primary human Oral Squamous Cell Carcinoma (OSCC), expression of lysyl oxidase (especially LOX and LOXL2) and lysyl hydroxylase is significantly increased, and is significantly elevated in advanced Regional Lymph Node Metastasis (RLNM) positive tumors. Both reducible or immature crosslinks (deH-DHLNL and deH-HLNL) and non-reducible or mature crosslinks (DPD and PYD) were significantly elevated in OSCC compared to normal tissue (J Dent Res, 2019; 98(5): 517-525).
The findings described herein provide a strong basis for combination therapy of patients involving LOX isozyme inhibitors and anti-tumor therapy.
Recently, CCT365623 is a reversible inhibitor of ubiquiX, and has been used in breast cancer models (MMTV-PyMT) to reduce metastasis and increase survival (Nat Commun 2017; 18(8): 14909).
The scientific and patent literature describes that small molecule inhibitors of lysyl oxidase isozymes and antibodies to LOX and LOXL2 have therapeutic effects in animal models of fibrosis and cancer metastasis. It has been reported that some known MAO inhibitors may also inhibit lysyl oxidase isozymes (e.g., the MAO-B inhibitor mofetiline (Mofegiline) shown below). This inhibitor is a member of the haloalkylamine family of MAO inhibitors. The halogen in mofiglan is fluorine. Fluoroallylamine inhibitors are described in U.S. patent No. 4,454,158. Patents claiming fluoroallylamine and chloroallylamine, such as MDL72274 (shown below) as lysyl oxidase inhibitors are disclosed (U.S. patent 4,943,593; 4,965,288; 5,021,456; 5,059,714; 5,182,297; 5,252,608). Many of the compounds claimed in these patents are reported to also be potent MAO-B and SSAO/VAP-1 inhibitors.
Additional fluoroallylamine inhibitors are described in U.S. patent 4,699,928. Other examples structurally related to mofetil can be found in WO 2007/120528.
WO2009/066152 discloses a family of 3-substituted 3-haloalkylamines as SSAO/VAP-1 inhibitors that are useful for the treatment of a variety of indications, including inflammatory diseases. None of these documents specifically discloses the fluoroallylamine compound of formula (I) according to the invention.
Antibodies against LOX and LOXL2 and methods for diagnostic and therapeutic applications have been disclosed in US 2009/0053224. anti-LOX and anti-LOXL 2 antibodies are useful for identifying and treating disorders, such as fibrotic disorders, angiogenesis, or for preventing a transition from an epithelial to a mesenchymal cell state: US 2011/0044907.
WO 2017/136871 and WO 2017/136870 disclose haloalkylamine indole and azaindole derivative inhibitors of lysyl oxidase and uses thereof.
WO 2018/157190 discloses halopropylamine pyrazole derivative inhibitors of lysyl oxidase and uses thereof.
WO 2017/141049 and WO 2019/073251 disclose the family of methylamine and bridged homopiperazine derivatives, respectively, as lysyl oxidase inhibitors and their use in the treatment of cancer and diseases associated with fibrosis.
WO 2003/097612, WO 2006/053555 and US 2008/0293936 disclose another class of lysyl oxidase inhibitors.
WO 2018/048930, WO 2017/015221, WO 2017/003862, WO 2016/144702 and WO 2016/144703 disclose other LOXL2 inhibitors.
Disclosure of Invention
The present invention provides substituted fluoroallylamine compounds that inhibit Lysyl Oxidase (LOX), lysyl oxidase-like 2(LOXL2), and other lysyl oxidase isozymes. Surprisingly, the previously described modification of the structure of 3-substituted 3-fluoroallylamines has led to the discovery of novel compounds that are potent inhibitors of the human LOX and LOXL isozymes. In addition, certain of these novel compounds also selectively inhibit certain LOX and LOXL isozymes relative to other enzymes in the amine oxidase family.
A first aspect of the invention provides a compound of formula I:
or a stereoisomer, pharmaceutically acceptable salt, polymorph form, solvate, hydrate or tautomeric form thereof; wherein:
a is aryl or heteroaryl;
each R1Independently selected from the group consisting of: X-R2Halogen, deuterium, C1-6Alkyl radical、O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R4And R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R7Selected from the group consisting of: halogen, OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having from 0 to 1 additional heteroatoms as ring members;
and is
n is 0, 1,2, 3,4,5 or 6.
A second aspect of the invention provides a pharmaceutical composition comprising a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient, carrier or diluent.
A third aspect of the invention provides a method of inhibiting amine oxidase activity of any of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.
A fourth aspect of the invention provides a method of treating a condition associated with any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to the second aspect of the invention.
A fifth aspect of the invention provides the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for the treatment of a condition associated with any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 protein.
A sixth aspect of the invention provides a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a condition associated with any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 protein.
In one embodiment of the methods and uses of the present invention, the disorder is selected from fibrosis, cancer and angiogenesis.
Contemplated herein are combination therapies, wherein the methods further comprise co-administering a therapeutic agent for the treatment of cancer, fibrosis, angiogenesis, inflammation, hypertension, immunosuppression, and metabolic disorders.
Definition of
The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to only these terms, but are set forth for a better understanding of the following description.
Integers, steps or elements of the invention recited herein are in the singular and the steps or elements clearly encompass both the singular and the plural of the recited integers, steps or elements unless the context specifically requires otherwise or indicates to the contrary.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
As used herein, the term "alkyl" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms, for example 1,2, 3,4,5, or 6 carbon atoms. Straight or branched chain alkyl groups are attached at any available point to produce stable compounds. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, pentyl, 1, 2-dimethylpropyl, 1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2, 2-dimethylbutyl, 3-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 1,2, 2-trimethylpropyl, 1, 2-trimethylpropyl, and the like.
As used herein, the term "alkoxy" or "alkyloxy" refers to a straight or branched chain alkoxy (i.e., O-alkyl) group, wherein alkyl is as defined above. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy and isopropoxy.
As used herein, the term "cycloalkyl" includes within its meaning monovalent ("cycloalkyl") and divalent ("cycloalkylene") radicals of saturated, monocyclic, bicyclic, polycyclic, or fused analogs. In the context of the present invention, cycloalkyl groups may have 3 to 10 carbon atoms. Fused analogs of cycloalkyl refer to a single ring fused to an aryl or heteroaryl group, wherein the point of attachment is on the non-aromatic moiety. Examples of cycloalkyl and fused analogues thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, adamantyl, and the like.
As used herein, the term "aryl" or variants such as "arylene" refers to mono ("aryl") and divalent ("arylene") mono-, polynuclear, conjugated and fused analogs of aromatic hydrocarbons having 6 to 10 carbon atoms. Fused analogs of aryl refer to aryl fused with a monocyclic cycloalkyl or monocyclic heterocyclyl, wherein the point of attachment is on the aromatic moiety. Examples of aryl and fused analogs thereof include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2, 3-dihydrobenzofuranyl, tetrahydrobenzopyranyl, 1, 4-benzodioxanyl, and the like. "substituted aryl" is aryl independently substituted with one or more, preferably 1,2, or 3 substituents attached at any available atom to produce a stable compound.
As used herein, the term "alkylaryl" includes within its meaning monovalent ("aryl") and divalent ("arylene"), mono-, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to a divalent, saturated, straight or branched chain alkylene radical. Examples of alkylaryl groups include benzyl.
As used herein, the term "heteroaryl" and variants such as "heteroaromatic group" or "heteroarylene group" includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), mono-, polynuclear, conjugated and fused heteroaromatic groups having 5 to 10 atoms, wherein 1 to 4 ring atoms or 1 to 2 ring atoms are heteroatoms independently selected from O, N, NH and S. Heteroaromatic compoundsRadicals are also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of the ternary ring nitrogen. The carbon or nitrogen atom is the point of attachment to the heteroaryl ring structure, thereby resulting in a stable compound. The heteroaromatic group may be C1-9A heteroaromatic. Fused analogs of heteroaryl refer to heteroaryl groups fused to a monocyclic cycloalkyl or a monocyclic heterocyclyl group, wherein the point of attachment is on the aromatic moiety. Examples of heteroaryl and fused analogs thereof include pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, triazinyl, thienyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothienyl, furo (2,3-b) pyridyl, indolyl, isoquinolyl, imidazopyridine, pyrimidinyl, pyridazinyl, pyrazinyl, pyridyl, phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl, pyrrolyl, furanyl, thienyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, and the like. "Nitrogen-containing heteroaryl" refers to heteroaryl wherein any heteroatom is N. "substituted heteroaryl" is heteroaryl independently substituted with one or more, preferably 1,2, or 3 substituents attached at any available atom to produce a stable compound.
As used herein, the term "heterocyclyl" and variants thereof, such as "heterocycloalkyl", include within its meaning monovalent ("heterocyclyl") and divalent ("heterocyclylene") groups having 3 to 10 ring atoms, saturated or partially saturated (non-aromatic), monocyclic, bicyclic, polycyclic or fused hydrocarbon groups in which 1 to 4 or 1 to 2 ring atoms are independently selected from O, N, NH or S, SO or SO2Wherein the point of attachment may be carbon or nitrogen. A fused analog of heterocyclyl refers to a monocyclic heterocycle fused to an aryl or heteroaryl group, wherein the point of attachment is on a non-aromatic moiety. The heterocyclic group may be C3-8A heterocyclic group. The heterocycloalkyl group may be C3-6A heterocyclic group. The heterocyclic group may be C3-5A heterocyclic group. Examples of heterocyclyl and fused analogues thereof include pyrrolidinyl, thiazolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2, 3-dihydrofuran (2,3-b) pyridinyl, benzoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, indolinyl, quinineCyclyl, azetidinyl, morpholinyl, tetrahydrothienyl, tetrahydrofuryl, tetrahydropyranyl and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2-or 4-pyridones linked by nitrogen or N-substituted uracils.
As used herein, the term "halogen" or variants such as "halide" or "halo" refers to fluorine, chlorine, bromine, and iodine.
As used herein, the term "heteroatom" or variants such as "hetero-" or "hetero-base" refers to O, N, NH and S.
Generally, "substituted" refers to an organic group (e.g., alkyl) as defined herein, wherein one or more bonds to a hydrogen atom contained therein is replaced with a bond to a non-hydrogen or non-carbon atom. Substituents also include groups in which one or more bonds to a carbon atom or a hydrogen atom are replaced by one or more bonds to a heteroatom, including double or triple bonds. Thus, unless otherwise specified, a substituent will be substituted with one or more substituents. In some embodiments, a substituted group is substituted with 1,2, 3,4,5, or 6 substituents.
As used herein, the term "optionally substituted" means that the group to which the term refers may be unsubstituted or may be substituted with one or more groups independently selected from: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halogen, haloalkyl, hydroxy, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, NO2NH (alkyl), N (alkyl)2Alkylamino, dialkylamino, acyl, alkenoyl, alkanoylalkynyl, amido, diamido, acyloxy, alkylsulfonyl, alkylsulfonyloxy, sulfonamido, heterocyclyloxy, heterocyclylamino, haloheterocyclylalkyl, alkylthio, alkylcarbonyloxy, phosphorus-containing groups such as phosphonyl and phosphinyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkylheteroaryl, cyano, CO2H、CO2Alkyl, C (O) NH2C (O) NH (alkyl) and-C (O) N (alkyl)2. Preferred substituents include halogen, C1-C6Alkyl radical, C1-C6Haloalkyl, C1-C6Alkoxy, hydroxy (C)1-6) Alkyl radical, C3-C6Cycloalkyl, C (O) OH, NHC (O) C1-C4Alkyl, C (O) C1-C4Alkyl, NH2、NHC1-C4Alkyl, N (C)1-C4Alkyl radical)2、SO2(C1-C4Alkyl), OH and CN. Particularly preferred substituents include C1-4Alkyl radical, C1-4Alkoxy, SO2(C1-C4Alkyl), halogen, OH, hydroxy (C)1-3) Alkyl (e.g. C (CH)3)2OH) and C1-3Haloalkyl (e.g. CF)3、CH2CF3)。
The present invention includes within its scope all stereoisomeric and isomeric forms of the compounds disclosed herein, including all diastereomers, racemates, enantiomers, and mixtures thereof. It is also understood that the compounds described by formula I may exist in the form of E and Z isomers, also referred to as cis and trans isomers. Thus, the present disclosure is understood to include compounds in, for example, E, Z, cis, trans, (R), (S), (L), (D), (+) and/or (-) forms. In each case as appropriate. Where no specific stereoisomer is indicated in the structure, it is understood to encompass any and all possible isomers. The compounds of the present invention encompass all conformational isomers. The compounds of the invention may also exist in one or more tautomeric forms, including individual tautomers and mixtures of tautomers. All polymorphic and crystalline forms of the compounds disclosed herein are also included within the scope of the present invention.
The present invention includes within its scope isotopes of different atoms. Any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Accordingly, the present disclosure should be understood to include deuterium and the deuterium isotopes of hydrogen.
All references cited in this application are specifically incorporated by cross-reference in their entirety. The reference to any such documents is not to be construed as an admission that the documents form part of the common general knowledge or are in the prior art.
In the context of the present specification, the term "administering" and variations of the term include "administering" and "administration" including contacting, administering, delivering or providing a compound or composition of the invention to an organism or surface by any suitable means. In the context of this specification, the term "treatment" refers to any and all uses for remedying a disease state or condition, preventing the establishment of a disease, or otherwise preventing, impeding, delaying or reversing the progression of a disease or any other form of adverse condition.
In the context of the present specification, the term "topical administration" or variations of this term including "topical application" includes within its meaning the application, contact, delivery or provision of a compound or composition of the present invention to the skin or to a local area of the body.
In the context of the present specification, the term "local administration" or variations of this term including "local application" includes within its meaning the application, contact, delivery or provision of a compound or composition of the invention to the skin or a local area of the body.
In the context of this specification, the term "effective amount" includes within its meaning a sufficient, but non-toxic, amount of a compound or composition of the invention sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" includes within its meaning a sufficient, but non-toxic, amount of a compound or composition of the present invention sufficient to provide the desired therapeutic effect. The exact dosage required will vary from subject to subject, depending on a variety of factors, such as the species being treated; the sex, age and general condition of the subject; the severity of the disease being treated; the particular drug administered; the mode of administration, and the like. Therefore, it is not possible to specify an exact "effective amount". However, for any given situation, one of ordinary skill in the art can determine the appropriate "effective amount" using only routine experimentation.
Drawings
FIGS. 1(a) and 1(b) depict survival curves comparing high and low gene expression in pancreatic cancer patients according to the TCGA dataset. Lox gene expression. LOXL2 gene expression.
FIG. 2(a-c) depicts dose-dependent blocking of the enzymatic activity of lysyl oxidase by Compounds 1 and 33. Rat tissues (a) ear (24 hours after single oral dose 10 and 30mg/kg, compound 1) compared to untreated control group; (b) ear (4, 24, 48 and 120 hours after a single oral dose of 30mg/kg, compound 33); and (c) determination of lysyl oxidase Activity in aorta (Single oral dose 5, 10 or 30mg/kg, Compound 33)
Figure 3(a) shows the reduction of collagen in the scar tissue of mice after treatment with topical compound 1.
Figure 3(b) histology shows thick, parallel collagen bundles in control scar tissue.
Figure 3(c) tissue treated with compound 1 showed a decrease in bundle density and a more "normal" structure of collagen.
Figure 4(a-b) shows the reduction in LOX activity and total collagen relative to control after 4 weeks of daily topical treatment with 3% compound 1 solution.
The gross morphology of FIG. 4(c-f) shows that similar wounds appeared to heal more (e control, f treatment) at injury (c control; d treatment) and at euthanasia treatment.
The histology of figure 4(g-h) shows thicker collagen bands in untreated scar tissue (arrows g highlighted). This appears to be reduced in the treated tissue (h).
FIG. 5(a-e) tumor growth data for orthotopic human pancreatic cancer xenograft models: efficacy data. A. Growth and treatment strategy diagrams. B. Tumor growth was monitored in vivo by bioluminescence imaging. C. Ex vivo bioluminescence signal for total tumor burden. D. Ex vivo bioluminescent signaling of primary tumors. E. Transferring the loaded ex vivo bioluminescent signal.
FIG. 6(a-c) histological analysis of a hardened mouse skin model treated topically with Compound 1. A. And (4) grading the compound skin. B. Mean collagen score. C. Mean LOX score.
FIG. 7(a-e) spleen analysis of a primary myelofibrosis model (GATA-1 low) treated with Compound 19. A. Gomori silver staining of spleen. B. Spleen weight. C. Quantification of spleen reticuloprotein fibrosis. D. H & E stained images of spleen. E. Quantification of megakaryocytes in the spleen.
FIG. 8(a-d) bone marrow analysis of a primary myelofibrosis model (GATA-1 low) treated with Compound 19. A. Gomori silver staining of bone marrow. B. Quantification of sclerostin fibrosis in bone marrow reticulum. C. H & E stained images of bone marrow. D. Quantification of megakaryocytes in bone marrow.
Figure 9 depicts changes in the fibrotic region in the mouse UUO model.
Figure 10 depicts the ability of compound 33 to reduce bleomycin-induced pulmonary fibrosis (Ashcroft score) and body weight gain.
FIGS. 11(a-d) depict Compound 33 in CCl under an in situ injected breast cancer cell line (4t)4The ability to reduce metastasis associated with fibrosis in an induced mouse liver fibrosis model. (a) A research design schematic diagram; (b) clinical measurement of liver fibrosis; (c) the concentration of cross-links in the liver; (d) measurement of liver metastasis.
Detailed Description
The present invention relates to substituted fluoroallylamine derivatives that inhibit Lysyl Oxidase (LOX), lysyl oxidase-like 2(LOXL2), and other lysyl oxidase isoenzymes. In particular, the present invention relates to substituted fluoroallylamine derivatives having a sulfone linkage.
In particular, the present invention relates to compounds of formula I:
or a stereoisomer, pharmaceutically acceptable salt, polymorph form, solvate, hydrate or tautomeric form thereof; wherein:
a is aryl or heteroaryl;
each R1Independently selected from the group consisting of: X-R2Halogen, deuterium, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl,Heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R4And R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R7Selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having from 0 to 1 additional heteroatoms as ring members;
and is
n is 0, 1,2, 3,4,5 or 6.
In one embodiment of the compounds of the present invention, a is selected from aryl and heteroaryl. In another embodiment of the compounds of the present invention, A is selected from phenyl,Naphthyl, pyridyl, quinolyl, benzothiazolyl, and indolyl. In further embodiments of the compounds of the present invention, a is selected from:in still further embodiments of the compounds of the present invention, a is selected from:in further embodiments, a is selected from the group consisting of:in yet a further embodiment, A isIn another embodiment, a is phenyl. In further embodiments, a is heteroaryl.
In one embodiment of the compounds of the invention, R1Independently selected from the group consisting of: X-R2Halogen, deuterium, C1-6Alkyl, OC1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3. In another embodiment of the compounds of the invention, each R is1Independently selected from the group consisting of: -X-R2、C1-6Alkyl radical, C1-6Haloalkyl, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6. In another embodiment of the compounds of the invention, each R is1Independently selected from the group consisting of: halogen, C1-6Alkyl radical, C1-6Haloalkyl and-S (O)2R6. In one embodiment of the compounds of the invention, R1At least one of (A) is X-R2. In another embodiment of the compounds of the invention, R1One is X-R2。
In one embodiment of the compounds of the present invention, X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO. In another embodiment of the compounds of the present invention, X is selected from the group consisting of: o, CH2、OCH2CONH and NHCO. In another embodiment of the compounds of the present invention, X is selected from the group consisting of: o, OCH2And CONH. In further embodiments of the compounds of the present invention, X is selected from the group consisting of: o, CH2And OCH2. In another embodiment of the compounds of the present invention, X is selected from the group consisting of: CONH and NHCO. In further embodiments of the compounds of the present invention, X is O. In another embodiment of the compounds of the invention, X is OCH2. In further embodiments of the compounds of the present invention, X is CONH.
In one embodiment of the compounds of the invention, R2Selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein each R is2Optionally substituted by one or more R7And (4) substitution. In another embodiment of the compounds of the invention, R2Selected from the group consisting of: aryl and cycloalkyl radicals, in which each R is2Optionally substituted by one or more R7And (4) substitution. In further embodiments of the compounds of the present invention, R2Is cycloalkyl, wherein each R is2Optionally substituted by one or more R7And (4) substitution. In another embodiment of the compounds of the invention, R2Is optionally substituted by one or more R7A substituted aryl group. In another embodiment of the compounds of the present inventionIn, R2Is represented by an R7A substituted phenyl group. In another embodiment of the compounds of the invention, R2Is adamantyl or phenyl, wherein each R2Optionally substituted by one or more R7And (4) substitution. In another embodiment, R2Is adamantyl or phenyl, optionally substituted by-S (O)2NR4R5And (4) substitution. In a further embodiment, R2Is an adamantyl group. In another embodiment, R2Is optionally substituted by-S (O)2NR4R5A substituted phenyl group.
In one embodiment of the compounds of the invention, R2Is substituted by one R7And (4) substitution. In another embodiment of the compounds of the invention, R2Is divided into two R7And (4) substitution. In further embodiments of the compounds of the present invention, R2Is divided into three R7And (4) substitution. In another embodiment of the compounds of the invention, R2By four or five R7And (4) substitution.
In one embodiment of the compounds of the invention, R3Selected from hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3. In another embodiment of the compounds of the invention, R3Is hydrogen. In further embodiments of the compounds of the present invention, R3Is C1-6Alkyl or C3-7A cycloalkyl group. In still further embodiments of the compounds of the present invention, R3Is hydrogen or C1-6An alkyl group. In another embodiment of the compounds of the invention, R3Is C1-6An alkyl group. In further embodiments of the compounds of the present invention, R3Is methyl or ethyl. In another embodiment of the compounds of the invention, R3Selected from the group consisting of: hydrogen, methyl and ethyl.
In one embodiment of the compounds of the invention, R4And R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4、-CF3、-CH2CF3and-O-CF3. In another embodiment of the compounds of the invention, R4And R5Independently selected from the group consisting of: hydrogen and C1-6An alkyl group. In another embodiment of the compounds of the invention, R4And R5Is hydrogen. In further embodiments of the compounds of the present invention, R4And R5Is C1-6An alkyl group. In another embodiment of the compounds of the invention, R4And R5Are all methyl. In further embodiments of the compounds of the present invention, R4And R5Are all isopropyl. In one embodiment of the compounds of the invention, R4Is hydrogen and R5Is isopropyl. In further embodiments of the compounds of the present invention, R4And R5Independently selected from the group consisting of: hydrogen and C3-7A cycloalkyl group. In another embodiment of the compounds of the invention, R4Is hydrogen and R5Is C1-6An alkyl group. In one embodiment of the compounds of the invention, R4Is hydrogen and R5Is methyl. In further embodiments of the compounds of the present invention, R4Is hydrogen and R5Is C3-7A cycloalkyl group.
In one embodiment of the compounds of the invention, R4And R5When attached to the same nitrogen atom combine to form a 4-to 7-membered ring having from 0 to 1 additional heteroatoms as ring members. In a further embodiment, R4And R5When attached to the same nitrogen atom combine to form a 4-to 7-membered ring having 1 additional heteroatom as a ring member. In another embodiment, R4And R5When attached to the same nitrogen atom combine to form a 4-to 7-membered ring having 0 additional heteroatoms as ring members.
In one embodiment of the compounds of the invention, R6Selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3. In another embodiment, R6Selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group. In another embodiment, R6Is C1-6An alkyl group. In a further embodiment, R6Is C3-7A cycloalkyl group.
In one embodiment of the compounds of the invention, R7Selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH. In another embodiment of the compounds of the invention, R7Selected from the group consisting of: halogen, C1-6Alkyl, -C (O) NR4R5、-S(O)2NR4R5and-S (O)2R6. In further embodiments of the compounds of the present invention, R7Selected from the group consisting of: -C (O) NR4R5、-S(O)2NR4R5and-S (O)2R6. In another embodiment of the compounds of the invention, R7is-S (O)2NR4R5. In another embodiment of the compounds of the present inventionIn, R7is-S (O)2N(CH3)2。
In one embodiment of the compounds of the invention, R8Is hydrogen or C1-6An alkyl group. In another embodiment of the compounds of the invention, R8Is hydrogen. In further embodiments of the compounds of the present invention, R8Selected from the group consisting of: hydrogen, methyl and ethyl. In another embodiment of the compounds of the invention, R8Is hydrogen or methyl.
In one embodiment of the compounds of the invention, R9Selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3. In another embodiment, R9Selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group. In another embodiment, R9Is C1-6An alkyl group. In a further embodiment, R9Is C3-7A cycloalkyl group.
In one embodiment of the compounds of the invention, R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member. In a further embodiment, R8And R9Combine to form a 5-to 7-membered ring with 1 additional heteroatom as a ring member. In another embodiment, R8And R9Combine to form a 5 to 7 membered ring with 0 additional heteroatoms.
In one embodiment of the compounds of the invention, n is 0, 1,2, 3,4 or 5. In another embodiment of the compounds of the present invention, n is 0. In further embodiments of the compounds of the present invention, n is 0, 1 or 2. In another embodiment of the compounds of the invention, n is 1,2 or 3. In another embodiment of the compounds of the invention, n is 1 or 2. In a further embodiment of the compounds of the invention, n is 1. In another embodiment of the compounds of the present invention, n is 2. In a further embodiment of the compounds of the invention, n is 3. In another embodiment of the compounds of the present invention, n is 4. In another embodiment of the compounds of the present invention, n is 5. In a further embodiment of the compounds of the invention, n is 6.
In one embodiment, the present invention also relates to compounds of formula Ia:
or a stereoisomer, pharmaceutically acceptable salt, polymorph form, solvate, hydrate or tautomeric form thereof; wherein:
each R1Independently selected from the group consisting of: X-R2Halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R7selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1,2 or 3.
In one embodiment, the invention also relates to compounds of formula Ib:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7Cycloalkyl radicals
R7Selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, and heteroaryl,C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2.
In one embodiment of the compounds of formula Ib of the present invention, X is selected from the group consisting of: o, OCH2And CONH; r2Selected from the group consisting of: adamantyl and phenyl; wherein each R2Optionally substituted by one or more R7Substitution; r4And R5Independently selected from the group consisting of: hydrogen and C1-6An alkyl group; r7is-S (O)2NR4R5(ii) a n is 0.
In another embodiment, the invention also relates to compounds of formula Ic:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, -O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2、CH2O、CH2S(O)2CONH and NHCO;
R2selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R7selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6Alkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2.
In one embodiment of the compounds of formula Ic of the present invention, X is selected from the group consisting of: OCH (OCH)2And CONH; r2Selected from the group consisting of: adamantyl and phenyl; wherein each R2Optionally substituted by one or more R7Substitution; r4And R5Independently selected from the group consisting of: hydrogen and C1-6An alkyl group; r7is-S (O)2NR4R5(ii) a And n is 0.
In another embodiment, the invention also relates to compounds of formula Id:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl,Heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
X is selected from the group consisting of: o, CH2、OCH2CONH and NHCO.
R2Selected from the group consisting of: cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; wherein each R2Optionally substituted by one or more R7Substitution;
R3selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R7selected from the group consisting of: halogen, -OH, C1-6Alkyl, O-C1-6Alkyl radical, C3-7Cycloalkyl, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6(ii) a Wherein each C1-6The alkyl group is optionally substituted with one or more substituents selected from the group consisting ofThe group consisting of: halogen and-OH;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2.
In one embodiment of the compounds of formula Id of the present invention, X is selected from the group consisting of: OCH (OCH)2And CONH; r2Selected from the group consisting of: adamantyl and phenyl; wherein each R2Optionally substituted by one or more R7Substitution; r4And R5Independently selected from the group consisting of: hydrogen and C1-6An alkyl group; r7is-S (O)2NR4R5(ii) a And n is 0.
In another embodiment, the invention also relates to compounds of formula Ie:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R3Selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2.
In another embodiment, the invention also relates to compounds of formula If:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R3Selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2.
In another embodiment, the invention also relates to a compound of formula Ig:
or a pharmaceutically acceptable salt or solvate thereof; wherein:
each R1Independently selected from the group consisting of: halogen, C1-6Alkyl, O-C1-6Alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -CN, -C (O) OR3、-C(O)NR4R5、-S(O)2NR4R5、-S(O)2R6、-NR8C(O)R9and-NR8S(O)2R9(ii) a Wherein each C1-6Alkyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -SO2CH3、-C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3；
R3Selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group;
R4and R5Independently selected from the group consisting of: hydrogen, C1-6Alkyl and C3-7A cycloalkyl group; or
R4And R5Combine when attached to the same nitrogen atom to form a 4-to 7-membered ring having 0 to 1 additional heteroatoms as ring members;
R6selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group;
R8is hydrogen or C1-6An alkyl group;
R9selected from the group consisting of: c1-6Alkyl and C3-7A cycloalkyl group; wherein each C1-6Alkyl and C3-7Cycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: halogen, -OH, -C1-4Alkyl, -O-C1-4Alkyl, -CF3、-CH2CF3and-O-CF3(ii) a Or
R8And R9Combine to form a 5-to 7-membered ring having 0 to 1 additional heteroatoms as a ring member;
and is
n is 0, 1 or 2:
in another embodiment of the compounds of formulae Ie, If, and Ig of the present invention, each R is1Is C1-6Alkyl, and n is 0 or 1.
In another embodiment of the compounds of formula I of the present invention, each R is1Independently selected from the group consisting of: halogen, C1-6Alkyl radical, C1-6Haloalkyl, C1-6Alkoxy radical, C1-6Haloalkoxy, -CN, -C (O) OR3、-C(O)NR4R5、-NR4C(O)R6、-S(O)2NR4R5、-NR4S(O)2R6and-S (O)2R6；R3Selected from the group consisting of: hydrogen, optionally substituted C1-6Alkyl and optionally substituted C3-7A cycloalkyl group; r4And R5Independently selected from the group consisting of: hydrogen, optionally substituted C1-6Alkyl and optionally substituted C3-7A cycloalkyl group; or R4And R5Combine to form a 4-to 7-membered ring member having 0 to 1 additional heteroatoms when attached to the same nitrogen atom; r6Selected from the group consisting of: optionally substituted C1-6Alkyl, optionally substituted C3-7Cycloalkyl and optionally substituted C1-6A haloalkyl group; and n is 0, 1,2 or 3.
In the invention, formula IIn another embodiment of the compounds, each R1Independently selected from the group consisting of: halogen, C1-6Alkyl radical, C1-6Haloalkyl and-S (O)2R6；R6Is C1-6An alkyl group; and n is 0, 1 or 2.
In another embodiment of the compounds of formula I of the present invention, each R is1Independently selected from the group consisting of: chlorine, fluorine, methyl, isopropyl, OCH3Phenyl and SO2CH3(ii) a And n is 1 or 2.
In a further embodiment of the compounds of the formula I according to the invention, A isAnd n is 0.
In the context of the present disclosure, any one or more aspects or embodiments may be combined with any other aspect or embodiment.
Exemplary compounds according to the present invention include the compounds listed in table 2:
table 2.
In one embodiment, the compound of the invention is selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the compounds of the present invention areOr a pharmaceutically acceptable salt or solvate thereof. In a further embodiment, the compounds of the invention areOr a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the compound of the invention is selected from the group consisting of:or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the compounds of the present invention areOr a pharmaceutically acceptable salt or solvate thereof. In a further embodiment, the compounds of the invention areOr a pharmaceutically acceptable salt or solvate thereof。
Preparation of Compounds of formula I
The compounds of formula I can be readily prepared by those skilled in the art using methods and materials known in the art and referring to standard textbooks such as Advanced Organic Chemistry (Advanced Organic Chemistry) by Jerry March (third edition, 1985, John Wiley and Sons) or Comprehensive Organic Transformations (1989, VCH publishers) by Richard c.
The compounds of formula I can be synthesized as described below. The following schemes provide an overview of representative non-limiting examples of the invention. One skilled in the art will recognize that analogs of formula I, including different isomeric forms, may also be prepared from similar starting materials.
Scheme 1:
x is-OCH2-The preparation of the compounds described by formula Ib is described in scheme 1 below. One skilled in the art will recognize that the compounds described by formulas Ic, Id, Ie, If and Ig can be prepared by analogous synthetic methods using suitable starting materials.
Scheme 1
P1Is a functional group for protecting the nitrogen functional group. P1Examples of (a) are urethane-forming groups such as t-Butyloxycarbonyl (BOC), 9-Fluorenylmethoxycarbonyl (FMOC) and benzyloxycarbonyl (CBZ).
In general scheme 1, R depicted in formula II1The substituted hydroxythiophenol starting materials may be obtained from commercial sources or may be prepared by a number of methods well known in the art. Although there are many ways to accomplish the reaction described in method a, one convenient method involves reacting the compounds described by formulas II and III with a base (such as potassium carbonate) in a solvent (such as N, N-dimethylformamide) at ambient temperature for several hours. According to standard extraction and purification methods, the formula IV can be obtained in good yield and purityThe product is described.
Although there are many ways to achieve the reaction described in method B, one convenient method involves reacting the compounds described by formulas IV and V (where Y is a suitable leaving group such as Br, I, OT and OM) with a base such as potassium carbonate in a solvent such as N, N-dimethylformamide at ambient temperature for several hours. The product described by formula VI can be recovered by standard work-up procedures.
A convenient method of converting the compound described by formula VI to the compound described by formula VII is method C, which involves treating a solution of the compound described by formula VI and a base (such as sodium bicarbonate) in a solvent (such as dichloromethane) with an oxidizing agent such as mCPBA (3-chloroperoxybenzoic acid) for several hours between 0 ℃ and ambient temperature. The product described by formula VII can be recovered by standard work-up procedures.
There are many well established chemical procedures for deprotecting a compound described by formula VII to a compound described by formula Ib (method D). For example, if P1Is a BOC protecting group, the compound described by formula VII may be treated with an acidic substance, such as dry hydrogen chloride, in a solvent, such as diethyl ether, to provide the compound described by formula Ib as a hydrochloride salt. Typically, the free amino compound is converted to an acid addition salt for ease of handling and improved chemical stability. Examples of acid addition salts include, but are not limited to, hydrochloride, hydrobromide, 2,2, 2-trifluoroacetate and methanesulfonate salts.
Scheme 2:
the preparation of compounds described by formula Ib, wherein X is-CONH-is depicted in scheme 2 below. One skilled in the art will recognize that the compounds described by formulas Ic, Id, Ie, If and Ig can be prepared by analogous synthetic methods using suitable starting materials.
Scheme 2
In general scheme 2, R1The substituted mercaptobenzoic acid starting material may be commercially availableThe source is obtained or may be prepared by a number of methods well known in the art.
Compounds of formula XI can be prepared by reacting an appropriately substituted benzoic acid fragment with a base such as triethylamine in a solvent such as N, N-dimethylformamide in the presence of a suitable coupling agent such as HATU for several hours at ambient temperature (method E). The product depicted in formula XI can be recovered by standard work-up procedures.
Scheme 3:
the preparation of compounds described by formula Ib, wherein X is- -O- -, is depicted in scheme 3 below. One skilled in the art will recognize that the compounds described by formulas Ic, Id, Ie, If and Ig can be prepared by analogous synthetic methods using suitable starting materials.
Scheme 3
In general scheme 3, R1The substituted hydroxythiophenol starting materials may be obtained from commercial sources or may be prepared by a number of methods well known in the art.
The compounds described in formulae IV and XIII can be coupled using a modification of the copper-catalyzed Ullmann (Ullmann) reaction (method F). Many variations of this type of reaction are described in the literature, one example being the Chan-Evans-Lam modification. The compounds described by formulae IV and XIII can be dissolved in a solvent such as dichloromethane in the presence of pyridine and then treated with copper (II) acetate for several hours at ambient temperature. The coupled product described by formula XIV can be obtained in good yield and purity according to standard extraction and purification procedures.
Scheme 4:
the preparation of the compounds described by formula Ia is described in scheme 4 below.
Scheme 4
In general scheme 4, R1Substituted thiol starting materials may be obtained from commercial sources or may be prepared by a number of methods well known to those skilled in the art.
Scheme 5:
the preparation of the compounds described by formula Ia is described in scheme 5 below.
Scheme 5
In general scheme 5, R depicted by formula XVIII1Substituted arylsulfinate starting materials can be obtained from commercial sources or can be prepared by a number of methods well known to those skilled in the art. One convenient scheme for achieving the transformations described by method E involves reacting the compounds described by formulas XVIII and III with a base (such as potassium carbonate) in a solvent such as N, N-dimethylformamide at ambient temperature. The product described by formula XVII can be obtained in good yield and purity according to standard extraction and purification procedures
It will be appreciated by those skilled in the art that compounds of formula I wherein a is heteroaryl may be prepared by methods analogous to those described above.
The cis/trans (E/Z) isomers may be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.
Therapeutic uses and formulations
Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable diluent, excipient or adjuvant.
The invention also relates to the use of compounds of formula I in therapy, in particular to inhibit the lysyl oxidase family members LOX, LOXL1, LOXL2, LOXL3 and LOXL 4. In one embodiment, the invention provides selective inhibition of specific lysyl oxidase isozymes. In another embodiment, the invention provides for simultaneous inhibition of 2,3 or 4 LOX isozymes. The relative inhibitory potency of a compound can be determined by the amount required to inhibit the amine oxidase activity of LOX, LOXL1, LOXL2, LOXL3, and LOXL4 in a variety of ways, such as in vitro purification with recombinant or purified human proteins or in assays of cells expressing normal rodent enzymes, in assays of cells that have been transfected with human proteins, in vivo assays in rodents and other mammalian species, with recombinant or purified non-human enzymes.
In one embodiment, the compounds of the invention are persistent inhibitors of lysyl oxidase family members LOX, LOXL1, LOXL2, LOXL3, and LOXL 4. In one embodiment, a compound of the invention is a persistent inhibitor of the LOX or LOXL1-4 enzyme if inhibition continues to be greater than 50% of the activity of the LOX or LOXL1-4 enzyme after the concentration of the compound is reduced below IC 50. In one embodiment, a compound of the invention exhibits sustained inhibition of the LOX or LOXL1-4 enzyme within 24 hours. In one embodiment, the compounds of the invention are irreversible inhibitors of lysyl oxidase family members LOX, LOXL1, LOXL2, LOXL3, and LOXL 4.
Accordingly, a further aspect of the invention relates to a method of inhibiting amine oxidase activity of any of LOX, LOXL1, LOXL2, LOXL3, or LOXL4 in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.
In one embodiment, the invention relates to a method of inhibiting amine oxidase activity of LOXL 2. In another embodiment, the invention relates to inhibiting the amine oxidase activity of LOX and LOXL 2. In a further embodiment, the invention relates to a method of inhibiting the amine oxidase activity of LOX.
As discussed previously, LOX and LOXL1-4 enzymes are members of a large class of flavin-dependent and copper-dependent amine oxidases, including SSAO/VAP-1, monoamine oxidase B (MAO-B), and diamine oxidase (DAO). In one embodiment, the compounds of the present invention selectively inhibit members of the lysyl oxidase isozyme family relative to SSAO/VAP-1, MAO-B, DAO, and other members of the amine oxidase family.
Also disclosed are methods of inhibiting one or more lysyl oxidase isozymes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) in a patient having a fibrotic disease using compounds described by formula I, and methods of treating a fibrotic disease. In addition, methods of inhibiting one or more lysyl oxidase isozymes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) in patients having cancer, including metastatic cancer, using compounds described by formula I, and methods of treating cancer and metastatic cancer are disclosed.
In a further aspect of the invention, there is provided a method of treating a disorder associated with any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 proteins, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.
In another aspect, there is provided a method of treating a disorder modulated by any one of LOX, LOXL1, LOXL2, LOXL3, or LOXL4, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof.
In one embodiment of the method of the invention, the disorder is selected from the group consisting of: fibrosis, cancer and angiogenesis.
In another aspect, the invention provides methods of reducing extracellular matrix formation by treating human subjects, companion animals and livestock with a fluoroallylamine inhibitor of the lysyl oxidase isozyme of formula I described herein.
The above method is suitable for the case where the condition is fibrosis. As used herein, "fibrosis" includes diseases such as cystic fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, scleroderma, radiation-induced fibrosis, pelonese disease, scarring, and other excessive fibrosis leading to disease pathology.
In one embodiment, the fibrosis is selected from the group consisting of: mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, crohn's disease, keloid, systemic sclerosis, joint fibrosis, dupuytren's contracture, adhesive joint capsulitis, pancreatic fibrosis, intestinal fibrosis, liver fibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis, fibrostenosis, cystic fibrosis, idiopathic lung fibrosis, radiation-induced fibrosis, peyronie's disease, and scleroderma, or is associated with: respiratory diseases, abnormal wound healing and repair, scarring, hypertrophic scarring/keloids, post-operative scarring, cardiac arrest, and all conditions in which excessive or abnormal deposition of fibrous material is associated with a disease, injury, implant, or surgery. In another embodiment, the fibrosis is selected from the group consisting of: liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis, scarring and scleroderma. In further embodiments, the fibrosis is selected from the group consisting of: myelofibrosis, systemic sclerosis, liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis and radiation-induced fibrosis.
In one embodiment, renal fibrosis includes, but is not limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renal fibrosis, glomerulonephritis or glomerulonephritis, including focal segmental glomerulosclerosis and membranous glomerulonephritis, IgA nephropathy and mesangial capillary glomerulonephritis. In one embodiment, liver fibrosis results in cirrhosis, and includes related disorders such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), Alcoholic Steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), Primary Biliary Cirrhosis (PBC), biliary cirrhosis, and autoimmune hepatitis.
In one embodiment, the fibrosis is selected from keloid, scar, ocular scar, hypertrophic scar, scleroderma, dupuytren's contracture, and pelmet's disease. In one embodiment, hypertrophic scars result from burns. In one embodiment, hypertrophic scars result from an external injury. In another embodiment, hypertrophic scars result from surgery. In one embodiment, the keloid is caused by an external injury. In another embodiment, the keloid is caused by surgery. In further embodiments, the keloid is the result of skin damage caused by acne, burns, chicken pox, ear pricks, scratches, surgical incisions, or vaccination sites.
The above methods are also applicable where the disorder is a proliferative disease (e.g., cancer). In one embodiment, the cancer is selected from the group consisting of: lung cancer; breast cancer; large bowel cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer; hepatobiliary cancer; esophageal cancer; mesothelioma; non-hodgkin lymphoma; bladder cancer; uterine cancer; gliomas, glioblastoma, medulloblastoma and other brain tumors; myelofibrosis, renal cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer, carcinoids of the gastrointestinal tract, breast and other organs; signet ring cell carcinoma; a mesenchymal tumor comprising sarcoma, fibrosarcoma, hemangioma, angiomatosis, hemangiopericytoma, pseudohemangiomatoid interstitial hyperplasia, myofibroblasts, fibromatosis, inflammatory myofibroblastoma, lipoma, angiolipoma, granulocytoma, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, or leiomyosarcoma.
In one embodiment, the cancer is selected from the group consisting of: breast cancer, head and neck squamous cell carcinoma, brain cancer, prostate cancer, renal cell carcinoma, liver cancer, lung cancer, oral cancer, cervical cancer, and tumor metastasis.
In one embodiment, lung cancer includes lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchoalveolar carcinoma, non-small cell carcinoma, and mesothelioma. In one embodiment, the breast cancer includes ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, and mucinous carcinoma. In one embodiment, colorectal cancer includes colon cancer and rectal cancer. In one embodiment, pancreatic cancer includes pancreatic cancer, islet cell carcinoma, and neuroendocrine tumors.
In one embodiment, the ovarian cancer comprises an ovarian epithelial cancer or superficial epithelial-mesenchymal tumor, including serous, endometrioid, and mucinous cystadenocarcinoma and gonadal-mesenchymal tumor. In one embodiment, liver and bile duct cancers include hepatocellular carcinoma, bile duct cancer, and hemangioma. In one embodiment, esophageal cancer includes esophageal adenocarcinoma and squamous cell carcinoma. In one embodiment, uterine cancer includes endometrial adenocarcinoma, papillary serous carcinoma of the uterus, clear cell carcinoma of the uterus, sarcoma of the uterus and leiomyosarcoma, and a mixed tumor of the muller tubes. In one embodiment, the renal cancer includes renal cell carcinoma, clear cell carcinoma, and wilm's tumor. In one embodiment, the head and neck cancer comprises squamous cell carcinoma. In one embodiment, gastric cancer includes gastric adenocarcinoma and gastrointestinal stromal tumors.
In one embodiment, the cancer is selected from the group consisting of: colon cancer, ovarian cancer, lung cancer, esophageal cancer, breast cancer, and prostate cancer. In one embodiment, the cancer is selected from the group consisting of: pancreatic cancer, liver cancer, breast cancer, myelofibrosis and mesothelioma.
In one embodiment, the compounds of the invention can be used to treat non-metastatic cancer. In another embodiment, the compounds of the invention can be used to treat metastatic cancer. In a further embodiment, the compounds of the invention may be used for the prevention or treatment of tumor metastasis.
The above method is applicable where the condition is angiogenesis.
In one embodiment of the method of the invention, the subject is selected from the group consisting of: humans, pets and livestock. In another embodiment of the methods of the invention, the subject is a human.
A further aspect of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of a condition associated with any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL4 protein.
Another aspect of the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for the treatment of a condition modulated by any one of LOX, LOXL1, LOXL2, LOXL3 or LOXL 4.
Pharmaceutical and/or therapeutic formulations
In another embodiment of the present invention, there is provided a composition comprising a compound having formula I and at least one pharmaceutically acceptable excipient, carrier or diluent thereof. The compounds of formula I may also be present as suitable salts, including pharmaceutically acceptable salts.
The phrase "pharmaceutically acceptable carrier" refers to any carrier known to those of skill in the art to be suitable for a particular mode of administration. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition, or may be combined with other active ingredients.
The phrase "pharmaceutically acceptable salt" refers to any salt formulation suitable for pharmaceutical use. Pharmaceutically acceptable salts are those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art and include acid addition salts and base salts. Half salts of acids and bases may also be formed. Pharmaceutically acceptable salts include amine salts of inorganic acids (e.g., hydrochloride, hydrobromide, sulfate, etc.); and amine salts of organic acids (e.g., formate, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, maleate, butyrate, valerate, fumarate, etc.).
For compounds of formula (I) having a basic site, suitable pharmaceutically acceptable salts may be acid addition salts. For example, suitable pharmaceutically acceptable salts of such compounds may be prepared by mixing a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid or citric acid, with a compound of the present invention.
Pharmaceutically acceptable salts are described in detail in SM Berge et al, J.pharmaceutical Sciences 1977,66: 1-19. These salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting the free base functionality with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, gluconate, glycerophosphate, hemisulfate, glucoheptonate, hydrobromide, hydrochloride, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, picrate, pivalate, picrate, etc, Propionate, stearate, succinate, sulfate, tartrate, tosylate, undecanoate, valerate, and the like. Suitable base salts are formed from bases which form non-toxic salts. Examples include arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine and zinc salts. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine, and the like.
Pharmaceutically acceptable salts of compounds of formula I can be prepared by methods known to those skilled in the art, including, for example:
(i) by reacting a compound of formula I with the desired acid or base;
(ii) by removing acid-or base-labile protecting groups from suitable precursors of compounds of formula I, or by ring-opening suitable cyclic precursors such as lactones or lactams using the desired acid or base; or
(iii) One salt of the compound of formula I is converted to another salt by reaction with a suitable acid or base or with the aid of a suitable ion exchange column.
The above reactions (i) to (iii) are usually carried out in solution. The resulting salt may precipitate out and be collected by filtration, or may be recovered by evaporation of the solvent. The degree of ionization in the resulting salt may vary from complete ionization to almost no ionization.
Thus, for example, suitable pharmaceutically acceptable salts of the compounds of the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, carbonic acid, tartaric acid or citric acid with the compounds of the present invention. Thus, suitable pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts.
The compounds of the invention may exist in non-solvated and solvated forms. As used herein, the term "solvate" is used to describe a molecular complex comprising a compound of the present invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydrate" is used.
In one embodiment, the compounds of formula I may be administered in a "prodrug" form. The phrase "prodrug" refers to a compound that is metabolized or otherwise converted to the biologically, pharmaceutically, or therapeutically active form of the compound by one or more steps or processes following in vivo administration. Prodrugs can be prepared by: the functional groups present in the compounds are modified such that the modifications can be cleaved, either in routine manipulation or in vivo, to the compounds described herein. For example, prodrugs include compounds of the present invention wherein a hydroxy, amino, or carbohydrate group is bonded to any group that, when administered to a mammalian subject, can cleave to form a free hydroxy, free amino, or free carboxylic acid group, respectively. Representative prodrugs include, for example, amide, ester, enol ether, enol ester, acetate, formate, benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention. Prodrug forms may be selected from, for example, -C (O) alkyl, -C (O) cycloalkyl, -C (O) aryl, -C (O) -arylalkyl, C (O) heteroaryl, -C (O) -heteroarylalkyl, and the like. With the aid of knowledge of the pharmacodynamic processes and drug metabolism in vivo, one skilled in the art, once aware of the pharmaceutically active compounds, can design prodrugs of said compounds (see, for example, Nogrady (1985) molecular and Biochemical methods of Medicinal Chemistry (Medicinal Chemistry A Biochemical Approach), New York university of Oxford Press, p. 388-.
The compositions herein comprise one or more compounds provided herein. In one embodiment, the compounds are formulated into suitable pharmaceutical formulations such as solutions, suspensions, tablets, creams, gels, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs for oral administration or for parenteral administration in sterile solutions or suspensions, as well as transdermal patches and dry powder inhalers. In one embodiment, the above compounds are formulated into Pharmaceutical compositions using techniques and methods well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, fourth edition, 1985,126).
In the compositions, an effective concentration of one or more compounds or pharmaceutically acceptable derivatives thereof is admixed with a suitable pharmaceutical carrier. As noted above, prior to formulation, the compounds may be derivatized as the corresponding salt, ester, enol ether or ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate, or prodrug. The concentration of the compound in the composition is effective to deliver, upon administration, an amount effective to treat, prevent, or ameliorate one or more symptoms of the disease or disorder being treated.
In one embodiment, the composition is formulated for single dose administration. To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration to thereby alleviate, prevent, or ameliorate one or more symptoms.
The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically effective effect without producing adverse side effects in the patient being treated. A therapeutically effective concentration may be empirically determined by: compounds were tested in the in vitro and in vivo systems described herein and then human dosages were extrapolated.
The concentration of the active compound in the pharmaceutical composition will depend on the rate of absorption, distribution, inactivation, and elimination of the active compound; the physicochemical properties of the compound; dosing regimen and amount of administration; and other factors known to those skilled in the art.
In one embodiment, a therapeutically effective dose should result in a serum concentration of the active ingredient of about 0.1ng/mL to about 50-100 μ g/mL. In another embodiment, the pharmaceutical composition should provide a dosage of about 0.001mg to about 2000mg of the compound per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 0.01mg, 0.1mg, or 1mg to about 500mg, 1000mg, or 2000mg, and in one embodiment, from about 10mg to about 500mg of the active ingredient or combination of essential ingredients per dosage unit form.
Administration may be at intervals of minutes, hours, days, weeks, months or years, or may be continuous during any of these periods. Suitable doses range from about 0.1ng per kilogram body weight per dose to 0.1g per kilogram body weight. The dose is preferably in the range of 10 μ g to 0.1g per kg body weight per dose, such as in the range of 0.1mg to 0.01g per kg body weight per dose. Suitably, the dose is in the range of 10 μ g to 50mg per kg body weight per dose, such as 10 μ g to 20mg per kg body weight per dose, or 10 μ g to 10mg per kg body weight per dose. Other suitable doses may range from 0.1mg to 25mg per kilogram body weight, including from 0.1mg to 10, 20, 50 or 100mg per dose per kilogram body weight.
Alternatively, an effective dose may be up to about 10mg/cm2Or alternatively, may be up to about 1mg/cm2About 0.5mg/cm2About 0.2mg/cm2About 0.1mg/cm2About 0.05mg/cm2About 0.02mg/cm2Or about 0.01mg/cm2. For example, it may be in the range of about 10. mu.g/cm2To about 1. mu.g/cm2About 10. mu.g/cm2To about 0.1mg/cm2About 10. mu.g/cm2To about 0.01mg/cm2About 10. mu.g/cm2To about 500. mu.g/cm2About 10. mu.g/cm2To about 200. mu.g/cm2About 10. mu.g/cm2To about 100. mu.g/cm2About 10. mu.g/cm2To about 50. mu.g/cm2About 20. mu.g/cm2To about 1mg/cm2About 50. mu.g/cm2To about 1mg/cm2About 100. mu.g/cm2To about 1mg/cm2About 200. mu.g/cm2To about 1mg/cm2About 500. mu.g/cm2To about 1mg/cm2About 50. mu.g/cm2To about 500. mu.g/cm2About 50. mu.g/cm2To about 200. mu.g/cm2About 100. mu.g/cm2To about 500. mu.g/cm2Or about 200. mu.g/cm2To about 500. mu.g/cm2Within the range of (1).
Suitable dosages and dosing regimens may be determined by the attending physician, and may depend on the particular condition being treated; the severity of the condition; and the overall health, age and weight of the subject.
In the case where the compound exhibits insufficient solubility, a method of solubilizing the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO); using surfactants, e.g.Dissolving in sodium bicarbonate water solution; formulating compounds of interest such as nanoparticles and the like. Derivatives of the compounds, such as prodrugs of the compounds, may also be used to formulate effective pharmaceutical compositions.
After mixing or adding the compounds, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition being treated and can be determined empirically.
Pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil and water emulsions containing suitable amounts of the compounds or pharmaceutically acceptable derivatives thereof. In one embodiment, the pharmaceutically therapeutically active compound and derivatives thereof are formulated and administered in unit dosage form or multiple dosage forms. The active ingredient may be administered at one time or may be divided into a number of smaller doses to be administered at intervals. As used herein, unit dosage forms refer to physically discrete units suitable for use in human and animal subjects, and packaged individually as is known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, together with a desired pharmaceutical carrier, vehicle or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. The unit dosage form may be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container for administration as separate unit dosage forms. Examples of multiple dosage forms include vials, tablets or capsules or pints or gallons. Thus, a multiple dosage form is a plurality of unit doses that are not divided in a package.
The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Iston, Pa., 15 th edition, 1975.
Dosage forms or compositions can be prepared containing in the range of 0.005% to 100% (by weight) of the active ingredient with the remainder being made up of non-toxic carriers. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain from 0.001% to 100% (wt.%) active ingredient, in one embodiment from 0.1% to 95% (wt.%), and in another embodiment from 75% to 85% (wt.%).
Mode of administration
Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral, inhalation, transdermal application, topical cream or gel or powder, vaginal or rectal administration. Depending on the route of administration, the formulation and/or the compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions that may inactivate the therapeutic activity of the compound. The compounds may also be administered parenterally or intraperitoneally.
Composition for oral administration
Oral pharmaceutical dosage forms may be solid, gel or liquid. The solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. The capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.
Solid composition for oral administration
In certain embodiments, the formulation is a solid dosage form, in one embodiment a capsule or tablet. Tablets, pills, capsules, lozenges and the like may contain one or more of the following ingredients or compounds of similar properties: a binder, a lubricant; a diluent; a glidant; a disintegrant; a colorant; a sweetener; a flavoring agent; a wetting agent; coating an emetic; and a film coating. Examples of binders include microcrystalline cellulose, tragacanth, dextrose solution, acacia mucilage, gelatin solutions, molasses, polyvinylpyrrolidone, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any approved certified water-soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on hydrated alumina. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic mixtures of compounds that produce a pleasant sensation, such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalate. The film coating comprises hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
The compound or pharmaceutically acceptable derivative thereof may be provided in the form of a composition that protects it from the acidic environment of the stomach. For example, the compositions may be formulated as an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The compositions may also be formulated in combination with antacids or other such ingredients.
When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. In addition, the dosage unit form may contain various other materials which modify the physical form of the dosage unit, such as sugars and other enteric solvent coatings. The compounds may also be administered as components of elixirs, suspensions, syrups, wafers, dispersions (sprinkle), chewing gums and the like. In addition to the active compounds, syrups may contain sucrose as a sweetening agent and certain preservatives, dyes, colorants and flavoring agents.
The active substance may also be mixed with other active substances which do not impair the desired action or with substances which supplement the desired action, such as antacids, H2 blockers and diuretics. The active ingredient is a compound described herein or a pharmaceutically acceptable derivative thereof. Higher concentrations, up to about 98% by weight of the active ingredient may be included.
In all embodiments, tablets and capsules may be coated as known to those skilled in the art to alter or maintain dissolution of the active ingredient. Thus, for example, they may be coated with conventional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.
Liquid composition for oral administration
Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent formulations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. The emulsion is oil-in-water or water-in-oil.
Pharmaceutically administrable liquid compositions can be formed, for example, by dissolving, dispersing or otherwise mixing an active compound as defined above and an optional pharmaceutical adjuvant in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to form a solution or suspension. If desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents.
Elixirs are clear, sweetened, hydroalcoholic formulations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed in the form of globules in another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable materials used in non-effervescent granules reconstituted into liquid oral dosage forms include diluents, sweeteners and wetting agents. The pharmaceutically acceptable materials used in the effervescent granules will be reconstituted into a liquid oral dosage form comprising an organic acid and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.
Solvents include glycerol, sorbitol, ethanol and syrup. Examples of preservatives include glycerol, methyl and propyl parabens, benzoic acid, sodium benzoate and ethanol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, gum acacia, gum tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspensions include sodium carboxymethylcellulose, pectin, gum tragacanth, magnesium salts of aluminium silicate and gum acacia. Sweetening agents include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved certified water soluble FD and C dyes and mixtures thereof. Flavoring agents include natural flavors extracted from plants such as fruits, and synthetic mixtures of compounds that produce a pleasant mouth feel.
For solid dosage forms, in one embodiment, a solution or suspension, such as propylene carbonate, vegetable oil, or triglycerides, is encapsulated in a gelatin capsule. For liquid dosage forms, such as solutions (e.g., in polyethylene glycol), it may also be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier (e.g., water) so as to be readily measured upon administration.
Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those listed in U.S. Pat. nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing the compounds provided herein, dialkylated mono-or polyalkylene glycols including, but not limited to, 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, where 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
Other formulations include, but are not limited to, aqueous alcoholic solutions including pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including but not limited to propylene glycol and ethanol. Acetals include, but are not limited to, di (lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.
Injections, solutions and emulsions
Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection in one embodiment, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injections, solutions and emulsions may also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancing agents and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
Implantation of sustained release or sustained release systems to maintain a constant dosage level is also contemplated herein. Briefly, the compounds provided herein are dispersed in a solid internal matrix, such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of acrylic and methacrylic esters, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate, surrounded by an outer polymer film, such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, poly (vinyl chloride), poly (vinyl acetate), poly (vinyl chloride), neoprene, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers, and ethylene/ethyleneoxyethanol copolymers that are insoluble in body fluids. In the release rate controlling step, the compound diffuses through the outer polymer membrane. The percentage of active compound contained in such parenteral compositions is highly dependent on its particular properties, as well as the activity of the compound and the needs of the subject.
Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administration. Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders, including subcutaneous tablets, combined with a solvent prior to use, sterile suspensions ready for injection, sterile dry insoluble products combined with a vehicle prior to use, and sterile emulsions. The solution may be aqueous or non-aqueous.
Suitable carriers, if administered intravenously, include physiological saline or Phosphate Buffered Saline (PBS), as well as solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.
Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable materials.
Examples of aqueous vehicles include sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, olive oil, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial or bacteriostatic concentrations of the antimicrobial agent must be added to parenteral formulations packaged in multi-dose containers comprising phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. The antioxidant comprises sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. The emulsifier comprises polysorbate 80Sequestering or chelating agents for metal ions include EDTA. The pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.
The concentration of the pharmaceutically active compound is adjusted so that the injection provides an effective amount to produce the desired pharmacological effect. The exact dosage will depend on the age, weight and condition of the patient or animal, as is known in the art.
The unit dose of parenteral formulation is packaged in ampoules, vials or needle syringes. All formulations for parenteral administration must be sterile, as is known and practiced in the art.
Illustratively, intravenous or intraarterial infusion of sterile aqueous solutions containing the active compounds is an effective mode of administration. Another example is a sterile aqueous or oily solution or suspension containing the active substance injected as required to produce the desired pharmacological effect.
Injections are designed for local and systemic administration. In one embodiment, a therapeutically effective dose is formulated to contain the active compound in a concentration of at least about 0.1% w/w to about 90% w/w or more, and in some embodiments, greater than 1% w/w of the pharmaceutically active ingredient relative to the treated tissue.
The compounds may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to alleviate symptoms of the disorder and can be determined empirically.
Freeze-dried powder
Also of interest herein are lyophilized powders, which can be reconstituted for administration in the form of solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solids or gels.
Sterile lyophilized powders are prepared by dissolving a compound provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. The solvent may contain excipients that may improve the stability or other pharmacological ingredients of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art at about neutral pH. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder may be stored under appropriate conditions, such as at about 4 ℃ to room temperature.
Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such an amount may be determined empirically.
Topical application
Topical mixtures are prepared as described for local and systemic administration. The resulting mixture may be in the form of a solution, suspension, emulsion, etc., and formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.
The compound or a pharmaceutically acceptable derivative thereof may be formulated as an aerosol for topical application (such as by inhalation). These formulations for respiratory administration may be in the form of an aerosol or solution of a spray, or in the form of a finely divided powder for insufflation, either alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation are less than 50 microns in diameter in one embodiment, and less than 10 microns in diameter in one embodiment.
The compounds may be formulated for topical or local (local or topical) administration, such as in the form of gels, creams and lotions for topical application to the skin and mucous membranes, such as the eye, and for application to the eye or for intracranial or intraspinal application. Topical administration is contemplated for transdermal delivery, as well as for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the active compounds may also be administered alone or in combination with other pharmaceutically acceptable excipients.
These solutions, especially those intended for ophthalmic use, can be formulated with appropriate salts as 0.01% to 10% (volume%) isotonic solutions having a pH of about 5-7.
Other administration route compositions
Other routes of administration are also contemplated herein, such as transdermal patches, including iontophoretic and electrophoretic devices, vaginal and rectal administration.
Transdermal patches including iontophoresis and electrophoresis devices are well known to those skilled in the art. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets with systemic action. Rectal suppositories are used herein mean solids for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or vehicles and agents that raise the melting point. Examples of bases include cocoa butter (cocoa butter), glycerol-gelatin, carbohydrates (polyoxyethylene glycols) and suitable mixtures of mono-, di-and triglycerides of fatty acids. Combinations of various bases may be used. Agents that increase the melting point of suppositories include acetogenin and waxes. Rectal suppositories may be prepared by compression methods or by molding. In one embodiment, the rectal suppository weighs between about 2 and 3 gm.
Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substances and by the same methods as the formulations for oral administration.
Target preparation
The compounds provided herein, or pharmaceutically acceptable derivatives thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the present invention.
In one embodiment, liposomal suspensions, including tissue-targeting liposomes, such as tumor-targeting liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes, such as multilamellar vesicles (MLVs), can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (at a 7:3 molar ratio) inside the flask. A solution of the compounds provided herein in Phosphate Buffered Saline (PBS) lacking divalent cations was added and the flask was shaken until the lipid film dispersed. The resulting vesicles were washed to remove unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.
Combined administration with other drugs
According to another aspect of the invention, it is contemplated that the compounds of formula I described herein may be administered to a subject in need thereof in combination with a drug recognized by those skilled in the art as being the current standard of care for the condition of interest. Such combinations provide one or more advantages to the subject, e.g., reduced dosage required to achieve similar benefits, desirable relief in less time, etc.
The compounds according to the invention may be administered with other drugs as part of a treatment regimen. For example, it may be desirable to administer a combination of active compounds in order to treat a particular disease or condition. Thus, within the scope of the present invention, two or more pharmaceutical compositions, at least one of which contains a compound of formula (I) according to the invention, may be combined in the form of a kit suitable for the combined administration of the compositions.
In one embodiment of the methods of the present invention, the compound of formula I may be administered with a second therapeutic agent. In one embodiment, the second therapeutic agent may be selected from one or more of:
(i) anticancer agents, such as cisplatin, oxaliplatin, carboplatin, cyclophosphamide, mechlorethamine, uracil mustard, bendamustine, melphalan, phenylbutyric acidNitrogen mustard, mechlorethamine hydrochloride, busulfan, temozolomide, nitrosourea, ifosfamide (ifosamide), pipobroman, tritylamine, triethylenethiophosphoramide, carmustine, lomustine, streptozotocin and dacarbazine, gemcitabine, fogercettabine palaapamide (fosgemicabine palabenamide), 5-fluorouracil, tegafur, raltitrexed, methotrexate, pemetrexed, leucovorin, cytosine arabinoside, floxuridine, cytarabine, 6-thioguanine, fludarabine phosphate, pentostatin (pentostatin), hydroxyurea, trifluridine, triflouracil, doxorubicin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, mitomycin, mithramycin, vincristine, vinblastine, vindesine and vinorelbine, Paclitaxel, taxotere, eribulin, carfilzomib, bortezomib, etoposide, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, camptothecin, actinomycin D, daunorubicin, doxorubicin, epirubicin, idarubicin, cytarabine, paclitaxel (Taxol)TM) Nabamine paclitaxel, docetaxel, desoxyzoomycin (deoxynoxamycin), L-asparaginase, IFN-alpha, azacitidine, decitabine, vorinostat, MS-275, panobinostat (panobinostat), romidepsin, valproic acid, moxifloxacin (mocetinostat), prasterone (pracetinostat), belinostat, elabepotidine (iractedin), tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene, idoxyfene (iodoxyfene), bicalutamide, flutamide, nilutamide, cyproterone acetate, goserelin, leuprorelin, buserelin, progestogen, megestrol acetate, anastrozole, letrozole, vorazozole, vorazone, amitrazone, elvalvine, lonamide (lipofectamine), penoxelane, penoxelargoline, penoxsulam, penoxetamide, and pelofuranine; and abiraterone, enzalutamide, lanreotide, dasatinib, bosutinib, trastuzumab, pertuzumab, panitumumab, cetuximab, gefitinib, erlotinib, afatinib, vandetanib (vandetanib), axitinib, rofitinib, lapatinib, nilotinib(iii) tinib, sorafenib, tipifarnib and lonafarnib, vemurafenib, dabrafenib, trametinib, cobitinib, panatinib, palbociclib, everolimus, ruxolitinib, pacritinib (pacritinib), jenkitinib (jaktinib), entastat (imetelstat), piridoxine (plitidipsin), povosita (pevonedistat), ibrutinib, ceritinib, crizotinib, exertinib (ectonib), cabozantinib (cabozaritisib), vegie (vismodegibib), sonegb (sonidegigib), sonidegeizumab (sonidegigib), regenamab (sonidegenib), regorafenib, vandetanib, vatamizumab, zemab, bizumab, arguzumab, gazerumab (bizumab), gazettuzumab, bizepindolastamicin, zerumatumab, zezumab (zepindol), zepindolizumab, zepindol (zepindol), zepindol, zepindolizumab, zepindolitumumab (zemab), zepindol, zepindolitumab (zepinbizumab, zepindol, zepindolitumab, zepin, Rituximab, ibritumomab, ofatumumab, peginterferon alpha-2 a, aldesleukin, Gardasil, ceriferin (Cervarix), Oncophage, sipulexel (Sipuleucel-T), nivalemab, pembrolizumab, altuzumab, indoximod, Nawurieuzumab, ipilimumab, bentuximab, trastuzumab, fludarabine, cladribine, pentostatin, Idelalisib, perifosine, birinapept, bortezomib, ixazozomib, carfilzomib, marizomib (marizomib), olaparipatinib, lucanib, nevutock, nevacipratrox (navitoclat), olbacura, glasediggiib (glasedegipib), pavidist, buclizib (paclobut), paclobutraib (paclobutraib), amitocin, cetirizine, fibrauretigabine, cetuiripib (arinib), cetirizine, nixib, nivalicarb (nabuipesx), nipazib, nixib, nixizanib, nixib, nixizanib, nixib, nixizanib, nixib, nixifibrauglibenicib, nixib, Vinflunine, napabusin, lubicantin, tasystat (tazemetostat), acartinib, lenvatinib, neratinib, pamipertib (pamiprarib), ecastat (epacadostat), enzastarin (enzastaurin), cerini (selinexor), masitinib, efamide (evofosfamide), glufosinate, roxastat (roxadustat), streptozotocin, tevista (devistat), galunivisertib, bimetinib(binimetinib), veliparib, entinostat, pexidinib (pexidartinib), talazoparib (talazoparib), and entrectinib (entretinib).
(ii) Anti-inflammatory agents such as meloxicam, fenoprofen, oxaprozin, salsalate, etoricoxib, tenoxicam, aspirin, nabumetone, flurbiprofen, fenamic acid, phenylbutazone, lornoxicam, indomethacin, etodolac, diflunisal, ketoprofen, valdecoxib, tolfenamic acid, piroxicam, sulindac, toluoylpyridine acetic acid, ketorolac, loxoprofen, acetaminophen, bromfenac, diclofenac, ibuprofen, meclofenamic acid, nabumetone, naproxen, nepafenac, celecoxib, triamcinolone acetonide, hydrocortisone pyruvate, hydrocortisone acetate, methylprednisolone, mefenaminoxidine, mefenamic acid, nabumetone, naproxen, celecoxib, triamcinolone, hydrocortisone acetate, and mefenoxanide,Alclometasone dipropionateEnricasan (emricasan), BI 1467335, namodenoson, GLPG-1,690, terguride.
(iii) Antihypertensives such as hydrochlorothiazide, chlorthalidone, furosemide, spironolactone, triamterene, amiloride, benazepril, captopril, lisinopril, enalapril, ramipril, fosinopril, moxapril, perindopril, quinapril, trandolapril, losartan, candesartan, valsartan, telmisartan, clonidine, methyldopa, propranolol, nadolol, timolol, prodolol, labetalol, metoprolol, atenolol, esmolol, betaxolol, carvedilol, prazosin, terazosin, doxazosin, phenoxybenzamine, phentolamine, verapamil, diltiazem, nifedipine, felodipine, amlodipine, nimodipine, diazoxide, minoxidil, pirnadil, nicorandil, pyridazine, diazapazone, propafenone, valsartan, iloprostenol, alprostadil, prostacyclin, alprostadil, pril, benazepril, benalol, beraprost, ebelaprost (esurprost), rilepag (ralinepag), macitentan, sitaxentan, ambrisentan, riociguat, treprostinil, ubenimex (ubenimex), selipipag (selexipag), levosimendan, udenafil, tadalafil and sildenafil.
(iv) Anti-fibrotic agentsSuch as pirfenidone (pirfenidone), nintedanib (nintedanib), cintixoc (cericiviroc), selectrib (selonsertib), raninfirono (laniferor), nimiximab (nimacimab), nitazoxanide, NGM282, aparenone (aparenone), and,Taibiflukast(tipelukast), Arthromus (Actimmune), ponatinib (ponatinib), lenvatinib (lenvatinib), dolvatinib (dovitinib), delvatinib (lucitanib), dannoutinib (danusertinib), brenvertinib (brivatinib),Ervatinib(erdafitinib), Belapine (belapectin) PD173074, PD166866, AZD4547, BGJ398, LY2874455, TAS-120, ARQ087, BLU9931, FGF401, BAY-1163877, ENMD-2076, IMCA1, FGF401, DEBIO1347, FIIN-2, GP-369, PRO-001, H3B-6527, BAY1187982, MFGR1877S, FP-1039, BLU554, PRN1371, S49076, SU6668, SU5416, PBI-4050, KD-025.
(v) Anti-angiogenic agents such as axitinib, bevacizumab, cabozantinib, lenalidomide, lenvatinib, pazopanib, ramucirumab, vandetanib, vatalanib, sunitinib, aflibercept, thalidomide, pomalidomide, lenalidomide.
(vi) Immunosuppressants such as prednisone, budesonide, prednisolone, tofacitinib, cyclosporine, tacrolimus, sirolimus, everolimus, azathioprine, leflunomide, mycophenolate, abacavir, adalimumab, anakinra, certolizumab ozogamicin, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tositumumab, ursinumab, vedolizumab, basiliximab, daclizumab, dimethyl fumarate, mycophenolate.
(vii) Metabolic drugs such as obeticholic acid, elafelbinar (elafibranor), aramchol, sladapar (seladelpar), MGL-3196, topiroxol (tropifexor), MSDC-0602K, BMS-986036, somaglutide (semaglutide), EDP-305, gemcabene (gemcabene), PF-05221304, PF-06865571, PF-06835919 LIK066, LMB763, vitamin E, acarbose, miglitol, pramine, alogliptan (alogliptan), linagliptin, saxagliptin, sitagliptin, arbitutide, dulaglutide, exenatide, liraglutide, lixide, lixivide, lisirade, insulin, nateglinide, and repaglinide. Metformin, canagliflozin (canagliflozin), dapagliflozin (dapagliflozin), engagliflozin (empagliflozin), chlorpropamide, glimepiride (glimepiride), glipizide (glipizide), glyburide (glyburide), tolazamide (tolazamide), tolbutamide (tolbutamide), rosiglitazone (rosiglitazone), pioglitazone (pioglitazone), atorvastatin (atorvastatin), amlodipine (amlodipine), simvastatin (simvastatin), ezetimibe (ezetimibe), lovastatin (lovastatin), sitagliptin (sitagliptin), cholestyramine (cholestyramine), lefluvelezetimibe (glietimibe), glietimibe (flagelliferol), fenofibrate (fenofibrate), fenofibrate (simvastatin), fluvastatin (fluvastatin), fenofibrate (simvastatin (fluvastatin), fenofibrate (fluvastatin), niacin (fluvastatin), fluvastatin (simvastatin, fluvastatin), fluvastatin (fulvestrant), fluvastatin (fenofibrate), fluvastatin (fluvastatin), fluvastatin (fluvastatin), fluvastatin (flu, Pitavastatin (pitavastatin), simvastatin (simvastatin), cerivastatin (cerivastatin), allopurinol (allopurinol), rexinide (lesinurad), pegolose (peglotinase), febuxostat (febuxostat), labrasase (rasburicase), ivacaitor (ivacaptor), velaglucerase alpha, galactosidase beta, sebelipase alpha, vestratonidase alpha, thiolase, elosufase alpha, eliglutastat (eliglustat), broussumab (busosumab), migalatat (migalatat), sapropterin, metreleptin, nitisinonide, phenylalanine ammonia lyase, asfotase alpha, einesen, meglumine (miglustat), orlistat (sodium butyrate), benzene butyrate (benzene sulfonate).
In one embodiment, the compounds of the present invention may be administered in combination with other methods of treatment. For example, the compounds of the present invention may be administered in combination with radiation therapy or chemotherapy. In one embodiment, the compounds of the present invention can be administered in combination with one or more additional antineoplastic agents and/or radiation therapy to treat cancer.
When two or more active ingredients are administered in combination, the active ingredients may be administered simultaneously, sequentially or separately. In one embodiment, the compound of formula I is administered in combination with a second therapeutic agent at the same time. In another embodiment, the compound of formula I and the second therapeutic agent are administered sequentially. In a further embodiment, the compound of formula I and the second therapeutic agent are administered separately.
The invention will now be described in more detail, by way of example only, with reference to the following non-limiting examples. The examples are intended to be illustrative of the invention and should not be construed as limiting the generality of the disclosure throughout the specification.
Experimental performance: general procedure
The commercially available solvents and reagents were used as such. The reaction is carried out under an argon atmosphere where appropriate. The reaction was monitored by analytical Thin Layer Chromatography (TLC) or by liquid chromatography-mass spectrometry (LCMS), which was recorded using reverse phase conditions on a Shimadzu LCMS 2020 instrument or an Agilent LC/MSD 1200 instrument. If necessary, column chromatography or preparative HPLC was used to purify the intermediate and final compounds. Normal phase column chromatography was performed on silica gel or pre-packed silica gel mini-columns at medium pressure using a flash chromatography system (CombiFlash Rf200, Teledyne Isco system, usa). Using a flash chromatography systemReverse phase column chromatography was performed on a pre-packed C18 mini-column at low pressure. The eluate was monitored by ultraviolet light (λ ═ 254/280 nm). Recording using a Bruker 300MHz NMR spectrometer, a Bruker Avance III plus 400MHz NMR spectrometer, or a Varian III plus 300MHz spectrometer1H-NMR and19F-NMR spectrum. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS; internal standard). The following abbreviations are used for multiplicity: s is singlet; br is wide singlet; d is bimodal; t is a triplet; q is quartet; m is multiplet; and br m broad multiplet. Low resolution Mass Spectra (MS) were obtained by electrospray-atmospheric pressure ionization (ES-API) mass spectrometry, recorded using reversed phase conditions on Shimadzu LCMS 2020 instrument or Agilent LC/MSD 1200 instrument. All animals performedThe experiments were in compliance with institutional guidelines and were approved by the local ethics committee.
Example 1
Preparation of (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamic acid tert-butyl ester
Procedure a: preparation of tert-butyl 2-oxoethylcarbamate
To a stirred solution of 3-amino-1, 2-propanediol (20.0g, 0.22mol) in water (200mL) was added di-tert-butyl dicarbonate (55.5mL, 0.24mol) at 0-5 ℃. After the alkalinity of the solution was adjusted to pH about 9 by addition of the aqueous solution, the mixture was stirred at room temperature (rt) for 18 hours by addition of NaOH (6N). The reaction mixture was cooled to 0-5 ℃ and then acidified to pH about 6, followed by the addition of sodium metaperiodate (56.3g, 0.26 mol). The resulting suspension was stirred at room temperature for 2 hours. The mixture was filtered to remove all solids, and the filtrate was transferred to a separatory funnel and extracted with ethyl acetate (200 mL). Sodium chloride was added to the aqueous layer until a saturated solution was obtained. The aqueous layer was then further extracted with ethyl acetate (100 mL). The combined organics were passed over Na2SO4Drying, followed by concentration in vacuo, gave crude tert-butyl 2-oxoethylcarbamate (45.7g) as a yellow gum. The crude material was used in the next step without purification.
Procedure B: preparation of (E) -4- (tert-Butoxycarbonylamino) -2-fluorobut-2-enoic acid ethyl ester and (Z) -4- (tert-Butoxycarbonylamino) Amino) -2-fluorobut-2-enoic acid ethyl ester
At 0 ℃ under N2To a stirred suspension of crude tert-butyl 2-oxoethylcarbamate (43.7g, 0.22mol) and magnesium sulfate (32.0g) in acetonitrile (200mL) was added ethyl 2-fluorophosphonoacetate (55.7mL, 0.27mol) followed by 1, 8-diazabicyclo [ 5.4.0%]Undec-7-ene (32.8mL, 0.22 mol). The reaction mixture was allowed to warm to room temperature and stirring was continued for 3 hours. After removing the solvent under reduced pressure, the residue was taken up in ethyl acetate (200mL) and then transferred to a separatory funnel. The organic phase was washed successively with aqueous HCl (2M; 100mL x 2), aqueous NaOH (2M; 100mL x 2) and brine (100 mL). Over MgSO4After drying, the organics were concentrated in vacuo to give the crude desired product as a mixture of E/Z isomers (2: 3; 57.0 g). This crude material was carried on to the next step without purification.
Procedure C: preparation of (E) -3-fluoro-4-hydroxybut-2-enylcarbamic acid tert-butyl ester and (Z) -3-fluoro-4-hydroxybut- 2-Alkenylcarbamic acid tert-butyl ester
At 0 ℃ under N2Next, to a stirred solution of crude E/Z-4- (tert-butoxycarbonylamino) -2-fluorobut-2-enoate (18.0g, 72.8mmol) in THF (150mL) was added dropwise diisobutylaluminum hydride (1M in toluene, 182mL, 182mmol) over 45 minutes. After the addition was complete, the mixture was kept under stirring at 0 ℃ for 3 hours. The reaction mixture was transferred to a separatory funnel and added dropwise to a stirred mixture of ice (100g) and aqueous NaOH (2M; 200 mL). After the addition, the mixture was stirred for 2 hours. The quenched reaction mixture was extracted with diethyl ether (100 mL. times.2) and the combined organics were washed with brine (100 mL). Over MgSO4After drying, the organics were concentrated in vacuo to give the crude alcohol as a mixture of E/Z isomers. This mixture was purified on silica gel (135g) eluting with 25% ethyl acetate in n-hexane to give tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate (6.20g, 30% in three steps) and tert-butyl (E) -3-fluoro-4-hydroxybut-2-enylcarbamate (1.85g, 8.9% in three steps). (E) -3-fluoro-4-hydroxybut-2-enylcarbamic acid tert-butyl ester:1H-NMR(200MHz；CDCl3) Δ ppm 1.43(9H, s),3.72(2H, dd, J7.5, 5.4Hz),4.25(2H, d, J21.5 Hz),4.85(1H, br.s),5.18(1H, dt, J19.2, 8.5 Hz). (Z) -3-fluoro-4-hydroxybut-2-enylcarbamic acid tert-butyl ester:1H-NMR(300MHz；CDCl3)δppm:1.46(9H,s),3.84(2H,dd,J 6.2,6.2Hz),4.13(2H,d,J 13.9Hz),4.68(1H,br.s),5.03(1H,dt,J 36.0,7.1Hz)。
the method D comprises the following steps: preparation of (Z) -4-bromo-3-fluorobut-2-enylcarbamic acid tert-butyl ester
To a stirred solution of tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate (6.20g, 30.2mmol) and triethylamine (6.32mL, 45.3mmol) in acetone (100mL) at 0 deg.C was added dropwise methanesulfonyl chloride (2.81mL, 36.3 mmol). After the addition was complete, the mixture was stirred at 0 ℃ for 30 minutes. Thereafter, lithium bromide (13.1g, 0.15mol) was added in portions, and the resulting suspension was stirred for a further 2 hours. The reaction mixture was filtered to remove all solids, and the filtrate was concentrated under reduced pressure. The residue was taken up in water (50mL) and CH2Cl2(50mL) and with additional CH2Cl2The aqueous layer was extracted (50 mL. times.2). The combined organics were passed over Na2SO4Dried and concentrated in vacuo. The crude residue was purified on silica gel (100g), eluting with n-hexane, followed by 25% ethyl acetate in n-hexane to give tert-butyl (Z) -4-bromo-3-fluorobut-2-enylcarbamate (7.00g, 86%) as a colorless solid.1H-NMR(300MHz；CDCl3)δppm:1.46(9H,s),3.85(2H,dd,J 6.2,6.2Hz),3.93(2H,d,J 19.5Hz),4.66(1H,br.s),5.16(1H,dt,J 34.0,6.5Hz)。
Example 2
The following compounds were prepared according to procedures E, F, G, H and I.
Preparation of (Z) -4- ((2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) -N, N-diisopropylbenzenesulfonamide hydrochloride (compound 11)
Procedure E: preparation of 4- (bromomethyl) -N, N-diisopropylbenzenesulfonamide
To a stirred solution of 4- (bromomethyl) benzenesulfonyl chloride (500mg, 1.86mmol) in CH at 0 deg.C2Cl2To the solution in (10mL) was added dropwise diisopropylamine (0.65mL, 4.63 mmol). After the addition, the resulting mixture was stirred at this temperature for 30 minutes, then allowed to warm to room temperature and stirred for an additional 48 hours. The reaction mixture was washed with HCl (1M, 20mL) and CH2Cl2(20 mL). The organic layer was washed with aqueous HCl (1M; 20mL) and water (20mL) over Na2SO4Dried and concentrated in vacuo to give the title compound (yellow oil, 190mg) as a mixture with 4- (chloromethyl) -N, N-diisopropylbenzenesulfonamide, which was used as such in the next step.
Procedure F: preparation of tert-butyl (Z) - (3-fluoro-4- ((2-hydroxyphenyl) thio) but-2-en-1-yl) carbamate
To a solution of 2-mercaptophenol (235mg, 1.86mmol) and tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (500mg, 1.86mmol) in acetone (3mL) was added potassium carbonate (387mg, 2.70mmol) at room temperature, and the resulting solution was stirred at room temperature for 16 h. The reaction mixture was then partitioned between EtOAc (20mL) and water (20mL) and the phases were separated. The aqueous phase was extracted with EtOAc (20 mL. times.2) and the organic phases were then combined and washed (brine; 20mL) and dried (Na)2SO4) And concentrated in vacuo to give tert-butyl (Z) - (3-fluoro-4- ((2-hydroxyphenyl) thio) but-2-en-1-yl) carbamate (580mg, 99%) as a pale yellow solid.1H-NMR(300MHz；CDCl3)δppm:1.45(9H,s),3.31(2H,d,J＝19.7Hz),3.69(2H,app.t,J＝6.7Hz),4.47(1H,dt,J＝34.6,7.2Hz),4.49(1H,br.s),6.67(1H,s),6.90(1H,ddd,J＝7.6,7.6 1.3Hz),7.02(1H,dd,J＝8.2,1.2Hz),7.31(1H,ddd,J＝8.2,7.3,1.6Hz),7.45(1H,dd,J＝7.7,1.7Hz)。
Procedure G: preparation of (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) thio) - 3-fluorobut-2-en-1-yl)Carbamic acid tert-butyl ester
To a stirred solution (107mg,0.32mmol) of tert-butyl (Z) - (3-fluoro-4- ((2-hydroxyphenyl) thio) but-2-en-1-yl) carbamate (100mg, 0.32mmol) and 4- (bromomethyl) -N, N-diisopropylbenzenesulfonamide in DMF (1mL) at room temperature was added potassium carbonate (66mg, 0.48 mmol). The resulting suspension was stirred at this temperature for 16 hours. The reaction mixture was then partitioned between EtOAc (10mL) and water (10mL) and the phases were separated. The aqueous phase was extracted with EtOAc (10 mL. times.2) and the organic phases were then combined and washed (saturated NH)4Aqueous Cl solution, then brine), dried (Na)2SO4) And concentrated in vacuo to give tert-butyl (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) thio) -3-fluorobut-2-en-1-yl) carbamate (180mg, 99%) as a yellow gum which was used in the next step without purification.
Procedure H: preparation of (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) sulfonyl) Yl) -3-Fluorobut-2-en-1-yl) carbamic acid tert-butyl ester
To a stirred solution of (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) thio) -3-fluorobut-2-en-1-yl) carbamate (180mg,0.32mmol) and sodium bicarbonate (133mg, 1.59mmol) in CH2Cl2To a solution of 3-chloroperoxybenzoic acid (178mg, 0.79mmol) in water (2mL) was added in three portions over 5 minutes. The resulting suspension was stirred at 0 ℃ for 2 hours and then saturated NaHCO was used3Diluted with aqueous solution (15ml) and CH2Cl2(10mL) extraction. The aqueous phase is further treated with CH2Cl2(10 mL. times.2) and the organic phases were combined and dried (Na)2SO4) And concentrated in vacuo. The crude material was purified by flash column, eluting with 40% EtOAc/hexanes followed by 2% MeOH in 50% EtOAc/hexanes to give tert-butyl (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) sulfonyl) -3-fluorobut-2-en-1-yl) carbamate (160mg, 84%) as a white solid.1H-NMR(300MHz；CDCl3) δ ppm:1.29(12H, d, J ═ 6.8Hz),1.43(9H, s),3.67-3.80(3H, m),4.15(2H, d, J ═ 18.9Hz),4.52(1H, br.s),4.93(1H, dt, J ═ 34.4,6.9Hz),5.33(2H, s),7.09(1H, d, J ═ 8.0Hz),7.18(1H, ddd, J ═ 8.3,7.8,0.8Hz),7.63(1H, ddd, J ═ 8.4,7.6,1.7Hz),7.66(2H, d, J ═ 8.4),7.93(2H, d, J ═ 8.5Hz),7.99(1H, ddh, 7.9 Hz). As an improvement of this process for the preparation of further compounds, at room temperature, by reactingAqueous solution (per mmol)At 1.2mL H2O4 equivalents) was slowly added to the mercaptoether starting material solution with MeOH: THF (1:1, about 3mL per mmol of mercaptoether) and allowed to react until LC-MS control indicated high conversion to the desired sulfone product. The mixture was then partitioned between an excess of saturated aqueous sodium metabisulfite and EtOAc, washed with brine and dried (Na)2SO4) Concentrated in vacuo and purified by column chromatography.
Procedure I: preparation of (Z) -4- ((2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) -N, n-diisopropylbenzenesulfonamide hydrochloride (Compound 11)
To a stirred solution of tert-butyl (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) benzyl) oxy) phenyl) sulfonyl) -3-fluorobut-2-en-1-yl) carbamate (160mg, 0.27mmol) in MeOH (1mL) at room temperature was added ethereal HCl (2M; 4.00mL, 8.00mmol), and the resulting mixture was stirred for 1 h. Thereafter, a white solid precipitated, which was collected by filtration and dried under high vacuum to give (Z) - (4- ((2- ((4- (amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) -methyl) -N, N-diisopropylbenzene-sulfonamide hydrochloride (79mg, 55%). white solid m.p.222-224 ℃;1H-NMR(300MHz；CD3OD)δppm:1.27(12H,d,J＝6.8Hz),3.59(2H,dd,J＝7.4,1.8Hz),3.79(2H,hept,J＝6.8Hz),4.45(2H,d,J＝19.2Hz),5.16(1H,dt,J＝32.8,7.4Hz),5.46(2H,s),7.23(1H,ddd,J＝7.4,7.4,0.9Hz),7.38(1H,d J＝7.9Hz),7.74(1H,ddd,J＝8.5,7.5,1.7Hz),7.79(2H,d,J＝8.6Hz),7.91-7.95(3H,m)。
example 3
The following compounds were prepared according to procedures E-I using appropriately functionalized thiol starting materials.
(Z) -4- ((2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 5)
A white solid; m.p.235-236 ℃;1H-NMR(300MHz；CD3OD)δppm:2.72(6H,s),3.60(2H,dd,J＝7.4,1.7Hz),4.47(2H,d,J＝19.2Hz),5.18(1H,dt,J＝32.9,7.4Hz),5.49(2H,s),7.24(1H,ddd,J＝7.9,7.9,0.9Hz),7.39(1H,dd,J＝8.5,0.7Hz),7.76(1H,ddd,J＝8.4,7.4,1.7Hz),7.86(4H,br.s),7.95(1H,dd,J＝7.9,1.8Hz)。
(Z) -4- ((2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) benzenesulfonamide hydrochloride (Compound 8)
An off-white solid; m.p.233-235 ℃;1H-NMR(300MHz；CD3OD)δppm:3.59(2H,dd,J＝7.4,1.6Hz),4.44(2H,d,J＝19.2Hz),5.14(1H,dt,J＝32.8,7.4Hz),5.45(2H,s),7.23(1H,dd,J＝7.3,7.3Hz),7.38(1H,d,J＝8.3Hz),7.74(1H,ddd,J＝8.6,8.6,1.7Hz),7.78(2H,d,J＝8.2Hz),7.94(1H,dd,J＝8.1,1.6Hz),7.97(2H,d,J＝8.5Hz)。
(Z) -4- ((3- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 9)
A white solid; m.p.211-213 ℃;1H-NMR(300MHz；d6-DMSO)δppm:2.63(6H,s),3.48(2H,br.s),4.65(2H,d,J＝19.6Hz),5.17(1H,dt,J＝34.6,7.2Hz),5.36(2H,s),7.45(1H,ddd,J＝8.1,2.5,1.0Hz),7.52-7.57(2H,m),7.63(1H,dd,J＝8.1,8.1Hz),7.75(2H,d,J＝8.5Hz),7.81(2H,d,J＝8.6Hz),8.11(3H,br.s)。
(Z) -4- ((4- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) methyl) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 10)
A white solid; m.p.216-218 ℃;1H-NMR(300MHz；d6-DMSO)δppm:2.63(6H,s),3.48(2H,br.s),4.55(2H,d,J＝19.7Hz),5.12(1H,dt,J＝34.8,7.1Hz),5.38(2H,s),7.29(2H,dd,J＝9.0,1.9Hz),7.74(2H,dd,J＝8.5,1.8Hz),7.81(2H,dd,J＝8.5,1.9Hz),7.88(2H,dd,J＝8.9,2.0Hz),8.03(3H,br.s)。
(Z) - (4- ((2- ((4- (amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) -methyl) -N-diisopropylbenzenesulfonamide hydrochloride (compound 14)
A white solid; m.p.248-250 ℃;1H-NMR(300MHz；CD3OD)δppm:1.05(6H,d,J＝6.6Hz),3.40(1H,hept,J＝6.6Hz),3.59(2H,app.d,J＝7.3Hz),4.45(2H,d,J＝19.1Hz),5.16(1H,dt,J＝33.0,7.4Hz),5.46(2H,s),7.23(1H,ddd,J＝8.0,8.0,1.0Hz),7.38(1H,d,J＝8.1Hz),7.74(1H,ddd,J＝8.4,7.4,1.8Hz),7.80(2H,d,J＝8.7Hz),7.91-7.95(3H,m)。
example 4
The following compounds were prepared according to procedure F-I using the appropriate thiol starting material.
(Z) -4- ((2- (benzyloxy) phenyl) sulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 7)
A white solid; m.p.205-207 ℃;1H-NMR(300MHz；CD3OD)δppm:3.57(2H,app.d,J＝7.0Hz),4.44(2H,d,J＝19.1Hz),5.14(1H,dt,J＝32.8,7.3Hz),5.36(2H,s),7.20(1H,dd,J＝7.4,0.9Hz),7.35-7.46(4H,m),7.55-7.60(2H,m),7.73(1H,ddd,J＝8.5,7.4,1.7Hz),7.92(1H,dd,J＝7.9,1.7Hz)。
example 5
The following compounds were prepared according to procedures F, H and I using appropriately functionalized thiol starting materials.
(Z) -4- ((3, 5-bis (trifluoromethyl) phenyl) sulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 2)
A white solid; m.p.217-220 ℃;1H-NMR(300MHz；d6-DMSO)δppm:3.48(2H.app.d,J＝7.1Hz),4.96(2H,d,J＝19.6Hz),5.19(1H,dt,J＝34.8,7.2Hz),8.10(3H,br.s),8.55(2H,s),8.67(1H,s)。
(Z) -4- (Biphenyl-2-ylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 20)
A white solid; m.p.170 ℃;1H NMR(300MHz,CD3OD)δppm:8.18(dd,J＝8.0,1.4Hz,1H),7.81(td,J＝7.5,1.4Hz,1H),7.68(td,J＝7.6,1.6Hz,1H),7.52–7.44(m,6H),5.03(dt,J＝32.9,7.4Hz,1H),3.83(d,J＝18.9Hz,2H),3.56(dd,J＝7.4,1.3Hz,2H)。
(Z) -3-fluoro-4- (2-isopropylbenzenesulfonyl) but-2-en-1-amine hydrochloride (Compound 21)
A white solid; m.p.205-215 ℃;1H NMR(300MHz,CD3OD)δppm:8.00–7.95(m,1H),7.77–7.67(m,2H),7.48–7.41(m,1H),5.25(dt,J＝32.8,7.4Hz,1H),4.34(d,J＝19.2Hz,2H),3.88(hept,J＝6.9Hz,1H),3.63(dd,J＝7.5,1.9Hz,2H),1.36(d,J＝6.8Hz,6H)。
(Z) -3-fluoro-4- (2-methoxyphenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 22)
A white solid; m.p.228-221 ℃;1H NMR(300MHz,CD3OD)δppm:7.89(dd,J＝7.9,1.7Hz,1H),7.75(ddd,J＝8.4,7.4,1.8Hz,1H),7.32(dd,J＝8.5,0.9Hz,1H),7.19(td,J＝7.6,1.0Hz,1H),5.23(dt,J＝32.8,7.4Hz,1H),5.17(t,J＝7.4Hz,0H),4.48(d,J＝19.3Hz,2H),4.05(s,3H),3.61(dd,J＝7.4,1.9Hz,2H)。
(Z) -3-fluoro-4- (Naphthalen-1-ylsulfonyl) but-2-en-1-amine hydrochloride (Compound 23)
A white solid; m.p.230-240 ℃;1H NMR(300MHz,CD3OD)δppm:8.76(ddd,J＝8.7,1.2,0.6Hz,1H),8.33(d,J＝7.8Hz,2H),8.13(ddd,J＝8.2,1.5,0.8Hz,1H),7.81(ddd,J＝8.6,6.9,1.5Hz,1H),7.72(td,J＝8.0,1.9Hz,2H),5.13(dt,J＝32.7,7.4Hz,1H),4.49(d,J＝19.2Hz,2H),3.57(dd,J＝7.4,1.9Hz,2H)。
(Z) -3-fluoro-4- (Naphthalen-2-ylsulfonyl) but-2-en-1-amine hydrochloride (Compound 24)
A white solid; m.p.215-220 ℃;1H NMR(300MHz,CD3OD)δppm:8.60(d,J＝1.9Hz,1H),8.18–8.11(m,2H),8.06(dd,J＝8.2,1.4Hz,1H),7.95(dd,J＝8.7,1.9Hz,1H),7.81–7.68(m,2H),5.19(dt,J＝32.8,7.4Hz,1H),4.46(d,J＝19.2Hz,2H),3.63(dt,J＝7.4,1.3Hz,2H)。
(Z) -4- (2, 4-dichlorophenylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 25)
A white solid; m.p.220 ℃;1H NMR(300MHz,CD3OD)δppm:8.10(d,J＝8.6Hz,1H),7.85(d,J＝2.0Hz,1H),7.66(dd,J＝8.6,2.0Hz,1H),5.28(dt,J＝32.9,7.4Hz,1H),4.60(d,J＝19.1Hz,2H),3.63(ddd,J＝7.4,2.0,0.6Hz,2H)。
(Z) -4- (3-Chlorophenylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 26)
A white solid; m.p.225-235 ℃;1H NMR(300MHz,CD3OD)δppm:8.00(t,J＝1.9Hz,1H),7.92(ddd,J＝7.8,1.8,1.1Hz,1H),7.82(ddd,J＝8.1,2.1,1.1Hz,1H),7.68(d,J＝8.3Hz,1H),5.22(dt,J＝32.9,7.4Hz,1H),4.44(d,J＝19.1Hz,2H),3.65(dd,J＝7.4,1.9Hz,2H)
(Z) -4- (4-Chlorophenylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 27)
A white solid; m.p.240 ℃;1H NMR(300MHz,CD3OD)δppm:7.96(dt,J＝8.8,2.3Hz,2H),7.71(dt,J＝8.3,1.9Hz,2H),5.20(dt,J＝32.9,7.4Hz,1H),4.40(d,J＝19.1Hz,2H),3.65(dd,J＝7.4,1.9Hz,2H)。
(Z) -4- (3, 5-dichlorophenylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 28)
A white solid; m.p.250 ℃;1H NMR(300MHz,CD3OD)δppm:7.97(d,J＝1.8Hz,2H),7.93(t,J＝1.9Hz,1H),5.27(dt,J＝33.0,7.4Hz,1H),4.50(d,J＝19.0Hz,2H),3.67(dd,J＝7.4,2.0Hz,2H)。
(Z) -3-fluoro-4- (pyridin-4-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 29)
A white solid; m.p.162-164 ℃;1H NMR(300MHz,CD3OD)δppm:9.07(dd,J＝4.9,1.7Hz,2H),8.22(dd,J＝4.6,1.6Hz,2H),5.31(dt,J＝33.1,7.4Hz,1H),4.63(d,J＝19.0Hz,2H),3.67(d,J＝7.4Hz,2H)
(Z) -3-fluoro-4- (quinolin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 32)
A white solid; m.p.203-205 ℃;1H NMR(300MHz,d6-DMSO)δppm:8.82(d,J＝8.6Hz,1H),8.29–8.20(m,2H),8.16(d,J＝8.5Hz,1H),8.12–7.93(m,3H),7.92–7.83(m,1H),5.26(dt,J＝34.8,7.2Hz,1H),4.92(d,J＝19.6Hz,2H),3.48(s,2H)。
(Z) -3-fluoro-4- (quinoline-8-sulfonyl) but-2-en-1-amine dihydrochloride (Compound 33)
A white solid; m.p.150-153 ℃;1H NMR(300MHz,CD3OD) δ ppm 9.18(d, J ═ 4.7Hz,1H),8.70(dd, J ═ 8.4,2.6Hz,1H),8.57(d, J ═ 7.4Hz,1H),8.45(d, J ═ 8.5Hz,1H), 7.99-7.68 (m,2H),5.22(dt, J ═ 32.9,7.4Hz,1H),5.00(d, J ═ 19.4Hz,2H),3.60(d, J ═ 7.7Hz, 2H); LCMS: for C13H13FN2O2S, calculated 280.1, found 281.1[ M +1]+。
(Z) -3-fluoro-4- (2-fluorophenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 37)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:8.01–7.90(m,1H),7.89–7.77(m,1H),7.53–7.39(m,2H),5.29(dt,J＝32.8,7.4Hz,1H),4.49(d,J＝19.1Hz,2H),3.63(dd,J＝7.4,1.9Hz,2H)。
(Z) -3-fluoro-4- (3-fluorophenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 38)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:7.83(ddd,J＝7.8,1.7,1.1Hz,1H),7.81–7.66(m,2H),7.56(tdd,J＝8.4,2.6,1.1Hz,1H),5.23(dt,J＝32.9,7.4Hz,1H),4.44(d,J＝19.1Hz,2H),3.65(dd,J＝7.4,1.9Hz,2H)。
(Z) -3-fluoro-4- (4-fluorophenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 39)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:8.13–7.99(m,2H),7.51–7.34(m,2H),5.19(dt,J＝32.8,7.5Hz,1H),4.39(d,J＝19.1Hz,2H),3.62(ddt,J＝7.4,1.9,0.6Hz,2H)。
(Z) -3-fluoro-4- (o-tolylsulfonyl) but-2-en-1-amine hydrochloride (Compound 40)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:7.99(dd,J＝7.6,1.1Hz,1H),7.71–7.59(m,1H),7.53–7.39(m,2H),5.22(dt,J＝32.8,7.4Hz,1H),4.35(dd,J＝19.3,0.5Hz,2H),3.63(ddt,J＝7.4,2.0,0.6Hz,2H),2.73(s,3H)。
(Z) -3-fluoro-4- (m-toluenesulfonyl) but-2-en-1-amine hydrochloride (Compound 41)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:7.84–7.72(m,2H),7.67–7.49(m,2H),5.44–5.08(m,1H),4.35(dq,J＝19.1,0.5Hz,2H),3.64(ddt,J＝7.4,2.0,0.6Hz,2H),2.56–2.38(m,3H)。
(Z) -3-fluoro-4- (p-toluenesulfonyl) but-2-en-1-amine hydrochloride (Compound 42)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:7.88–7.82(m,2H),7.54–7.45(m,2H),5.17(dt,J＝32.8,7.4Hz,1H),4.32(dd,J＝19.2,0.5Hz,2H),3.64(ddt,J＝7.4,2.0,0.6Hz,2H),2.49(s,3H)
(Z) -3-fluoro-4- ((3-fluoroquinolin-8-yl) sulfonyl) but-2-en-1-amine dihydrochloride (Compound 51)
1H NMR(300MHz,CD3OD)δppm:9.09(dd,J＝2.9,0.6Hz,1H),8.49(ddd,J＝7.4,1.4,0.4Hz,1H),8.38(ddd,J＝8.3,1.4,0.4Hz,1H),8.31(dd,J＝8.8,2.9Hz,1H),7.87(ddd,J＝8.3,7.3,0.8Hz,1H),5.20(dt,J＝32.9,7.4Hz,1H),4.99(d,J＝19.3Hz,2H),3.59(d,J＝7.4Hz,2H)。
Example 6
The following compounds were prepared according to procedures F, J, H and I using appropriately functionalized thiol starting materials.
Procedure J: preparation of (Z) - (3-fluoro-4- ((4- (methylsulfonyl) phenyl) sulfonyl) but-2-en-1-yl) carbamic acid methyl ester Tert-butyl ester
To a stirred mixture of tert-butyl (Z) - (3-fluoro-4- (4- (methylthio) phenyl) thio) but-2-en-1-yl) carbamate (120mg, 0.35mmol) and sodium bicarbonate (150mg, 1.79mmol) in CH at 0 deg.C2Cl2To a solution of (4mL) and water (2mL) was added 3-chloroperoxybenzoic acid (378mg, 2.19mmol) in three portions over 5 minutes. The resulting suspension was stirred at 0 ℃ for 1.5 hours, then treated with aqueous NaOH (2M; 1mL), water (10mL) and CH2Cl2And (6) diluting. The phases are then separated, the aqueous phase is treated with CH2Cl2(10 mL. times.2). The organic phases are combined and dried (Na)2SO4) And concentrated in vacuo. The crude material was purified by flash column eluting with 30% EtOAc/hexanes to give tert-butyl (Z) - (3-fluoro-4- ((4- (methylsulfonyl) phenyl) sulfonyl) but-2-en-1-yl) carbamate (17mg, 12%) as a white solid。1H-NMR(300MHz；d6-DMSO)δppm:1.37(9H,s),3.33(3H,s),3.54(2H,app.t,J＝5.6Hz),4.62(2H,d,J＝19.4Hz),4.93(1H,dt,J＝36.4,6.8Hz),7.05(1H,t,J＝5.8Hz),8.15(2H,dd,J＝8.7,2.1Hz),8.21(2H,dd,J＝8.7,2.1Hz)。
(Z) -4- ((3, 5-bis (trifluoromethyl) phenyl) sulfonyl) -3-fluorobut-2-en-1-amine trifluoroacetate (Compound 3)
A white solid; m.p.155-157 ℃;1H-NMR(300MHz；d6-DMSO)δppm:3.35(3H,s),3.50(2H,app.d,J＝6.7Hz),4.80(2H,d,J＝19.7Hz),5.12(1H,dt,J＝34.7,7.3Hz),7.88(3H,br.s),8.19(2H,dd,J＝8.8,2.5Hz),8.24(2H,dd,J＝8.9,2.4Hz)。
example 7
The following compounds were then prepared according to procedures K, L and M, followed by F, N, H and I.
Preparation of (Z) -4- (2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 6)
Procedure K: preparation of 4-bromo-N, N-dimethylbenzenesulfonamide
To a stirred solution of dimethylamine (12mL, 40% w/w aq.) in THF (20mL) at 5 deg.C was added a solution of 4-bromobenzenesulfonyl chloride (5.00g, 19.6mmol) in THF (10mL) over 5 minutes. After the addition, the mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated in vacuo, and the resulting residue was washed with water (25mL) and CH2Cl2(20mL) and use additional CH2Cl2(20mLX 2) extract the aqueous layer. The combined organics were passed over Na2SO4Dried and concentrated in vacuo to give 4-bromo-N, -dimethylbenzenesulfonamide (4.83g, 93%) as a white solid.1H-NMR(300MHz；CDCl3)δppm:2.74(6H,s),7.64-7.73(4H,m)。
Procedure L: preparation of N, N-dimethyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzenesulfonamide
Stirred 4-bromo-N, N-dimethylbenzenesulfonamide (1.00g, 3.79mmol), 4,5, 5-tetramethyl-2- (4,4,5, 5-tetramethyl-1, 3, 2)-A solution of dioxaborolan-2-yl) -1,3, 2-oxaborane (1.15g, 4.54mmol) and potassium acetate (1.11g, 11.4mmol) in 1, 4-dioxane (25mL) was purged with nitrogen for 15 minutes, then 1,1' -bis (diphenylphosphino) ferrocene-palladium (II) dichloride dichloromethane complex (155mg, 0.19mmol) was added. The resulting suspension was heated at 80 ℃ under nitrogen for 16 hours. The mixture was cooled to room temperature, partitioned between EtOAc (40mL) and water (30mL), and filtered through celite. The organic layer was separated and the aqueous layer was extracted with additional EtOAc (20 mL. times.2). The combined organics were then washed with brine and dried (Na)2SO4) And concentrated in vacuo to give N, N-dimethyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -benzenesulfonamide (1.60g, 68%) as a grey solid.1H-NMR(300MHz；CDCl3)δppm:1.38(12H,s),2.71(6H,s),7.77(2H,dd,J＝8.4,1.0Hz),7.98(2H,dd,J＝8.4,0.9Hz)。
Program M: preparation of (4- (N, N-dimethylsulfonylamino) phenyl) boronic acid
To a stirred solution of N, N-dimethyl-4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzenesulfonamide (1.00g, 2.25mmol) in THF (20mL) and water (5mL) at 0 deg.CmL), sodium periodate (2.06g, 9.64mmol) was added. The mixture was stirred at this temperature for 5 minutes, then allowed to warm to room temperature and stirred for a further 30 minutes. Aqueous HCl (1M; 1.57mL, 1.57mmol) was added and the resulting mixture was stirred at room temperature for an additional 1 hour. The reaction mixture was diluted with water (30mL) and extracted with EtOAc (20 mL. times.3). The organic layers were then combined and washed (brine) and dried (Na)2SO4) And concentrated in vacuo. The crude material was purified by flash column, eluting with 50% EtOAc/hexanes followed by 10% MeOH in 50% EtOAc/hexanes to give (4- (N, N-dimethylsulfonamido) phenyl) boronic acid (470mg, 91%) as a brown solid.1H-NMR(300MHz；CD3OD)δppm:2.69(6H,s),7.75(2H,d,J＝8.2Hz),7.88-7.98(2H,m)。
Procedure N: preparation of (Z) - (4- ((2- (4- (N, N-dimethylsulfonylamino) phenoxy) phenyl) thio) -3-fluorobutane- 2-En-1-yl) carbamic acid tert-butyl ester
To a stirred solution of tert-butyl (Z) - (3-fluoro-4- ((2-hydroxyphenyl) thio) but-2-en-1-yl) carbamate (150mg, 0.48mmol), (4- (N, N-dimethylsulfonylamino) phenyl) boronic acid (219mg, 0.96mmol) and pyridine (0.19mL, 2.39mmol) in CH at room temperature2Cl2To the solution in (6mL), copper (II) acetate (87mg, 0.48mmol) was added in one portion. The resulting mixture was stirred at this temperature for 16 hours. Thereafter, by adding CH2Cl2The reaction was diluted (30mL), filtered through celite, and washed with aqueous HCl (1M; 20mL) and then saturated NaHCO3Aqueous solution (20mL) and brine (20 mL). The organic phase is then dried (Na)2SO4) And concentrated in vacuo. The crude material was purified by flash column eluting with 25% EtOAc/hexanes to give tert-butyl (Z) - (4- ((2- (4- (N, N-dimethylsulfonylamino) phenoxy) phenyl) thio) -3-fluorobut-2-en-1-yl) carbamate (80mg, 34%).1H-NMR(300MHz；CDCl3)δppm:1.45(9H,s),2.73(6H,s),3.55(2H,d,J＝17.1Hz),3.73(2H,app.t,J＝5.6Hz),4.46(1H,br.s),4.80(1H,dt,J＝34.8,6.8Hz),7.02(2H,d,J＝8.7Hz),7.06(1H,dd,J＝8.2,1.0Hz),7.24(1H,ddd,J＝7.5,7.5,1.1Hz),7.35(1H,ddd,J＝7.6,7.6,1.6Hz),7.52(1H,dd,J＝7.7,1.5Hz),7.75(2H,d,J＝8.6Hz)。
(Z) -4- (2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) phenoxy) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 6)
A white solid; m.p.153-156 ℃;1H-NMR(300MHz；CD3OD)δppm:2.73(6H,s),3.64(2H,app.d,J＝6.9Hz),4.57(2H,d,J＝19.1Hz),5.30(1H,dt,J＝32.8,7.3Hz),7.25(1H,dd,J＝8.3,0.7Hz),7.30(2H,dd,J＝8.9,2.0Hz),7.49(1H,ddd,J＝7.9,7.9,1.0Hz),7.81(1H,ddd,J＝7.8,8.3,1.7Hz),7.86(2H,dd,J＝8.6,2.0Hz),8.07(1H,dd,J＝7.9,1.6Hz)。
example 8
The following compounds were prepared according to procedures K, L, M, N, H and I.
(Z) -4- (3- (4-amino-2-fluorobut-2-enylsulfonyl) phenoxy) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 16)
A white solid; 1H NMR (300MHz, CD)3OD)δppm:7.89–7.82(m,3H),7.76(td,J＝8.0,0.5Hz,1H),7.68(t,J＝2.4,1.7Hz,1H),7.53(ddd,J＝8.1,2.5,1.1Hz,1H),7.29–7.22(m,2H),5.28(dt,J＝33.2,7.4Hz,1H),4.90(s,6H),4.43(d,J＝19.1Hz,2H),3.65(dd,J＝7.4,1.9Hz,2H)。
(Z) -3- (3- (4-amino-2-fluorobut-2-enylsulfonyl) phenoxy) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 17)
A white solid; m.p.220 ℃;1H NMR(300MHz,CD3OD)δppm:7.81(ddd,J＝7.8,1.7,1.2Hz,1H),7.77–7.69(m,2H),7.66–7.59(m,2H),7.50(ddd,J＝8.0,2.5,1.2Hz,1H),7.46–7.38(m,2H),5.24(dt,J＝32.9,7.4Hz,1H),4.90(s,6H),4.41(d,J＝19.1Hz,2H),3.65(dd,J＝7.4,1.9Hz,2H)。
(Z) -3- (2- (4-amino-2-fluorobut-2-enylsulfonyl) phenoxy) -N, N-dimethylbenzenesulfonamide hydrochloride (Compound 18)
A white solid; m.p.205 ℃;1H NMR(300MHz,CD3OD)δppm:8.06(ddd,J＝7.9,1.7,0.3Hz,1H),7.83–7.73(m,1H),7.70(dd,J＝7.7,0.7Hz,1H),7.65(dt,J＝7.8,1.4Hz,1H),7.51–7.42(m,3H),7.20(dd,J＝8.3,1.0Hz,1H),5.31(dt,J＝33.0,7.4Hz,1H),4.90(s,6H),4.60(d,J＝19.2Hz,2H),3.64(dd,J＝7.4,1.9Hz,2H)。
example 9
The following compounds were then prepared according to procedures O and P, and then according to F, Q, H and I.
Preparation of (Z) -2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonylamino) -N- (4- (N, N-diisopropylsulfonylamino) -phenyl) benzamide hydrochloride (Compound 15)
Procedure O: preparation of N, N-diisopropyl-4-nitrobenzenesulfonamide
To a stirred solution of diisopropylamine (3.16mL, 22.6mmol) in THF (10mL) at 0-5 deg.C was added a solution of 4-nitrobenzenesulfonyl chloride (2.00g, 9.02mmol) in THF (5mL) over 5 minutes. After the addition, the mixture was stirred at room temperatureFor 16 hours. The reaction mixture was then concentrated in vacuo, and the resulting residue was washed with water (25mL) and CH2Cl2(20 mL). Separating the phases and using the other CH2Cl2The aqueous layer was extracted (20 mL. times.2). The combined organics were then passed over Na2SO4Dried and concentrated in vacuo. The crude material was purified by flash column eluting with 20% EtOAc in hexanes to give N, N-diisopropyl-4-nitrobenzenesulfonamide (285mg, 11% yield) as a yellow solid.1H-NMR(300MHz；CDCl3)δppm:1.30(12H,d,J＝6.7Hz),3.79(1H,hept,J＝6.9Hz),8.07(2H,dd,J＝9.2,2.2Hz),8.35(2H,dd,J＝8.9,1.9Hz)。
Procedure P: preparation of 4-amino-N, N-diisopropylbenzenesulfonamide
To a stirred solution of N, N-diisopropyl-4-nitrobenzenesulfonamide (260mg, 0.91mmol) in methanol (10mL) was added a slurry of palladium on carbon (10% w/w; 50mg) in water (50. mu.L) at room temperature under a nitrogen blanket. The resulting mixture was stirred under a hydrogen atmosphere for 3 hours. The mixture was filtered through celite and concentrated in vacuo to give 4-amino-N, N-diisopropylbenzenesulfonamide (200mg, 86%) as a brown solid.1H-NMR(300MHz；CDCl3)δppm:1.27(12H,d,J＝6.8Hz),3.66(2H,hept,J＝6.8Hz),4.04(2H,br.s),6.67(2H,dd,J＝8.7,2.1Hz),7.65(2H,dd,J＝8.6,1.9Hz)。
Procedure Q: preparation of (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) phenyl) -carbamoyl) phenyl) Thio) -3-fluorobut-2-en-1-ylcarbamic acid tert-butyl ester
To stirred (Z) -2- ((4- ((tert-butoxycarbonyl) amino) -2-fluorobut-2-en-1-yl) thio) benzoic acid (180mg, 0.53mmol) at room temperature,To a solution of 4-amino-N, N-diisopropylbenzenesulfonamide (203mg, 0.79mmol) and triethylamine (0.26mL, 1.85mmol) (0.8mL) in DMF was added HATU (301mg, 0.79mmol), and the resulting solution was stirred at this temperature for 16 hours. The reaction mixture was then partitioned between water (10mL) and EtOAc (10mL) and the phases were separated. The aqueous phase was extracted again with EtOAc and the organic phases were combined and washed with HCl (1M; 20mL) and then with water (20 mL. times.3) and brine (20 mL). The organic phase was dried (Na)2SO4) And concentrated in vacuo. The crude material was purified by flash column eluting with 50-100% EtOAc/hexanes to give tert-butyl (Z) - (4- ((2- ((4- (N, N-diisopropylsulfonamido) phenyl) carbamoyl) phenyl) thio) -3-fluorobut-2-en-1-yl) carbamate (93mg, 30%) as pale lotus gray oil.1H-NMR(300MHz；CDCl3)δppm:1.31(12H,d,J＝6.7Hz),1.44(9H,s),3.57(2H,d,J＝18.5Hz),3.63(2H,app.t,J＝5.6Hz),3.74(2H,hept,J＝6.7Hz),4.40(1H,dt,J＝34.8,6.9Hz),4.75(1H,br.s),7.41(1H,dd,J＝7.1,7.1Hz),7.47(1H,ddd,J＝7.8,7.8,1.8Hz),7.62(1H,dd,J＝7.8,1.2Hz),7.75(1H,dd,J＝7.3Hz),7.88(4H,br.s)。
(Z) -2- ((4-amino-2-fluorobut-2-en-1-yl) sulfonyl) -N- (4- (N, N-diisopropylsulfonamido) phenyl) benzamide hydrochloride (Compound 15)
A white solid; m.p.248-250 ℃;1H-NMR(300MHz；CD3OD)δppm:1.29(12H,d,J＝6.9Hz),3.64(2H,dd,J＝7.4,1.5Hz),3.78(2H,hept,J＝6.7Hz),4.69(2H,d,J＝19.2Hz),5.23(1H,dt,J＝32.4,7.4Hz),7.76-7.82(2H,m),7.85-7.93(5H,m),8.10-8.13(1H,m)。
example 10
The following compounds were prepared according to procedures Q, H and I using the appropriate thiol and amine starting materials.
N- (adamantan-1-yl) -4- (((Z) -4-amino-2-fluorobut-2-en-1-yl) sulfonyl) benzamide hydrochloride (Compound 4)
An off-white solid; m.p.231-233 ℃;1H-NMR(300MHz；d6-DMSO)δppm:1.67(6H,br.s),2.08(9H,br.s),3.46(2H,br.s),4.67(2H,d,J＝19.7Hz),5.10(1H,dt,J＝34.6,7.1Hz),7.97(2H,dd,J＝8.6,1.5Hz),8.00(2H,dd,J＝8.8,1.4Hz),8.06(3H,br.s)。
n- (adamantan-1-yl) -2- (((Z) -4-amino-2-fluorobut-2-en-1-yl) sulfonyl) benzamide hydrochloride (Compound 12)
A white solid;1H-NMR(300MHz；CD3OD)δppm:1.77-1.80(6H,m),2.09-2.15(3H,m),2.16-2.21(6H,m),3.61(2H,app.d,J＝7.2Hz),4.62(2H,d,J＝19.5Hz),5.16(1H,dt,J＝32.6,7.4Hz),7.56(1H,dd,J＝7.5,1.0Hz),7.67(1H,ddd,J＝7.7,7.7,1.2Hz),7.78(1H,ddd,J＝7.5,7.5,1.1Hz),8.01(1H,dd,J＝7.8,1.0Hz)。
n- (adamantan-1-yl) -3- (((Z) -4-amino-2-fluorobut-2-en-1-yl) sulfonyl) benzamide hydrochloride (Compound 13)
A white solid; m.p.230-232 ℃;1H-NMR(300MHz；d6-DMSO)δppm:1.68(6H,br.s),2.05-2.12(9H,m),3.49(2H,br.s),4.68(2H,d,J 19.8Hz),5.12(1H,dt,J 34.7,7.0Hz),7.73(1H,dd,J 7.7,7.7Hz),7.98(3H,br.s),8.03(1H,ddd,J 7.8,1.7,0.9Hz),8.16(1H,ddd,J 7.8,1.4,1.0Hz),8.28(1H,dd,J 1.6,1.1Hz)。
example 11
The following compounds were prepared according to procedures R and I.
(Z) -3-fluoro-4- (phenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 1)
Procedure R: preparation of (Z) - (3-fluoro-4- (phenylsulfonyl) but-2-en-1-yl) carbamic acid tert-butyl ester
To a stirred solution of tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (60.0g, 224mmol) in DMF (300mL) was added sodium benzenesulfinate (44.1g, 269mmol) portionwise at room temperature over 15 minutes. The resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (2.7L) and stirring was continued at room temperature for a further 15 minutes. The resulting precipitated solid was filtered, washed with water (50 mL. times.2) and then dried in an oven at 60 ℃ to give tert-butyl (Z) - (3-fluoro-4- (phenylsulfonyl) but-2-en-1-yl) carbamate (74.0g, 100%) as a white solid, which was used directly in the next step.1H NMR(300MHz,CDCl3)δppm:7.98–7.91(m,2H),7.76–7.67(m,1H),7.61(ddt,J＝8.3,6.6,1.3Hz,2H),4.94(dt,J＝34.3,7.0Hz,1H),4.59(s,1H),3.94(d,J＝18.4Hz,2H),3.79(t,J＝6.5Hz,2H),1.46(s,9H)。
(Z) -3-fluoro-4- (phenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 1)
An off-white solid; m.p.209-211 ℃;1H-NMR(300MHz；CD3OD) δ ppm 3.64(2H, dd, J ═ 7.3,1.2Hz),4.36(2H, d, J ═ 19.1Hz),5.18(1H, dt, J ═ 32.7,7.4Hz),7.65-7.71(2H, m),7.79(1H, tt, J ═ 7.4,1.2Hz),7.96-8.00(2H, m); LCMS: for C10H12FNO2S, calculated 229.1, found 230.1[ M +1 ]]+。
Example 12
The following compounds were prepared according to procedures R and I.
(Z) -4- (2-Chlorophenylsulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 19)
A white solid; m.p.205-207 ℃;1H NMR(300MHz,CD3OD) δ ppm 8.13(ddd, J ═ 7.9,1.5,0.6Hz,1H), 7.81-7.69 (m,2H),7.62(ddd, J ═ 7.9,6.4,2.2Hz,1H),5.27(dt, J ═ 32.8,7.4Hz,1H),4.59(d, J ═ 19.1Hz,2H),3.62(dd, J ═ 7.4,1.9Hz, 2H); LCMS: for C10H11ClFNO2S, calculated 263.0, found 264.0[ M +1 ]]+。
(Z) -3-fluoro-4- (pyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 30)
A white solid; m.p.153-155 ℃;1H NMR(300MHz,CD3OD)δppm:8.88–8.73(m,1H),8.25–8.10(m,2H),7.77(ddd,J＝6.8,4.7,1.9Hz,1H),5.26(dt,J＝32.9,7.4Hz,1H),4.59(d,J＝19.1Hz,2H),3.63(dd,J＝7.3,1.8Hz,2H)。
(Z) -3-fluoro-4- (pyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 31)
A white solid; m.p.168-170 deg.c;1H NMR(300MHz,CD3OD)δppm:9.23(dd,J＝2.3,0.8Hz,1H),9.04(dd,J＝5.2,1.5Hz,1H),8.64(dt,J＝8.2,1.9Hz,1H),7.94(ddd,J＝8.2,5.2,0.8Hz,1H),5.30(dt,J＝33.1,7.4Hz,1H),4.59(d,J＝19.1Hz,2H),3.68(d,J＝7.4Hz,2H)。
example 13
The following compounds were prepared according to procedures S, F, H and I.
(Z) -3-fluoro-4- (3-methylpyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 43)
And a procedure S: preparation of 3-methylpyridine-2-thiol
2-chloro-3-methylpyridine (500mg, 3.93mmol) and sodium hydrosulfide hydrate (2.21g, 39.36mmol) were dissolved in DMF (2.0 mL). The resulting mixture was heated at 120 ℃ for 12 hours. After completion of the reaction (TLC), the reaction mixture was cooled to room temperature, diluted with water (20mL), acidified (pH ═ 5) by addition of acetic acid, and extracted with EtOAc (20mL × 3). The combined organic extracts are purified over Na2SO4Dried and concentrated under reduced pressure to give 3-methylpyridine-2-thiol (1.50g, 50.5%).1H NMR(600MHz,d6-DMSO)δppm:13.5-13.3(m,1H),7.6-7.58(m,1H),7.51(d,J＝6Hz,1H)。2.21(s,3H)
(Z) -3-fluoro-4- (3-methylpyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 43)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:8.53(dd,J＝4.7,1.3Hz,1H),7.92(d,J＝7.6Hz,1H),7.60(dd,J＝7.8,4.5Hz,1H),5.37(dt,J＝32.9,7.2Hz,1H),4.75(d,J＝19.0Hz,2H),3.67(d,J＝7.1Hz,2H),2.68(s,3H)。
example 14
The following compounds were prepared according to procedures S, F, H and I
(Z) -3-fluoro-4- (2-methylpyridin-4-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 44)
A light yellow solid;1H NMR(300MHz,CD3OD)δ9.00(d,J＝5.6Hz,1H),8.35(s,1H),8.23(d,J＝5.4Hz,1H),5.36(dt,J＝33.0,7.1Hz,1H),4.68(d,J＝18.9Hz,2H),3.69(d,J＝6.7Hz,2H),2.90(s,3H)。
(Z) -3-fluoro-4- (5-isopropylpyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 34)
A white glassy solid;1H NMR(300MHz,CD3OD)δ8.70(t,J＝1.3Hz,1H),8.12–7.94(m,2H),5.28(dt,J＝32.9,7.4Hz,1H),4.55(d,J＝19.1Hz,2H),3.64(dd,J＝7.6,1.7Hz,2H),3.15(hept,J＝6.9Hz,1H),1.36(d,J＝6.9Hz,6H)。
(Z) -3-fluoro-4- (5-methylpyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 35)
A white solid; m.p.155-157 ℃;1H NMR(300MHz,d6-DMSO)δ8.67(d,J＝0.9Hz,1H),8.09(s,3H),7.98(t,J＝1.4Hz,2H),5.20(dt,J＝34.8,7.2Hz,1H),4.70(d,J＝19.6Hz,2H),3.46(t,J＝5.9Hz,2H),2.45(s,3H)。
(Z) -3-fluoro-4- (6-methylpyridin-2-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 36)
A light yellow solid; m.p.174-176 ℃;1H NMR(300MHz,d6-DMSO)δ8.18(s,3H),8.06(t,J＝7.8Hz,1H),7.88(d,J＝7.7Hz,1H),7.66(d,J＝7.5Hz,1H),5.23(dt,J＝34.8,7.1Hz,1H),4.70(d,J＝19.6Hz,2H),3.46(t,J＝5.9Hz,2H),2.61(s,3H)。
example 15
The following compounds were prepared according to procedures T, U and V, then according to F, H and I
(Z) -3-fluoro-4- (6-isopropylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 49)
And a program T: preparation of methyl 3- ((6-chloropyridin-3-yl) thio) propionate
2-chloro-5-iodopyridine (2.50g, 10.5mmol) was dissolved in degassed 1, 4-dioxane (25mL) at room temperature in a 50mL resealable reaction tube under a nitrogen atmosphere. Sequentially adding Pd under nitrogen atmosphere2(dba)3(100mg, 0.11mmol), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (Xantphos) (125mg, 0.22mmol), methyl 3-mercaptopropionate (1.25g, 10.5mmol) and DIPEA (2.50mL, 14.4 mmol). The solution was degassed by purging nitrogen for 15 minutes and then gradually heated to 70 ℃. The resulting reaction mixture was stirred at this temperature for 6 hours. After completion of the reaction (TLC), the reaction mixture was cooled to room temperature, diluted with cold water, and extracted with ethyl acetate (3 × 30 mL). The combined organics were washed with brine solution and concentrated under reduced pressure. The obtained residue was purified by column chromatography on silica gel (100-200 mesh) eluting with 10% EtOAc-hexane to give methyl 3- ((6-chloropyridin-3-yl) thio) propanoate (2.40g, 99%) as an off-white solid.1H NMR(400MHz,CDCl3)δppm:8.36(d,J＝2.4Hz,1H),7.67(dd,J＝8.4,2.4Hz,1H),7.28(d,J＝9.2Hz,1H),3.69(s,3H),3.17(t,J＝7.2Hz,2H),2.64(t,J＝7.2Hz,2H)。
And a program U: preparation of methyl 3- ((6-isopropylpyridin-3-yl) thio) propionate
Methyl 3- ((6-Chloropyridin-3-yl) thio) propanoate (5.00g, 21.6mmol) was dissolved in anhydrous THF (200mL) and 1-methyl-2-pyrrolidone (25mL) in a 1000mL round bottom flask under a nitrogen atmosphere. The solution was cooled to-55 ℃ and a solution of iron (III) acetylacetonate (1.70g, 4.80mmol) in THF (50mL) was added while maintaining a nitrogen atmosphere. The resulting mixture was stirred at-55 ℃ for 15 minutes under a nitrogen atmosphere, at which time a solution of isopropyl magnesium chloride in THF (2M, 50mL) was added dropwise at-55 ℃. The resulting reaction mixture was stirred at-40 ℃ for a further 1 hour. After completion of the reaction (TLC), the reaction mixture was cooled to 0 ℃ with saturated NH4The Cl solution (50mL) was quenched and the product was extracted with ethyl acetate (100 mL. times.3). The combined organics were passed over Na2SO4Dried and concentrated under reduced pressure. The obtained residue was purified by column chromatography on silica gel (100-200 mesh) eluting with 10% EtOAc-hexane to give methyl 3- ((6-isopropylpyridin-3-yl) thio) propanoate (2.60g, 50%) as a pale yellow liquid.
Procedure V: preparation of 6-isopropylpyridine-3-thiol
Methyl 3- ((6-isopropylpyridin-3-yl) thio) propanoate (860mg, 3.59mmol) was dissolved in anhydrous THF (20mL) in a 100mL round bottom flask under a nitrogen atmosphere and the solution was cooled to-78 ℃. A solution of potassium tert-butoxide in THF (1.0M, 3.5mL, 3.59mmol) was added to the mixture under a nitrogen atmosphere. The resulting mixture was stirred at-78 ℃ for 1 hour. After completion of the reaction (TLC), the reaction mixture was warmed to room temperature and concentrated under reduced pressure. The obtained residue was washed with n-hexane to give 6-isopropylpyridine-3-thiol (655mg) as a pale brown solid. The compound was used in the next step without further purification.
(Z) -3-fluoro-4- (6-isopropylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 49)
1H NMR(300MHz,CD3OD)δppm:9.29(dd,J＝2.1,0.7Hz,1H),9.02(ddd,J＝8.6,3.5,2.1Hz,1H),8.32(dd,J＝8.6,3.2Hz,1H),5.48(t,J＝7.3Hz,1H),4.74(d,J＝19.0Hz,2H),3.81–3.65(m,2H),3.55(dq,J＝7.0,2.2Hz,1H),1.53(dd,J＝7.0,0.8Hz,6H)。
Example 16
The following compounds were then prepared according to procedures T, U and V, followed by F, H and I.
(Z) -3-fluoro-4- (2-isopropylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 45)
1H NMR(300MHz,CD3OD)δppm:8.95(dd,J＝5.2,1.7Hz,1H),8.66(dd,J＝8.2,1.8Hz,1H),7.78(dd,J＝8.0,5.0Hz,1H),5.39(dt,J＝33.2,7.3Hz,1H),4.58(d,J＝19.0Hz,2H),4.08(p,J＝6.8Hz,1H),3.72–3.62(m,2H),1.45(d,J＝6.7Hz,6H)。
Example 17
The following compounds were then prepared according to procedures T and V, and then F, H and I.
(Z) -3-fluoro-4- (6-methylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 46)
1H NMR(300MHz,CD3OD)δppm:9.17(d,J＝2.2Hz,1H),8.67(d,J＝8.5Hz,1H),7.96(d,J＝8.4Hz,1H),5.33(dt,J＝33.0,7.3Hz,1H),4.62(d,J＝19.1Hz,2H),3.75–3.63(m,2H),2.85(s,3H)。
(Z) -3-fluoro-4- (2-methylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 47)
1H NMR(300MHz,CD3OD)δppm:9.00(dd,J＝5.6,1.5Hz,1H),8.91(dd,J＝8.1,1.5Hz,1H),8.05(dd,J＝8.1,5.6Hz,1H),5.42(dt,J＝33.2,7.3Hz,1H),4.67(d,J＝19.0Hz,2H),3.79–3.64(m,2H),3.12(s,3H)。
(Z) -3-fluoro-4- (4-methylpyridin-3-ylsulfonyl) but-2-en-1-amine dihydrochloride (Compound 48)
An off-white solid;1H NMR(300MHz,CD3OD)δppm:9.25(s,1H),8.97(d,J＝5.8Hz,1H),8.07(d,J＝5.9Hz,1H),5.42(dt,J＝33.2,7.3Hz,1H),4.68(d,J＝19.1Hz,2H),3.70(dd,J＝7.4,1.9Hz,2H),2.99(d,J＝0.6Hz,3H)。
(Z) -3-fluoro-4- ((2-methylbenzo [ d ] thiazol-4-yl) sulfonyl) but-2-en-1-amine hydrochloride (Compound 50)
A light yellow solid; m.p.243-245 ℃;1H NMR(300MHz,CD3OD)δppm:8.37(dd,J＝8.1,1.2Hz,1H),8.10(dd,J＝7.7,1.2Hz,1H),7.63(t,J＝7.9Hz,1H),5.20(dt,J＝32.8,7.4Hz,1H),4.82(d,J＝19.0Hz,2H),3.60(d,J＝7.4Hz,2H),2.97(s,3H)。
example 18
The following compounds were then prepared according to procedure W, X, Y, Z, followed by H and I
(Z) -4- ((2, 3-dimethyl-1H-indol-7-yl) sulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 52)
Procedure W: preparation of (2-iodophenyl) hydrazine
In a 1L round bottom flask, a solution of 2-iodoaniline (40.0g, 0.182mmol) in concentrated HCl (250mL) was treated with NaNO at 0 deg.C2(15.1g, 0.22mmol) in water (40 mL). The reaction mixture was stirred at 0 ℃ for 2 hours and slowly SnCl at 0 ℃2(86.6g, 0.46 mmol). The reaction temperature was gradually raised to room temperature and stirred for another 6 hours. After completion of the reaction (TLC), the reaction mixture was filtered and washed with n-pentane (50mL) and diethyl ether (50mL) to give the title compound (42g, 98.26%).1H NMR(300MHz,d6-DMSO)δppm:10.2(br.s,2H),7.79-7.76(m,1H),7.7-7.45(br,1H),7.36(t,J＝8.1Hz,1H),7.02(d,J＝7.8Hz,1H),6.75(t,J＝7.8Hz,1H)。
Procedure X: preparation of 7-iodo-2, 3-dimethyl-1H-indole
In a 250mL round bottom flask, a solution of (2-iodophenyl) hydrazine (10g, 42.73mmol) in acetic acid (80mL) was gradually heated to 60 ℃ and stirred for 1 hour. 2-butanone (6.18g, 85.36mmol) was added slowly at 60 ℃. The resulting reaction mixture was then heated at 80 ℃ for 5 hours. After completion of the reaction (TLC), the reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The obtained residue was diluted with cold water and extracted with ethyl acetate (100mL × 2). The combined organic extracts were washed with brine and concentrated under reduced pressure. The resulting residue was purified by silica gel eluting with 10% EtOAc-hexanes to give the title compound as an off-white solid (2.00g, 18%).1H NMR(400MHz,d6-DMSO)δppm:10.54(s,1H),7.37-7.24(m,2H),6.73(t,J＝8Hz,1H),2.32(s,3H),2.12(s,3H)。
Procedure Y: preparation of methyl 3- ((2, 3-dimethyl-1H-indol-7-yl) thio) propionate
A solution of 7-iodo-2, 3-dimethyl-1H-indole (1.60g, 10.5mmol) in 1, 4-dioxane (10mL) was degassed under nitrogen in a 50mL resealable reaction tube. Sequentially adding Pd under nitrogen atmosphere2(dba)3(50.0mg, 0.06mmol), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (100mg, 0.18mmol), methyl 3-mercaptopropionate (0.70g, 5.90mmol) and DIPEA (2.00mL, 11.80 mmol). The solution was degassed by purging argon for 15 minutes, then gradually heated to 110 ℃ and stirred at this temperature for 12 hours. After completion of the reaction (TLC), the reaction mixture was cooled to rt, diluted with cold water and extracted with EtOAc (2 × 20 mL). The combined organic extracts were washed with brine, over anhydrous Na2SO4Dried and concentrated under reduced pressure. The resulting residue was purified on silica gel eluting with 10% EtOAc-hexanes to give the title compound as an off-white solid (1.50g, 96%).1H NMR(400MHz,CDCl3)δppm:8.6(br,1H),7.44(d,J＝8Hz,1H),7.22(d,J＝7.6Hz,1H),7.03(t,J＝7.6Hz,1H),3.7(s,3H),3.08(t,J＝6.8Hz,2H),2.57(t,J＝7.2Hz,2H),2.41(s,3H),2.22(s,3H)。
Procedure Z: preparation of (Z) - (4- ((2, 3-dimethyl-1H-indol-7-yl) thio) -3-fluorobut-2-en-1-yl) amino Benzoic acid tert-butyl ester
Cesium carbonate (1.49g, 4.56mmol) was added to a stirred solution of methyl 3- ((2, 3-dimethyl-1H-indol-7-yl) thio) propionate (0.40g, 1.52mmol) and tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (0.41g, 1.53mmol) in DMF (15mL) at room temperature in a 100mL round bottom flask. The reaction mixture was stirred at room temperature for 5 hours. After completion of the reaction (TLC), the reaction mixture was quenched by addition of ice-cold water (10mL) and quenched with waterExtraction with ethyl acetate (10 mL. times.3). The combined organic extracts were extracted with anhydrous Na2SO4Dried and concentrated under reduced pressure. The crude material was purified on silica gel eluting with 20% ethyl acetate hexanes to give the title compound as a pale yellow liquid (350mg, 63%).1H NMR(300MHz,d6-DMSO-)δppm:10.65(s,1H),7.31(d,J＝7.5Hz,1H),7.07(d,J＝7.8Hz,1H),6.93-6.83(m,2H),4.66(dt,J＝36.3,7.2Hz,1H),3.67(d,J＝19.5Hz,2H),3.47(t,J＝6Hz,2H),2.32(s,3H),2.13(s,3H),1.34(s,9H)。
(Z) -4- ((2, 3-dimethyl-1H-indol-7-yl) sulfonyl) -3-fluorobut-2-en-1-amine hydrochloride (Compound 52)
1H NMR(300MHz,CD3OD)δppm:10.25(s,1H),7.80(dd,J＝7.8,1.0Hz,1H),7.55(dd,J＝7.7,1.1Hz,1H),7.20(t,J＝7.7Hz,1H),5.05(dt,J＝32.7,7.4Hz,1H),4.34(d,J＝19.2Hz,2H),3.55(dd,J＝7.4,1.9Hz,2H),2.49–2.39(m,3H),2.26(d,J＝0.8Hz,3H)。
Example 19
Preparation of (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamic acid tert-butyl ester
Procedure AA: preparation of tert-butyl 2-oxoethylcarbamate
A vessel containing 3-amino-1, 2-propanediol (50.0kg, 549mol) and ethyl acetate (150L) was cooled to 0-5 ℃. To this solution di-tert-butyl dicarbonate (120kg, 550mol) is added in portions. The resulting mixture was stirred at 20-25 ℃ for 12 hours. After cooling the reaction mixture to 0-5 ℃, sodium periodate (120kg, 561mol) was added in portions. Will be provided withThe suspension was stirred at this temperature for 1 hour. The reaction mixture was then filtered, and the filter "cake" was washed with ethyl acetate (180L). The combined filtrates were washed with aqueous NaCl (10% w/w, 300L) and Na2SO4Drying, followed by concentration in vacuo, gave crude tert-butyl 2-oxoethylcarbamate (70.0kg, 80%). The crude material was used in the next step without purification.
Procedure AB: preparation of (E) -4- (tert-butoxycarbonylamino) -2-fluorobut-2-enoic acid ethyl ester
A vessel containing ethyl 2-fluorophosphonoacetate (46.0kg, 190mol), acetonitrile (250L) and 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) (38.0kg, 250mol) was cooled to 0-10 ℃. Tert-butyl 2-oxoethylcarbamate (68.0kg, 427mol) was added dropwise thereto, and the resulting mixture was stirred at 0 to 10 ℃ for 4 hours. The reaction mixture was diluted with tert-butyl methyl ether (500L) and water (500L) and stirring was continued for 30 min. After standing for another 30 minutes, the aqueous layer was removed. The organic layer was washed with water (250L) and then concentrated in vacuo. The residue was dissolved in ethyl acetate (56L) and petroleum ether (240L) and purified by silica gel (40kg, mesh size: 100-200) eluting with ethyl acetate (1:10) in petroleum ether to give crude (E) -ethyl 4- (tert-butoxycarbonylamino) -2-fluorobut-2-enoate (90 kg). The crude material was used in the subsequent steps without further purification.
Procedure AC: preparation of (Z) -3-fluoro-4-hydroxybut-2-enylcarbamic acid tert-butyl ester
Sodium borohydride (16.6kg, 439mol) was added portionwise over 4 hours at 0-10 ℃ in a reaction vessel charged with crude (E) -4- (tert-butoxycarbonylamino) -2-fluorobut-2-enoic acid ethyl ester (90.0kg, 364mol), THF (314L) and methanol (36L). After the addition was complete, the resulting mixture was stirred at 0-10 ℃ for 3 hours. The reaction was quenched by the addition of aqueous HCl (0.5N, 900L). The product was then extracted with ethyl acetate (720 L.times.2). The combined organics were washed with water (450L) and concentrated in vacuo. The residual water was removed by co-evaporation with THF (200L X3) to give crude tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate (85 kg). This material was taken to the next step without further purification.
Procedure AD: preparation of (Z) -4- ((tert-butoxycarbonyl) amino) -2-fluorobut-2-en-1-yl 3, 5-dinitrobenzyl Acid esters
Charging crude oil at 0-20 deg.C(Z) -3-fluoro-4-hydroxybutane-2-3, 5-dinitrobenzoyl chloride (190kg, 824mol) was added in portions to a reaction vessel of tert-butyl alkenylcarbamate (170kg, 828mol), THF (765L) and triethylamine (174L, 1245 mol). The resulting mixture was stirred at 0-20 ℃ for 2 hours, then diluted with water (850L) and ethyl acetate (1700L). Removing water layer, and adding Na to organic matter2CO3The aqueous solution (10% w/w, 850 L.times.2) was washed, then with water (850L). After concentration in vacuo (to about 190L), ethyl acetate (245L) was added. The mixture was stirred at 55 ℃ until a clear solution was obtained. The mixture was cooled to 10-20 ℃, n-heptane (730L) was added, and the mixture was stirred for 6 hours. The solid thus formed was isolated by filtration and washed with n-heptane/ethyl acetate (4:1, 170L). HPLC analysis indicated a content of 30% E-isomer. The E-isomer content was reduced to 4.3% by the following precipitation procedure. The solid was dissolved in ethyl acetate (570L). N-heptane was added thereto, and the resulting slurry was stirred at 20-30 ℃ for 4-6 hours. The solid was isolated by filtration and the filter "cake" was washed with n-heptane/ethyl acetate (4: 1). This procedure was repeated again to give (Z) -4- ((tert-butoxycarbonyl) amino) -2-fluorobut-2-en-1-yl 3, 5-dinitrobenzoate (84kg as a wet "cake"). The data has proceeded to the next step.
Procedure AE: system for makingPreparation of tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate
A reaction vessel charged with (Z) -4- ((tert-butoxycarbonyl) amino) -2-fluorobut-2-en-1-yl 3, 5-dinitrobenzoate (84kg, 210mol), THF (187L) and aqueous NaOH (1.00M, 420L) was stirred at 15-25 ℃ for 2-5 hours. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with isopropyl acetate (966L) and stirring was continued for 10-30 minutes. After standing for 10-30 minutes, the layers were separated and the aqueous layer was extracted with isopropyl acetate (483L). With Na2CO3The combined organics were washed with aqueous (10% w/w, 420L X2), aqueous NaCl (10% w/w, 420L) and then concentrated to about 84L. Residual water was removed by co-evaporation with THF (468L) to give tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate (29.0kg, 67%). HPLC analysis indicated a 3.8% E-isomer content.
Procedure AF: preparation of (Z) -4-bromo-3-fluorobut-2-enylcarbamic acid tert-butyl ester
The reaction vessel charged with tert-butyl (Z) -3-fluoro-4-hydroxybut-2-enylcarbamate (29.0kg, 141mol), THF (290L) and Diisopropylethylamine (DIPEA) (74.0L, 425mol) was cooled to 0-10 ℃. To this was added dropwise a solution of methanesulfonic anhydride (50.0kg, 287mol) in THF (145L). After the addition was complete, lithium bromide (74.0kg, 852mol) was added in portions while maintaining the temperature between 0-10 ℃. The resulting mixture was stirred at 0-10 ℃ for 4 hours. TLC thereafter indicated complete consumption of starting material. The reaction mixture was diluted with water (290L) and the product was extracted with ethyl acetate (290L + 145L). The combined organics were washed with water (150L) and then concentrated to dryness. The crude residue was taken up in ethyl acetate (67L) and n-heptane (440L) and passed through silica gel (40 kg; mesh size 100-Purification was carried out with ethyl acetate/n-heptane (1: 5). All fractions containing the desired product were concentrated to dryness. Ethyl acetate (56L) was added and stirring was continued at 40-50 ℃ until a clear solution was obtained. N-heptane (280L) was added dropwise thereto. The mixture was cooled to 10-15 ℃ and stirred for 8 hours. The resulting solid was collected by filtration. HPLC analysis indicated an E-isomer content of 0.9%. To further reduce the E-isomer content, the precipitation process was repeated as follows. Ethyl acetate (33L) was added and stirring was continued at 40-50 ℃ until a clear solution was obtained. N-heptane (164L) was added dropwise thereto. The mixture was cooled to 10-15 ℃ and stirred for 8 hours. The resulting solid was collected by filtration and dried to give tert-butyl (Z) -4-bromo-3-fluorobut-2-enylcarbamate (29.5kg, 68%). HPLC analysis indicated an E-isomer content of 0.1%.1H-NMR(300MHz；CDCl3)δppm:1.46(9H,s),3.85(2H,dd,J 6.2,6.2Hz),3.93(2H,d,J 19.5Hz),4.66(1H,br.s),5.16(1H,dt,J＝34.0,6.5Hz)。
Example 20
The following compounds were prepared according to procedures AG and AH.
Preparation of (Z) -3-fluoro-4- (phenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 1)
Procedure AG: preparation of (Z) - (3-fluoro-4- (phenylsulfonyl) but-2-en-1-yl) carbamic acid tert-butyl ester
The vessel containing tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (2.40kg, 8.95mol) and DMF (12.0L) was cooled to 15-20 ℃. Sodium benzenesulfinate (2.20kg, 13.4mol) was added thereto, and the resulting mixture was stirred at 15-20 ℃ for 4 h. The reaction mixture was diluted with water (12.0L) and stirring was continued at room temperature for an additional 1 hour. The solid thus formed was separated by filtration and the filter "cake" was further washed with water (6.0L). The solid was then dried under vacuum at 50-55 ℃ for 20 hours to give (Z) - (3-fluoro-4- (phenylsulfonyl) but-2-en-1-yl) carbamate (2.70kg, 92%). Retention Time (RT) ═ 13.75 minutes; method-Agilent LC/MSD 1200 series; a chromatographic column: ZORBAX SB-C18, ODS 2000 (50X 4.6mm, 5 μm), operating in ES (+) or (-) ionization mode; flow rate 1.5 mL/min; the temperature (T) is 30 ℃; detection wavelength: 214 nm; mobile phase: from 5% acetonitrile (containing 0.05% trifluoroacetic acid (TFA)) and 95% water (containing 0.05% TFA) to 90% acetonitrile and 10% water in 24 minutes.
Procedure AH: preparation of (Z) -3-fluoro-4- (phenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 1)
To a vessel containing HCl (4.00M in ethyl acetate; 13.5L, 54.0mol) and cooled to 10-20 deg.C was added a filtered solution (hyflo) of tert-butyl (Z) - (3-fluoro-4- (phenylsulfonyl) but-2-en-1-yl) carbamate (2.70kg, 8.20mol) in ethyl acetate (27.0L). The reaction mixture was stirred at 10-20 ℃ for 6 hours. The resulting solid was isolated by filtration and the filter "cake" was washed with ethyl acetate (8.0L). The solid was then dried under vacuum at 50-55 deg.C for 40 hours to give (Z) -3-fluoro-4- (phenylsulfonyl) but-2-en-1-amine hydrochloride (Compound 1) (2.10 kg; 96%).1H-NMR(300MHz；CD3OD) δ ppm 3.64(2H, dd, J ═ 7.3,1.2Hz),4.36(2H, d, J ═ 19.1Hz),5.18(1H, dt, J ═ 32.7,7.4Hz),7.65-7.71(2H, m),7.79(1H, tt, J ═ 7.4,1.2Hz),7.96-8.00(2H, m); LCMS for C10H12FNO2S, calculated 229.1, found 230.1[ M +1 ]]+。
Example 21
The following compounds were prepared according to the procedures AI, AJ and AK.
Preparation of (Z) -3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-amine dihydrochloride monohydrate (Compound 33)
Procedure AI: preparation of quinoline 8-sulfinic acid sodium salt
At room temperature, will contain Na2SO3A vessel of (6.70kg, 53.2mol) and water (21.0L) was stirred for 20 minutes. Adding Na into the container2SO3(5.50kg, 51.9mol) and stirring was continued at room temperature for 20 minutes. Quinoline-8-sulfonyl chloride (6.00kg, 26.4mol) was then added in portions while maintaining the temperature below 40 ℃. The resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and the filter "cake" was washed with methanol (7.0L). The filtrate was concentrated to dryness in vacuo and methanol (7.0L) was added to the resulting residue. After stirring at room temperature for 1 hour, the mixture was filtered and the filtrate was concentrated to dryness. In the second and final washing cycle, the residue was taken up in methanol (10.0L) and stirring was continued at room temperature for 1 hour. The mixture was filtered and the filtrate was concentrated in vacuo to give sodium quinoline-8-sulfinate (4.10kg, 72%).
Process AJ: preparation of (Z) - (3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-yl) carbamic acid tert-butyl ester
The contents of tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (3.50kg, 13.1mol), sodium quinoline-8-sulfinate (4.20kg, 19.5mol) and DMF (17.5L) were cooled to 15-20 ℃. The resulting mixture was stirred at this temperature for 20 hours. The mixture was then diluted with ethyl acetate (35.0L) and water (35.0L) and stirring was continued for another 10 minutes. The organic layer was then separated and washed with water (20.0 L.times.2). After concentrating the organic layer to about 20L, n-heptane (42.0L) was added dropwise. The resulting suspension was stirred at 20-30 ℃ for 20 hours. The solid was isolated by filtration, washed with n-heptane and then dried under vacuum at 50-55 deg.C for 20 hours to give tert-butyl (Z) - (3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-yl) carbamate (3.80kg, 77%). RT ═ 12.97 minutes; method-Agilent LC/MSD 1200 series; a chromatographic column: ZORBAX SB-C18, ODS 2000 (50X 4.6mm, 5 μm), operating in ES (+) or (-) ionization mode; flow rate 1.5 mL/min; the temperature (T) is 30 ℃; detection wavelength: 214 nm; mobile phase: from 5% acetonitrile (containing 0.05% trifluoroacetic acid (TFA)) and 95% water (containing 0.05% TFA) to 90% acetonitrile and 10% water in 24 minutes.
Procedure AK: preparation of (Z) -3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-amine dihydrochloride monohydrate (Compound 33)
To a vessel containing HCl (1.5M in ethyl acetate; 53L) was added tert-butyl (Z) - (3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-yl) carbamate (5.3kg, 14mol) at 10-20 ℃. The mixture was stirred at 15 ℃ for 4 hours. The resulting solid was isolated by filtration and washed with ethyl acetate (20L). Ethyl acetate (53L) was added to the flask containing the solids. The suspension was then stirred at 10-20 ℃ for 2 hours. The solid was isolated by filtration and washed with ethyl acetate (20L). The solid was dissolved in methanol (53L) and the solution was filtered. Water (500mL) and tert-butyl methyl ether (53L) were then added dropwise thereto and stirring was continued at 15 ℃ for another 20 hours. The solid was collected by filtration and dried under vacuum at 55-60 ℃ to give (Z) -3-fluoro-4- (quinolin-8-ylsulfonyl) but-2-en-1-amine dihydrochloride monohydrate (Compound 33) (4.4kg, 90%).1H NMR(300MHz,CD3OD) δ ppm 9.18(d, J ═ 4.7Hz,1H),8.70(dd, J ═ 8.4,2.6Hz,1H),8.57(d, J ═ 7.4Hz,1H),8.45(d, J ═ 8.5Hz,1H), 7.99-7.68 (m,2H),5.22(dt, J ═ 32.9,7.4Hz,1H),5.00(d, J ═ 19.4Hz,2H),3.60(d, J ═ 7.7Hz, 2H); LCMS: for C13H13FN2O2S, calculated 280.1, found 281.1[ M +1]+。
Example 22
The following compounds were prepared according to procedures AL and AM.
(Z) -3-fluoro-4- ((quinolin-8-yl-d)6) Preparation of sulfonyl) but-2-en-1-amine dihydrochloride (Compound 53)
6Process AL: preparation of (Z) - (3-fluoro-4- ((quinolin-8-yl-d) sulfonyl) but-2-en-1-yl) carbamic acid tert-butyl Butyl ester
To a stirred solution of tert-butyl (Z) - (4-bromo-3-fluorobut-2-en-1-yl) carbamate (606mg, 2.26mmol) in DMF (4.0mL) was added sodium 2,3,4,5,6, 7-hexadeuterated quinoline-8-sulfinate (500mg, 2.26mmol) in one portion. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then diluted with water (40mL) and the product extracted with ethyl acetate (20 mL. times.3). The combined organic layers were saturated with NH4Aqueous Cl (20 mL. times.3) and brine (20mL) and washed with Na2SO4Dried and then concentrated in vacuo. The crude product was purified by normal phase chromatography (reveirs) eluting with 20-50% ethyl acetate in hexane to give (Z) - (3-fluoro-4- ((quinolin-8-yl-d)6) Sulfonyl) but-2-en-1-yl) carbamic acid tert-butyl ester (470mg, 53%) as an off-white solid.
6Program AM: preparation of (Z) -3-fluoro-4- ((quinolin-8-yl-d) sulfonyl) but-2-en-1-amine dihydrochloride (Compound Thing 53)
To the stirred (Z) - (3-fluoro-4- ((quinolin-8-yl) at room temperature-d6) Sulfonyl) but-2-en-1-yl) carbamic acid tert-butyl ester (450mg, 1.22mmol) in methanol (1.0mL) was added HCl (2.0M in ether; 4.0mL, 8.0 mmol). The reaction mixture was stirred at room temperature for 1 hour, during which time a solid precipitated. The reaction mixture was transferred to a vial and centrifuged in a centrifuge (4000rpm, 4 minutes). The supernatant was carefully decanted and the remaining solid "cake" was dried under high vacuum to give (Z) -3-fluoro-4- ((quinolin-8-yl-d)6) Sulfonyl) but-2-en-1-amine dihydrochloride (355mg, 81%) as a white solid.1H NMR(300MHz,CD3OD)δppm:5.20(dt,J＝32.8,7.4Hz,1H),5.08–4.96(m,2H),3.59(d,J＝7.4Hz,2H)。
Example 23
Methods of determining the ability of compounds of the invention to inhibit LOX and LOXL1-4 from different sources
Lysyl Oxidase (LOX) is an extracellular copper-dependent enzyme that oxidizes peptidyl-lysine and hydroxylysine residues in collagen and lysine residues in elastin, producing peptidyl- α -aminoadipate- δ -semialdehyde. This catalytic Reaction can be irreversibly inhibited by beta-aminopropionitrile (BAPN) bound to the active site of LOX (Tang SS, Trackman PC and Kagan HM, "Reaction of aortic lysyl oxidase with beta-aminopropionitrile)". Journal of biochemistry 1983; 258:4331-4338). There are five members of the LOX family; they are LOX, LOXL1, LOXL2, LOXL3 and LOXL 4. LOX and LOXL family members are commercially available as recombinant active proteins, and can be extracted from animal tissues such as bovine aorta, tendon, and pig skin; or prepared from cell culture. Application in detecting the activity of phagocytes NADPH oxidase and other oxidases (A stable non-fluorescent derivative of resorufin for the detection of the activity of the phagocytes in order to detect the presence of the fluorescent protein of the gene of interest in the production of the protein of interest in detecting the activity of the gene of the protein of interest NADPH oxidases and other oxidases, analytical biochemistry (anal. biochem.), (1997); 253,162-168), tested for inhibition of a given LOX-LOXL formulation. The assay is performed using 384 or 96 well format. Briefly, in A standard black clear bottom 384-well plate assay, 25 μ L of A dilution of any isoenzyme and ortholog in 1.2M ureA, 50mM sodium borate buffer (pH 8.2) was added to each well in the presence of 1 μ M of mofetil and 0.5mM of pargyline (inhibiting SSAO and MAO-B and MAO-A, respectively; not necessary if the enzyme was from recombinant or purified form). Test compounds were dissolved in DMSO and, after incubation with enzyme for 30 minutes at 37 ℃, tested in a Concentration Response Curve (CRC) at 11 data points, typically in the micromolar or nanomolar range. Then putrescine-containing K prepared in 1.2M urea, 50mM sodium borate buffer (pH 8.2)mTwenty-five microliters of reaction mixture, 120 μ M Amplex Red (Sigma Aldrich) and 1.5U/ml horseradish peroxidase at twice the concentration of values (Sigma Aldrich), e.g. 20mM for LOX, or 10mM for LOXL2 and LOXL3) were added to the respective wells. In the case of 96-well plates, the volume is doubled. Fluorescence (RFU) was read every 2.5 minutes for 30 minutes under conditions of excitation 565nm and emission 590 (Optima; BMG Labtech) at a temperature range of 37 ℃. The kinetic slope of each well was calculated using the MARS data analysis software (BMG Labtech) and this value was used to derive the IC50Value (Dotmatics). Table 3 shows the ability of the compounds of the invention to inhibit amine oxidase activity LOX and other family members.
TABLE 3
LOX and LOXL2 inhibitory Activity of Compounds examples of the invention
Example 24
The compounds of the invention show sustained inhibition of LOXL1 and LOXL2
Compounds that provide sustained, long-term inhibition of LOX and LOXL1-4 have a greater advantage over competitive inhibitors for a meaningful pharmacological effect in the presence of high substrate concentrations, because the pharmacological effect makes the presence of unbound inhibitor more persistent. In a preferred embodiment, the compounds of the invention exhibit sustained inhibition of LOX and LOXL 1-4.
Methods of determining sustained inhibition of LOX and LOXL1-4 by compounds of the invention
Jump dilution experiment: the assay was performed using a 96-well format, and the starting enzyme concentration was set at 100-fold inhibition studies. Enzyme at 10-fold or (it is desirable to determine inhibitor concentration over enzyme concentration) or 30-fold IC50Incubate at 37 ℃ for 40 minutes in the presence of the concentration of test inhibitor. After incubation, the mixture was diluted 50-fold in assay buffer and then 2-fold more diluted in Amplex Red horseradish peroxidase-tyrosine reaction mixture (same as inhibition studies) before fluorescence measurement. Results are expressed as percentage of signal recovery after the indicated time compared to uninhibited controls. The results are shown in Table 4.
TABLE 4
Measurement of sustained inhibition of LOXL1 and LOXL2 by compounds of the invention
Compound (I)
LOXL 1-Activity was restored. (%)
LOXL 2-Activity was restored. (%)
Reversible standard (control)
80.9
96.8
1
2.4
9.5
4
16.7
21.2
5
3.6
10.0
6
2.5
10.4
7
3.0
8
35.1
9
4.4
15.1
10
15.2
11
10.2
12
2.6
9.3
13
3.5
8.9
14
1.9
15
26.1
16
14.4
17
5.3
18
6.9
19
7.8
7.9
29
8.6
30
1.9
31
8.2
33
4.5
6.9
38
1.6
45
6.6
46
1.0
Example 25
Pharmacokinetics in vivo
Test compounds (10mg/kg and 30mg/kg in PBS solution, oral, n-3) were administered to male Wistar rats of 7-9 weeks of age. Collecting a test compound blood sample at a time point (compound 1) of 0.25 hours to 8.00 hours; 0.5-8.00 hours after administration (compound 33), starting from the tail vein.
Sample preparation: mu.L of calibration sample (single), QC sample (in duplicate) and rat plasma sample were mixed with 60. mu.L of acetonitrile containing internal standards (IS; 200ng/mL tosylbutamide and 50ng/mL propanol). The mixture was vortexed for 1 min and then centrifuged for 10 min, and 50 μ L of the supernatant was transferred to a 96-well plate containing 100 μ L of water. After shaking for 10 minutes, 10. mu.L was injected into a liquid chromatography mass spectrometry (LC-MS/MS) system for analysis.
LC method (Compound 1)
HPLC: agilent 1100; mass spectrometry: API 4000
A chromatographic column: phenomenex Gemini C185 μm 50x4.6mm
Mobile phase: 0.1% formic acid in water, 0.1% formic acid in acetonitrile
LC method (Compound 33)
HPLC: shimadzu LC30 AD; mass spectrometry: API 4000
A chromatographic column: agilent SB C181.8 μm 50x2.1mm
Mobile phase: 0.1% formic acid in water, 0.1% formic acid in acetonitrile
For the diluted samples: 10 μ L of the sample was added to 90 μ L of blank rat plasma. The mixture was precipitated in an Eppendorf tube with 300. mu.L acetonitrile containing IS. The mixture was vortexed for 1 min and then centrifuged for 10 min, and 50 μ L of the supernatant was transferred to a 96-well plate containing 100 μ L of water. After shaking for 10 minutes, 10. mu.L was injected into the LC-MS/MS system for analysis. Tables 5 and 6 show the determined mean plasma concentrations of compounds 1 and 33, respectively.
TABLE 5
Mean plasma concentrations of Compound 1 after oral administration of 10 and 30mg/kg
TABLE 6
Mean plasma concentrations of Compound 33 after oral administration of 10 and 30mg/kg
Example 26
Target engagement
A single dose of a mechanism-based inhibitor may be sufficient to block enzymatic activity in vivo for a prolonged period of time.
Assay for lysyl oxidase Activity-ear assay
Ears collected immediately after sacrifice (treated or untreated with LOX inhibitor) were snap frozen in dry ice for storage at-80 ℃. For analysis, a 5 × 5mm sample was cut (still semi-molten), embedded in agarose gel, and cut into 200 μ M thick cross sections (vibrating microtome). Thin sections were collected in ice cold PBS containing protease inhibitors and allowed to stand in a bath for 2-3 hours to allow soluble contaminants to diffuse out before the assay. For the 96-well plate format: three sheets were collected, gently blotted dry on a Kimwipe, and then transferred to each well containing 100. mu.l of assay buffer (1.2M urea, 50mM sodium borate buffer, pH 8.2), pre-incubated in the presence of 1. mu.M of mofetil and 500. mu.M of pargyline for 30 minutes at 37 ℃. BAPN (500. mu.M) has been added to all "low control" wells. At least three copies are executed per condition.
After pre-incubation, 100 μ l Amplex Red horseradish peroxidase (HRP) mix (120 μ M Amplex Red, 1.5U/mL HRP, 20mM putrescine) was added to all wells and the plates were read 13 times every 2.5 min at 37 ℃ (544 nm excitation and 590nm emission on BMG Clariostar in orbital, top reading mode). The slope of the kinetic curve minus the value obtained at the low control (in the presence of BAPN) is considered a measure of the activity of a particular lysyl oxidase in the sample.
Example 27
Measurement of lysyl oxidase Activity in aorta
Sample preparation: all preparative activities were performed on ice in buffer in the presence of protease inhibitors (PMSF: Sigma P7626, 0.25mM, aprotinin: Sigma A6279, 1mL/mL) ("Methods in Cell Biology" 2018; 143: 147-156). Under a dissecting microscope, the adventitia and muscle/fat surrounding the frozen aortic sample were removed in ice-cold wash buffer (0.15M NaCl, 50mM sodium borate, pH 8.0, containing protease inhibitors) using fine forceps. The aorta was blotted dry on a Kimwipe and weighed in a 1.5mL Eppendorf tube and then placed on ice. After the aorta was snap frozen in liquid nitrogen, the tissue was crushed with a mortar and pestle (stored at-80 ℃). The crushed tissue was transferred to a designated tube containing metal beads, 10x v/w wash buffer + protease inhibitor. The tissue was homogenized using a bead mill for 5 seconds. The homogenate was centrifuged at 10,000Xg for 10 minutes at 4 ℃ and the supernatant was discarded. The homogenization and washing steps were repeated two more times. The resulting pellet was resuspended in 3x v/w 6M urea buffer (50mM sodium borate, 6M urea, pH 8.2) + protease inhibitor and then vortexed. The sample was then placed on a roller for 3 hours extraction at 4 ℃. After extraction, the samples were centrifuged (10,000Xg, at 4 ℃ for 20 minutes) and the supernatant was retained. An aliquot of all samples was transferred to a fresh tube for dilution (minimum requirement 33 μ L). The samples were diluted to 2.4M urea (about 4.5M urea at this stage) with sodium borate buffer (50mM sodium borate, pH 8.2) + protease inhibitor and used for subsequent assays.
And (3) determination: baragiline (final concentration of 0.5mM) and Mofeijran (final concentration of 1. mu.M) were added to the samples. Duplicate wells were placed in a black 384 plate at 25 μ L per well. To duplicate, 0.5 μ L of 30mM BAPN (a pantysine oxidase inhibitor) was added; background values (low control) are provided. The samples were then incubated at 37 ℃ for 30 minutes. The reaction mixture (25. mu.L) was added to each well (concentration shown as 2-fold of the final concentration in the assay: 120. mu.M Amplex red, 1.5U/mL HRP, 20mM putrescine). Then in FluostarTMPlate fluorescence was measured every 2.5 minutes (e.g., 544 nm/Em: 590 nm/gain: 1260; 37 ℃).
Young male Wistar rats were given a single oral dose (10 or 30mg/kg) of compound 1 or 33 (see tables 5 and 6, respectively) and measured for enzyme activity in tissues with high basal activity in the ear at this age (fig. 2a and b; compounds 1 and 33, respectively) and aorta (fig. 3 c; compound 33 only). The reactions were normalized to the activity measured in animals treated with saline (control).
Compound 1 described herein has a long lasting inhibitory effect on LOX. Although the plasma concentration of Compound 1 in rat plasma after 8 hours was much lower than IC50(table 5), but a sustained decrease in dose-dependent LOX activity was measured 24 hours after a single oral dose (fig. 2 a);>after 20 hours, the plasma concentration of compound 1 dropped below IC50。
It was found that a single high (30mg/kg) dose of compound 33 completely abolished lysyl oxidase activity. Although the plasma concentration of compound 33 after 8 hours (table 6) was much lower than IC50But the half-life of recovery was between 2-3 days (ear) and 24 hours (aorta) (fig. 2b and 2 c). Thus, compound 33 elicits a long-lasting inhibitory effect, persisting the presence of the active compound in the plasma.
Example 28
Method for determining the ability of a compound of formula I to inhibit human recombinant SSAO/VAP-1
Human recombinant SSAO/VAP-1 amine oxidase activity was measured using a coupled colorimetric assay described for monoamine oxidase, copper-containing amine oxidase and related enzymes (Holt A. and Palcic M. "A peroxidase-coupled continuous absorption microplate reader assay for flavin monoamine oxidase, copper-containing amine oxidase and related enzymes, (" A peroxidase-coupled continuous absorption plates for flavin monoamine oxidases, ", copper-containing amine oxidases and related enzymes") according to the Nature's laboratory Manual (Nat Protoc) 2006; 1: 2498-. Briefly, a cloned cDNA template corresponding to residues 34-763 of human SSAO/VAP-1 and incorporating the mouse Ig kappa (kappa) signal sequence, the N-terminal marker epitope tag, and the Tobacco Etch Virus (TEV) cleavage site was assembled in a mammalian expression vector (pLO-CMV) by Geneart AG. This vector containing human SSAO/VAP-1 residues was transfected into the CHO-K1 glycosylation mutant cell line Lec 8. Clones stably expressing human SSAO/VAP-1 were isolated and grown on a large scale. Immunoaffinity chromatography was used to purify and recover active human SSAO/VAP-1. This was used as a source of SSAO/VAP-1 activity. Development using 96 or 384 well formatA high throughput colorimetric assay is provided. Briefly, in a standard 96-well plate assay, 50 μ L of purified human SSAO/VAP-1(0.25 μ g/mL) in 0.1M sodium phosphate buffer (pH 7.4) was added to each well. Test compounds were dissolved in DMSO and tested at 4-11 data points, typically in the micromolar or nanomolar range, in a Concentration Response Curve (CRC) after 30 minutes incubation with human SSAO/VAP-1 at 37 ℃. After 30 min incubation, 50 μ L of reaction mixture containing 600 μ M benzylamine (sigma aldrich) prepared in 0.1M sodium phosphate buffer (pH), 120 μ M Amplex Red (sigma aldrich) and 1.5U/mL horseradish peroxidase (sigma aldrich) 7.4) were added to the corresponding wells. Fluorescence Units (RFU) were read every 2.5 min at 37 ℃ for 30 min at excitation 565nm, emission 590 (Optima; BMG labtech). The kinetic slope of each well was calculated using the MARS data analysis software (BMG Labtech) and this value was used to derive the IC50Value (Dotmatics). Table 7 shows the ability of compounds of formula I to inhibit SSAO/VAP-1.
Example 29
Method for determining the ability of compounds of formula I to inhibit human recombinant MAO-B
The specificity of the compounds of the invention was tested by determining their ability to inhibit MAO-B activity in vitro. Recombinant human MAO-B (0.06 mg/mL; Sigma Aldrich) was used as the source of MAO-B enzyme activity. The assay was performed in a similar manner to human SSAO/VAP-1 (example 28) except that 100. mu.M of the substrate benzylamine was used. Table 7 shows the ability of compounds of formula I to inhibit MAO-B.
TABLE 7
The selectivity of the compound of formula I for LOX and LOXL2 compared to SSAO/VAP-1 and MAO-B
The LOX and LOXL1-4 enzymes are members of a large class of flavin-dependent and copper-dependent amine oxidases, including SSAO/VAP-1 and monoamine oxidase B (MAO-B). With respect to SSAO/VAP-1, MAO-B and other family members amine oxidases, the compounds of the present invention selectively inhibit LOX family members of the enzyme. An example of the magnitude of selectivity can be seen in table 7.
Example 30
Rodent injury model
Mice were injured by excision of skin tissue. In the treatment group, a 1% solution of compound 1 was applied topically from 24 hours after injury to 1 week after injury. The wound was then allowed to heal for another week. Mice were euthanized at day 14 post-injury and tissues were analyzed for collagen I content, as well as gross morphological and histological changes.
The amount of collagen I (measured using LCMS and normalized to protein content) in the treated tissue was reduced compared to the control (fig. 3 a). Histology showed parallel collagen bundles thick in the control scar tissue. The tissue treated with compound 1 showed a decrease in bundle density and a more "normal" structure of collagen (fig. 3b and 3 c).
Example 31
Pig burn model
The pigs received 4x25cm22 on each side of the deep skin burn. Starting from the epithelial regeneration, each pig was treated with 3% of compound 1 cream daily for 2 wounds for 4 weeks, and 2 received the control cream. Pigs were euthanized and tissues were processed for analysis.
LOX activity was significantly reduced in treated tissues (FIG. 4a), as was total collagen I (FIG. 4 b). Gross morphology (fig. 4c-f) and histology (fig. 4g-h) support a reduction in the density of thick collagen fibers in treated wounds.
Example 32
Pancreatic cancer mouse model
Female athymic nude mice (4-5 weeks old) were directly inoculated with MiaPaca-2luc (human pancreatic cell line) in the pancreas. On day 14 after the initial vaccination, the measured bioluminescent signal intensity and body weight were used to classify the tumors into treatment groups. Treatment groups included 1 vehicle, intraperitoneal injections, 3 times a week. Abraxane (albumin-bound paclitaxel) (10mg/kg, i.p. injection, 2 times per week) and gemcitabine (initial dose 100mg/kg, i.p. injection; second dose drops to 60mg/kg due to acute toxicity, i.p. injection, twice per week). (Albumin-bound paclitaxel) (administered as described above) and gemcitabine (administered as described above) and Compound 33(10mg/kg, i.p. injection, 3 times weekly) (see FIG. 5 a). Mice were euthanized on day 43 post tumor inoculation. Mice were monitored twice weekly for condition and weight changes. Throughout the study, tumor growth was monitored in vivo by bioluminescence imaging (fig. 5b), and organs were monitored ex vivo at the time of tissue collection (fig. 5 c-e).
Example 33
Mouse model of sclerosis
Female C57BL/6 was administered subcutaneous bleomycin every other day (20 days total) to induce skin fibrosis as a sclerosing model. Treatment of lesions with vehicle, 0.5%, 1% or 3% compound 1. Histology was complete after 21 days. Histological analysis is shown in FIG. 6 (a-c).
Example 34
Mouse model of primary myelofibrosis
Fifteen to sixteen weeks old GATA1 low male and female mice were injected intraperitoneally with vehicle (olive oil) or compound 19 at a dose of 15mg/kg four times a week for 10 weeks. The mice were then sacrificed and the spleen and femur collected for histology and analysis (FIGS. 7(a-e) and 8 (a-d)).
Spleen weights of mice treated with compound 19 were adjusted to significantly lower relative body weights compared to vehicle group (242.25 + -18 mg vs 305.11 + -22.4 mg, p < 0.05). There was no difference in hematological parameters before treatment, however, the platelet count was significantly lower in the treated group compared to vehicle mice after treatment (77.5 ± 4.4K/uL vs 106 ± 12, p < 0.05). Bone Marrow (BM) and spleen fibrosis were significantly reduced in the treated mice compared to vehicle. H & E stained morphological BM Megakaryocyte (MK) counts were reduced in treated mice compared to vehicle (24.65 ± 0.6 pairs 32.91 ± 0.71 per 20x field, p < 0.001).
Example 35
Single side transfusionUreteral Obstruction (UUO) model
A 14-day Unilateral Ureteral Obstruction (UUO) model of acute renal fibrosis in mice was performed to model, in an accelerated manner, the different stages of obstructive renal disease leading to interstitial fibrosis in the tubules. The UUO procedure was performed by ligation of the left ureter and resulted in a reduction in left kidney thickness and atrophy (as indicated by a significant decrease in kidney weight and kidney/body weight ratio compared to the contralateral side) and a significant increase in kidney fibrosis. During the study period, the treatment group received compound 33 orally (10mg/kg per day). Compound 33 increased kidney weight and thickness and decreased area of fibrosis measured with sirius red (figure 9). Captopril (about 32 mg/kg/day in drinking water) was used as a positive control.
Example 36
Bleomycin-induced pulmonary fibrosis model
C57Bl/6 mice were given 1.5U/kg of universal clinical bleomycin (Blenoxane) by oropharyngeal administration. Compound 33 was administered daily by gavage for 21 days, and tissues were collected and analyzed. As shown in FIG. 10, compound 33 significantly reduced the Ashcroft score and lung weight. As expected for lysyl oxidase inhibitors, the number of immature (DHLNL) and mature (PYD) cross-links in each lung was also reduced by 37% and 45%, respectively.
Example 37
Compound 33 reduces fibrosis-related transfer
There is increasing evidence that to develop cancer metastasis, a pre-metastatic niche (niche) needs to be established to aid in extravasation and metastatic colonization. The fibrotic microenvironment is thought to enhance tumor invasion and metastasis.
Twice weekly using 1mg/kg carbon tetrachloride (CCl)4) BALB/c mice were treated to induce hepatic fibrosis for 8 weeks. Also, treatment with compound 33 (20 mg/kg injected intraperitoneally daily) was provided to significantly reduce liver fibrosis (FIGS. 11a and b). At the end of week 4, a mouse breast cancer cell line (4t1) was injected in situ (as shown in fig. 11 a). Treatment with Compound 33 significantly reduced liver fibrosis (FIG. 11b), collagen crossingCombined with the metastatic load in the liver (fig. 11c and 11 d). No difference in primary tumor growth or collagen crosslinking was observed.
Example 38
The compounds of the invention have reduced substrate activity for SSAO as compared to the E-olefin, isomer counterpart.
Minimizing off-target activity is a key consideration in designing and developing compounds for therapeutic applications. Compounds comprising E-1 have been exemplified as inhibitors of semicarbazide-sensitive amine oxidase (SSAO) [ WO2009/066152 ]. It is reported that this type of molecule can also be transformed as a substrate for SSAO, producing potentially toxic metabolites (Foot et al, 2012). In a preferred embodiment, the compounds of the present invention are neither inhibitors nor substrates of SSAO.
Measurement of substrate conversion by SSAO
In short, the assay used determines the substrate propensity of the compound against background (dimethyl sulfoxide only). Compound oxidation by rhSSAO was measured by fluorimetry (Holt and Palcic, 2006). Briefly, rhSSAO was incubated in HEPES buffer at 37 ℃ for 2 hours, followed by the addition of equal volumes of Amplex Red (20mM), horseradish peroxidase (4U/ml) and compound (2.5mM) in the same buffer. The kinetics of the formation of resorufin was measured immediately using an Optima reader (BMG Labtech GmbH, Ortenburg, germany) at 37 ℃. Representative results are shown in table 6.
TABLE 6
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