HYDRAZINE CONTAINING NUCLEAR TRANSPORT MODULATORS AND THEIR USES

专利摘要:
hydrazine containing nuclear transport modulators and their uses. the invention relates generally to nuclear transport modulators, for example, crm1 inhibitors, and more particularly to a compound represented by structural formula (i): or a pharmaceutically acceptable salt thereof, in which the alternative values and values for the variables are as defined and described herein. the invention also includes the synthesis and use of a compound of formula i, or a pharmaceutically acceptable salt or a composition thereof, for example, in the treatment, modulation and / or prevention of physiological conditions associated with crm1 activity.
公开号:BR112014001934B1
申请号:R112014001934-7
申请日:2012-07-26
公开日:2020-12-15
发明作者:Vincent P. Sandanayaka；Sharon Shacham；Dilara McCauley；Sharon Schechter
申请人:Karyopharm Therapeutics, Inc；
IPC主号:

专利说明:

Related requests
[0001] The present application claims the benefit of provisional application US no. 61 / 513,428, filed on July 29, 2011, provisional application US no. 61 / 513,432, filed on July 29, 2011, provisional application US no. 61 / 610,178, filed on March 13, 2012, provisional application no. 61 / 654,651, filed on June 1, 2012, and provisional application US no. 61 / 653,588, deposited on May 31, 2012. The contents of the above requests are hereby incorporated by reference in full. Background of the invention
[0002] Cells from most major human solid and hematological malignancies exhibit abnormal cellular localization of a variety of oncogenic proteins, tumor suppressor proteins, and cell cycle regulators (Cronwshaw et al., 2004, Falini and others 2006). For example, certain p53 mutations lead to localization in the cytoplasm instead of the nucleus. This results in loss of normal growth regulation, despite the tumor suppressor's intact function. In other tumors, wild type p53 is either sequestered in the cytoplasm or rapidly degraded, again leading to loss of its suppressive function. The recovery of appropriate nuclear localization of functional p53 protein can normalize some properties of neoplastic cells (Cai and others. 2008; Hoshino and others. 2008; Lain and others. 1999a; Lain and others. 1999b; Smart and others. 1999), can recover the sensitivity of cancer cells to DNA damaging agents (Cai et al. 2008), and can lead to regression of established tumors (Sharpless & De Pinho 2007, Xue et al. 2007). Similar data were obtained for other tumor suppressor proteins such as fork (Turner and Sullivan 2008) and c-Abl (Vignari and Wang 2001). In addition, the abnormal location of several growth-regulating and tumor-suppressing proteins may be involved in the pathogenesis of autoimmune diseases (Davis 2007, Nakahara 2009). Inhibition of CRM1 can provide particularly interesting utility in familial cancer syndromes (for example, Li-Fraumeni Syndrome due to the loss of a p53 allele, BRCA1 or 2 cancer syndromes) where specific tumor suppressor proteins (TSP) are eliminated or dysfunctional and where increasing TSP levels by systemic (or local) administration of CRM1 inhibitors could help restore normal tumor suppressor function.
[0003] Specific proteins and RNAs are carried in and out of the nucleus by specialized transport molecules, which are classified as importins if they transport molecules into the nucleus, and exportins if they transport molecules out of the nucleus (Terry et al. 2007 ; Sorokin et al. 2007). Proteins that are transported into or out of the nucleus contain nuclear localization / import (NLS) or export (NES) sequences that allow them to interact with the relevant transporters. Maintenance of chromosomal region 1 (Crm1 or CRM1) which is also called exportin-1 or Xpo1 is a main exportin.
[0004] Crm1 overexpression has been reported in several tumors, including human ovarian cancer (Noske et al. 2008), cervical cancer (van der Watt et al. 2009), pancreatic cancer (Huang et al. 2009), hepatocellular carcinoma (Pascale et al. 2005) and osteosarcoma (Yao et al. 2009) and is independently correlated with poor clinical outcomes in these types of tumors.
[0005] Crm1 inhibition blocks the exodus of tumor suppressor proteins and / or growth regulators such as p53, c-Abl, p21, p27, pRB, BRCA1, IkB, ICp27, E2F4, KLF5, YAP1, ZAP, KLF5, HDAC4, HDAC5 or fork proteins (eg, FOXO3a) from the nucleus that are associated with gene expression, cell proliferation, angiogenesis and epigenetics. Crm1 inhibitors have been shown to induce apoptosis in cancer cells even in the presence of activation of growth-stimulating or oncogenic signals, while sparing normal (untransformed) cells. Most Crm1 inhibition studies used the natural product Crm1 inhibitor Leptomycin B (LMB). LMB itself is highly toxic to neoplastic cells, but insufficiently tolerated with marked gastrointestinal toxicity in animals (Roberts and others 1986) and humans (Newlands and others, 1996). Derivatization of LMB to improve drug-like properties leads to compounds that retain antitumor activity and are better tolerated in animal tumor models (Yang et al. 2007, Yang et al. 2008, Mutka et al. 2009). Therefore, nuclear export inhibitors could have beneficial effects on neoplastic disorders and other proliferative disorders.
[0006] In addition to tumor suppressor proteins, Crm1 also exports several major proteins that are involved in many inflammatory processes. These include IkB, NF-kB, Cox-2, RXRα, Commd1, HIF1, HMGB1, FOXO, FOXP and others. The nuclear factor kappa B (NF-kB / rel) family of transcription activators, named for the discovery that it triggers immunoglobulin kappa gene expression, regulates mRNA expression of a variety of genes involved in inflammation, proliferation, immunity and survival of cells. Under baseline conditions, an NF-kB protein inhibitor, called IkB, binds to NF-kB in the nucleus and the IkB-NF-kB complex makes the NF-kB transcriptional function inactive. In response to inflammatory stimuli, IkB dissociates from the IkB-NF-kB complex, which releases NF-kB and unmasks its potent transcriptional activity. Many signals that activate NF-kB do this by directing IkB for proteolysis (phosphorylation of IkB makes it “marked” for ubiquitination and then proteolysis). The nuclear IkBa-NF-kB complex can be exported to the cytoplasm by Crm1 where it dissociates and NF-kB can be reactivated. Ubiquitinated IkB can also dissociate from the NF-kB complex, recovering the NF-kB transition activity. Inhibition of Crm1 induced export in human neutrophils and macrophage-like cells (U937) by LMB not only results in accumulation of nuclear IkB-NF-kB complex, inactive in transcription mode but also prevents the initial activation of NF-kB even after cell stimulation (Ghosh 2008, Huang 2000). In a different study, treatment with LMB inhibited IL-1β-induced NF-kB DNA binding (the first step in activating NF-kB transcription), IL-8 expression and intercellular adhesion molecule expression in pulmonary microvascular endothelial cells (Walsh 2008). COMMD1 is another nuclear inhibitor of both NF-kB and hypoxia-inducible factor 1 (HIF1) transcription activity. Blocking the nuclear export of COMMD1 by inhibiting Crm1 results in increased inhibition of NF-kB and HIF1 transcription activity (Muller 2009).
[0007] Crm1 also mediates transport of α retinoid X receptor (RXRα). RXRα is highly expressed in the liver and plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. During liver inflammation, nuclear RXRα levels are significantly reduced, mainly due to nuclear export mediated by RXRα inflammation by Crm1. LMB is able to prevent IL-1β-induced cytoplasmic increase in RXRα levels in cells derived from human liver (Zimmerman 2006).
[0008] The role of Crm1-mediated nuclear export in NF-kB, HIF-1 and RXRα signaling suggests that nuclear export blockade may be potentially beneficial in many inflammatory processes across multiple tissues and organs including vasculitis (vasculitis, arteritis, polymyalgia rheumatica, atherosclerosis), dermatological (see below), rheumatological (rheumatoid and related arthritis, psoriatic arthritis, spondyloarthropathies, crystal arthropathies, systemic lupus erythematosus, mixed connective tissue disease, myositis syndromes, dermatomyositis, dermatomyositis inclusion, non-differentiated connective tissue disease, Sjogren's syndrome, overlapping syndromes and scleroderma, etc.).
[0009] Inhibition of CRM1 affects gene expression by inhibiting / activating a number of transcription factors such as ICp27, E2F4, KLF5, YAP1 and ZAP.
[00010] Crm1 inhibition has potential therapeutic effects through many dermatological syndromes including inflammatory dermatoses (atopy, allergic dermatitis, chemical dermatitis, psoriasis), sun damage (ultraviolet (UV) damage) and infections. The inhibition of CRM1, studied better with LMB, showed minimal effects on normal keratinocytes, and exerted anti-inflammatory activity on keratinocytes submitted to UV, TNFα or other inflammatory stimuli (Kobayashi & Shinkai 2005, Kannan & Jaiswal 2006). Inhibition of Crm1 also upwardly regulates NRF2 activity (factor 2 related to nuclear factor erythroid) that protects keratinocytes (Schafer et al. 2010, Kannan & Jaiswal 2006) and other cell types (Wang et al. 2009) against oxidative damage . LMB induces apoptosis in keratinocytes infected with strains of human oncogenic papilloma virus (HPV) such as HPV16, but not in uninfected keratinocytes (Jolly et al. 2009).
[00011] Crm1 also mediates the transport of major neuroprotective proteins that may be useful in neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis (ALS). For example, by (1) forcing nuclear retention of major neuroprotective regulators such as NRF2 (Wang 2009), FOXA2 (Kittappa et al. 2007), parking in neuronal cells, and / or (2) inhibiting NFkB transcription activity by hijacking IkB for the nucleus in glial cells, Crm1 inhibition could decrease or prevent the strong neuronal cell found in these disorders. There is also evidence linking abnormal proliferation of glial cells with abnormalities in levels of CRM1 or function of CRM1 (Shen 2008).
[00012] Intact nuclear export, mainly mediated through CRM1, is also required for the intact maturation of many viruses. Viruses where nuclear export, and / or CRM1 itself, has been involved in its life cycle include human immunodeficiency virus (HIV), adenovirus, simian retrovirus type 1, Borna disease virus, influenza (common strains as well as H1N1 and strains Bird H5N1), hepatitis B virus (HBV) and C (HCV), human papillomavirus (HPV), respiratory syncytial virus (RSVA), Dungee, severe acute respiratory syndrome coronavirus, yellow fever virus, West Nile virus, herpes simplex virus (HSV), cytomegalovirus (CMV), and Merkel cell polyomavirus (MCV). (Bhuvanakantham 2010, Cohen 2010, Wittaker 1998). Additional viral infections based on intact nuclear exports are expected to be discovered in the future.
[00013] HIV-1 Rev protein, which transits through nucleoli and switches between the nucleus and cytoplasm, facilitates the export of unjoined and individually joined HIV transcripts containing Rev Response Element RNA (RRE) via the CRM1 export path. Inhibition of Rev-mediated RNA transport using CRM1 inhibitors such as LMB or PKF050-638 can halt the process of HIV-1 transcription, inhibit the production of new HIV-1 virions, and thereby reduce HIV-1 levels (Pollard 1998 , Dalemans 2002).
[00014] Dengue virus (DENV) is the causative agent of the common arthropod-borne viral disease, Dengue fever (DF), and potentially fatal and more serious hemorrhagic fever dengue (DHF). DHF appears to be the result of an excessive exuberant inflammatory response to DENV. NS5 is the largest and most conserved protein in DENV. CRM1 regulates the transport of NS5 from the nucleus to the cytoplasm, where most NS5 functions are mediated. CRM1-mediated export inhibition of NS5 results in altered virus production kinetics and reduces the induction of inflammatory chemokine interleukin-8 (IL-8), presenting a new path for the treatment of diseases caused by DENV and other medically important flaviviruses including hepatitis C virus (Rawlinson 2009).
[00015] Other virus-encoded RNA binding proteins that use CRM1 to exit the nucleus include the HSV type 1 integument protein (VP13 / 14, or hUL47), human CMV protein pp65, the ORF Protein 3b from Coronavirus SARS and the matrix protein (M) RSV (Williams 2008, Sanchez 2007, Freundt 2009, Ghildyal 2009).
[00016] Interestingly, many of these viruses are associated with specific types of human cancer including hepatocellular carcinoma (HCC) due to chronic HBV or HCV infection, cervical cancer due to HPV, and MCV-associated Merkel cell carcinoma. CRM1 inhibitors could therefore have beneficial effects on both the viral infectious process and the neoplastic transformation process due to these viruses.
[00017] CRM1 controls the nuclear localization and therefore activity of multiple enzymes to metabolize DNA including histone deacetylases (HDAC), histone acetyl transferases (HAT), and histone methyl transferases (HMT). The suppression of cardiomyocyte hypertrophy with irreversible CRM1 inhibitors has been demonstrated and is believed to be linked to the nuclear retention (and activation) of HDAC 5, an enzyme known to suppress a hypertrophic genetic program (Monovich et al. 2009). Thus, inhibition of CRM1 can have beneficial effects in hypertrophic syndromes, including certain forms of congestive heart failure and hypertrophic cardiomyopathies.
[00018] CRM1 has also been linked to other disorders. Leber's disorder, an inherited disorder characterized by degeneration of retinal ganglion cells and visual loss, is associated with the inaction of the CRM1 switch (Gupta N 2008). There is also evidence linking neurodegenerative disorders to abnormalities in nuclear transport.
[00019] To date, however, small-molecule Crm1 inhibitors similar to the drug for in vitro and in vivo use are uncommon. Summary of the invention
[00020] The present invention relates to compounds, or pharmaceutically acceptable salts thereof, useful as nuclear transport modulators. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using the compounds and compositions in the treatment of various disorders, such as those associated with abnormal cellular responses triggered by inadequate nuclear transport.
[00021] In an embodiment of the invention, compounds are represented by formula I:
or a pharmaceutically acceptable salt thereof, where the values and alternative values for each variable are as defined and described here.
[00022] Another embodiment of the invention is a composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[00023] Yet another embodiment of the invention is a method of treating a disorder associated with CRM1 activity, the method comprising administering to a subject in need of a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof.
[00024] Another embodiment of the invention is the use of a compound of the invention to treat a disorder associated with CRM1 activity in a subject.
[00025] Another embodiment of the invention is the use of a compound of the invention for the manufacture of a medicament to treat a disorder associated with CRM1 activity in a subject.
[00026] The nuclear transport modulators of the present invention, and pharmaceutically acceptable salts and / or compositions thereof, provide excellent exposure in vivo as measured by AUC in mice, rats, dogs and monkeys, while having low levels of brain penetration. Therefore, the compounds of the present invention, and pharmaceutically acceptable salts and / or compositions thereof, are useful for treating a variety of diseases, disorders or conditions, associated with abnormal cellular responses triggered by inappropriate nuclear transport, such as those diseases, disorders or conditions described here. The compounds provided by the present invention are also useful for studying the modulation of nuclear transport in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by kinases; and the comparative assessment of nuclear transport modulators. Brief description of the figures
[00027] Figure 1 is a graph of tumor volume as a function of time and shows the effect of compound I-3 on tumor volume in a triple negative Breast Cancer (TNBC) mouse xenograft model.
[00028] Figure 2A is a Western blot image showing the effect of increasing concentrations of compound I-1 in CRM1 and apoptosis marker proteins in TNBC MDA-MB-468 cells.
[00029] Figure 2B is a Western blot image showing the effect of increasing concentrations of compound I-3 on CRM1 and apoptosis marker proteins in DU4475 BC cells.
[00030] Figure 2C is a Western blot image showing the effect of increasing concentrations of Compound I-3 in CRM1 and apoptosis marker proteins in TNBC HS578T cells.
[00031] Figure 3 are Western blot images showing the effect of increasing concentrations of Compound I-3 in cell cycle and anti-apoptosis proteins in TNBC cell lines MDA-MB-468 and HSS578T.
[00032] Figure 4 is a graph of average body weight versus time for days 0 to 12 in antibody-induced male Balb / c mice undergoing the indicated treatment.
[00033] Figure 5 is a graph of clinical arthritic markings of mean total paw versus time to days - to 12 in male BALB / c antibody-induced mice submitted to the indicated treatment.
[00034] Figure 6 is a bar graph for average ear thickness, flaking and fold determined from day 0 to 7 in PMA-induced male BALB / c mice submitted to the indicated treatment.
[00035] Figure 7 is a set of graphs showing preference for the object of mice treated as indicated in the new Object Recognition Model.
[00036] Figure 8A is a set of graphs showing cumulative and mean food intake versus time in obese and lean Zucker rats treated as indicated.
[00037] Figure 8B is a set of graphs that shows mean and percentage of body weight versus time in obese and lean Zucker rats treated as indicated. Detailed Description
[00038] The new features of the present invention will become apparent to those skilled in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and specific examples presented, while indicating certain modalities of the present invention, are provided for purposes of illustration only because various changes and modifications understood in the spirit and scope of the invention will become evident to those skilled in the art. in the art from the detailed description of the invention and claims that follow. Compounds of the invention
[00039] One embodiment of the invention are the compounds represented by formula I:
Or a pharmaceutically acceptable salt of the same where:
[00040] R1is selected from hydrogen and methyl;
[00041] R2is selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, and quinoxalin-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophen-3 -il and cyclopropyl, where R2 is optionally substituted with one or more independent substituents selected from methyl and halogen; or
[00042] R1 and R2 are taken together with their intermediate atoms to form 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3 -hydroxyzetidin-1-yl, or morpholin-4-yl;
[00043] R3is selected from hydrogen and halo; and
[00044] »AA represents a single bond in which a carbon-carbon double bond attached to it is in an (E) - or (Z) - configuration.
[00045] As generically described above, R1 is selected from hydrogen and methyl. In some embodiments, R1 is hydrogen. In some embodiments, R1 is methyl.
[00046] As generically described above, R2 is selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, quinoxalin-2-yl, pyrimidin-4-yl, 1,1 - dioxotetrahydrothiophen-3-yl and cyclopropyl, where R2 is optionally substituted with one or more independent substituents selected from methyl and halogen. In some embodiments of formula I, R2 is pyridin-2-yl. In some embodiments of formula I, R2 is pyridin-3-yl. In some embodiments of formula I, R2 is pyridin-4-yl. In some embodiments of formula I, R2 is pyrazin-2-yl. In some embodiments of formula I, R2 is pyrimidin-4-yl. In some embodiments of formula I, R2 is quinoxalin-2-yl. In some embodiments of formula I, R2 is selected from pyridin-2-yl, pyridin-3-yl and pyridin-4-yl. In some embodiments of formula I, R2 is selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl and pyrimidin-4-yl. In some embodiments of formula I, R2 is selected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl and pyrimidin-4-yl.
[00047] In some modalities, R2 is selected from:

[00048] In some modalities of formula I, R2 is optionally substituted with a single substituent selected from methyl and chlorine. In some embodiments of formula I, R2 is optionally substituted with a methyl group. In some embodiments of formula I, R2 is optionally substituted with a chlorine group. In some modalities, R2 is selected from:

[00049] In some modalities, R2 is selected from:

[00050] In some modalities, R2 is selected from:

[00051] In some embodiments of formula I, R1 and R2 are taken together with their intermediate atoms to form 4-hydroxypiperidin-1-yl, pyrrolidin-1-yl, azepan-1-yl, 4-benzylpiperazin-1-yl, 4 -ethylpiperazin-1-yl, 3-hydroxyazetidin-1-yl, or morpholin-4-yl. In some embodiments of formula I, R1 and R2 are taken together with their intermediate atoms to form 4-hydroxypiperidin-1-yl.
[00052] As generically described above, R3 is selected from hydrogen and halogen. In some embodiments, R3 is hydrogen. In some embodiments, R3 is halogen (for example, chlorine, bromine, iodine or fluorine). In some of these modalities, R3 is chlorine.
[00053] As generically described above, carbon carbon double bond between the triazole fraction and the carbonyl fraction is in an (E) or a (Z) configuration. in some modalities, the double bond is in a configuration (E). in some modalities, this double bond is in a configuration (Z) and the compound is represented by the formula (II):
or a pharmaceutically acceptable salt thereof, wherein R1, R2 and R3 are as defined above and described herein.
[00054] An additional embodiment of the invention is a compound represented by formula II, or a pharmaceutically acceptable salt thereof, wherein the alternative values and values for the variables are as defined above for a compound of the formula I.
[00055] In a first aspect of this additional modality, R1 is as defined above; and R2 is selected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl and pyrimidin-4-yl, wherein R2 is optionally substituted with a single substituent selected from methyl and chlorine; or R1 and R2 are taken together with their intermediate atoms to form 4-hydroxypiperidin-1-yl.
[00056] In a specific aspect of the first aspect, R3 is hydrogen. The alternative values and values for the remaining variables are as described above for a compound of formula I or in the additional modality, or first aspect thereof.
[00057] Exemplary compounds of formula I are shown in table 1.
[00058] Table 1. Exemplary compound of the formula



[00059] In some embodiments, the compound of the invention is selected from any of compounds I-3 to I26. In one aspect of these embodiments, the compound is selected from compounds I-3, I-4, I-5, I-7, I-8, I-10, I12, I-18, I-19 and I-24. In a more specific aspect, the compound of the invention is selected from I-3 and I-4.
[00060] Pharmacokinetics (PK) plays an increasing role in drug discovery and development. Pharmacokinetics is the quantitative study of the time course of drug absorption, distribution, metabolism and / or excretion. When a drug is administered, it rapidly distributes from its site of administration into the systemic bloodstream. A measure of the extent of distribution of a therapeutic agent is the curve of the area under the plasma-time concentration (AUC), calculated to the last measured concentration (AUCt) and extrapolated to infinity (AUCInf). AUC is thus a useful metric for quantifying drug exposure.
[00061] Generally, the higher the exposure of a therapeutic agent, the greater the effects of the agent. However, high exposure to a therapeutic agent can have detrimental effects on certain tissues such as the brain. While the brain-blood barrier (BBB), a protective network consisting of tight junctions between endothelial cells, limits the diffusion of hydrophilic and / or large molecules, drugs with high AUC are still able to penetrate the BBB and / or cerebrospinal fluid . Such penetration is often undesirable and can lead to undesirable side effects. Current drug discovery efforts are directed, in part, to achieving a balance between maximizing exposure to the drug (eg, AUC) while minimizing brain penetration.
[00062] The brain to plasma ratio (B: P) is a method of quantifying the relative distribution of a therapeutic agent in brain tissue to that in circulation and, as such, provides an indication of the penetration of the brain of a therapeutic agent given away. A high brain to plasma ratio is preferred when targeting diseases located in the central nervous system (CNS), including the brain and cerebrospinal fluid. However, a lower brain-to-plasma ratio is generally preferable for non-CNS therapeutic agents to minimize brain penetration and avoid potential side effects caused by undesirable build-up of therapeutic agents in the brain and CNS tissue.
[00063] As set out in more detail in the Exemplification, the compounds of the present invention require a higher AUC and / or a lower B: P in comparison to other nuclear transport inhibitors, such as those disclosed in copending patent application no. 13 / 041,377, deposited on March 5, 2011 and filed with US 2009/0275607 on November 10, 2011. In some embodiments of the present invention, the compound of formula I has a nuclear export activity less than approximately 1 μ M , an AUCinf greater than approximately 3300 (for example, greater than approximately 3500), and a B: P ratio less than approximately 2.5 when dosed in a mouse at 10 mg / kg po. Synthetic methods of the invention
[00064] According to the present invention, there is provided a method of preparing (Z) -olefin derivatives of a compound of the formula Z useful in the preparation of the compound of the invention (for example, precursors for the compounds of the invention):
Or a pharmaceutically acceptable salt thereof, where:
[00065] Ring A is an optionally substituted ring selected from phenyl, an 8-10 membered bicyclic aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur , and a 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur;
[00066] Y is a covalent bond or -L-;
[00067] L is a saturated or unsaturated, linear or branched C1-8 bivalent hydrocarbon radical, where one or two methylene units of L is optionally substituted by -NR-, -N (R) C (O) -, -C (O) N (R) -, -O-, -C (O) -, -OC (O) -, -C (O) O-, -S-, -SO-, -SO2-, - C (S) -, -C (NOR) - or -C (NR) -;
[00068] Each R is independently hydrogen or an optionally selected group of C1-6 aliphatic, phenyl, a 4-7 membered, saturated or partially unsaturated carbocyclic ring, a saturated or partially unsaturated 4-7 membered heterocyclic ring having 1- 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, a 5-6 membered monocyclic heteroaryl ring having 1-4 independently selected nitrogen, oxygen and sulfur heteroatoms, an 8-10 membered bicyclic aryl ring, and a ring of 8-10 membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; or
[00069] two R groups on the same nitrogen are taken together with the nitrogen atom to which they are attached to form a saturated or partially unsaturated 4-7 membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur;
[00070] each of V1, V2 and V3 is independently C (Ry) or N;
[00071] each Rx and Ry is independently selected from -R, halogen, -OR, -SR, -N (R) 2, -CN, -NO2, -N3, -SOR, -SO2R, -SO2NR, -C ( O) R, -CO2R, -C (O) OR, -C (O) N (R) 2, -NRC (O) R, -OC (O) R, -OC (O) N (R) 2, -NRC (O) OR, -NRC (O) NR2 and -NRSO2R;
[00072] each R1 and R2 are independently hydrogen, deuterium, tritium or halogen;
[00073] W is -CN, haloalkyl, -NO2 or -C (= Z) R3;
[00074] Z is O, S, or NR;
[00075] R3is selected from hydrogen, -R, OR, -SR and -N (R4) 2;
[00076] each R4 is independently -R; or
[00077] two R4 in the same nitrogen are taken together with the nitrogen atom to which they are attached to form a saturated or partially unsaturated 4-7 membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur or a ring of 5-6 membered heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the ring thus formed is optionally substituted with - (R5) n;
[00078] each R5 is independently selected from -R, halogen, -OR, -SR, -N (R) 2, -CN, -NO2, -N3, -SOR, -SO2R, -SO2NR, -C (O) R , -CO2R, -C (O) OR, -C (O) N (R) 2, -NRC (O) R, -OC (O) R, -OC (O) N (R) 2, -NRC ( O) OR, -NRC (O) NR2 and -NRSO2R; and
[00079] each m and n is independently selected from 0, 1, 2, 3 and 4.
[00080] Compounds of the formula Z have been described, for example, in US 13 / 041,377, filed on March 5, 2011, and in U.S. provisional application Nos. 61 / 513,428, deposited on July 29, 2011 and 61 / 653,588, deposited on June 1, 2012. The compounds of formula Z are generally synthesized as a mixture of isomers of (E) - and (Z) olefin, which must be separated. The separation of (E) - and (Z) -olefin isomers requires extensive chromatography and results in a 50% loss of advanced intermediate A, since the undesirable isomer cannot typically be converted into the desired isomer. A 50% yield is inefficient and expensive at any stage of a synthesis, but such unacceptable yields are even more problematic at the end of a multi-stage synthesis. It has now surprisingly been discovered that the use of sterically hindered bases in a 1,4-nucleophilic addition can effect (Z) -selectivity of the reaction, thereby providing the cis-olefin isomer as the main or exclusive product. Accordingly, the present invention provides for a selective synthesis- (Z) of compounds of formula Z, and methods of preparing synthetic intermediates useful for preparing compounds of formula Z. A major step in the synthesis of compounds of formula Z is shown in scheme I.
[00081] In certain embodiments, the compounds of formula Z are prepared according to scheme I, set out below: Scheme I
Where LG is a leaving group and each of ring A, Y, V1, V2, V3, Rx, R1, R2, W in is as defined above with respect to a compound of formula Z and described in the embodiments of the present invention.
[00082] In some embodiments of step S-1.1, intermediate A is coupled to intermediate B through a 1,4-nucleophilic addition / elimination reaction. In some embodiments of step S-1.1, LG is an appropriate exit group. In some of these modalities of step S-1.1, LG is a halogen. In some embodiments, LG is iodine. In some modalities of step S-1.1, LG is bromine. In some modalities of step S-1.1, LG is sulfonated. In some of these modalities, LG is methanesulfonate (mesylate).
[00083] In some embodiments of step S-l.1, intermediate A is coupled with intermediate B in the presence of a sterically hindered nucleophilic base. A person skilled in the art will be able to select a suitable sterically hindered base. Suitable esterophilic nucleophilic bases for use in the present invention include 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU), 1,5-diazabicyclo [4,3,0] non-5-ene ( DBN), 1,4-diazabicyclo (2,2,2) octane (DABCO), N, N-dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1,2,2, 6,6-pentamethylpiperidine (PMP), 7-methyl-1, 5,7-triazabicyclo (4,4,0) -dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine.
[00084] In certain embodiments, the compounds of formula Y are prepared according to Scheme II, defined below: Scheme II
Where LG is a leaving group and each of R x, R y, R 1, R 2, W and m is as defined above with respect to a compound of the formula Z and described in embodiments here.
[00085] In some embodiments of step S-2.1, intermediate C is reacted with a thiolate salt to provide intermediate D. In some embodiments of step S-2.1, the thiolate salt is sodium thiolate. In some embodiments of step S-2.1, the thiolate salt is potassium thiolate.
[00086] In step S-2.2, intermediate D is reacted with an hydrazine equivalent in order to provide intermediate E.
[00087] In step S-2.3, intermediate E is coupled with intermediate B to provide a compound of formula Y. In some embodiments of step S-2.3, LG is a suitable leaving group. In some of these modalities of step S-2.3, LG is a halogen. In some embodiments, LG is iodine. In some modalities of step S-2.3, LG is bromine. In some embodiments of step S-2.3, LG is a sulfonate. In some of these modalities, LG is methanesulfonate (mesylate).
[00088] In some embodiments of step S-2.3, intermediate E is coupled with intermediate B in the presence of a sterically hindered nucleophilic base. A person skilled in the art will be able to select a suitable sterically-hindered base. Suitable esterophilic nucleophilic bases for use in the present invention include 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU), 1,5-diazabicyclo [4,3,0] non-5-ene ( DBN), 1,4-diazabicyclo (2,2,2) octane (DABCO), hexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1,2,2,6,6-pentamethylpiperidine ( PMP), 7-methyl-1, 5,7-triazabicyclo (4,4,0) dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine.
[00089] According to one aspect, the present invention provides a method for providing a compound of formula Z:
or a pharmaceutically acceptable salt thereof, wherein each of ring A, Y, V1, V2, V3, Rx, R, R1, R2, W in is as defined above with respect to a compound of the formula Z, comprising the steps of : (a) provide a compound of the formula A:
wherein each of ring A, Rx, Y, V1, V2, V3 and m is as defined above for a compound of formula Z; and (B) reacting the compound of formula A with an olefin of formula B:
where LG is halogen, -OSO2R or -OSO2CF3; and each of R, W, R1 and R2 are as defined above for a compound of formula Z; in the presence of a sterically hindered nucleophilic base, to form a compound of formula Z.
[00090] As described above, a compound of formula A is coupled with intermediate B via a 1,4-nucleophilic addition / elimination reaction. In some embodiments, a compound of formula A is coupled with intermediate B in the presence of a sterically hindered nucleophilic base. Suitable sterically hindered bases include tertiary amine bases. In some embodiments, a sterically hindered base includes sterically hindered secondary amine bases. In some embodiments, the sterically hindered nucleophilic base is selected from 1,8-diazabicycles [5,4,0] undec-7-ene (DBU), 1,5-diazabicycles [4,3,0] non-5 -ene (DBN), 1,4-diazabicyclo (2,2,2) octane (DABCO), N, N-dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1,2 , 2,6,6-pentamethylpiperidine (PMP), 7-methyl-1, 5,7-triazabicyclo (4,4,0) -dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine. In some embodiments, the sterically hindered nucleophilic base is 1,4-diazabicyclo (2,2,2) octane (DABCO). In some embodiments, the sterically hindered nucleophilic base is 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU).
[00091] In some embodiments, the sterically hindered nucleophilic base is a phosphine. In some of these embodiments, the sterically hindered nucleophilic base is triphenylphosphine.
[00092] In some embodiments, step (b) above is carried out in a temperature range of approximately 0 ° C to approximately 100 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 0 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 25 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 50 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 100 ° C.
[00093] A person of ordinary skill in the art will recognize that the 1,4-nucleophilic addition / elimination reaction of a compound of formula A and intermediate B requires the use of a polar, aprotic organic solvent. Suitable polar aprotic organic solvents include ethers, such as dioxane, tetrahydrofuran and methyl tert-butyl ether (MTBE), and amides, such as dimethylformamide (DMF) and dimethylacetamide (DMA). A person of ordinary skill in the art is able to select the appropriate solvent for the desired reaction temperature.
[00094] According to another aspect, the present invention provides a method of providing a compound of formula Y:
Or a pharmaceutically acceptable salt thereof, wherein each of R, Rx, Ry, R1, R2, W and s are as defined above with respect to a compound of the formula Z, Comprising the steps of: (a) Providing a compound of the formula AND:
Wherein each of Rx, Rye m are as defined above for a compound of the formula Y; and (b) Reacting the compound of formula E with an olefin of formula B:
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, W, R1 and R2 are as defined above for a compound of the formula Y, in the presence of a nucleophilic base sterically hindered to form a compound of the formula Y.
[00095] As described above, a compound of formula E is coupled with intermediate B via a 1,4-nucleophilic addition / elimination reaction. In some embodiments, a compound of formula E is coupled with intermediate B in the presence of a sterically hindered nucleophilic base. Suitable sterically hindered bases include tertiary amine bases. In some embodiments, a sterically hindered base includes sterically hindered secondary amine bases. In some embodiments, the sterically hindered nucleophilic base is selected from 1,8-diazabicycles [5,4,0] undec-7-ene (DBU), 1,5-diazabicycles [4,3,0] non-5-ene (DBN), 1,4-diazabicyclo (2,2,2) octane (DABCO), N, N-dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1,2,2 , 6,6-pentamethylpiperidine (PMP), 7-methyl-1,5,7-triazabicyclo (4,4,0) -dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine. In some embodiments, the sterically hindered nucleophilic base is 1,4-diazabicyclo (2,2,2) octane (DABCO). In some embodiments, the sterically hindered nucleophilic base is 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU).
[00096] In some embodiments, the sterically hindered nucleophilic base is a phosphine. In some of these embodiments, the sterically hindered nucleophilic base is triphenylphosphine.
[00097] In some embodiments, step (b) above is carried out in a temperature range of approximately 0 ° C to approximately 100 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 0 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 25 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 50 ° C. In some embodiments, step (b) is carried out at a temperature of approximately 100 ° C.
[00098] A person of ordinary skill in the art will recognize that the 1,4-nucleophilic addition / elimination reaction of a compound of formula E and intermediate B requires the use of a polar, aprotic organic solvent. Suitable polar aprotic organic solvents include ethers, such as dioxane, tetrahydrofuran and methyl tert-butyl ether (MTBE), and amides, such as dimethylformamide (DMF) and dimethylacetamide (DMA). A person of ordinary skill in the art is able to select the appropriate solvent for the desired reaction temperature.
[00099] In some embodiments of a compound of formula Y, W represents CN. In some embodiments, W is haloalkyl. In some of these modalities, W is -CF3. In some embodiments, W is -NO2.
[000100] In some modalities, W is -C (O) R3, where R3 is selected from -OR, -SR or -N (R4) 2. In some embodiments, W is -C (O) OR. In some embodiments, W is - C (O) OR, where R is selected from methyl, ethyl, isopropyl, butyl, tert-butyl and sec-butyl. In some embodiments, W is -C (O) OCH3. In some embodiments, W is -C (O) OCH2CH3. In some embodiments, W is -C (O) OCH (CH3) 2.
[000101] In some embodiments, W is -C (O) N (R4) 2. In some embodiments, W is - (O) NH (R4). In some embodiments, W is -C (O) NH2. In some embodiments, W is - C (= O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a saturated 4-7 membered heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the ring thus formed is optionally replaced with - (R5) n. in some embodiments, W is -C (O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a 4-7 membered saturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the ring thus formed is optionally replaced with - (R5) n. in some embodiments, W is -C (O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a 4-7 membered saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, where the ring thus formed is optionally replaced with - (R5) n. In some embodiments, W is -C (O) N (R4) 2, in which the two groups R4 are taken together with the nitrogen atom to which they are attached to form a 4-7 membered saturated heterocyclic ring having 1 nitrogen atom , wherein the ring thus formed is optionally replaced with - (R5) n.
[000102] In some embodiments, W is -C (O) N (R4) 2, in which the two groups R4 are taken together with the nitrogen atom to which they are attached to form a 4-6 membered saturated heterocyclic ring having 1 nitrogen atom, where the ring thus formed is optionally replaced with - (R5) n. In some embodiments, W is -C (O) N (R4) 2, where the two groups R4 are taken together with the nitrogen atom to which they are attached to form a 4-6 membered saturated heterocyclic ring having 1 nitrogen atom , wherein the ring thus formed is optionally replaced with - (R5) n. In some embodiments, W is -C (O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a saturated 4-membered heterocyclic ring having 1 nitrogen atom, wherein the ring thus formed is optionally replaced with - (R5) n. In some embodiments, W is -C (O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a saturated 4-membered heterocyclic ring having 1 nitrogen atom, wherein the ring thus formed is replaced with fluorine. In some embodiments, W is -C (O) N (R4) 2, where the two R4 groups are taken together with the nitrogen atom to which they are attached to form a saturated 4-membered heterocyclic ring having 1 nitrogen atom, in that the ring thus formed is replaced with at least two fluors. In some embodiments, W is.

[000103] In some embodiments, R1 is hydrogen. In some embodiments, R1 is deuterium. In some embodiments, R2 is hydrogen. In some embodiments, R2 is deuterium. In some embodiments, R1 and R2 are individually hydrogens.
[000104] In some modalities, m is 1. In some modalities, m is 2. In some of these modalities, Rx is haloalkyl. In some embodiments, Rx is -CF3.
[000105] In some modalities, Ryé hydrogen.
[000106] In some embodiments, the present invention provides a method of providing a compound of the formula E:
wherein Rx, Rye m are as described for a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula D:
Where each of Rx and m are as defined above for a compound of the formula E; and (b) reacting the compound of formula D to form a compound of formula E.
[000107] In some embodiments, conditions effective for forming a compound of formula D include an equivalent of hydrazine. Thus, in some embodiments, step (b) of the method of delivering a compound of formula E includes reacting the compound of formula D with an equivalent of hydrazine in the form of the compound of formula E. In some embodiments, intermediate D is reacted with hydrazine hydrate to provide a compound of formula E. In some embodiments, intermediate D is reacted with a protected form of hydrazine, such as tert-butyl hydrazinecarboxylate and subsequently deprotected to provide intermediate D.
[000108] A person of ordinary skill in the art will recognize that the addition of hydrazine to intermediate D requires a polar, aprotic organic solvent. Suitable polar aprotic organic solvents include ethers, such as dioxane, tetrahydrofuran and methyl tert-butyl ether (MTBE), alcohols such as isopropyl alcohol, and amides, such as dimethylformamide (DMF) and dimethylacetamide (DMA). A person of ordinary skill in the art is able to select the appropriate solvent for the desired reaction temperature.
[000109] In some embodiments, the present invention provides a method for the preparation of a compound of formula D:
where Rx and m are as defined above for a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula (C): αCN
where each of Rx and m are as defined above for a compound of the formula D; and (b) Reacting the compound of formula C to form a compound of formula D.
[000110] As described above, in some embodiments, intermediate C is treated with a thiolate salt to provide intermediate D. In some embodiments, the thiolate salt is sodium thiolate. A person of ordinary skill in the art will recognize that the reaction of intermediate C, with a thiolate salt, requires the use of a polar, aprotic solvent. Suitable polar aprotic solvents include ethers, such as dioxane, tetrahydrofuran and methyl tert-butyl ether (MTBE).
[000111] In some embodiments, the present invention provides a method for preparing a compound of formula B:
On what:
[000112] LG is halogen, -OSO2R or -OSO2CF3; and each of R, R1, R1 and W are as defined above for a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula F: R2 = w (F) where each of the R2e W is as defined above for a compound of formula B; and (b) reacting the compound of formula F to form a compound of formula B.
[000113] As described above, in some intermediate B modalities, LG is a halogen. In some of such embodiments, a compound of formula F is treated with a halide salt. In some embodiments, a compound of formula F is treated with a sodium halide. In some of these embodiments, a compound of formula F is treated with sodium iodide. In some embodiments, intermediate F is treated with a halide salt in the presence of an acid. Suitable acids include mineral acids and organic acids. In some embodiments, intermediate F is treated with a halide salt and an organic acid such as acetic acid. In some embodiments, intermediate F is treated with sodium iodide in the presence of acetic acid to provide a compound of formula B.
[000114] A person of ordinary skill in the art will recognize that the addition of an intermediate F halide salt requires a polar, aprotic organic solvent. Suitable aprotic polar organic solvents include ethers, such as dioxane, tetrahydrofuran and methyl tert-butyl ether (MTBE).
[000115] According to another aspect, the present invention provides a method of providing a compound of formula X:
Or a pharmaceutically acceptable salt thereof, wherein each of R, Rx, Ry, R1, R2, R4e m is as defined above with respect to a compound of the formula Z, Comprising the steps of: (a) providing a compound of the formula AND:
Wherein each of Rx, Rye m are as defined above for a compound of the formula X; and (b) reacting the compound of formula E with an olefin of formula G:
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, R1, R2 and R4 are as defined above In the presence of a sterically hindered nucleophilic base to form a compound of the formula X. According to another aspect the present invention provides a method of providing a compound of the formula W:
Or a pharmaceutically salt in which each of R, Rx, Ry, R1, R2, R5, men are as defined above with respect to a compound of the formula Z, Comprising the steps of: (a) providing a compound of the formula E:
Where each of Rx, Ry and m are as defined above for a compound of the formula W; and (b) reacting the compound of formula E with an olefin of formula H:
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, R1, R2, R5 and n are as defined above for a compound of the formula W, In the presence of a sterically hindered nucleophilic base to form a compound of the formula W. According to another aspect, the present invention provides a method to provide a compound of formula V:
Or a pharmaceutically acceptable salt thereof, wherein each of R, Rx, Ry, R1, R2, is as defined above with respect to a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula E :
Wherein each of Rx, Rye m are as defined above for a compound of the formula V; and (b) reacting the compound of formula E with an olefin of formula J:
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, R1 and R2 is as defined above for a compound of the formula V, In the presence of a nucleophilic base sterically hindered to form a compound of the formula V. In some embodiments, the present invention provides a method for preparing a compound of the formula G :
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, R1, R2 and R4 are as described here with respect to a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula K:
Wherein each of R2 and R4 is as defined above for a compound of the formula G; and (b) reacting the compound of formula K to form a compound of formula G.
[000116] As described above, in some modalities of intermediate G, LG is a halogen. In some of such embodiments, a compound of formula K is treated with a halide salt. In some embodiments, a compound of formula K is treated with a sodium halide. In some of such embodiments, a compound of the formula is treated with sodium iodide. In some embodiments, intermediate K is treated with a halide salt in the presence of an acid. Suitable acids include mineral acids and organic acids. In some embodiments, intermediate K is treated with a halide salt and an organic acid such as acetic acid. In some embodiments, intermediate K is treated with sodium iodide in the presence of acetic acid to provide a compound of formula G.
[000117] In some embodiments, the present invention provides a method for the preparation of a compound of formula K:
Where each of R2 and R4 is as defined above with respect to a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula L:
Where R2 is hydrogen, deuterium, tritium or halogen; and (c) reacting the compound of formula L with HN (R4), wherein each R4 is as defined above with respect to a compound of formula K, to form a compound of formula K.
[000118] In some embodiments, a compound of formula L is treated with an amide coupling agent in the presence of HN (R4) 2 to form a compound of formula K. Suitable amide coupling agents include HOBt, HOAt, HAMDU , HAMTU, PyBOP, PyBrOP, TBTU, HATU and T3P. One skilled in the art will recognize that the use of such amide coupling reagents requires the use of a base. Suitable bases include organic bases, such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylpyridine (DMAP), and the like.
[000119] In some embodiments, a compound of formula L is reacted with a chlorinating agent such as thionyl chloride to form an acyl chloride, which is then reacted with HN (R4) 2 to form a compound of formula K.
[000120] In some embodiments, the present invention provides a method for preparing a compound of formula G:
Where: LG is halogen, -OSO2R or -OSO2CF3; and Each of R, R1, R2 and R4 are as defined above with respect to a compound of the formula Z, comprising the steps of: (a) providing a propargylic acid of the formula L:
Where R2 is as defined above for a compound of the formula G; (b) reacting the compound of formula L with an alcohol having the formula HO-R to form a propargyl ester of formula M:
Wherein each of R and R2 is as defined above for a compound of the formula G; (c) reacting the propargyl ester of formula M to form a compound of formula N:
Wherein each of R, R1, R2 and LG are as defined above for a compound of the formula G; (d) hydrolyze the compound of formula N to form a compound of formula Q:
Where each R, R1, R2 and LG are as defined above for a compound of the formula G; and (e) reacting the compound of formula Q with HN (R4) 2, wherein each R4 is as defined above for a compound of formula G, to form a compound of formula G.
[000121] In some embodiments, a propargylic acid of formula L is treated with an alcohol to form a propargyl ester of formula M. Suitable alcohols include methanol, ethanol and isopropanol. A person skilled in the art will recognize that the esterification of a propargylic acid of formula L can be carried out by catalytic acid. Thus, in some embodiments, a propargylic acid of formula L is treated with methanol or ethanol, in the presence of catalytic sulfuric acid to provide a propargyl ester of formula M.
[000122] A person skilled in the art will recognize that such esterification can be carried out at temperatures of approximately 25 ° C to approximately 100 ° C, or up to the boiling point of alcohol. In some embodiments, the esterification of a propargylic acid of formula L is heated to reflux (boiling point of alcohol).
[000123] As described above, in some embodiments of a compound of formula N, LG is a halogen. In some of such embodiments, a compound of formula M is treated with a halide salt. In some embodiments, a compound of formula M is treated with a sodium halide. In some of these embodiments, a compound of formula M is treated with sodium iodide. In some embodiments, a compound of formula M is treated with a halide salt in the presence of an acid. Suitable acids include mineral acids and organic acids. In some embodiments, a compound of formula M is treated with a halide salt and an organic acid such as acetic acid. In some embodiments, a compound of formula M is treated with sodium iodide in the presence of acetic acid to provide a compound of formula N.
[000124] In some embodiments, the ester of a compound of formula N is hydrolyzed with acrylic acid. Suitable hydrolysis conditions are known to those skilled in the art and include hydroxide, such as lithium hydroxide, sodium hydroxide, potassium hydroxide and cesium hydroxide, in the presence of water. A person of ordinary skill in the art will recognize that such hydrolysis can be carried out at temperatures of approximately 25 ° C to approximately 100 ° C. In some embodiments, the hydrolysis of an acrylate of formula N is heated to reflux.
[000125] In some embodiments, an acrylic acid of formula Q is reacted with HN (R4) 2 to form a compound of formula G. In some embodiments, an acrylic acid of formula Q is treated with an amide coupling agent in the presence of HN (R4) 2 to form a compound of formula G. Suitable amide coupling agents include HOBt, HOAt, HAMDU, HAMTU, PyBOP, PyBrOP, TBTU, HATU and T3P. A person of ordinary skill in the art will recognize that the use of such amide coupling reagents requires the use of a base. Suitable bases include organic bases, such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylpyridine (DMAP), and the like.
[000126] In some embodiments, a compound of formula Q is reacted with a chlorinating agent such as thionyl chloride to form an acyl chloride, which is then reacted with HN (R4) 2 to form a compound of formula G.
[000127] In some embodiments, the present invention provides a method of delivering a compound of formula V:
or a pharmaceutically acceptable salt thereof, wherein each of R, Rx, Ry, R1, R2 and m are as defined above with respect to a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula L:
wherein R2 is as defined above for a compound of formula V; (b) reacting the compound of formula L with to form a compound of formula R:

wherein R2 is as defined above for a compound of formula V; (c) reacting the compound of formula R to provide a compound of formula J:
Where: LG is halogen, -OSO2R or -OSO2CF3; and each of R, R1 and R2 is as defined above for a compound of formula V; and (d) reacting the compound of formula J with a compound of formula E:
wherein each of Rx, Rye m is as defined above for a compound of formula V, in the presence of a sterically hindered nucleophilic base to provide a compound of formula V.
[000128] In some embodiments, the present invention provides a method of providing a compound of formula V:
or a pharmaceutically acceptable salt thereof, wherein each of R, Rx, Ry, R1, R2 and m are as defined above with respect to a compound of the formula Z, comprising the steps of: (a) providing a compound of the formula L:
wherein R2 is as defined above for a compound of formula V; (b) reacting the compound of formula L with an alcohol having the formula HO-R to form a compound of the formula M:
wherein each of R and R2 is as defined above for a compound of formula V, (c) reacting the compound of formula M to provide a compound of formula N:
where: LG is halogen, -OSO2R or -OSO2CF3; and each of R, R1 and R2 is as defined above for a compound of formula V; (d) hydrolyze the compound of formula N to form a compound of formula Q:
wherein each of R1, R2 and LG is as defined above for a compound of formula V; (e) reacting the compound of formula Q with F> ■ / to form a compound of formula J:

where: LG is halogen, -OSO2R or -OSO2CF3; and each of R, R1 and R2 is as defined above for a compound of formula V; and (f) reacting the compound of formula J with a compound of formula E:
wherein each of Rx, Rye m is as defined above for a compound of formula V, in the presence of a sterically hindered nucleophilic base to provide a compound of formula V. Definitions
[000129] The compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses and species disclosed herein. As used herein, the following definitions apply unless otherwise indicated. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75aEd. In addition, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, Science University Books, Sausalito: 1999, and "March’s Advanced Organic Chemistry", 5th Ed, Ed :. Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire content of which is incorporated by reference.
[000130] Unless otherwise specified in this specification, the nomenclature used in this specification generally follows the examples and rules set out in the Organic Chemistry Nomenclature, sections A, B, C, D, E, F and H, Pergamon Press, Oxford, 1979, which is incorporated by reference, for its exemplary chemical structure names and rules on how to name chemical structures. Optionally, a compound name can be generated using a chemical nomenclature program: ACD / ChemSketch, Version 5.09 / September 2001, Advanced Chemistry Development, Inc., Toronto, Canada.
[000131] The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (for example, as described in: EL Eliel and SH Wilen, Stereo-chemistry of carbon compounds, by John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, or enantiomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention.
[000132] The term "aliphatic" or "aliphatic group", as used herein, designates a monovalent hydrocarbon radical, which is linear (i.e., unbranched), branched or cyclic (including fused, bonded, and polycyclic spiro -fused). An aliphatic group may be saturated or may contain one or more units of unsaturation, but it is not aromatic. Unless otherwise specified, aliphatic groups contain 1-6 carbon atoms. However, in some embodiments, an aliphatic group contains 1-10 or 2-8 carbon atoms. In some embodiments, the aliphatic groups contain 1-4 carbon atoms, and in still other embodiments, the aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl, and their hybrids such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl.
[000133] The term "alkyl", as used herein, means an saturated, straight chain or branched aliphatic group. In one respect, an alkyl group contains 1-10 or 2-8 carbon atoms. Alkyl includes, but is not limited to, methyl, ethyl, propyl, iso-propyl, n-butyl, sec-butyl, t-butyl, and the like.
[000134] The term "alkenyl", as used herein, means a straight or branched chain aliphatic group having one or more carbon-carbon double bonds (i.e., -CH = CH-). In one aspect, an alkenyl group has two to eight carbon atoms, and includes, for example, and is not limited to, ethylene, 1-propenyl, 1-butenyl and the like. The term "alkenyl" embraces radicals that have carbon-carbon double bonds in "cis" and "trans" or, alternatively, "E" and "Z" configurations. If an alkenyl group includes more than one carbon-carbon double bond, each carbon-carbon double bond is, independently, a cis or trans double bond, or a mixture thereof.
[000135] The term "alkynyl", as used herein, means a straight or branched chain aliphatic radical having one or more carbon-carbon triple bonds (i.e., -C = C-). In one aspect, an alkyl group has two to eight carbon atoms, and includes, for example, and is not limited to, 1-propynyl (propargyl), 1-butynyl and the like.
[000136] The terms "cycloaliphatic", "carbocyclyl", "carbocycle" and "carbocyclic", used alone or as part of a larger fraction, refer to a saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring system, such as described here, having 3 to 10 members, wherein the aliphatic ring system is optionally substituted as defined above and described here. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyla, cycloheptenyl, cyclooctyl, cyclooctenyl and cyclooctadienyl. The terms "cycloaliphatic", "carbocyclyl", "carbocycle" and "carbocyclic" also include aliphatic rings that are fused with one or more aromatic or non-aromatic rings, such as decahydronaphil, tetrahydronaphil, decalin, or bicycles [2 , 2.2] octane.
[000137] The term "cycloalkyl", as used herein, means a cyclic saturated aliphatic monocyclic or bicyclic ring system with 3-10 members. A cycloalkyl can be optionally substituted as described herein. In some embodiments, a cycloalkyl group has 3-6 carbons.
[000138] The term "heterocycloalkyl", as used herein, means an aliphatic, saturated or unsaturated ring system, in which at least one carbon atom is replaced with a heteroatom selected from N, S and O. The heterocycloalkyl can contain one or more rings, which can be attached together in a pendant manner or can be fused. In one aspect, a heterocycloalkyl is a three to seven-membered ring system and includes, for example, and is not limited to, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrofuranyl and the like.
[000139] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus or silicon, and includes any oxidized form of nitrogen, sulfur, phosphorus, or silicon, the quaternized form of any basic nitrogen, and a nitrogen substitutable of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl).
[000140] The term "unsaturated", as used herein, means that a fraction has one or more units of unsaturation.
[000141] The term "halo" or "halogen", as used herein, means halogen and includes, for example, and is not limited to, fluorine, chlorine, bromine, iodine and the like, in both radioactive and non-radioactive forms radioactive.
[000142] The term "haloalkyl", as used herein, means an aliphatic group, which is replaced with one or more halogen atoms. In some embodiments, haloalkyl refers to a perhalogenated aliphatic group. In some embodiments, haloalkyl refers to an alkyl group that is replaced with one or more halogen atoms. Exemplary haloalkyl groups include -CF3, -CCl3, -CF2CH3, -CH2CF3, -CH2 (CF3) 2, -CF2 (CF3) 2, and the like.
[000143] The term "aryl", alone or in combination, as used herein, means an aromatic carbocyclic system containing one or more rings, which can be attached together in a pendant manner or can be fused. In particular modalities, arila is one, two or three rings. In one aspect, the aryl has five to twelve ring atoms. The term "aryl" encompasses aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl and acenaftyl. An "aryl" group can have 1 to 4 substituents, such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino and the like.
[000144] The term "heteroaryl", alone or in combination, as used herein, means an aromatic system in which at least one carbon atom is replaced by a heteroatom selected from N, S and O. A heteroaryl may contain one or more rings, which can be attached together in a pendant manner or can be fused. In particular modalities, heteroaryl is one, two or three rings. In one aspect, heteroaryl has five to twelve ring atoms. The term "heteroaryl" encompasses heteroaromatic groups such as triazolyl, imidazolyl, pyrrolyl, pyrazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, furyl, benzofuryl, thienyl, benzothienyl, quinolyl, oxazolyl and oxazolyl, oxazolyl and oxazolyl, oxazolyl and oxazolyl, oxazolyl and oxazolyl, oxazolyl and oxazolyl. A "heteroaryl" group can have 1 to 4 substituents, such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy, lower alkylamino and the like.
[000145] It is understood that substituents and substitution patterns in the compounds of the invention can be selected by a person skilled in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as the methods described below . In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated fraction are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent in each replaceable position in the group, and when more than one position in a given structure may be replaced with more than one substituent selected from a specified group, the substituent can be the same or different at each position. Alternatively, an "optionally substituted" group may be unsubstituted.
[000146] The combinations of substituents provided for by this invention are preferably those that result in the formation of stable or chemically feasible compounds. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon atom or on different carbon atoms, as long as it results in a stable structure. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions that permit their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more more of the purposes revealed here.
[000147] Suitable monovalent substituents on a replaceable carbon atom of an "optionally substituted" group are, independently, halogen, - (CH2) o-4R °; - (CH2) o-4OR °; -O (CH2) o-4Ro, -O- (CH2) o-4C (O) OR °; - (CH2) o- 4CH (OR °) 2; - (CH2) O-4SR °; - (CH2) 0-4Ph, which can be replaced with R °; - (CH2) o-4O (CH2) o-1Ph that can be replaced with R °; -CH = CHPh, which can be replaced with R °; - (CH2) o-4O (CH2) o-1-pyridyl which can be substituted with R °; -NO2; -CN; -N3; - (CH2) o-4N (R °) 2; - (CH2) o-4N (R °) C (O) R °; -N (R °) C (S) R °; - (CH2) o-4N (R °) C (O) NR ° 2; -N (R °) C (S) NR ° 2; - (CH2) o-4N (R °) C (O) OR °; -N (R °) N (R °) C (O) R °; -N (R °) N (R °) C (O) NR ° 2; -N (R °) N (R °) C (O) OR °; - (CH2) o-4C (O) R °; -C (S) R °; - (CH2) o-4C (O) OR °; - (CH2) o-4C (O) SR °; - (CH2) o-4C (O) OSiR ° 3; - (CH2) o-4OC (O) R °; -OC (O) (CH2) o-4SR-, SC (S) SR °; - (CH2) o-4SC (O) R °; - (CH2) o-4C (O) NR ° 2; -C (S) NR ° 2; -C (S) SR °; -SC (S) SR °, - (CH2) o-4OC (O) NR ° 2; -C (O) N (OR °) R °; -C (O) C (O) R °; -C (O) CH2C (O) R °; -C (NOR °) R ° ;-( CH2) o-4SSR °; - (CH2) o-4S (O) 2R °; - (CH2) o-4S (O) 2OR °; - (CH2) o-4OS (O) 2R °; -S (O) 2NR ° 2; - (CH2) o-4S (O) R °; -N (R °) S (O) 2NR ° 2; -N (R °) S (O) 2R °; -N (OR °) R °; -C (NH) NR ° 2; -P (O) 2R °; -P (O) R ° 2; -OP (O) R ° 2; -OP (O) (OR °) 2; SiR ° 3; - (straight or branched C1-4 alkylene) O-N (R °) 2; or - (linear or branched C1-4 alkylene) C (O) ON (R °) 2, where each R ° can be substituted as defined below and is independently hydrogen, aliphatic C1-6, -CH2Ph, -O (CH2 ) o-1Ph, -CH2- (5-6 membered heteroaryl ring) or a 5-6 membered partially saturated aryl ring having o-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, despite the above definition, two independent occurrences of R °, taken together with their intermediate atom (s), form a partially unsaturated, partially saturated, monocyclic or bicyclic aryl ring of 3-12 members with 0-4 heteroatoms selected independently from nitrogen, oxygen and sulfur, which can be replaced as defined below.
[000148] Suitable monovalent substituents on R ° (or the ring formed taking two independent occurrences of R ° together with their intervening atoms), are independently halogen, - (CH2) O-2R *, - (haloR *) , - (CH2) o — 2θH, - (CH2) O-20R *, - (CH2) O-2CH (OR *) 2; -O (haloR *), -CN, -N3, - (CH2) O-2C (0) R *, - (CH2) o-2C (O) OH, - (CH2) o-2C (O) OR * , - (CH2) o-2SR *, - (CH2) o-2SH, - (CH2) o-2NH2, - (CH2) o-2NHR *, - (CH2) o-2NR * 2, -NO2, -SiR * 3, -OSiR * 3, -C (O) SR *, - (straight or branched C1-4 alkylene) C (O) OR *, or -SSR * where each R * is unsubstituted or when preceded by " halo "is replaced with just one or more halogens, and is independently selected from C1-4 aliphatic, -CH2Ph, -O (CH2) o-1Ph, or a 5-6 partially unsaturated saturated aryl ring members having o-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R ° include = O and = S.
[ooo149] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: = O, = S, = NNR * 2, = NNHC (O) R *, = NNHC (O) OR *, = NNHS (O) 2R *, = NR *, = NOR *, -O (C (R * 2)) 2-3O-, and -S (C (R * 2)) 2-3S-, where each independent occurrence of R * is selected from hydrogen, aliphatic C1-6 that can be substituted, as defined below, or an unsubstituted, 5-6 membered, saturated, partially unsaturated aryl ring having selected O-4 hetero atoms regardless of nitrogen, oxygen and sulfur. Suitable divalent substituents that are attached to substitutable vicinal carbon atoms of an "optionally substituted" group include: -O (CR * 2) 2-3O-, where each independent occurrence of R * is selected from hydrogen, C1 -6 aliphatic which can be replaced as defined below, or an 5-6 membered, unsubstituted, partially unsaturated, aryl ring with 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
[000150] Suitable substituents on the aliphatic group of R * include halogen -R *, - (haloR *), -OH, - OR *, -OChaloR *), -CN, -C (O) OH, -C (O) OR *, -NH2, —NHR *, —NR * 2, and -NO2, where each R * is unsubstituted or where preceded by “halo” is replaced only with one or more halogens, and is independently C1-4 aliphatic , -CH2Ph, -O (CH2) 0-1Ph, or a 5-6- membered, saturated, partially unsaturated aryl ring, having 0-4 heteroatoms selected independently of nitrogen, oxygen and sulfur.
[000151] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group, include -R +, -NR + 2, -C (O) R +, -C (O) OR +, -C (O) C (O) Rt, -C (O) CH2C (O) R +, -S (O) 2Rf, -S (O) 2NRf2, -C (S) NR + 2, -C (NH) NRf2, and -N (Rt) S (O) 2Rt; wherein each Rfé independently hydrogen, C1-6 aliphatic which can be substituted as defined below, unsubstituted -OPh, or a 5-6 membered saturated, partially unsaturated aryl ring, having 0-4 heteroatoms selected independently from nitrogen, oxygen, and sulfur, or, despite the above definition, two independent occurrences of Rftomates together with their intervening atom (s) form an unsubstituted 3-12 membered monocyclic or bicyclic aryl ring , partially unsaturated, with 0-4 heteroatoms selected independently from nitrogen, oxygen and sulfur.
[000152] Suitable substituents on the R's aliphatic group are independently halogen, -R *, - (haloR *), -OH, -OR *, -O (haloR *), -CN, -C (O) OH, - C (O) OR *, -NH2, -NHR *, -NR * 2, or -NO2, where each R * is unsubstituted or where preceded by "halo" is replaced only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -O (CH2) 0-1Ph, or a saturated, partially unsaturated 5-6-membered aryl ring, having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.
[000153] As used herein, "hydrazine equivalent" means a chemical reagent that can be used to introduce an N-N-fraction into a molecule. Hydrazine equivalents include hydrazine hydrate, as well as protected forms of hydrazine, such as hydrazine tert-butyl carboxylate.
[000154] As used herein, "leaving group" refers to a functional group that is displaced from a molecule during a chemical reaction. The leaving groups include halogens, as well as sulfonate groups, such as tosylate and mesylate.
[000155] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are, within the scope of good medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicities, irritation , allergic response and the like, and are commensurable with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge and others describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from appropriate inorganic and organic acids and bases that are compatible with the treatment of patients.
[000156] Examples of pharmaceutically acceptable non-toxic acid addition salts are the salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid , oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or using other methods used in the art such as ion exchange. Other different pharmaceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanopropionate, digluconate, dodecylsulfate, formate, fumarate, ethanosulfonate glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, iodhydrate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate oxide, nitrate, nitrate oxide, nitrate, nitrate , pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.
[000157] In some embodiments, exemplary inorganic acids that form appropriate salts include, but are not limited to, hydrochloric, hydrobromic, sulfuric and phosphoric acid and metallic acid salts such as sodium ortho-phosphate and hydrogen sulfate of potassium. Illustrative organic acids that form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroximalic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenolic. , p-toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Either the mono- or diacid salts can be formed and these salts can exist either in a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of these compounds are more soluble in water and in various hydrophilic organic solvents, and in general, show higher melting points compared to their free base forms.
[000158] In some embodiments, the acid addition salts of the compounds of formula I are more suitably formed from pharmaceutically acceptable acids, and include, for example, those formed with inorganic acids, for example, hydrochloric, sulfuric or phosphoric acid and organic acids, for example, succinic, maleic, acetic or fumaric acid.
[000159] Other non-pharmaceutically acceptable salts, for example, oxalates can be used, for example, in the isolation of compounds of formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are base addition salts (such as sodium, potassium and ammonium salts), solvates and hydrates of the compounds of the invention. The conversion of a given compound salt to a desired compound salt is accomplished by applying conventional techniques, well known to a person skilled in the art.
[000160] A "pharmaceutically acceptable base addition salt" is any non-toxic organic or inorganic base addition salt of the acid compounds represented by formula I or any of its intermediates. Illustrative inorganic bases that form suitable salts include, but are not limited to, lithium, sodium, potassium, calcium, magnesium or barium hydroxides. Illustrative organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt may be important so that an ester functionality, if any, in other positions of the molecule is not hydrolyzed. The selection criteria for the appropriate salt will be known to a person skilled in the art.
[000161] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1-4 alkyl) 4 salts. Representative alkaline or alkaline earth salts of earth metal include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate .
[000162] Unless otherwise indicated, the structures illustrated here are also intended to include all isomeric (eg, enantiomeric, diastereoisomeric and geometric (or conformational) forms) of the structure, for example, the R and S configurations for each center asymmetric, double bond Z and E isomers, and Z and E conformation isomers. Therefore, the individual stereochemical isomers as well as enantiomeric, diastereoisomeric and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
[000163] Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by replacing a hydrogen atom with deuterium or tritium, or a carbon atom with a 13C or 14C enriched carbon are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention.
[000164] The term "stereoisomers" is a general term for all isomers of an individual molecule that differ only in the orientation of their atoms in space. It includes mirror image isomers (enantiomers), geometric (cis / trans) isomers and isomers of compounds with more than one chiral center that are not mirror images of each other (diastereomers).
[000165] The term "treatment" or "treating" means to alleviate one or more symptoms, eliminate the cause of one or more symptoms, either on a temporary or permanent basis, or to prevent or delay the onset of one or more symptoms associated with a disorder or condition.
[000166] The term "therapeutically effective amount" means an amount of a compound that is effective in treating or decreasing the severity of one or more symptoms of a disorder or condition.
[000167] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant or other material that is mixed with the active ingredient in order to allow the formation of a pharmaceutical composition, that is, a form of dosage capable of being administered to a patient. An example of such a vehicle is the pharmaceutically acceptable oil typically used for parenteral administration. Pharmaceutically acceptable vehicles are well known in the art.
[000168] By presenting the elements disclosed herein, the articles "one", "one", "o", and "referred" are intended to mean that one or more of the elements exist. The terms "comprising", "having" and "including" are intended to be unlimited and mean that there may be additional elements in addition to the listed elements. Formulation and Administration Pharmaceutically acceptable compositions
[000169] Another embodiment of the invention is a composition comprising a compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or carrier. The amount of compound in a composition of the invention is an amount that is effective to measurably inhibit CRM1 in a biological sample or a patient. In certain embodiments, a composition of the invention is formulated for administration to a patient in need of the composition. The term "patient", as used herein, means an animal. In some embodiments, the animal is a mammal. In certain embodiments, the patient is a veterinary patient (that is, a non-human mammalian patient). In some embodiments, the patient is a dog. In other modalities, the patient is a human being.
[000170] The phrase "pharmaceutically acceptable vehicle, adjuvant or vehicle" refers to a non-toxic vehicle, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable vehicles, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl-pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene polymers, polyethylene glycol and wool fat.
[000171] The compositions of the present invention can be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously and intradermally), by inhalation spray, topically, rectally, nasal, buccally, vaginally or via an implanted reservoir . In some embodiments, the compounds or compositions provided are administrable intravenously and / or intraperitoneally.
[000172] The term "parenteral", as used herein, includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic injection, intralesional intraperitoneal injection or infusion techniques and intracranial. Preferably, the compositions are administered orally, subcutaneously, intraperitoneally or intravenously. Sterile injectable forms of the compositions of the present invention can be an aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing agents or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a parenterally acceptable, non-toxic diluent, or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium.
[000173] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, suspensions and aqueous solutions. In the case of tablets for oral use, commonly used vehicles include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifiers and suspending agents. If desired, certain sweetening, flavoring or coloring agents can also be added. In some embodiments, a presented oral formulation is formulated for immediate release or continuous / delayed release. In some embodiments, the composition is suitable for oral or sublingual administration, including tablets and lozenges. A compound provided can also be in microencapsulated form.
[000174] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. The pharmaceutically acceptable compositions of the present invention can also be administered topically, especially when the target of treatment includes areas or organs easily accessible by topical application, including diseases of the eyes, the skin, or the lower intestinal tract. Appropriate topical formulations are easily prepared for each of these areas or organs.
[000175] Topical application to the lower intestinal tract can be carried out in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically transdermal patches can also be used.
[000176] For ophthalmic use, pharmaceutically acceptable compositions can be formulated as micronized suspensions or in an ointment, such as petrolatum.
[000177] The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or inhalation.
[000178] In some embodiments, the pharmaceutically acceptable compositions of the present invention are formulated for intraperitoneal administration.
[000179] The amount of compounds of the present invention that can be combined with vehicle materials to produce a composition in a single dosage form will vary depending on the treated host and the particular mode of administration. In one embodiment, a composition is formulated so that a dosage between 0.01-100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving the composition. In another embodiment, the dosage is approximately 0.5 to approximately 100 mg / kg of body weight, or between 1 mg and 1000 mg / dose, every 4 to 120 hours, or according to the requirements of the particular drug. Typically, the pharmaceutical compositions of this invention will be administered approximately 1 to approximately 6 times a day.
[000180] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, schedule of administration, the rate of excretion, combination of drugs, the decision of the treating physician and the severity of the particular disease to be treated. The amount of a compound of the present invention in the composition will also depend on the particular compound in the composition.
[000181] In some embodiments, the composition further includes one or more additional therapeutic or prophylactic agents. When the compositions of the present invention comprise a combination of a compound of the formulas described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent must be present at dosage levels of between approximately 1 to 100%, and more preferably between approximately 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents can be administered separately, as part of a multiple dose regimen, of the compounds of the present invention. Alternatively, the additional agents can form part of a single dosage form, mixed together with a compound of the present invention in a single composition.
[000182] After the improvement of a patient's condition, a maintenance dose of a compound, composition or combination of the present invention can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been relieved to the desired level. Patients may, however, need intermittent treatment on a long-term basis after any recurrence of disease symptoms. Uses of pharmaceutically acceptable compounds and compositions
[000183] The compounds and compositions described herein are generally useful for the inhibition of CRM1 and are, therefore, useful for the treatment of one or more diseases associated with the activity of CRM1. Thus, in certain embodiments, the present invention provides a method for treating a disorder mediated by CRM-1 comprising the step of administering to a patient in need of it, a compound of the present invention, or a pharmaceutically acceptable salt or composition the same. The compounds and compositions described herein can also be administered to cells in culture, for example, in vitro or ex vivo, or to a subject, for example, in vivo, to treat, prevent and / or diagnose a variety of disorders, including those described here below.
[000184] The activity of a compound used in the present invention as a CRM1 inhibitor can be assayed in vitro, in vivo or in a cell line. Detailed conditions for testing a compound used in the present invention as a CRM1 inhibitor are shown in the Exemplification.
[000185] As used herein, the term "disorder or condition mediated by CRMl" or "disorder or condition associated with CRMl activity" means any disease or other harmful condition in which CRMl plays a role. Therefore, another embodiment of the present invention relates to the treatment or reduction of the severity of one or more diseases in which CRM1 plays a role. In some embodiments, the present invention provides methods of treating a disease associated with the expression or activity of the p53, p73, p21, pRb, p27, IKB, NFkB, c-Abl, FOXO proteins, COX-2 proteins in one subject, which comprises administering to the patient a therapeutically effective amount of a compound described herein. In another embodiment, the present invention relates to a method of treating or decreasing the severity of a disease or condition selected from a proliferative disorder (eg, cancer), an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder or a neurodegenerative disease, the method comprising administering to a patient in need thereof, a compound or composition according to the present invention. In a more specific embodiment, the present invention relates to a method of treating or decreasing the severity of the cancer. The specific examples of diseases above are presented in detail below.
[000186] Cancers treatable by the compounds of the present invention include, but are not limited to, hematological malignancies (leukemias, lymphomas, myelomas, myelodysplastic and myeloproliferative syndromes) and solid tumors, such as carcinomas (prostate, breast, lung, colon, pancreas, kidney, ovary as well as soft tissue and osteosarcomas, and stromal tumors). Breast cancer (BC) can include, Basal-like breast cancer (BLBC), triple negative breast cancer (TNBC) and breast cancer that is BLBC and TNBC. In addition, breast cancer can include invasive or non-invasive, tubular, medullary, mucinous, papillary duct or lobe carcinoma, cribriform breast carcinoma, male breast cancer, recurrent or metastatic breast cancer, breast tumor phyllodes, Paget's disease of the nipple.
[000187] Inflammatory disorders that can be treated by the compounds of the present invention include, but are not limited to, multiple sclerosis, rheumatoid arthritis, degenerative joint disease, systemic lupus, systemic sclerosis, vasculitis syndromes (small, medium and large vessels) , atherosclerosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucous colitis, ulcerative colitis, gastritis, sepsis, psoriasis and other dermatological inflammatory diseases (such as eczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma, psoriasis and dermatoses with acute inflammatory components, pemphigus, pemphigoid, allergic dermatitis), and urticaria syndromes. In some embodiments, the disorder or condition associated with CRM1 activity is multiple sclerosis, irritable bowel syndrome, rheumatoid arthritis, psoriasis or other inflammatory skin diseases.
[000188] Viral diseases that can be treated by the compounds of the present invention include, but are not limited to, acute febrile pharyngitis, conjunctival pharyngitis, epidemic keratoconjunctivitis, childhood gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt's lymphoma, acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (eg, gingivostomatitis, in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (eg, cold sores and wounds), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, cytomegalic inclusion disease, Kaposi's sarcoma, multicentric Castleman's disease, primary effusion lymphoma, AIDS, influenza, Reye's syndrome, measles, encephalomyelitis postinfectious, mumps, hyperplastic epithelial lesions (eg, common, smooth, plantar and anogenital warts, laryngeal papillomas, epidermod isplasia verruciformis), cervical carcinoma, squamous cell carcinoma, croup, pneumonia, bronchiolitis, common flu, polio, rabies, influenza-like syndrome, severe pneumonia bronchiolitis, measles, congenital rubella, chickenpox and herpes zoster. Viral diseases that can be treated by the compounds of the present invention also include chronic viral infections, including hepatitis B and hepatitis C.
[000189] Exemplary eye disorders include, but are not limited to, macular edema (diabetic and non-diabetic macular edema), age-related macular degeneration (wet and dry forms), age-varying macular degeneration, cystoid macular edema, eyelid edema, retinal edema, diabetic retinopathy, chorioretinopathy, neovascular maculopathy, neovascular glaucoma, uveitis, iritis, retinal vasculitis, endophthalmitis, panophthalmitis, metastatic ophthalmia, choroiditis, retinal pigment epithelitis, cyclitis, neuritis, scleritis, neuritis, scleritis, neuritis , keratitis, blepharitis, exudative retinal detachment, corneal ulcer, conjunctival ulcer, chronic umular keratitis, ophthalmic disease associated with hypoxia or ischemia, prematurity retinopathy, proliferative diabetic retinopathy, polypoidal choroidal vasculopathy, angiomatous retinal proliferation retina, retinal vein occlusion, Coats' disease, vitreore exudative familial tinopathy, pulseless disease (Takayasu's disease), Eales disease, antiphospholipid antibody syndrome, leukemic retinopathy, blood hyperviscosity syndrome, macro globulinemia, interferon-associated retinopathy, hypertensive retinopathy, radiation retinopathy, epithelial stem cell deficiency of the cornea or cataract.
[000190] Neurodegenerative diseases that can be treated by a compound of the invention include, but are not limited to, Parkinson's disease, Alzheimer's disease, and Huntington's disease and amyotrophic lateral sclerosis (ALS / Lou Gehrig disease). In some embodiments, the disorder or condition associated with a CRM activity is ALS.
[000191] The compounds and compositions described herein can also be used to treat abnormal tissue growth disorders and fibrosis, including dilatable cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pulmonary fibrosis, liver fibrosis, glomerulonephritis, and other renal disorders.
[000192] The compounds and compositions described herein can also be used for diseases related to food intake, such as obesity and hyperphagia. In some modalities, the disorder or condition associated with the CRM1 activity is obesity.
[000193] In some embodiments, the disorder or condition associated with CRM1 activity is muscular dystrophy, arthritis, for example, osteoarthritis and rheumatoid arthritis, ankylosing spondylitis, traumatic brain injury, spinal cord injury, sepsis, rheumatic disease, atherosclerosis cancer, type 1 diabetes, type 2 diabetes, leptospirosis kidney disease, glaucoma, retinal disease, aging, headache, pain, complex regional pain syndrome, cardiac hypertrophy, muscle wasting, catabolic disorders, obesity, growth retardation fetal, hypercholesterolemia, heart disease, chronic heart failure, ischemia / reperfusion, stroke, cerebral aneurysm, angina pectoris, lung disease, cystic fibrosis, acid-induced lung injury, pulmonary hypertension, asthma, chronic obstructive pulmonary disease, Sjogren, hyaline membrane disease, kidney disease, glomerular disease, alcoholic liver disease, bowel diseases, the endome peritoneal triosis, skin diseases, nasal sinusitis, mesothelioma, anhydrous ecodermal dysplasia-ID, Behcet's disease, pigmentary incontinence, tuberculosis, asthma, Crohn's disease, colitis, eye allergy, appendicitis, Paget's disease, pancreatitis, periodonitis, endometriosis, inflammatory bowel disease, inflammatory lung disease, silica-induced diseases, sleep apnea, AIDS, HIV-1, autoimmune diseases, antiphospholipid syndrome, lupus, lupus nephritis, familial Mediterranean fever, hereditary periodic fever syndrome, diseases from psychosocial stress, neuropathological diseases, familial amyloid polyneuropathy, inflammatory neuropathy, Parkinson's disease, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cataracts, or hearing loss.
[000194] In other modalities, the disorder or condition associated with CRM1 activity is head trauma, uveitis, inflammatory pain, allergen-induced asthma, non-allergen-induced asthma, glomerular nephritis, ulcerative colitis, necrotizing enterocolitis, hyperimmunoglobulinemia D with recurrent fever (HIDS), periodic syndrome associated with TNF receptor (TRAPS), periodic syndromes associated with cryopyrin, Muckle-Wells syndrome (urticaria deafness amyloidosis), familial urticaria, neonatal onset multisystem inflammatory disease (NOMID), fever disease, aphthous stomatitis, pharyngitis and adenitis (PFAPA syndrome), Blau syndrome, sterile pyogenic arthritis, gangrenous pyoderma, acne (PAPA), interleukin -1 receptor antagonist (DIRA) deficiency, subarachnoid hemorrhage, polycystic kidney disease, transplantation , organ transplantation, tissue transplantation, myelodysplasia syndrome, irritant-induced inflammation, irritant-induced inflammation plant, inflammation induced by poison ivy oil / urushiol, inflammation induced by chemical irritant, inflammation induced by bee sting, inflammation induced by insect sting, sunburn, burns, dermatitis, endotoxemia, lung injury, acute respiratory distress syndrome , alcoholic hepatitis, or kidney damage caused by parasitic infections.
[000195] In another embodiment, a compound or composition described herein can be used to treat or prevent allergies and respiratory diseases, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome; and any chronic obstructive pulmonary disease (COPD).
[000196] Another embodiment of the invention is the use of a compound of formula I in the manufacture of a medicament for the treatment of a disorder or condition associated with CRM1 activity. In further aspects, the present invention provides the use of a compound of formula I for the manufacture of a medicament for the treatment of a disease associated with the expression or activity of p53, p73, p21, pRb, p27, IKB, NFKB, c- Abl, FOXO or COX-2 proteins in a subject. In some embodiments, the present invention provides a use of a compound of formula I in the manufacture of a medicament for the treatment of any cancer and / or neoplastic diseases, angiogenesis, autoimmune disorders, inflammatory diseases and / or diseases, epigenetics, disorders and / or hormonal diseases, viral diseases, neurodegenerative diseases and / or eye diseases and disorders.
[000197] In some embodiments, the present invention provides a method for inhibiting CRM1 in a biological sample or in a patient comprising contacting the biological sample with, or administering to a patient, a pharmaceutically acceptable salt of a compound of formula I, or a pharmaceutically acceptable composition. Neoplastic Disorders
[000198] A compound or composition described herein can be used to treat a neoplastic disease. A "neoplastic disorder" is a disease or disorder characterized by cells that have the capacity for autonomous growth or replication, for example, an abnormal state or condition characterized by a cell growth of benign or malignant proliferation. Exemplary neoplastic disorders include: carcinoma, sarcoma (eg, soft tissue), osteosarcoma, metastatic disorders (eg, tumors resulting from the prostate, brain, bone, gastrointestinal, lung, breast, ovary, cervical, pancreas, kidney, head and neck, and liver), haematopoietic neoplastic disorders (eg, leukemias, lymphomas, myeloma and other malignant plasma cell disorders) and metastatic tumors. In one embodiment, the cancer to be treated is selected from the breast, ovary, cervical, gastrointestinal, prostate, colon, lung, kidney, brain, liver and pancreatic cancer. Treatment with the compound can be in an amount effective to ameliorate at least one symptom of the neoplastic disorder, such as reduced cell proliferation, reduced tumor mass, etc.
[000199] In one embodiment, the neoplastic disorder is a basal-like breast cancer (BLBC). BLBCs account for up to 15% of breast cancers (BC) and are usually triple negative breast cancer (TNBC), characterized by a lack of ER, progesterone receptor PR and HER-2 amplification. In a specific embodiment, breast cancer is TNBC. In addition, most BCs associated with BRCA1 are BLBC and TNBC, expressing basal cytokeratins and EGFR. BLBC is characterized by an aggressive phenotype, high histological grade, and poor clinical results with recurrence and high metastasis rates. Combination therapies
[000200] In some embodiments, a compound described herein is administered in conjunction with a "second" therapeutic agent or additional treatment. The choice of the second therapeutic agent can be made from any agent that is typically used in monotherapy to treat the indicated disease or condition. As used herein, the term "jointly administered" and related terms refer to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a compound of the present invention can be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a compound of formula I, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant or carrier.
[000201] In an embodiment of the invention, in which a second therapeutic agent is administered to a subject, the effective amount of the compound of the present invention is less than its effective amount would be if the second therapeutic agent were not administered. In another embodiment, the effective amount of the second therapeutic agent is less than its effective amount would be if the compound of the invention was not administered. In this way, the undesirable side effects associated with high doses of either agent can be minimized. Other potential advantages (including, without limitation, improved dosing regimens and / or reduced drug cost) will be evident to those skilled in the art.
[000202] Exemplary additional cancer treatments include, for example: chemotherapy, target therapies, such as antibody therapies, kinase inhibitors, immunotherapy and hormonal therapy, epigenetic therapy, proteasome inhibitors and anti-angiogenic therapies. Examples of each of these treatments are provided below.
[000203] Examples of chemotherapeutic agents used in cancer therapy include, for example, anti-metabolites (for example, folic acid, purine and pyrimidine) and alkylating agents (for example, nitrogen mustards, nitrosoureas, platinum, alkylsulfonates, hydrazines, triazenes, aziridines, spindle poison, cytotoxic agents, topoisomerase inhibitors and others). Exemplary agents include aclarrubicin, actinomycin, alitretinoin, altretamine, aminopterin, aminolevulinic acid, amrubicin, amsacrine, anagrelide, arsenic trioxide, asparaginase, tardentan, belotecan, bexarotene, bendamustin, carcinogen, carbohydrate, carcinogen, busulfine, carcinogen, bromide , carmustine, celecoxib, chlorambucil, chlormetin, cisplatin, cladribine, clofarabine, chrysantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, demecolcine, docetaxel, doxorubicin, ethoxycitine, ethanol, efaproxiral , floxuridine, fludarabine, fluorouracil (5-FU), photemustine, gemcitabine, gliadel implants, hydroxycarbamide, hydroxyurea, idarubicin, ifosfamide, irinotecan, irofulven, ixabepilone, larotaxeline, liporomine, lorinexine, lipid, lorine, lipid mannosulfan, masoprocol, melphal year, mercaptopurine, mesna, methotrexate, methyl aminolevulinate, mitobronitol, mitoguazone, mitotane, mitomycin, mitoxantrone, nedaplatin, nimustine, oblimersen, omacetaxine, ortataxel, oxaliplatin, paclitaxel, piperatine, piperatamine, pegaspar, pegaspar sodium, prednimustine, procarbazine, raltitrexed, ranimustine, rubitecan, sapacitabine, semustina, sitimagene ceradenovec, strataplatin, streptozocin, talaporfin, tegafur-uracil, temoporfin, temozolomide, teniposido, tetone, tetone, tetone, tetone, tetone, tetone, tetanone , trabectedin, triaziquone, triethylene melamine, triplatin, tretinoin, treosulfan, trophosphamide, uramustine, valrubicin, verteporfin, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vorinostat, zorubicin and other cytotoxic agents described here.
[000204] Since some drugs work better together than alone, two or more drugs are often given at the same time. Often, two or more chemotherapy agents are used as the chemotherapy combination. In some embodiments, chemotherapy agents (including combination chemotherapy) can be used in combination with a compound described herein.
[000205] Targeted therapy is the use of specific agents for deregulated proteins in cancer cells. Molecular targeted therapy drugs are generally inhibitors of enzyme domains in mutant, overexpressed, or otherwise critical proteins within a cancer cell. Prominent examples are tyrosine kinase inhibitors, such as axitinib, bosutinib, cediranib, desatinib, erolotinib, imatinib, gefitinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib and vandetanib, and also kinase inhibitors, and kinase inhibitors and kinase inhibitors, seliciclib. Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody that specifically binds to a protein on the surface of cancer cells. Examples include the anti-HER2 / neu trastuzumab antibody (Herceptin®) typically used in breast cancer, and the anti-CD20 antibody rituximab and tositumomab typically used in a variety of B-cell malignancies. Other exemplary antibodies include cetuximab, panitumumab, trastuzumab, alemtuzumab, bevacizumab, edrecolomab and gemtuzumab. Examples of fusion proteins include aflibercept and denileucine diftitox. In some embodiments, the target therapy can be used in combination with a compound described herein, for example, Gleevec (Vignari and Wang, 2001).
[000206] Targeted therapy can also involve small peptides as "home" devices, which can bind to receptors on the affected cell surface or extracellular matrix surrounding a tumor. The radionuclides that are attached to these peptides (for example, RGDS) eventually kill the cancer cell if the nuclide is in proximity to the cell. An example of such therapy includes BEXXAR®.
[000207] Anti-angiogenic therapy may include kinase inhibitors that target vascular endothelial growth factor (VEGF) such as sunitinib, sorafenib, or monoclonal or “attractive” antibodies to VEGF receptors or VEGF receptors including bevacizumab or VEGF-Trap, or thalidomide or its analogs (lenalidomide, Pomalidomide), or agents that target non-VEGF angiogenic targets such as fibroblast growth factor (FGF), angiopoietins, angiostatin or endostatin.
[000208] Epigenetic therapies include enzyme inhibitors that control epigenetic modifications, specifically DNA methyltransferases and histone deacetylases, which have shown promising antitumor effects for some malignancies, as well as antisense oligonucleotides and siRNA.
[000209] Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor. Contemporary methods for generating an immune response against tumors include intravesicular BCG immunotherapy for superficial bladder cancer, the Provenge prostate cancer vaccine and the use of interferons and other cytokines to induce an immune response in patients with renal cell carcinoma and melanoma.
[000210] Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy, since donor immune cells often attack the tumor in a graft versus tumor effect. In some embodiments, immunotherapy agents can be used in combination with a compound described herein.
[000211] Hormone therapy agents include the administration of hormone agonists or hormone antagonists and include retinoids / retinoic acid, compounds that inhibit estrogen or testosterone, as well as the administration of progestogens.
[000212] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described for illustrative purposes only and are not intended to limit the scope of the invention. Changes in the form and replacement of equivalents are considered as circumstances may suggest or make convenient. Although specific terms have been used here, these terms are intended in a descriptive sense and not for purposes of limitation. EXEMPLIFICATION Abbreviations
[000213] atm Atmosphere
[000214] aq. Aqueous
[000215] BINAP 2,2'-bis (diphenyl phosphine) -1, 1'-binafty
[000216] Boc tert-butoxycarbonyl
[000217] CDI N, N'-carbonyldiimidazole
[000218] CH2CI2 Dichloromethane
[000219] DCC N, N-dicyclohexylcarbodiimide
[000220] DCM Dichloromethane
[000221] DBU diazan (1,3) -bicycle [5.4.0] undecane
[000222] DIC N, N'-Diisopropylcarbodiimide
[000223] DIPEA N, N-diisopropylethylamine
[000224] DMAP N, N-Dimethyl-4-aminopyridine
[000225] DMF N, N-Dimethylformamide
[000226] DMSO dimethyl sulfoxide
[000227] DPPF diphenylphosphinoferrocene
[000228] EA ethyl acetate
[000229] EDCI N- [3- (dimethyl amino) propyl] -N’-ethylcarbodiimide hydrochloride
[000230] EDC 1-ethyl-3 - (3-dimethylaminopropyl) carbodiimide
[000231] eq. equivalent (s)
[000232] ET20 Diethyl ether
[000233] EtOAc Ethyl acetate
[000234] EtOH Ethanol
[000235] Iodine TSI
[000236] Et Etila
[000237] Fmoc 9-fluorenylmethyloxycarbonyl
[000238] GC gas chromatography
[000239] hour (s)
[000240] Hetero Heteroaryl
[000241] HOBt N-hydroxybenzotriazole
[000242] HBTU 0 - (benzotriazol-l-yl) -N, N, N ', N'-hexa tetramethyluronium fluorophosphate
[000243] HPLC High Performance Liquid Chromatography
[000244] LAH lithium aluminum hydride
[000245] LCMS Liquid Chromatography Mass Spectrometry
[000246] MCPBA m-Chloroperbenzoic acid
[000247] MeCN acetonitrile
[000248] MeOH Methanol
[000249] Min. Minutes
[000250] MeI Iodometano
[000251] MeMgCl Methyl magnesium chloride
[000252] Metila me
[000253] NaOAc sodium acetate
[000254] NMR Nuclear magnetic resonance
[000255] NMP N-methyl pyrrolidinone
[000256] O.n. During the night
[000257] RT ambient temperature or Retention Time
[000258] T3P propylphosphonic anhydride
[000259] TEA triethylamine
[000260] THF tetrahydrofuran
[000261] TLC Thin Layer Chromatography
[000262] Throughout the following description of processes, it is to be understood that, where appropriate, the appropriate protecting groups will be added to, and subsequently removed from, various reagents and intermediates in a manner that will be readily understood by a person skilled in the art. of organic synthesis. Conventional procedures for using these protecting groups, as well as examples of suitable protecting groups, are described, for example, in "Protective Groups in Organic Synthesis", TW Green, PGM Wuts, Wiley-Interscience, New York, (1999). It is also to be understood that a transformation of one group or substituent into another group or substitute by chemical manipulation can be carried out in any intermediate or final on the synthesis path for the final product, in which the possible type of transformation is only limited by incompatibility inherent in other functionalities developed by the molecule at that stage for the conditions or reagents used in the transformation. Such inherent incompatibilities and the ways to get around them by carrying out appropriate transformations and synthetic steps in an appropriate order, will be readily understood by a person versed in the technique of organic synthesis. Examples of transformations are given below, and it should be understood that the transformations described are not limited only to the generic or substituent groups for which the transformations are exemplified. References and descriptions of other suitable transformations are given in "Comprehensive Organic Transformations - a guide to functional group preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in organic chemistry textbooks, for example, "Advanced Organic Chemistry", March, 4aed. McGraw Hill (1992) or, "Organic Synthesis", Smith, McGraw Hill, (1994). Techniques for the purification of intermediates and final products include, for example, normal and reverse phase chromatography on a column or rotating plate, recrystallization, distillation and extraction of liquid-liquid or solid-liquid, which will be easily understood by a skilled person in the technique. The definitions of substituents and groups are as described for formula I, except when defined differently. The terms "room temperature" and "room temperature" shall mean, unless otherwise stated, a temperature between 16 and 25 ° C. The term "reflux" shall mean, unless otherwise stated, in reference to the solvent, a temperature equal to or above the boiling point of the solvent. Example 1: Synthesis of Intermediate (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylic acid.
Synthesis of 3,5-bis (trifluoromethyl) benzothioamide

[000263] A 2-L, 3-neck, round-bottomed flask was loaded with a solution of 3,5-bis (trifluoromethyl) benzonitrile (200 g) in DMF (1 L). The solution was then treated with NaSH (123.7 g, 2.0 eq.) And MgCl 2 (186.7 g, 1.0 eq.) And the reaction mixture was stirred at room temperature for 3 hours. The mixture was poured into an ice-water paste (10 L) and the compound was extracted with EtOAc (3 x 1 L). The combined organic layers were washed with saturated aqueous brine (3 x 100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain 205 g of the desired crude 3,5-bis (trifluoromethyl) benzothioamide (yield: 90 %), which was used without purification in the next step. Synthesis - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole:

[000264] A 3-neck, 5-L, round-bottom flask was loaded with a solution of 3,5-bis (trifluoromethyl) benzothioamide (205.65 g) in DMF (1.03 L). Hydrazine hydrate (73.2 ml, 2.0 eq.) Was added dropwise and the reaction mixture was stirred at room temperature for 1 h. HCOOH (1.03 L) was added dropwise and the reaction mixture was refluxed at 90 ° C for 3 hours. After allowing to cool to room temperature, the reaction mixture was poured into a saturated aqueous sodium bicarbonate solution (7 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with saturated aqueous brine (3 x 500 ml), dried over anhydrous Na2 SO4, filtered and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 180 g of crude compound. This crude material was stirred with petroleum ether (3 x 500 ml), filtered and dried to obtain 160 g. 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole obtained as a pale yellow solid (yield: 75%). Synthesis of (Z) -isopropyl acrylate 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl):

[000265] A 2-L, 3-neck, round bottom flask was loaded with a solution of 3 - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole (160 g) in DMF (960 ml). The solution was treated with DABCO (127.74 g, 2 eq.) And stirred for 30 min. before adding (Z) -isopropyl-3 iodoacrylate (150.32 µg, 1.1 eq.) in drops. After approximately 1 hour, the reaction mixture was poured into an ice-water paste (5 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with saturated aqueous brine (3 x 100 ml), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 250 g of crude compound which was purified by chromatography. column (60/120 silica gel), using a gradient of ethyl acetate / n-hexane (the column was packed in hexane and the desired compound started eluting from 2% EtOAc / n-hexane). Fractions containing the desired compounds were combined to obtain 138 g of the pure desired compound (yield: 61%). Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 2,4-triazol-1-yl-1 H-1) acrylic acid:

[000266] In a 5 L flask, with 3 bottlenecks, round bottom, (Z) -isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazole- 1-yl) acrylate (130 g, 1.0 eq.) was dissolved in THF (1.3 L). A solution of LiOH (69.3 g, 5.0 eq.) In water (1.3 L) was added dropwise to the solution and the reaction mixture was stirred at room temperature for 4 h before being quenched with 400 mL of ice-water paste and made acidic (pH = 2-3) with dilute aqueous HCl. The mixture was extracted with EtOAc (3 x 1 L) and the combined organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide 110 g of desired carboxylic acid (yield: 94%) (cis content = 90.0%, trans content = 8.2% by LCMS). Example 2: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (pyrazin-2-yl ) acrylhydrazide (1-3).

[000267] A 50 ml, 3-neck, round-bottom flask was loaded with a suspension of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4 -triazol-1-yl) -acrylic acid (0.200 g) in 1: 1 CH2 Cl2: AcOEt (25 mL). 2-Hydrazinopyrazine (0.062 g) was added at -40 ° C, followed by T3P (50%) (0.432 g) and DIPEA (0.147 g). The reaction mixture was stirred for 30 min at -40 ° C before being concentrated under reduced pressure (35 ° C, 20 mmHg). The crude oil was purified by preparative TLC using 5% MeOH in CH2Cl2 as a mobile phase (under ammonia atmosphere) to obtain 40 mg (yield: 16%) of (Z) -3 - (3 - (3 5 5 - bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) -N '- (pyrazin-2-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 10.53 (s, 1H), 9.59 (s, 1H), 9.14 (s, 1H), 8.53 (s, 2H), 8, 29 (s, 1H), 8.13 (s, 1H), 8.06-8.07 (m, 1H), 7.92-7.93 (d, J = 2.8 Hz, 1H), 7 , 51-7.53 (d, J = 10.4 Hz, 1H), 6.07-6.10 (d, J = 10.4 Hz, IH); LCMS for C17H12F6N7O [M + H] + predicted: 444.31, found: 444.49 (RT 2.70 min, purity: 95.78%). Example 3: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-yl) -N'- (pyridin-2-yl) acrylhydrazide hydrochloride (I4).

[000268] A 500 ml, 3-neck, round-bottom flask was loaded with a suspension of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1, 2,4 -triazole- 1-yl) acrylic acid (10 g, 1.0 eq) in 1: 1 CH2 Cl2: AcOEt (200 mL). 2-hydrazinopyridine (3.11 g) was added at - 40 ° C. T3P (50% in ethyl acetate) (21.75 g) was added dropwise, followed by DIPEA (7.36 g) and the reaction mixture was stirred for 30 min at -40 ° C before being concentrated under reduced pressure (35 ° C, 20 mm Hg) to obtain a crude brown oil which was purified by column chromatography (the compound eluted with 1.3% MeOH in CH2Cl2). Fractions containing the desired compound were combined to produce 6.0 g (yield: 48%) (Z) -3 - (3 - (3,5-bis- (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) -N '- (pyridin-2-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 10.41 (s, 1H), 9.66 (s, 1H), 8.59 (s, 1H), 8.53 (s, 2H), 8, 28 (s, 1H), 8.06-8.08 (d, J = 5.2 Hz, 1H), 7.48-7.53 (m, 1H), 7.49-7.52 (d, J = 10.4, 1H), 6.71-6.75 (m, 1H), 6.66-6.68 (d, J = 8.4 Hz, LH), 6.07-6.09 ( d, J = 10.4, 1H). LCMS for C18H12F6N6O [M + H] + predicted: 443.33, found: 443.44 (RT 2.45 min, purity: 100%). Synthesis of hydrochloride (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) -N '- (pyridin-2-yl) acrylhydrazide:

[000269] A 500 mL, 3-neck, round-bottom flask was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2, 4-triazol - 1-yl) -N '- (pyridin-2-yl) acrylhydrazide (5.5 g) in Et20 (250 ml). The solution was cooled to 5 ° C, treated with HCl in 1,4-dioxane, allowed to warm to RT and stirred until completion, as shown by TLC analysis (approximately 1 h). The solids were filtered through a Buchner funnel, washed with Et20 and dried in vacuo to obtain 5.5 g (yield: 92%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) hydrochloride) phenyl) -1H-1,2,4-triazol-1-yl) -N '- (pyridin-2-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 11.26 (s, 1H), 10.89 (s, 1H), 9.55 (s, 1H), 8.52 (s, 2H), 8, 28 (s, 1H), 8.03-8.07 (m, 2H), 7.62-7.59 (d, J = 10.4 Hz, 1H), 7.21-7.24 (m, 1H), 7.05-7.09 (m, 1H), 6.16 6.19 (d, J = 10.4 Hz, 1H), LCMS for C18H13F6N6O [M + H] +443.33, found 443 , 44 (RT 3.54 min, purity: 99.0%). Example 4: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - 1 - (4-hydroxypiperidin-1 - il) prop-2-en-1-one (1-5).

[000270] A 50 mL flask, 3 necks, round bottom was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazole-1-yl) acrylic acid (0.20 g) in CH2 Cl2 (10 ml). Piperidin-4-ol (0.07 g, 1.2 eq.) Was added and the solution was cooled to -60 ° C during the addition of T3P (phosphonic propyl anhydride) (0.40 ml, 1.2 eq. .) And DIPEA (0.19 mL, 2.0 eq.). The reaction mixture was stirred for 30 min before being poured into water (50 ml) and extracted with CH2Cl2 (2 x 50 ml). The combined organic layers were washed with saturated aqueous brine (50 ml), dried over MgSO4, filtered, and concentrated under reduced pressure (25 ° C, 20 mmHg). Purification by column chromatography using silica 60/120 and MeOH: CH 2 Cl 2 as the mobile phase, (desired compound started eluting with 3.0% MeOH / CH2Cl2) provided 0.025 g (yield: 10%) of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) - 1 - (4-hydroxypiperidin-1-yl) prop-2-en -1-one. 1H NMR (400 MHz, CDC13) δ, 8.75 (s, LH), 8.58 (s, 2H), 7.93 (s, IH), 7:08 - 07:11 (d, J = 10 , 4 Hz, IH), 6.01-6.04 (d, J = 10.4 Hz, IH), 4:02 - 4:14 (m, IH), 3.98-4.01 (m, IH), 3 , 78-3.85 (m, IH), 03: 47-03: 52 (s, IH), 3:32 - 3:38 (w, IH), 1.96 (s, IH), 1.83 (s, IH), 1.27 (s, IH), 0.90 (s, IH); LCMS for the Chemical Formula: C18H17F6N4O2 [M + H] + 435.34, found 435.24 (RT 2.408 min, purity: 89.6%). Example 5: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) - N- (pyrrolidin-1-yl) acrylamide (1-6).

[000271] A cold (-40 ° C) solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazol-l-yl) - acrylic (0.35 g) in 1: 1 CH 2 Cl 2: EtOAc (200 ml) was treated with 1-aminopyrrolidine HCl (0.134 g). The mixture was then treated with T3P (50%> in EtOAc; 0.77 ml, 1.3 eq) followed by slow addition of DIPEA (0.51 ml, 3.0 eq.). The reaction mixture was stirred for 30 min at -40 ° C before being quenched with ice water, and extracted with EtOAc (3 x 20 ml). The combined organic layers were washed with an aqueous saturated saline solution, dried with anhydrous Na2SO4 and concentrated under reduced pressure (35 ° C, 20 mmHg) to provide 0.275 g of crude solid. Purification by column chromatography on silica gel (60-120 mesh) using MeOH in CH 2 C1 2 as the mobile phase provided (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 Pure H-1, 2,4-triazol-1-yl) -N- (pyrrolidin-1-yl) -acrylamide (7.0 mg yield: 1.7%): 1H NMR (400 MHz, DMSO-d6 ) δ, 9.49 (s, IH), 8.95 (s, IH), 8.53 (s, 2H), 8.28 (s, IH), 7.4-7.38 (d, J = 7.6 Hz, IH), 5.87-5.84 (d, J = 10.4 Hz, IH), 2.86-2.81 (m, 4H), 1.74-1.73 ( m, 4H); LCMS for C17H16F6N5O [M + H] +420.33, found 420.13 (RT 7.76 min, purity: 92.4%). Example 6: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N'-methyl-N '- ( pyridin-2-yl) acrylhydrazide (17).
Synthesis of 2- (1-methyl hydrazinyl) pyridine:
[000272] A 25 ml bottle, 3 necks, round bottom was loaded with 2-bromopyridine (0.31 g) and methylhydrazine, under an atmosphere of nitrogen and the mixture was stirred and heated to reflux temperature at 80 ° C (5.09 g, 34.2 eq.) - 85 ° C for 1 hour. The reaction mixture was concentrated under reduced pressure (40 ° C, 20 mmHg) to provide a yellow oil that was treated with 10% w / v aqueous Na2C03 solution and extracted with EtOAc. The organic layer was washed with a saturated aqueous saline solution, dried over Na2S04, filtered and concentrated under reduced pressure (40 ° C, 20 mmHg) to provide a yellow oil (0.40 g), which was used as such in the following step .
[000273] A 50 mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole-1 -yl) acrylic acid (0.43 g), 2 - (1-methyl hydrazinyl) pyridine (0.15 g, 1.0 eq) in EtOAc (10 ml). T3P (50% in EtOAc, 1.1 g, 1.5 eq) and DIPEA (0.40 g, 2.5 eq) were added under nitrogen at a temperature of -60 ° C and the reaction progressed monitored by TLC (using 10% MeOH: CH2Cl2 as the mobile phase and visualization with UV light). The reaction mixture was concentrated under reduced pressure (25 ° C, 20 mmHg) to provide 0.65 g of crude solid. Purification was performed by Combi-Flash column chromatography in CH2C12 and MeOH (desired compound started eluting to 3.3% MeOH in CH2C12). Fractions containing the desired compound were combined and concentrated under reduced pressure (35 ° C, 20 mm Hg) to provide 90.0 mg (yield: 18%) 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s , 1H), 9.79 (brs, 1H), 8.57-8.62 (d, 2H), 7.92-7.94 (d, J = 11.2 Hz, 1H), 7.59- 7.64 (m, 1H), 7.19-7.25 (q, 1H), 6.75-6.89 (m, 2H), 5.85-5.88 (d, J = 10.8 Hz, 1H), 3.46 (d, 3H); LCMS for C19H15F6N6O [M + H] +457.35, found 456.26 (RT 2.52 min, Purity: 100.0%).
[000274] Example 7: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N'-methyl-N '- (pyrazin-2-yl) acrylonidrazide (1-8).
Synthesis of 2 - (l-methylhydrazinyl) pyrazine

[000275] In a 25 mL flask, 3 necks, round bottom, 2-chloropyrazine (0.5 g) was dissolved in methylhydrazine (0.5 g, 1.5 eq.) Under a nitrogen atmosphere at temperature environment. K2C03 Solid (0.9 g, 1.5 eq.) Was added and the reaction mixture was stirred and heated to reflux at 80-85 ° C for 1.0 h. The reaction mixture was then allowed to cool to room temperature and was concentrated under reduced pressure (40 ° C, 20 mmHg) to obtain a yellow oily residue which was treated with 10% w / v aqueous Na2C03 solution and extracted with EtOAc. The organic extract was washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (40 ° C, 20 mmHg) to provide 0.43 g of a yellow oil which was used as such in the next step.
[000276] A 50 mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole-1 -yl) acrylic acid (0.3 g), 2- (1-methyl hydrazinyl) pyrazine (0.12 g, 1.1 eq) and CH2 Cl2 (10 ml). T3P (50% in EtOAc, 0.38 g, 1.5 eq) and DIPEA (0.50 g, 3.5 eq) were added under a nitrogen atmosphere at -60 ° C. monitoring the progress of the reaction by TLC (using 10% MeOH: CH2C12 as mobile phase and visualization under UV light). The reaction mixture was concentrated under reduced pressure (25 ° C, 20 mmHg) to provide 0.265 g of crude solid. Purification using Combi-Flash column chromatography using CH2C12: MeOH as eluent (desired compound started eluting at 1.5% MeOH in CH2C12) provided 75.0 mg of pure compound (23% yield), (Z) - 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) -N'-methyl-N '- (pyrazin-2-yl) acrylhydrazide: 1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.40-9.36 (wide s, 1H), 8.52 (s, 2H), 8.29-8.27 (d, 2H), 8.15 (s, 1H), 7.925-7.92 (d, 1H), 7.56-7.54 (d, J = 10.4 Hz, 1H), 6.13- 6.10 (d, J = 10.4 Hz, 1H), 3.43 (d, 3H); LCMS for C18H14F6N7O [M + H] +458.34; found 458.24 (RT 2.83 min; Purity: 96.31%). Example 8: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N'-methyl-N '- ( 3-methylpyridin-2-yl) acrylonidrazide (1-9).

[000277] A 50 mL, 3-neck, round-bottomed flask was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) acrylic acid (0.25 g) in EtOAc (20 ml). The solution was cooled to -70 ° C and treated successively with 3-methyl-2- (1-methyl hydrazinyl) pyridine, (0.135 g, 1.0 eq), T3P (50% in EtOAc, 1.4 mL, 4 eq) and DIPEA (0.6 ml, 6 eq.). The clear reaction mixture was stirred at -60 ° C for 4 h. The progress of the reaction was followed by TLC analysis using 2.5% MeOH in CH2Cl2 as the mobile phase and visualization under UV. The reaction mixture was concentrated under reduced pressure (25 ° C, 20 mm Hg) to obtain a crude compound that was purified by column chromatography (60/120 Si02 mesh and eluting with a MeOH: CH2Cl2 gradient). The desired compound started eluting with 0.3-0.4% MeOH in dichloromethane. The fractions containing the desired material were combined to obtain 0.21 g (yield: 40%) 1H NMR (400 MHz, DMSO-d6) δ = 10.73 (s, 1H), 9.32 (s, 1H) , 8.52 (s, 2H), 8.45-8.46 (d, J = 4.4 Hz, 1H), 8.29 (s, 1H), 7.97-7.99 (d, J = 8 Hz, 1H), 7.48-7.50 (d, J = 10 Hz, 1H), 7.01-7.05 (m, 1H), 5.86-5.88 (d, J = 10 Hz 3H); LCMS for C20H14F9N6O [M + H] +525.35, found 525.19 (RT 3.31 min, purity 99.40%). Example 9: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (2-5-methylpyridine -yl) acryloyrazide (I-10).

[000278] One 50 mL flask, 3 round-bottomed necks, loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole -1-yl) -acrylic (0.25 g) in EtOAc (10 ml) was treated with 2-hydrazinyl-5-methylpyridine (0.97 g, 1.1 eq.). The mixture was cooled to -60 ° C and treated with T3P (phosphonic propyl anhydride; 0.85 ml, 2.0 eq.) And DIPEA (0.5 ml, 4.0 eq.). The mixture was stirred for 30 min, then poured into water (50 ml) and extracted with CH2Cl2 (2 x 50 ml). The combined organic layers were washed with brine (50 ml), dried over MgS04, filtered, and concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain a crude compound that was purified by column chromatography (S1O2, 60 / 120 mesh, MeOH: CH2Cl2 as mobile phase). The desired compound started eluting with 2.5% MeOH: CH2Cl2. Fractions containing the desired compound were combined and concentrated under reduced pressure to obtain 0.130 g (yield: 40%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2 , 4-triazol-1-yl) -N '- (5-methylpyridin-2-yl) acrylhydrazide. 1H NMR (400 MHz, CDC13) δ, 10.38 (s, 6.61-6.63 (d, J = 8.4 Hz, 1H), 6.20-6.23 (d, J = 10, 4 Hz, 1H), 2.15 (s, 3H); LCMS for C19H15F6N6O [M + H] +457.35, found 457.24 (RT 2.61 min, purity: 99.13%). Example 10: Synthesis of (Z) -3- (3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N1-methylN '- (pyridine-3 -yl) acryl hydrazide (I-11).

[000279] A 50 mL bottle, with 3 round-bottomed necks loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole -l-yl) - acrylic (0.25) in CH2Cl2 (12 ml) was treated with 3 - (1-methyl hydrazinyl) pyridine (0.105 g, 1.2 eq). The mixture was cooled to -60 ° C and treated with T3P (phosphonic propyl anhydride; 0.50 ml, 1.2 eq.) And DIPEA (0.24 ml, 2.0 eq.) And stirred for 1 h. The progress of the reaction was followed by TLC analysis using 10% MeOH: CH2Cl2 as the mobile phase and visualization under UV light. The reaction mixture was then poured into water (50 ml) and extracted with CH2Cl2 (2 x 50 ml). The combined organic layers were washed with brine (50 ml), dried over MgS04, filtered, and concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain the impure compound which was purified by column chromatography (Si02, 60 / 120 mesh, MeOH: CH2Cl2 as mobile phase). The desired compound started eluting to 3.0% MeOH: CH2Cl2. Fractions containing the compound were collected and concentrated under reduced pressure to obtain 140 mg (yield: 43%) (Z) -3 - (3 - (3,5-bis (Trifluoromethyl) phenyl) - 1 H-1, 2 , 4-triazol-1-yl) -N'-methyl-N '- (pyridin-3-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-D6) δ, 10.55 (s, 1H), 9.41 (s, 1H), 9.15 (s, 2H), 8.58 (s, 1H), 8, 53 (s, 1H), 8.29 (s, 1H), 7:51 - 07:54 (d, J = 10.4 Hz, 1H), 7:18 - 07:22 (m, 2H), 06 : 05-06: 07 (d, J = 10.4 Hz, 1H), 3.20 (s, 3H); LCMS for C19H15F6N6O [M + H] + 457.35, found 457.19 (RT 2.43 min, purity: 83.48%). Example 11: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-yl) -N'- (6-chloropyrimidin-4-yl ) acrylhydrazide (1-12).

[000280] A 25 mL flask, 3 necks, round bottom was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) - acrylic acid (0.5 g) and 4-chloro-6-hydrazinopyrimidine (0.20 g, 1.0 eq.) in EtOAc (5.0 ml). The mixture was cooled to -40 ° C and treated with T3P (2.3 ml, 2.5 eq.) And DIPEA (0.98 ml, 4.0 eq.). TLC analysis (using 5% MeOH-CH2Cl2 as the eluant) revealed that the starting material had been consumed after 30 min. The reaction mixture was then diluted with CH2Cl2, washed with water, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to provide the crude material that was subjected to preparative TLC purification using 5% MeOH-CH2Cl2, as the mobile phase. This provided 250 mg (yield: 36.74%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) -N '- (6-chloropyrimidin-4-yl-) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6), δ = 10.59 (br s, exchangeable, 1H), 9.85 (br s, exchangeable, 1H), 9.52 (s, 1H), 8.50 ( s, 2H), 8.38 (s, 1H), 8.27 (s, 1H), 7.52-7.55 (d, 1H, J = 10.4 Hz), 6.69 (s, 1H ), 6.05-6.08 (d, 1H, J = 10.4 Hz); LCMS: Calculated for C17H11ClF6N7O (M + H) +478.76, found: 478.09 (RT 2.79 min, purity: 97.51%). Example 12: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (pyridin-3-yl ) acrylhydrazide (I-13).

[000281] A 5-mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazole- 1-yl) acrylic acid (0.25 g) and 3-hydrazinopyridine (0.077 g, 1.0 eq.) in EtOAc (10 ml). T3P (50% in EtOAc, 0.52 g, 1.2 eq) and DIPEA (0.27 g, 2.0 eq) were added under a nitrogen atmosphere at -55 to -60 ° C. The progress of the reaction was followed by TLC analysis using 10% MeOH: CH2Cl2 as the mobile phase and visualization under UV light. The reaction mixture was concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain 0.475 g of a crude solid. Purification was performed using Combi-Flash column chromatography (with MeOH: CH2Cl2). The desired compound started eluting to 2.3% MeOH in CH2Cl2. The fractions containing the compound were combined and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 20.0 mg (yield: 6%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl ) phenyl) -H-1,2,4-triazol-1-yl) -N '- (pyridin-3-yl) acrylonidrazide. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.66 (s, 1H), 8.53 (s, 2H), 8.28 (s, 1H), 8.24 (s, 1H), 8.13 (s, 1H), 7, 93-7, 95 (m, 1H), 7.52-7 .54 (d, J = 10.4 Hz, 1H), 7.09 - 7.15 (m, 2H), 6.04-6.07 (d, J = 10.4 Hz, 1H), LCMS for C18H13F6N6O [M + H] + found 443.33 443.19 (RT 2.19 min, purity: 99.60%). Example 13: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (quinoxalin-2-yl ) acrylhydrazide (1-14).
Synthesis of 2-hydrazinyl quinoxaline:

[000282] In a sealed 30 mL tube, 2-chloroquinoxaline (1.0 g) was dissolved in ethanol (8 mL) and hydrazine hydrate (8 mL) was added under a nitrogen atmosphere at room temperature. The mixture was stirred and heated to reflux temperature (80 ° C) for 1 hour. The progress of the reaction was followed by TLC analysis using 10% MeOH: CH2Cl2 as the mobile phase and visualization under UV light and / or with ninhydrin. The reaction mixture was concentrated under reduced pressure (40 ° C, 20 mmHg) to obtain 240 mg of a white solid, which was used as such in the next step.
[000283] A 50 mL, 3-neck, round-bottomed flask was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) - acrylic (0.25 g) and 2-hydrazinylquinoxaline (0.14 g, 1.2 eq.) in EtOAc. T3P (50% in EtOAc, 0.83 mL, 2.0 eq) and DIPEA (0.5 mL, 4.0 eq) were added under a nitrogen atmosphere at -55 to -60 ° C and the reaction mixture it was stirred for 2 h before being concentrated under reduced pressure (25 ° C, 20 mmHg) to provide 0.150 g of crude solid. Purification using Combi-Flash column chromatography (elution with MeOH: CH2Cl2; desired compound started eluting with 5% MeOH in CH2Cl2) provided 60 mg (yield: 20%) 1H NMR (400 MHz, DMSO-d6) δ = 10.851 (s, 1H), 9.89-9.87 (s, 1H), 9.67 (s, 1H), 8.49-8.54 (m, 3H), 8.26 (s, 1H) , 8.28 (s, 1H), 7.86-7.88 (d, J = 8 Hz, 1H), 7.45-7.66 (m, 4H), 6.17-6.20 (d , J = 10.4 Hz, 1H); LCMS for C21H14F6N7O [M + H] +494.37, found 494.19 (RT 2.88 min, purity: 100%). Example 14: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (1,1-dioxotetra -hydrothiophen-3-yl) acryloyrazide (I-15).

[000284] A 50 mL bottle, 3 necks, round bottom loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole -l -yl) - acrylic (0.5 g) in EtOAc (20.0 mL) was treated with 2 - (1,1-dioxotetrahydrothiophen-3-yl) hydrazine (0.3 g, 1.2 eq ). The mixture was cooled to -60 ° C and treated simultaneously with T3P (50% in EtOAc, 2.0 ml, 2 eq.) And DIPEA (1 ml, 4 eq.). The reaction mixture was stirred for 30 min at -60 ° C before being concentrated under reduced pressure (35 ° C, 20 mmHg) to provide 0.60 g of a solid residue. Purification by column chromatography (Si02; elution with MeOH: CH2Cl2; desired compound eluted with 5% MeOH in CH2Cl2) gave 100 mg (yield = 15%) (Z) -3 - (3 - (3, 5-bis (trifluoromethyl) phenyl) - 1 H-1,2,4-triazol-1-yl) -N '- (tetrahydrothiophene-1 - 1-dioxide-3-yl) acrylhydrazide. 1H NMR (400 MHz, CD30D) δ = 9.57 (s, 1H), 8.64 (s, 2H), 8.10 (s, 1H), 7.34-7.36 (d, J = 10 , 4 Hz, 1H), 5.89-5.92 (d, J = 10.8 Hz, 1H), 4.01 (m, 1H), 3.04-3.26 (m, 4H), 2 , 27-2.34 (m, 2H). LCMS for C17H15F6N5O3S [M + H] + 484.40, found 483.39 (RT 2.63 min, purity: 66.39%). Example 15: Synthesis of (Z) -N- (azepan-1-yl) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1,2,4-triazol-1-yl) acrylamide ( I-16).

[000285] A 500 mL bottle, 3 necks, round bottom was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) -acrylic (0.3 g) in CH2 Cl2: EtOAc (1: 1, 200 ml) and the solution was treated with azepan-1-amine (0.137 g) at room temperature. The mixture was cooled to -60 ° C and treated first with T3P (50% in EtOAc, 0.78 ml) and then with DIPEA (0.58 ml). The reaction mixture was stirred for 30 min at -60 ° C before being quenched with ice water and extracted with EtOAc (3 x 20 ml). The combined organic extracts were washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (35 ° C, 20 mmHg) to provide 0.57 g of solid. Purification by column chromatography (Si02, MeOH: CH2Cl2 as a mobile phase; compound started eluting with 0.1% MeOH in CH2Cl2) provided 90 mg (yield: 24%) (Z) -N- (azepan-1- yl) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylamide. 1H NMR (400 MHz, DMSO-D6) δ, 9.61 (s, IH), 9.49 (s, IH), 9.14 (s, IH), 8.52 (s, 2 h), 8 , 28 (s, IH), 7,397.97 (d, J = 10 Hz, IH), 6.52-6.49 (d, J = 10.4 Hz, IH), 5.86-5.83 ( d, J = 10.4 Hz, IH), 3.00-2.97 (m, 4H), 1.581.54 (m, 8h) LCMS for C19H19F6N5O [M + H] +448.39; Pvt found at 448.30 3.22 min Purity (96.48%). Example 16: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-yl) -N'- (2,6-dimethylpyrimidin-4 -yl) acrylonidrazide (1-17).

[000286] A 50 mL flask, 3 necks, round bottom was loaded with a solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4- triazol-1-yl) acrylic acid (0.20 g) dissolved in ethyl acetate (15 ml). The solution was cooled to -40 ° C and treated with 4-hydrazinyl-2,6-dimethylpyrimidine (0.078 g, 1 eq.). T3P (50% in EtOAc, 0.7 g, 3.0 eq) and DIPEA (0.367 g, 4.0 eq) were added simultaneously and the reaction mixture was stirred for 30 min at -40 ° C. The reaction mixture was then allowed to warm to room temperature and was concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 0.340 g of the crude oily compound which was purified by flash-combi using MeOH: CH2Cl2 as the mobile phase ( the desired compound was eluted with 7-8% MeOH in CH2Cl2) to obtain 50 mg (yield: 18%) (Z) -3 - (3 - (3, 5-bis (trifluoromethyl) phenyl) - 1 H- 1,2,4-triazol-1-yl) -N'- (2,6-dimethylpyrimidin-4-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 10.54 (s, IH), 9.19 (b, IH), 8.54 (s, 2H), 8.30 (s, IH), 7.52- 7.55 (d, J = 10.4, IH), 6.29 (s, IH), 6.06-6.08 (d, J = 10.4, IH), 2.33 (s, 3H ), 2.13 (s, 3H), LCMS for C19H15F6N7O [M + H] +472.37, found 472.24 (RT 2.88 min, purity: 99.59%). Example 17: Synthesis of (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) - N'- (pyrazin-2-yl ) acrilohydrazide
Synthesis of 3,5-bis (trifluoromethyl) benzothioamide:

[000287] A 2-L, 3-neck, round-bottomed flask, loaded with a solution of 3,5-bis (trifluoromethyl) benzonitrile (200 g) in DMF (1 L), was treated with NaSH (123.7 g, 2.0 eq.) and MgCl 2 (186.7 g, 1 eq.). The reaction mixture was stirred at RT for 3 h before being poured into an ice-water slurry (10 L) and was extracted with EtOAc (3 x 1 L). The combined organic extracts were washed with brine (3 x 100 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain 205 g of crude compound (yield: 90%), which was used in the next step without further purification. Synthesis of 3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1,2,4-triazole:

[000288] A 5-L, 3-neck, round bottom flask, loaded with a solution of 3,5-bis (trifluoromethyl) benzothioamide (205.65 g) in DMF (1.03 L) was treated with hydrate hydrazine (73.16 mL, 2.0 eq.) dropwise. The reaction mixture was stirred at room temperature for 1 h before being treated with HCOOH (1,028 L) added dropwise. The reaction mixture was refluxed at 90 ° C for 3 h, then cooled to room temperature and poured into saturated aqueous NaHC03 solution (7 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with brine (3 x 500 ml), dried over anhydrous Na2 SO4, filtered and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 180 g of a solid. The solid was suspended in petroleum ether and the suspension was stirred, filtered and dried to provide the desired triazole as a pale yellow solid (160 g, yield: 75%). Synthesis of (Z) -isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) methyl acrylate and (E) -isopropyl 3 - ( 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate:

[000289] One 2-L flask, 3 round-bottomed necks, loaded with a solution of 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole (160 g) in DMF (0.96 L, 6V), was treated with DABCO (127.74 g, 2 eq.) And stirred for 30 min. (Z) -isopropyl-3 iodoacrylate (150.32 g, 1.1 eq.) Was added dropwise to the above reaction mixture and stirred for 1 h before being poured into an ice-water paste (5 L) and extracted with EtOAc (3 x 1 L). The combined organic extracts were washed with brine (3 x 100 ml), dried over anhydrous Na2So4, filtered and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 250 g of crude compound. Purification by column chromatography (Si02, 60/120 mesh, elution with EtOAc: hexanes; desired compounds started eluting in 2-2.5% EtOAc in hexanes) provided the pure cis ester (138 g, yield: 61 , 6%) and pure trans ester (11.6 g, yield: 5.2%).
[000290] Synthesis of (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl acid

[000291] A 500 mL bottle, 3 necks, round bottom was loaded with a solution of (E) - isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH- 1, 2.4 -triazol-1-yl) acrylate (5.0 g) in THF (50 ml). The solution was treated with a solution of LiOH (2.66 g, 5.0 eq.) In water (50 ml) and the reaction mixture was stirred at room temperature for 4 h. before being diluted with 40 ml of water, acidified (pH = 2-3) with diluted aqueous HCl and extracted with EtOAc (3 x 100 ml). The organic extract was washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to obtain 2.75 g of the desired unsaturated carboxylic acid (yield: 61.6%, purity: 99.0% by CL-EM) . Synthesis of (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N '- (pyrazin-2-yl) acryloyrazide acid :

[000292] To a solution of (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -acetic acid (0.75 g ,) in EtOAc (25 ml) and THF (12.5 ml) a solution of 2-hydrazinopyrazine (0.23 g) in 12 ml of THF was added at room temperature. T3P (50% in ethyl acetate, 1.52 ml) and DIPEA (1.46 ml) were added dropwise and simultaneously, and the reaction mixture was stirred for 30 minutes at room temperature before being quenched with ice water and extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous Na2S04 and concentrated under reduced pressure (35 ° C, 20 mmHg), yielding 0.698 g of a crude solid. Trituration first with petroleum ether, then with Et20 provided 275 mg (yield: 29%) of (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1,2 , 4-triazol-1-yl) -N '- (pyrazin-2-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 10.3 (s, 1H), 9.15 (s, 2H), 8.59 (s, 2H), 8.30-8.26 (d, J = 14.8 Hz, 1H), 8.13 (s, 1H), 8.06-8.07 (m, 1H), 6.98-6.95 (d, J = 13.4 Hz, 1H) ; LCMS for C17H12F6N7O [M + H] +443.31, found 444.19 (RT 2.625 min, purity: 99.06%). Example 18: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol - yl) - N '- (pyridin-4-yl) hydrochloride acryloyrazide (1-19).

[000293] A 50-mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole- 1-yl) acrylic acid (0.25 g) and EtOAc (10.0 ml). 4-Hydrazinylpyridine hydrochloride (0.16 g, 1.2 eq.) Was added at -40 ° C, followed by the simultaneous addition of T3P (50% in EtOAc, 0.85 mL, 2.0 eq.) And DIPEA (0.49 mL, 4.0 eq.). The reaction mixture was stirred for 30 min at -40 ° C before being concentrated under reduced pressure (35 ° C, 20 mmHg) to provide 0.35 g of crude material. Purification by column chromatography, using MeOH: CH2Cl2 as the mobile phase (compound was eluted with 4% MeOH in CH2Cl2) provided 80 mg (yield: 29.85%) (Z) -3 - (3 - (3, 5-bis (trifluoromethyl) phenyl) - 1H-1, 2,4-triazol-1-yl) -N'- (pyridin-4-yl) acrylhydrazide 1H NMR (400 MHz, DMSO-d6) δ, 10.53 (s wide, exchangeable NH, 1H), 9.58 (s, 1H), 8.88 (s wide, exchangeable NH, 1H), 8.84 (s, 2H), 8.29 (s, 1H), 8.09-8.11 (d, 2H), 7.52-7.54 (d, J = 10.4 Hz, 1H), 6,666.69 (m, 2H), 6.06-6.10 ( d, J = 14.4 Hz, H); LCMS for C18H13F6N6O [M + H] +443.33, found 443.24 (TA 2.241 min, purity: 90.17%).
[000294] A 25 mL bottle, 3 necks, round bottom was loaded with a cold (0 ° C) solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH- 1,2,4-triazol-l-yl) -N '- (pyridin-4-yl) acrylonidrazide (0.08 g) in CH2Cl2 (5.0 mL) and treated with HCl4N in dioxane (0.5 mL) . The reaction mixture was allowed to warm to room temperature and stirred for 4 h before being concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 0.05 g (yield: 40.81%) (Z) - 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N '- (pyridin-4-yl) acrylhydrazide salt HCl. 1H NMR (400 MHz, DMSO-d6) δ 13.67 (br s, exchangeable, 1H), 10.67 (s, exchangeable, 1H), 9.43 (s, 1H), 8.58 (s, 2H ), 8.35-8.38 (m, 4H), 7.60-7.62 (d, J = 10.4 Hz, 1H), 6.92-6.96 (m, 2H), 611- 6.13 (d, J = 10.4 Hz, 1H); LCMS for C18H13F6N6O [M + H] +443.33, found 443.24 (RT 3.00 min, purity: 90.97%). Example 19: Synthesis of (Z) -N- (4-benzylpiperazin-1-yl) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazole-1 -yl) -acrylamide (1-20).
Synthesis of 4-benzylpiperazine-1-amine.

[000295] A 50 ml bottle, 3 necks, round bottom was loaded with conc. and water, and the solution was cooled to 0-5 ° C during the addition of NaN02 and benzyl piperazine (5.0 g) under an atmosphere of nitrogen. The reaction mixture was stirred for 2.5 h at 0-5 ° C, before being diluted with water and extracted with EtOAc (3 x 100 ml). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (40 ° C, 20 mmHg) to obtain 4.40 g of a colorless solid. Purification using combi flash chromatography (elution with 25.5% EtOAc: hexane) gave 2.0 g of the desired compound (yield: 34.3%).
[000296] A cold solution (-70 ° C) of 1-benzyl-4-nitrous-4-piperazine (0.8 g) in THF was treated with excess LAH under an atmosphere of nitrogen. The reaction mixture was allowed to warm to room temperature and stirred 1.0 h, before being quenched with water and extracted with EtOAc (3 x 10 ml). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (40 ° C, 20 mmHg) to provide 0.70 g of 4-benzylpiperazin-1-amine as a colorless solid. Synthesis of (Z) -N- (4-benzylpiperazin-1-yl) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1 H-1, 2,4-triazol-1-yl) acrylamide.
[000297] A 50 mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazole-1 -yl) acrylic acid (0.220 g, 1.2 eq.), 4-benzylpiperazin-1-amine (0.10 g, 1.0 eq.) and EtOAc (15 ml). T3P (50% in 0.99 g EtOAc, 3.0 eq.) And DIPEA (0.27 mg, 4.0 eq.) Were added under a nitrogen atmosphere to the cold solution (-60 ° C). The progress of the reaction was followed by TLC analysis (Si02, 15% MeOH: CH2Cl2 as a mobile phase, visualization under UV light). The reaction mixture was quenched in water and extracted with ethyl acetate (3 x 15 mL). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to provide 0.35 g of crude solid. Combi-flash purification (eluting with 10% MeOH / CH2Cl2) gave 20 mg (yield: 6%) 1H NMR (400 MHz, DMSO-d6) δ 9.44-9.48 (t, 3H), 9 , 10 (s, IH), 8.51 (s, 2H), 7.23- 7.41 (m, 6H), 6.46-6.49 (d, J = 10.4 Hz, IH), 5.83-5.86 (d, J = 10.4 Hz, IH), 3.47 (s, 2H), 2.81 (s, 4H), 2.23-2.33 (d, 2H) LCMS for C24H23F6N6O [M + H] +525.47, found 525.20 (RT 9.87 min, purity: 100%). Example 20: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N-ethylpiperazin-1-yl) acrylamide (1-21).

[000298] A cold (-40 ° C) solution of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazol-l-yl) -acid acrylic (0.25 g) in EtOAc (20 ml) was treated with 4-ethylpiperazin-1-amine (0.12 g). T3P (50% in EtOAc, 0.84 ml) and DIPEA (0.24 ml) were added simultaneously and the reaction mixture was stirred for 30 min at -40 ° C before being brusquely cooled with ice water and extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2S04 and concentrated under reduced pressure (35 ° C, 20 mmHg) to obtain 0.280 g of crude compound. Purification by combined flash chromatography (elution with 2% MeOH in CH2Cl2) followed by purification on a preparative TLC plate (elution with 10% MeOH in CH2Cl2) provided 60 mg of (Z) -3 - (3 - (3 , 5-bis (trifluoromethyl) phenyl) - 1 H-1,2,4-triazol-1-yl) -N- (4-ethylpiperazin-1-yl) acrylamide. 1H NMR (400 MHz, CF3COOD) δ: 10.75 (s, IH), 8.31-8.29 (d, J = 10.2 H), 7.98 (s, IH), 7.21- 7.23 (d, IH), 6.08-6.10 (d, IH), 3.52-3.54 (m, 3H), 3.36 (s, IH), 3.11 (m, 8H), 1.19-1.22 (m, 3H); LCMS for C19H21F6N6O [M + H] +463.40, found 463.23 (RT 2.43 min, purity: 98.63%). Example 21: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -Nmorpholinoacrylamide (1-22).

[000299] A 50 mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -H-1,2,4-triazole-1 -yl) acrylic acid (0.250 g), morpholin-4-amine (0.072 g, 1.0 eq.) and EtOAc (10 ml). The solution was cooled to -60 ° C and treated with T3P (50% in EtOAc, 0.63 ml, 1.5 eq.) And DIPEA (0.24 ml, 2.0 eq.) Under a nitrogen atmosphere. The progress of the reaction was followed by TLC analysis using 10% MeOH: CH2Cl2 as the mobile phase and visualization under UV light. Upon completion, the reaction mixture was quenched with water and extracted with EtOAc (3 x 15 mL). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to provide 0.35 g of a crude solid. Purification (Combi-Flash, elution with 3% MeOH: CH2Cl2) gave 100 mg (yield: 33%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-l 2 , 4-triazol-1-yl) -N-morpholinoacrylamide. 1H NMR (400 MHz, DMSO-d6) δ = 9.52 (s, 8.51 (s, 2H), 8.28 (s, 1H), 7.38-7.42 (m, 1H), 6 , 50-6.53 (d, J = 10.4 Hz, 1H), 5.84-5.86 (d, J = 10.4 Hz, 1H), 3.63 (s, 4H), 2, 87 (s, 4H); LCMS for C17H16F6N5O2 [M + H] +436.33, found 436.18 (RT 2.64 min, purity: 100%). Example 22: Synthesis of (Z) -3 (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (pyrimidin-4-yl) acrylonidrazide (1-23).
Synthesis of 4-hydrazinyl pyrimidine:

[000300] A solution of 2,4-dichloropyrimidine (2.0 g) in EtOH (25 ml) was cooled to 0-20 ° C and treated with hydrazine (2.8 ml). The progress of the reaction was followed by TLC using 10% MeOH: CH2C12 as the mobile phase and visualization under UV light. The mixture was concentrated under reduced pressure to produce 3.1 g of 2-chloro-4-hydrazinyl-pyrimidine chloride (yield = 94.8%).
[000301] To a solution of 2-chloro-4-hydrazinyl-pyrimidine (200 mg) dissolved in MeOH (10 ml) was added 10% Pd / C (200 mg) and the suspension was stirred under an atmosphere of hydrogen until which was shown to be complete by TLC analysis (using 10% MeOH: CH2Cl2 as the mobile phase and viewing under UV light). The mixture was filtered through Celite ® and concentrated under reduced pressure to obtain 250 mg of 4-hydrazinylpyrimidine. Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) -N '- (pyrimidin-4-yl) acrylhydrazide .
[000302] A 50 mL flask, and 3 round-necked bottlenecks, was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazole- 1-yl) acrylic acid (250 mg, 1.0 eq.) and AcOEt (20.0 ml). 4- Hydrazinylpyrimidine (231 mg, 3 eq) was added at -60 ° C, followed by the simultaneous addition of T3P (50% in EtOAc, 0.84 mL, 2.0 eq.) And DIPEA (0.24 mL , 2.0 eq.). The reaction mixture was stirred for 30 min at -60 ° C before being concentrated under reduced pressure (35 ° C, 20 mmHg) to provide 0.20 g of a solid. Purification by column chromatography (elution with 5% MeOH in CH2C12) yielded 75 mg of material which was purified by preparative TLC (using MeOH: CH2Cl2, as mobile phase) to provide 13 mg (yield = 5%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1,2,4-triazol-1-yl) -N'- (pyrimidin-4-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ = 10.59 (s, 1H), 9.68 (s, exchange NH, 1H), 9.47 (s, exchange NH, 1H), 8.53 -8.59 (t, 2H), 8.30 (s, 1H), 8.19-8.20 (d, 1H), 7.53-7.56 (d, J = 11.2 Hz, 1H ), 6.66-6.67 (d, 1H), 6.06-6.09 (d, J = 10.4 Hz, 1H); LCMS for C17H12F6N7O [M + H] + 444.31, found 444.19 (RT 2.39 min, purity: 94.97%). Example 23: Synthesis of (Z) -3 - (3 - (4-chloro-3,5-bis (trifluoromethyl) phenyl) -1H-1, 2,4-triazol-1-yl) -N '- (pyrazin -2-yl) acryloyrazide (1-24).
Synthesis of 4-chloro-3,5-bis (trifluoromethyl) benzamide:

[000303] A solution of 4-chloro-3,5-bis (trifluoromethyl) benzonitrile (1.0 g) in DMSO (10 ml) was treated with solid K2C03 (0.55 g, 1.1 eq.) And H202 (30% v / v, 1.0 ml). The reaction mixture was stirred at room temperature for 3 h before being poured into ice water (20 ml). The precipitate was filtered and washed with petroleum ether to obtain 1.0 g of crude desired primary amide (yield: 90%). Synthesis of 4-chloro-3,5-bis (trifluoromethyl) benzothioamide:

[000304] To a solution of 4-chloro-3,5-bis (trifluoromethyl) benzamide (1.2 g) in toluene (20 ml) was added Lawesson's reagent (3.32 g, 2.0 eq.). The reaction mixture was stirred at 90 ° C for 8 h before being cooled to room temperature and filtered. The filtrate was poured into water and extracted with EtOAc (3 x 100 ml). The combined organic extracts were washed with brine (3 x 50 ml), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain 2 g of crude compound. The crude compound was purified by flash-combi chromatography (eluting with 7% EtOAc: hexane) to obtain 1.0 g of the desired compound (yield: 79%). Synthesis of 3 - (4-chloro-3,5 - bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole:

[000305] A solution of 4-chloro-3,5-bis (trifluoromethyl) benzothioamide (1 g) in DMF (10 ml) was treated with hydrazine hydrate (0.32 g, 2.0 eq.) And the mixture The reaction mixture was stirred at room temperature for 1 h before the addition of formic acid (3 ml). The reaction mixture was refluxed at 90 ° C for 3 h, then cooled to room temperature, poured into saturated aqueous NaHC03 solution (slowly, maintaining the temperature at 25-30 ° C) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with brine (3 x 50 mL), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, Synthesis of (Z) -isopropyl 3 - (3- (4-chloro-3, 5 -bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) acrylate:

[000306] A solution of 3 - (4-chloro-3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole (2.1 g) in DMF (20 ml) was treated with DABCO ( 1.5 g, 2 eq.) And the mixture was stirred for 30 min before the addition of (Z) -isopropyl-3 iodoacrylate (1.76 g, 1.1 eq.). The reaction mixture was stirred at room temperature for 5 h, then poured into ice water (50 ml) and extracted with EtOAc (3 x 15 ml). The combined organic extracts were washed with brine (3 x 10 ml), dried over anhydrous Na2S04, filtered and concentrated under reduced pressure (25 ° C, 20 mmHg) to obtain 3.0 g of crude compound. Purification by column chromatography, using (60/120 Si02 mesh, lution with 1-1.2% MeOH in CH2 Cl2) provided the desired unsaturated ester (1.33 g, yield: 52%). Synthesis of (Z) -3 - (3 - (4-chloro-3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - acrylic acid:

[000307] A 25 mL bottle, 3 necks, round bottom was loaded with a solution of (Z) - isopropyl 3 - (3 - (4-chloro-3,5-bis (trifluoromethyl) phenyl) -lH-l , 2,4-triazol-1-yl) acrylate (1.33 g) in 1: 1 THF: water (26 ml). The solution was treated with solid LiOH (0.53 g, 4 eq.) And stirred at room temperature for 4 h before being diluted with 400 ml of water, acidified to pH = 2-3 with diluted aqueous HCl, and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2S04, filtered and concentrated under reduced pressure to obtain 0.8 g of crude compound (yield: 66%). Synthesis of (Z) -3 - (3 - (4-chloro-3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N '- (pyrazin-2- il) acrilohydrazide:

[000308] In a 50 ml, 3-neck, round-bottom flask loaded with a solution of (Z) -3 - (3 - (4-chloro-3,5, bis-trifluoromethyl) phenyl) -lH-l , 2,4-triazol-1-yl) -acetic acid (0.8 g) in 1: 1 EtOAc: THF (20 mL). The solution was cooled to -70 ° C and sequentially treated with 2-hydrazinopyrazine (0.275 g, 1.2 eq), T3P (50% in EtOAc, 2.5 ml, 2.0 eq.) And DIPEA (1.44 mL, 4.0 eq.) added dropwise. The clear reaction mixture was stirred at -60 ° C for 1 h before being concentrated under reduced pressure (25 ° C, 20 mm Hg) to obtain the crude compound. Purification by column chromatography using (60/120 Si02 mesh, eluting with 3-4% MeOH in CH2 Cl2) provided 0.30 g (yield: 30%) (Z) -3 - (3 - (4-chloro- 3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N '- (pyrazin-2-yl) acrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ = 10.53 (s, 1H), 9.58 (s, 1H), 9.11 (s, 1H), 8.47 (s, 1H), 8, 32 (s, 1H), 8.13 (s, 1H), 8.06 (s, 1H), 7.97 (s, 1H), 7.52-7.55 (d, J = 10.4 Hz , 1H), 6.08-6.11 (d, J = 10.4 Hz, 1H); LCMS for C17H11ClF6N7O [M + H] +478.76 found 478.1 (RT 2.64 min, purity: 100%). Example 24: Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) - N'-cyclopropylacrylhydrazide (1-25) .

[000309] A 100-mL, 3-neck, round-bottomed flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazole- 1-yl) acrylic acid (0.50 g.) and CH2 Cl2 (25 ml). DCC (0.29 g, 1.0 eq.) Was added and the mixture was cooled to 0 ° C during the sequential addition of cyclopropyl hydrazine hydrochloride (0.15 g, 1.0 eq.) And DIPEA (0, 24 mL, 1.0 eq.). The reaction mixture was stirred for 1H before being poured into water (50 ml) and extracted with CH2 Cl2 (2 x 50 ml). The combined organic extracts were washed with brine (50 ml), dried over anhydrous MgS04, filtered, and concentrated under reduced pressure (25 ° C, 20 mmHg) to provide the crude compound. Purification by flash-combi chromatography (elution with 1.5-2.5% MeOH in CH2C12) followed by further purification on a preparative TLC plate (elution with 70% EtOAc in hexane) gave 15 mg (yield: 2 , 6%) (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1,2,4-triazol-1-yl) -N'-cyclopropylacrylhydrazide. 1H NMR (400 MHz, DMSO-d6) δ, 9.16 (s, 1H), 8.52 (s, 1H), 8.28 (s, 1H), 7.23-7.26 (d, J = 10.4 Hz, 1H), 6.40-6 .43 (d, J = 10.4 Hz, 1H), 4.97 (s, 1H), 4.63 (s, 1H), 3.18-3 , 20 (m, 1H), 0.830.87 (m, 2H), 0.65-0.69 (m, 2H); LCMS for the Chemical Formula: C16H14F6N5O [M + H] +406.31 406.19 found (RT 2.74 min, purity: 98.85%). Example 25: Synthesis of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -N- (3-hydroxy azetidin-1 -yl) acrylamide (1-26).
Synthesis of l-amino-azetidin-3-ol:

[000310] A cooled solution (15-20 ° C) of azetidin-3-ol hydrochloride (2.0 g) in water (20 ml) was treated with NaOH (0.8 g in 10 ml of water) and the The mixture was stirred at 15-20 ° C for 1 h. The reaction mixture was then cooled to 0 ° C and sequentially treated with a solution of NaN02 (1.89 g in 10 ml of water) and acetic acid (1.3 ml). After being stirred for 2 h at 0-5 ° C, the reaction mixture was poured into water (20 ml), acidified to pH = 2-3 with dilute aqueous HCl and extracted with EtOAc (3 x 25 ml). The combined organic extracts were washed with brine (20 ml), dried over anhydrous Na2S04 and concentrated under reduced pressure to obtain 0.26 g of the desired compound, which was used as such in the next step (LCMS purity: 59.84 %).
[000311] A solution of l-nitrosoazetidin-3-ol (0.25 g) in MeOH (15 ml) was cooled to -75 ° C and treated with dilute aqueous HCl (1.5 ml). Zinc powder (1.35 g) was then added in portions and the reaction mixture was stirred at approximately -70 ° C for 3 h before being filtered through Celite ® and concentrated under reduced pressure to obtain 90 mg of 1 -amino-azetidin-3-ol, which was used as such in the next step. Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1,2,4-triazol-1-yl) -N- (3-hydroxy-azetidin-l- il) acrylamide.
[000312] A 50 mL, 3-neck, round-bottom flask was loaded with (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -HH-1,2,4-triazole- 1-yl) acrylic acid (200 mg) and THF (20.0 ml). The solution was cooled to -60 ° C and treated with a solution of 1-amino-azetidin-3-ol (65 mg, 1.3 eq.) In THF. T3P (50% in EtOAc, 0.67 mL, 2.0 eq) and DIPEA (0.51 mL, 2.0 eq) were added simultaneously and the reaction mixture was stirred for 30 min at -60 ° C before allowed to warm up to room temperature. The reaction mixture was then concentrated under reduced pressure (35 ° C, 20 mmHg), giving 100 mg of solid. Purification by column chromatography (elution with 3% EOH in CH2C12) provided 20 mg of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4- triazol - 1-yl) -N- (3-hydroxyazetidine-1-yl) acrylamide. 1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, IH), 6.38 (s, IH), 8.52 (s, 2H), 8.26 (s, IH), 7.32 -7.35 (d, J = 10.8 Hz, IH), 6.40 (d, exchangeable, IH), 5.78-5.81 (d, J = 10.8 HZ, IH), 4, 14-4.15 (d, IH), 3.82 (m, 2H), 3.71 (m, 2H); LCMS for Chemical Formula C16H14F6N5O2 [M + H] + found 422.31; 422.19 (RT 2.46 min, purity: 91.49%).
[000313] EXAMPLES 26-31: Examples 26-31 describe new methods of synthesis useful in the preparation of the compounds of the invention (for example, as precursors of compounds of the invention, such compounds described by formula Z above). Example 26.
Synthesis of isopropyl propiolate:

[000314] A 20-L flask, with four necks, with a round bottom, equipped with an addition funnel, thermometer socket and a mechanical stirrer, was loaded with propiolic acid (1,000 g, 1 equiv.) And IPA (8 L, 8 vol.). BF3-etherate (4.54 kg, 2.0 equiv.) Was added slowly from an addition funnel at 25 ° C over a period of 30 minutes. The temperature of the reaction mixture was gradually increased to 90 ° C and the reaction mass was maintained at that temperature for 3 hours. GC monitoring after 3 hours showed the completion of the reaction. The reaction mixture was cooled to room temperature, quenched with 20 L of cold DM water and stirred for 30 minutes. 10L of dichloromethane was added to the reaction mixture and the reaction mass was stirred for another 30 minutes. The organic layer was separated and the aqueous layer was re-extracted with 5 L of dichloromethane. The combined organic layers were washed with 10 L of saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo at 35 ° C to 40 ° C (product is volatile) to produce the product as a brown liquid (1.32 kg , 81.25%). Purity 89.67% (GC), 1H NMR (300 MHz, CDC13) δ: 1.22 (d, 6H, J = 6.6 Hz), 2.85 (s, 1H), 4.98-5, 05 (m, 1H). Synthesis of (3-isopropyl-iodoacrylate):

[000315] A 20-L, four-necked, round-bottomed flask, equipped with an addition funnel, a thermometer socket and a mechanical stirrer was loaded with propionic isopropyl ester (1,000 g, 1 equiv.) And acetic acid ( 3.7 L, 3.7 vol.) At 25 ° C and the reaction mass was stirred for 10 minutes. Sodium iodide (2.138 kg, 1.6 vol.) Was added and the reaction mixture was stirred (a dark brown color was observed). The temperature was raised to 110 ° C and the reaction was maintained at that temperature for 1.5 hours. GC monitoring showed the completion of the reaction after 1.5 hours. The reaction mixture was cooled to room temperature, quenched with ice cold DM water (18.75L, 18.75 V) and stirred for 30 minutes. MTBE (5 L) was added to the reaction mass and stirred for another 30 minutes. The organic layer was separated and the aqueous layer was re-extracted with MTBE (5 L). The combined organic layer was washed with NaHC03 (2 x 10 L), NaHS03 (2 x 5 L), saturated saline (5.2 L, 5.2 V), dried over sodium sulfate and concentrated in vacuo at 35 °. C to provide (Z) -isopropyl-3 iodoacrylate as a brown liquid (1.49 kg, 70%). Purity 87.34% (GC), 1H NMR (300 MHz, CDC13) δ: 1.28 (d, 6H, J = 6.3 Hz), 5.08-5.131 (m, 1H), 6.83 ( d, 1H, J = 8.7 Hz), 7.38 (d, 1H, J = 8.7 Hz). Synthesis of 3,5-bis (trifluoromethyl) benzothioamide:

[000316] A 20-L, multi-necked flask equipped with an overhead stirrer, and thermometer socket was loaded with bis (trifluoromethyl) benzonitrile (1.25 kg, 1.0 equiv.) And DMF (6.25 1.5V), and the resulting mixture was stirred under nitrogen at room temperature (28 ° C). The reaction mixture was cooled to 10 ° C and 0.775 g NaSH.H20 (2 equiv.) Was added over a period of 10 minutes. After stirring for 15 minutes, MgCl2.6H20 (1,169 kg, 1.1 equiv.) Was added over a period of 15 minutes and the reaction was stirred for another period of 35 minutes.The progress of the reaction (green colored solution) was monitored by HPLC, which revealed 99.6% product and 0.03% benzonitrile. The reaction mixture was cooled to 0-5 ° C and 30% dil. (3.75 L) was added dropwise to adjust the pH to 2-3. The resulting mass was extracted with MTBE (5 L x 1). The layers were separated and 1 L of DM water was added to the aqueous layer, which was re-extracted with MTBE (2.5 L x 1). The combined organic layers were washed with brine (4.5 L x 3), dried and concentrated in vacuo. Hexane was added to the obtained solid, followed and the product was isolated as a yellow solid (1,400 kg, 98.0%). Purity: 99.28% (HPLC). 1H NMR (300 MHz, CDCl3) δ: 8.27 (s, 1H), 8.53 (s, 2 h), 10.0 (s, 1H), 10.38 (s, 1H). Synthesis of 3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1,2,4-triazole:

[000317] A 20-L, multi-necked flask equipped with an overhead stirrer and thermometer socket, was loaded with thioamide (1,378 g, 1 equiv.) And DMF (6.89 L, 5V), and the mixture was stirred under nitrogen at room temperature (28 ° C). The reaction mass was cooled to 10 ° C and hydrazine hydrate (505.4 g, 2.0 equiv.) Was added dropwise over 2 hours with stirring. The reaction mass was cooled to 0 ° C to 5 ° C and formic acid was added over a period of 1 hour (6.89 L, 5 V) (exothermic reaction was observed and the temperature increased to 20 ° C). The reaction mixture was then heated to 95 to 100 ° C for an additional 12 hours. The progress of the reaction was monitored by HPLC which showed the formation of 99.5% of the product. The reaction mass was cooled to 35 to 40 ° C, added to 20.6 liters of pre-cooled DM water (10 to 15 ° C) and stirred for 30 minutes. The reaction mass was extracted with MTBE (8.26 L). The aqueous layer was extracted again with MTBE (5.512 L) and the combined organic layers were washed with 10% sodium bicarbonate (6.89 L, 2V), brine (6.89 L x 3), dried with sodium sulfate and concentrated in vacuo. Dichloromethane (2V) was added to the obtained yellow solid and stirred at 0 to 5 ° C for 1 hour, which, in filtration, gave the product as a yellow solid (1.156 g, 82.2%). Purity: 99.7% (HPLC), 1H NMR (300 MHz, DMSO) δ: 8.15 (s, 1H), 8.55 (s, 2H), 8.79 (s, 1H), 14.5 (s, 1H, NH). Synthesis of (Z) -isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) acrylate

[000318] A 10-L, four-necked round-bottom flask, equipped with an addition funnel, thermometer socket, mechanical stirrer, stopper was loaded with 3 - (3,5-bis (trifluoromethyl) phenyl) -lH- 1,2,4-triazole (600 g, 1.0 eq.), DABCO (480 g, 2.0 eq) and DMF (3.0 L). The reaction mixture was stirred for 30 minutes. After 30 minutes, a solution of iodine ester (1024.8 g, 2.0 eq) in DMF (1200 ml) was added dropwise over a period of 1 hour. The progress of the reaction was monitored by HPLC and showed (Z) -isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate: 62, 36% and 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole: 15.1%. After an additional 1 hour, an equivalent of DABCO (258 g) was added and the reaction was continued for another hour. HPLC analysis showed the conversion of 75.63% of (Z) - isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate and 2% 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazole. The reaction mixture was quenched with cold DM water (12 L), stirred for 15 minutes, and extracted with ethyl acetate (2 x 6 L). The combined organic layers were washed with saturated brine solution (30% 2 x 3 L), dried over anhydrous sodium sulfate (100 g) and concentrated. The crude mass (840 g) was received in a 10 L round-bottom flask and methanol (1200 mL) was added. The solution was maintained at 0-5 ° C and stirred for 30 minutes. The obtained solid was filtered and washed with methanol (200 ml), which provided the product as a white solid (550 g, 65.0%). Purity: 87.34% (HPLC); 1H NMR (300 MHz, CDC1 3) δ: 1.30 (d, 6H, J = 6.0 Hz), 5.12 (m, 1H), 5.73 (d, 1H, J = 10.8 Hz ), 7.24 (d, 1H, J = 10.8 Hz), 7.91 (s, 1H), 8.58 (s, 2H), 9.70 (s, 1H). cis Isomer: transisomer ratio is 83: 8. Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylic acid

[000319] A 5L flask, with four necks, with a round bottom, equipped with an addition funnel, thermometer socket, mechanical stirrer and stopper, was loaded with THF (1.25 L) and (Z) -isopropyl 3 - ( 3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate (125 g, 1 eq.) At room temperature. The reaction mixture was cooled to 0 ° C. To the stirring solution was added a cold lithium hydroxide solution (66.58 g in 1.25 L of water) over a period of 30 minutes via an addition funnel. The reaction temperature was slowly raised to 25 ° C and the reaction mass was maintained at that temperature for 2 hours. HPLC monitoring showed the following state: (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazol-l-yl) -acrylic acid: 87.66 %, (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -acrylic acid: 9.91%, (Z) - isopropyl 3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1 H-1, 2,4-triazol-1-yl) 2% acrylate. The reaction continued for another 30 minutes and subjected to HPLC monitoring ((Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acid acrylic: 88.20%, (E) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) -acrylic acid: 11,03% After completion of the reaction, the reaction mixture was cooled sharply with ice water (385 ml) and stirred for 30 minutes, the pH was adjusted to 1-2 with diluted hydrochloric acid (30%, 400 ml) and the reaction mass was extracted with ethyl acetate (3 x 625 ml). The combined organic layers were washed with saturated saline solution (30%, 650 ml), dried over anhydrous sodium sulfate (12.5 g) and concentrated under reduced pressure at 30 ° C. -35 ° C Hexane was added to the crude material and stirred for 30 minutes The obtained solids were filtered through a Buchner funnel and washed with hexane (250 ml) The obtained solid was dried for 30 minutes under vacuum and at room temperature for 3-4 hours. The product was isolated with a white powder (92.8 g, 84.36%). Purity: 93% (HPLC), 1H NMR (300 MHz, DMSO-d6) δ: 5.98 (d, 1H, J = 10.2 Hz), 7.48 (d, 1H, J = 10.2 Hz ), 8.2 (s, 1H), 8.50-8.54 (m, 2H), 9.39 (s, 1H). Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1, 2,4-triazol-1yl) - 1 - (3, 3-1-difluoroazetidin -yl) -2-en-1-one:

[000320] To a 3-L flask, with four necks, round bottom, equipped with nitrogen inlet, addition funnel, thermometer socket, mechanical stirrer, was added (Z) -3 - (3 - (3, 5-bis (trifluoromethyl) phenyl) - 1H-1,2,4-triazol-1-yl) -acrylic acid (100 g, 1.0 eq.) In DCM (1.8 L, 18 V). The reaction mixture was cooled to -10 ° C. HOBT (4.4 g, 0.1 eq.), EDC-HC1 (80.6 g, 1.5 eq.) And 3,3-difluoroazetidine hydrochloride (44 g, 1, 2 eq.). To the resulting mixture at -10 ° C, DIPEA (72 ml, 1.5 eq) was added dropwise over a period of 1.5 hours. The progress of the reaction was monitored by HPLC analysis which showed that the completion of the reaction at the end of the addition of DIPEA. The reaction temperature was slowly raised to 15 ° C to 20 ° C (~ 2 h). The reaction mixture was quenched with 1 L of ice-water paste. The organic layer was separated and the aqueous layer was extracted with DCM (400 ml x 2). The organic layer was washed with saturated brine solution (2 x 500 ml), dried over anhydrous Na2 SO4 (10 g) and concentrated under reduced pressure (~ 35 ° C) to obtain the crude compound. The crude compound thus obtained was dissolved in 5 vol. of DIPE and stirred at room temperature for 30 min. and then filtered. The gross weight was 100 g (yield = 82.39%) [cis-85.07% by HPLC, Trans-14.36% by HPLC].
[000321] The crude compound thus obtained was further purified by recrystallization from ethyl acetate according to the following procedure. To a 500 mL flask, with four necks, round bottom, equipped with a mechanical stirrer, thermometer socket and stopper, 100 g of (Z) -3- (3- (3,5-bis (trifluoromethyl) phenyl) - were added - 1H- 1,2,4-triazol-1-yl) -1- (3,3-difluoroazetidin-1-yl) prop-2-en-1-one. To this compound at room temperature was added ethyl acetate (7 volumes), under stirring. However, the compound was not completely soluble. Thus, the resulting solution was heated to 60 ° C to obtain a clear solution and then it was slowly cooled to - 30 ° C. At -30 ° C, the solution was stirred for 20 min. and filtered under suction. The obtained compound was dried under vacuum at 40-45 ° C for 3 h - 4 h to provide the product as a white solid. (Cis-98.9% by HPLC), (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) -lH-1,2,4-triazol-1-yl) -l- ( 3, 3-difluoroazetidin-1-yl) prop-2-en-1-one. 1H NMR (300 MHz, CDC1 3) δ 9.57 (s, 1H), 8.56 (s, 2H), 7.90 (s, 1H), 7.187.21 (d, J = 10.8 Hz, 1H), 5.61 - 5.65 (d, J = 10.8 Hz, 1H), 4.39 - 4.45 (m, 4H). Example 27 Synthesis of (Z) -3-iodoacrylic acid:

[000322] A 250 ml flask with three bottle flasks, round bottom equipped with nitrogen input was added propiolic acid (7.0 g, 1.0 eq) dissolved in acetic acid (70 ml, 10 V) and iodide sodium (29.96 g, 2.0 eq). The reaction mixture was refluxed at 1000 C for 2-3 h. The progress of the reaction was followed by TLC analysis on silica gel with 10% MeOH: DCM as the mobile phase. SM Rf = 0.3 and the product of Rf = 0.5. The reaction mixture was poured into ice water (700 ml) and neutralized with a saturated solution of bicarbonate solidum. The reaction mixture was extracted with EtOAc (3 x 100 ml). The combined organic layers were washed with brine solution (3 x 100 ml), dried over MgS04, filtered and concentrated by rotary evaporation (25 ° C, 20 mmHg) to obtain 12.0 g of the crude compound which was purified by chromatography. column using 60/120 silica using MeOH: DCM as mobile phase. The column (5 x 10 cm) was conditioned in DCM and the elution in MeOH gradient started, starting with the collection of fractions (50 mL fractions) of 2% to 5% MeOH in DCM. The compound started eluting with 2% MeOH in DCM. Fractions containing such a TLC profile were combined to obtain 8.0 g of the desired compound (40.44% yield). Synthesis of - 1 - (3, 3-difluoroazetidin-1-yl) -3-iodoprop-2-en-1-one:

[000323] In a 25 mL flask, with three round bottom bottlenecks, equipped with nitrogen inlet and a rubber septum, (Z) - 3 iodoacrylic acid (0.250 g, 1.0 eq.) Was dissolved in DCM (10 mL, 40 V). The reaction mixture was cooled to 0 ° C, and DIPEA (0.168 g, 1.1 eq), HATU (0.494 g, 1.1 eq) and 3,3-difluoroazetidine hydrochloride (0.179 g, 1.1) were added. The reaction mixture was stirred at 0 ° C for 2-3 hours. The progress of the reaction was followed by TLC analysis on silica gel with 40% ethyl acetate in hexane. The reaction mixture was filtered and concentrated by rotary evaporation (25 ° C, 20 mmHg) to obtain 0.3 g of crude compound, which was purified by column chromatography using silica 60/120 using 40% ethyl acetate in hexane as a mobile phase. The column (5 10 cm) was conditioned in 5% ethyl acetate in hexane and the gradient eluted in ethyl acetate starting with the collection of fractions (50 mL fractions) of 20% to 30% acetate of ethyl in hexane. The compound started eluting with 20% ethyl acetate in hexane. The fractions containing such a TLC profile were combined to obtain 0.18 g of the desired compound (52.33% yield). Mass: [M + H] +: 273.8. Synthesis of (Z) -3 - (3 - (3,5-bis (trifluoromethyl) phenyl) - 1H-1,2,4-triazol-1-yl) - 1 - (3, 3-difluoroazetidine-1-yl ) prop-2-en-1-one:

[000324] In a 25 mL flask, with three round-bottomed necks, equipped with nitrogen inlet, (3,5-bis (trifluoromethyl) phenyl) -H-1, 2,4-triazole (0.18 g, 1.0 eq.) Was dissolved in DMF (5.0 mL, 27.0 V), and DABCO (0.143 g, 2.0 eq) and (Z) -l- (3,3-difluoroazetidin-l-yl ) -3- iodoprop-2-en-1-one (0.192 g, 1.1 eq) were added. The reaction mixture was stirred at room temperature for 2-3 hours. The progress of the reaction was followed by TLC analysis on silica gel with 80% ethyl acetate-hexane as the mobile phase, SM Rf = 0.60 and product Rf = 0.4. The reaction mixture was poured into ice water (50 ml) and extracted with EtOAc (3 x 25 ml). The combined organic layers were washed with brine solution (3 x 25 ml), dried over MgS04, filtered, and concentrated by rotary evaporation (25 ° C, 20 mmHg) to obtain 0.3 g of crude compound, which was purified by column chromatography using silica 60/120, using ethyl acetate: hexane as the mobile phase. The column (5 x 10 cm) was conditioned in hexane and eluted in ethyl acetate in the form of a gradient starting with the collection of fractions (fractions of 50 ml) from 40% to 45%) of ethyl acetate in hexane . The compound started eluting with 40% ethyl acetate in hexane. Fractions containing such a TLC profile were combined to obtain 70 mg of the desired compound (25.64% yield). Example 28. Synthesis of (Z) -isopropyl 3 - (3 - ((3-isopropoxy-5 trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate:

[000325] Synthesis of isopropyl propiolate. To a mixture of propiolic acid (500 g, 7.1 moles) in isopropanol (4000 ml) was added BF3 etherate (2015 g, 14.2 moles) at 10 ° C. After stirring for 10 minutes, the reaction mixture was heated to 90 ° C and stirred for 2 hours. The completion of the reaction was monitored by TLC. The reaction mixture was brought to 25 to 30 ° C and quenched with crushed ice followed by extraction with dichloromethane. The organic layer was washed with water and then with brine solution. The organic layer was dried over sodium sulfate and concentrated in vacuo to provide isopropyl propiolate (440 g, 55%). The product was confirmed by 1H NMR.
[000326] Synthesis of (Z) -isopropyl-3 iodoacrylate. To a mixture of isopropyl propiolate (350g, 3.1 moles) in AcOH (1,300 ml) was added Nal (930 g, 6.2 moles) at 25 ° C. The reaction mixture was heated to 115 ° C and stirred for 1.5 hours. The reaction mixture was cooled to 25 to 30 ° C and quenched with water, followed by extraction with MTBE. The organic layer was washed with saturated bicarbonate, bisulfite and a brine solution. The organic layer was dried over sodium sulfate and concentrated in vacuo to provide the product (Z) - isopropyl-3 iodoacrylate (626 g; 83.5%) The product was confirmed by 1H NMR. Synthesis of 3-isopropoxy-5 (trifluoromethyl) benzonitrile:

[000327] To a mixture of propan-2-ol (102.96 g 1.76 moles) in DMF (3200 ml, 8 V) at 5 ° C was added NaH (122 g, 5.08 moles). The mixture was stirred for 2 hours. To this mixture was added 3-fluoro-5- (trifluoromethyl) benzonitrile (400, 2.1 mol) dropwise. The temperature of the dough was raised to 25 to 30 ° C and maintained at the same temperature for 1 hour. The reaction was monitored by HPLC. After completion, the reaction mixture was quenched with ice water and extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over sodium sulfate and then concentrated in vacuo to provide 530 g (2.31 moles, 110%) of 3-isopropoxy-5 (trifluoromethyl) benzonitrile, which was taken as such for the next step without further purification. HPLC purity - 96.5. % By area (a / a). Synthesis of 3-isopropoxy-5 (trifluoromethyl) benzothioamide:

[000328] 3-Isopropoxy-5- (trifluoromethyl) benzonitrile (1,000 g, 4.3 moles) was dissolved in DMF (4000 ml) and sodium hydrogensulfide hydrate (636 g, 8.6 moles) was added followed by hexahydrate magnesium chloride (960.2 g, 4.7 moles). The reaction mixture was stirred for 1 hour at 25 to 30 ° C. The completion of the reaction was monitored by TLC using ethyl acetate: hexane (2: 8) as the mobile phase. The reaction mixture was quenched in an ice-water paste (250 ml) and the pH was adjusted to 5 by the addition of 10% aqueous HCl. The reaction mixture was extracted with MTBE and was washed with 20% brine solution. The organic layer was concentrated in vacuo to provide 1136 g (4.3 moles, 100%) of the title compound, which was taken as such for the next step. HPLC purity - 97.37% w / w. Synthesis of 3 - (3-isopropoxy-5- (trifluoromethyl) phenyl) -1H-1,2,4-triazole:

[000329] 3-Isopropoxy-5- (trifluoromethyl) benzothioamide (646 g; 2.74 moles) was combined with hydrazine hydrate (140 g, 4.4 moles) and DMF (3,200 ml; 5V). The mixture was stirred for 30 minutes and cooled to 10 ° C. To this reaction mixture was added formic acid (3,200 ml) dropwise. The reaction mixture was heated to 90 to 100 ° C and maintained for 12 hours. After the completion of the reaction by HPLC, the reaction mass was cooled to 25 to 30 ° C and quenched with ice water. The mixture was extracted in MTBE. The organic layer was washed with brine, followed by aqueous sodium bicarbonate, and concentrated in vacuo. The residue was expelled using hexane, the resulting residue was formed in paste at 10 ° C for 1 hour. The obtained solid was filtered and dried for 12 hours at 25 to 30 ° C to provide 550 g (2.26 moles: 82%) of the product 3 - (3-methoxy-5- (trifluoromethyl) phenyl) -H-1, 2,4-triazole. HPLC purity -95.24% w / w. Synthesis of (Z) -isopropyl 3 - (3 - (((3-isopropoxy-5 trifluoromethyl) phenyl) -1H-1,2,4-triazol-1-yl) acrylate:

[000330] A mixture of 3 - (3-methoxy-5- (trifluoromethyl) phenyl) -H-1,2,4-triazole (500 g, 1.8 moles) and DABCO (417.6 g; 3.6 moles) in DMF (1200 ml) was stirred for 30 minutes. To this mixture was added (Z) - isopropyl-3 iodoacrylate (864 g; 3.6 moles) in DMF (1200 ml), slowly, at 25 to 30 ° C and the reaction mixture was stirred for 1 hour. After 1 hour, DABCO (208 g, 1 eq) was added and the reaction mixture was stirred for 1 hour. HPLC analysis showed 3 - (3-methoxy-5- (trifluoromethyl) phenyl) - 1H-1,2,4-triazole 9.59%, (Z) - isopropyl 3 - (3 - (3-isopropoxy-5 - (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) acrylate: 73.76%, (E) - isopropyl 3 - (3 - (((3-isopropoxy-5 trifluoromethyl) phenyl) - 1 H-1,2,4-triazol-1-yl) methyl acrylate: 6.66%. The reaction mass was quenched with water, extracted with dichloromethane and concentrated in vacuo to provide the crude product. The crude product was subjected to chromatography using ethyl acetate-hexane system on 60-120 silica gel to provide 310 g (0.8 moles, 44%). HPLC purity - 99% w / w. Example 29. Synthesis of (Z) -isopropyl 3 - (3 - (3-methoxy-5- (trifluoromethyl) phenyl) -H-1,2,4-triazol-1-yl) acrylate:

[000331] To a solution of 3 - (3-methoxy-5- (trifluoromethyl) phenyl) -1H-1,2,4-triazole (0.50 g) (prepared according to Example 3) in DMF (1 , 5 mL) DABCO (2 equiv.) Was added. The resulting reaction mixture was stirred for 30 min at room temperature, then (Z) -isopropyl-3 iodoacrylate (2.0 equiv .; prepared according to Example 3) has been added. The resulting mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with ice water, and extracted with ethyl acetate (3 times). The organic layers were separated and the combined organic layer was dried over anhydrous sodium sulfate. LC-MS and HPLC analysis revealed 62% cis isomer and 36% transisomer. 1H NMR (400 MHz, CDC1 3) δ: 9.72 (s, 1H), 8.02 (s, 1H), 7.86 (s, 1H), 7.30 (s, 1H), 7.28 (d, J = 8.8 Hz, 1H), 5.71 - 5.73 (d, J = 10.8 Hz, 1H), 5.12-5.18 (m, 1H), 3.94 (s, 3H), 1.34 (d, 6 H): LCMS for C16H16F3N3O3 [M + 1] +355.31 355.92 found in 4.317 min (LCMS 99.82%). Example 30. Synthesis of (Z) -isopropyl 3 - (3 - (2-chloro-6-isopropoxypyridin-4-yl) -1H-1,2,4-triazol-1-yl) acrylate:

[000332] 2-chloro-6-isopropoxy-4- (1H-1,2,4-triazol-3-yl) pyridine (0.5 g) (prepared as in Example 3) in 3 ml of DMF, added DABCO (0.467g, 2 equiv.) was added and the resulting mixture was stirred for 30 min. A solution of (Z) -isopropyl-3 iodoacrylate (0.990 g, 2 equiv.) (Prepared as in Example 3) was added to the reaction mixture, and the resulting mixture was stirred for 3 h at room temperature. The reaction mixture was made as in Example 3, to obtain 53% cis isomer and 34% 34% trans isomer. Example 31. Synthesis of (Z) -isopropyl 3 - (3 - (3 - (cyclobutylamino) -5 - (trifluoromethyl) phenyl) - 1H-1,2,4-triazol-1-yl) acrylate:

[000333] to N-cyclobutyl-3- (1H-1,2,4-triazol-3-yl) -5 - (trifluoromethyl) aniline (0.5 g) (prepared as in Example 3) in 1.5 ml of DMF, DABCO (0.188 g) was added and the resulting mixture was stirred for 30 min. A solution of (Z) -isopropyl-3 iodoacrylate (0.404 g) (prepared as in Example 3) was added to the reaction mixture, and the resulting mixture was stirred for 3 h at room temperature. The reaction mixture was made as in Example 3, to obtain 44% cis isomer and 20% transisomer.
[000334] Example 32: Tests. Exemplary compounds of the invention were tested in parallel with compounds X1, X2 and X-3 (illustrated in Table 2), in several tests. The results are shown in Table 2 below. Nuclear Export Inhibition
[000335] The ability of exemplary compounds of the invention to inhibit CRM-1 mediated nuclear export was evaluated in a RevGFP assay. Rev is a human immunodeficiency virus type 1 (HIV-1) protein and contains a nuclear export signal (NES) in its C-terminal domain and a nuclear localization signal (NLS) in its N-terminal domain. Nuclear export of Rev protein is dependent on the classic NES / CRM1 pathway (Neville et al. 1997). Nuclear accumulation of Rev can be observed in cells treated with specific CRM1 inhibitors, such as LMB (Kau et al. 2003).
[000336] In this assay, RevGFP-U20S cells were seeded in plates with 384 wells, light-colored, black, the day before the experiment. The compounds were serially diluted 1: 2 in DMEM, starting from 40 μM in a separate 384-well plate, and then transferred to the cells. The cells were incubated with the compound for approximately 1 hour before fixation with 3.7% formaldehyde and staining the nuclei with Hoechst 33258. The amount of GFP in the cell nucleus was measured and the IC50 of each compound was determined (Kau and others 2003). The compounds of the invention are considered active in the Rev-GFP assay described above, if they have an IC 50 less than approximately 10 μM, with the most preferred compounds having an IC 50 less than approximately 1 μM. The results of the RevGFP assay appear in Table 2. Cell Proliferation Assay
[000337] The CellTiter® AQueous One Solution cell proliferation assay (Promega) was used in MM.1S multiple myeloma cell lineage to study the cytotoxic and cytostatic properties of the compounds. The assay is based on the cleavage of the tetrazolium salt, MTS, in the presence of an electron coupling reagent PES (phenazine ethosulfate). The tetrazolium compound MTS is bioreduced by cells in a colored formazan product, which is soluble in tissue culture medium. This conversion is performed, presumably by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. The tests are performed by adding a small amount of CellTiter® AQueous One Solution reagent directly to the culture wells, incubating for 1-4 hours and then recording the absorbance at 490 nm with a 96 well plate reader. Absorbance revealed a direct correlation with the number of cells and their metabolic activity.
[000338] The cells were seeded at 5 x 10 3 to 1.5 x 10 4 cells in each well of a 96 well plate in 100 μL of fresh culture medium and the adherent cells were allowed to bind overnight. The material solutions of the compounds were diluted in cell culture medium to obtain eight concentrations of each drug, ranging from 1 nM to 30 μM and DMSO less than 1% v / v was used as a negative control. The resulting drug solutions were transferred to the cells. After 72 h of treatment, 20 μl of CellTiter 96® Aqueous reagent was added to each well of the 96-well assay plates and the plate was incubated at 37 ° C for 1-4 hours in a humid, 5% C02. Then, the absorbance of each well was recorded at 490 nm, using a 96 well plate reader. In most cases, the assay was performed in triplicate and the results were presented as half of the maximum inhibitory concentration (IC50). Optical density versus compound concentration was plotted and analyzed using non-linear regression equations (IDBS XLfit) and the IC50 for each compound was calculated. Pharmacokinetics (PK) and brain assay: Plasma ratio determination
[000339] AUC. Blood was collected from mice (n = 3) to contribute to the total of 10 time points (before dose, 5 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours hours after the dose). The mice were bled on a rotating basis, each mouse contributing three time points to blood collection. At the designated time points, the animals were anesthetized with isoflurane, and approximately 110 μL of blood per time point was collected by retro-orbital puncture in pre-cooled K2EDTA (anticoagulant) tubes. Blood samples were placed on wet ice and centrifuged (2000g, 5 min at 4 ° C) to obtain plasma within 30 minutes after sample collection. All samples were stored frozen at approximately - 80 ° C until analysis. Before analysis, the samples were mixed with the internal standard (dexamethasone) in acetonitrile, vortexed, centrifuged, and the supernatant was injected for analysis. The concentration of compounds in plasma was determined using instrumentation LC-MS-MS (API 4000, triple quadrupole with electrospray ionization; liquid chromatography column of ultra-sharp performance Cl8, with MeOH and formic acid as organic solvents). AUC values were calculated using WinNonlin Professional software package 6.2, NCA200 non-compartment pharmacokinetic model.
[000340] Brain to Plasma Ratio (B: P). A separate group of mice (N = 3) was dosed (PO at 10 mg / kg) and then sacrificed at the time of maximum plasma concentration (Tmax estimated at 2 hours after dose), in which brain tissue and terminal plasma of time were collected. After collection, brain tissue was rinsed with cold saline, dried on filter paper, weighed and frozen by placing on dry ice. All samples were stored frozen at approximately -80 ° C until analysis. At the time of analysis, the brain tissue was homogenized (PBS homogenize solution, pH 7.4), mixed with internal standard (dexamethasone) in acetonitrile, stirred, centrifuged, and the supernatant was injected for analysis of the compound concentration using LC-MS-MS (API 4000, Triple Quadruple with electrospray ionization; Acuity C18 ultra-performance liquid chromatography column, with MeOH and formic acid as organic solvents). The plasma samples were treated with the identical method (except the homogenization step) and the concentration of the compound in each matrix was calculated based on generated standard curves. The results of the PK test and the determination of the ratio B: P determination are shown in Table 2. Table 2. Test results for compounds of formula I and comparators thereof (A = IC50 value of <= 1 μ M; B = IC50 value of 1-10 μ M; C = IC50 value of> 10 μ M; NT = not tested).




* Dosed in mice at 10 mg / kg po. ** Compound 26 of US 2009/0275607. *** Compound 44 of US 2009/0275607. ‡ AUCInf values for compound X-1 dosed in mice at 10 mg / kg po were below the limit of quantification. The data reported for 5 mg / kg iv. † Dosed in rats at 10 mg / kg po.
[000341] The AUCinf for compound X-1 was below the limit of detection when administered to mice at 10 mg / kg po. When administered at doses of 5 mg / kg iv, compound X-I showed minimal exposure, as indicated by the low AUCinf of 209 hr * mg / mL. The brain-to-plasma ratio for compound X-1 has not been determined due to its negligible exposure levels when administered po.
[000342] The AUCinf for compound X-2 was calculated to be 68.3 hr * ng / ml when administered to rats at doses of 10 mg / kg po. Such exposure levels are extremely low when compared to compound X-3 and the compounds of formula I of the present invention. However, compound X-2 exhibits a moderate brain to plasma ratio. Low AUCinf together with a non-negligible brain-to-plasma ratio suggests that compound X-2 can cross BBB, despite low exposure levels. Compound X-2 is believed to have a significantly higher brain-to-plasma ratio if AUCinf were increased.
[000343] The AUCinf for compound X-3 was calculated to be 12300 hr * ng / mL when administered to rats at 10 mg / kg po, indicating good exposure. However, compound X-3 demonstrated a high B: P ratio of 5.0.
[000344] The compounds of Formula I are characterized by AUCinf greater than approximately 3300 hr * ng / mL, in most cases, greater than approximately 3500 hr * ng / mL, and a relatively low B: P ratio (<2, 5). In general, higher levels of exposure to a therapeutic agent increase the likelihood of penetration into the brain. It is therefore surprising and unexpected that the compounds of formula I have high levels of AUCinf and relatively low brain to plasma ratios. In vivo and in vitro activity of compounds of the invention against breast cancer
[000345] Basal-like breast cancers (BLBC) make up up to 15% breast cancer (BC) and are generally triple negative breast cancer (TNBC) and characterized by a lack of ER, PR progesterone receptor and HER amplification -two. In addition, most of the BCs associated with BRCA-1 are BLBC and TNBC, expressing basal cytokeratins and EGFR. BLBC is characterized by an aggressive phenotype, high histological grade, and poor clinical results with recurrence and high metastasis rates. Additional therapies are needed. The activity of the compounds of the invention, for example, Compound 1-3 was evaluated in various breast cancer cell lines, in vitro and in vivo. Inhibition of TNBC Xenograft (Triple negative breast cancer) In Vivo
[000346] MDA-MB-468 triple negative breast cancer cells (ATCC # HTB-132) were obtained from the ATCC. These cells were cultured in Leibovitz's L-15 medium supplemented with 10% fetal calf serum (FCS), 1% penicillin and streptomycin, and 2 mM L-glutamine. The cells were subcultured by dilution, at a 1: 3 ratio. Fifty (50) female SCID mice (Charles River Labs), aged 5 to 6 weeks, with an average pre-treatment body weight of 19.2 grams were used. SCID mice were inoculated s.c. on the left flank with 5 x 10 6 MDA-MB-468 cells. When the tumors reached an average size between 100 and 200 mm3, the mice were randomly and prospectively divided into a vehicle control group of ten mice (10) and five treatment groups of eight (8) mice per vehicle control group. . The groups were as follows: Vehicle (1% Pluronic, 1% PVP in distilled water) 5 FU 50mg / kg Compound I-3 5mg / kg Monday (M), Wednesday (W), Friday Monday (F) Compound I-3 15 mg / kg, M, W, F Compound I-3 25 mg / kg M, W, F. Compound 1-3 25 mg / kg M, Thursday (Th).
[000347] All administrations were orally. The animals were fed with sterile Labdiet ® 5053 rodent ration (pre-sterilized) and sterile water was provided ad libitum. The tumors were measured once every two days, using micro-tweezers, and the tumor volume was calculated as (length x width x width) / 2. All animals were weighed daily in order to assess weight differences between the animals. treatment groups and monitor animal welfare. Any animals that lost more than 20% of initial weight during the course of the study were euthanized. Any animals with a tumor above 1500 mm3 in volume were also euthanized. Survival was recorded daily. Dosing solutions were freshly prepared daily. Compound 1-3 was supplied as a lyophilized powder containing 67.8% of the drug product with the remainder of Pluronic F-68 and PVP K29 / 32. This was prepared by dissolving the lyophilized powder at a rate of 6.64 mg / 90 uL in sterile water, and diluting as necessary in the vehicle (1% Pluronic F-68 and 1% PVP K29 / 32) in sterile water. All Compound 1-3 dosing solutions were dosed at 0.1 ml / 10 g. Statistical differences between treatment groups were determined using Mann-Whitney Rank Sum or ANOVA tests with a critical value of 0.05.
[000348] On day 33 after inoculation, the tumors were removed. FIG. 1 is a graph of tumor volume as a function of time and shows that Compound 1-3 exhibited efficacy in a dose-dependent manner, inhibiting inhibition of approximately 60% (5 mg / kg on Monday, Wednesday Friday, Friday) at approximately 100% tumor growth (to 25 mg / kg on Monday, Thursday regimen) compared to vehicle treated animals. In addition, compound 1-3 was well tolerated.
[000349] After excision, tumors were also labeled for the tumor suppressor proteins (TSP) FOX03a, IKB, and p27, and the nuclear location of the TSPs was confirmed by immunohistochemistry. Inhibition of proliferation and cytotoxicity in BC TNBC and Luminal cell lines
[000351] The cell proliferation assay CellTiter 96® AQueous One Solution (Promega) was used to study the cytotoxic and cytostatic properties of Compound 1-3 in various BC TNBC and luminal cell lines.
[000352] The cells were seeded in 5x103 to 1.5x104 cells (depending on the type of cells) in each well of a 96 well plate in 100 µL of fresh culture medium and the adherent cells were allowed to bind overnight. The material solutions of the compounds were diluted in cell culture medium to obtain eight concentrations of each drug, ranging from 1 nM to 30 μM and DMSO in less than 1% v / v was used as a negative control. The resulting drug solutions were transferred to the cells. After 72 h of treatment, 20 μl of CellTiter96® Aqueous reagent was added to each well of the 96-well assay plates and the plate was incubated at 37 ° C for 1 - 4 hours in a humid atmosphere, 5% CO2 atmosphere. Then, the absorbance of each well was recorded at 490 nm using a 96 well plate reader. In most cases, the assay was performed in triplicate and the results were presented as half of the maximum inhibitory concentration (IC50). Optical density versus compound concentration was plotted and analyzed using non-linear regression equations (Excel Fit) and the IC50 for each cell line against Compound 1-3 was calculated.
[000353] The results of the cell proliferation assay are shown in Table 3. The results demonstrate the potent cytotoxicity of Compound 1-3 in nine of fifteen BC cell lines tested. The compound was considered potent in a cell line if it had an IC50 value less than approximately 1.0 μM. Cell lines in which Compound 1-3 had an IC50 value less than 1.0 μM were considered sensitive cell lines, while cell lines in which Compound 1-3 had an IC50 value greater than 1, 0 μM were considered resistant cell lines. Seven of the nine sensitive cell lines were TNBC. Genomic analyzes in all BC strains indicated that p53, PI3K / AKT and BRCA1 or status 2 did not affect cytotoxicity. Table 3. IC 50 values for Compound 1-3 in various breast cancer cell lines.

Compound I-3 induces apoptosis and inhibits long-term BC growth
[000354] The ability of compound I-3 to induce apoptosis and inhibit long-term growth of selected BC cell lines was evaluated.
[000355] MDA-MB-468 TNBC, DU4475 and Hs578T TNBC cells were exposed to concentrations of Compound 1-3 ranging from 0 to 10 μM for 24 hours. After 24 hours, the extracts of total protein cells were processed in immunoblots and were exposed to antibodies against the proteins indicated in figures 2A-2C.
[000356] FIGS. 2A-2C are images of immunoblots obtained from some of the most resistant and sensitive breast cancer cell lines described above, including MDA-MB-468 TNBC, DU4475 and Hs578T TNBC. The study shows that Compound 1-3 induces apoptosis in BC TNBC and luminal sensitive cell lines (MDA-MB-468 and DU4475, respectively) after 24 hours, as indicated by decreased PARP and caspase 3, two markers of apoptosis, and the increase in cleaved PARP and cleaved caspase 3. In contrast, only an insignificant increase in cleaved PARP and cleaved caspase 3 was observed when a resistant cell line, Hs578T, was treated with Compound 1-3.
[000357] Long-term growth assays were also performed, in which Hs578T cells MDA-MB-468, MDA-MB-231 and were treated with 1 μM of Compound 1-3 and incubated for 7 (Hs578T) or 10 (MDA-MB- 468 and MDA-MB-231) days. At the end of the test, the medium was removed from the cells and the remaining cells were stained with crystal violet. The study showed that Compound 1-3 inhibited long-term growth of all three cell lines, including both sensitive (MDA-MB-468 and MDA-MB-231) and resistant (Hs578T) cell lines. Compound 1-3 Increases Nuclear FOX03a and IKB in TNBC cell lines
[000358] Basal A TNBC MDA-MB-468 and basal B TNBC BT-20 cells were exposed to DMSO or 1 μM Compound 1-3 for 24 hours and then stained for FOX03a or IKB with or without DAPI nuclear dye . The colored cells were examined for nuclear location. After treatment with compound 1-3, both FOX03a and IkB were located in the cell nucleus, whereas in cells treated with DMSO, both FOX03a and IkB were located in the cytoplasm. Effect of Compound 1-3 on Anti-apoptosis and Cell Cycle Proteins in two TNBC strains
[000359] The effect of increasing concentrations of Compound 1-3 on MDA-MB-468 and Hs578T cells was examined. MDA-MB-468 and Hs578T cells were exposed to increasing concentrations of Compound 1-3 for 24 hours and the total cell protein levels of various proteins were probed with antibodies against the proteins indicated in FIG. 3.
[000360] FIG. 3 shows that, despite the approximately 100-fold difference in the IC50 of Compound 1-3 in two cell lines after 72 hours (lOnM versus 1.5μM), a reduction in MCL-1 is observed in both cell lines in response to increasing concentrations of Compound 1-3.
[000361] The experiments described in Example 32 indicate that inhibition of CRM1-mediated nuclear export by the compounds of the invention, including Compound 1-3, induces the localization and activation of proteins from nuclear tumor suppressor genes, resulting in selective apoptosis , the cytotoxicity of cancer cells and the inhibition of tumor growth. EXAMPLE 33: MONOCLONAL ANTIBODY-INDUCED ARTHRITIS (CAIA)
[000362] BalbC mice were randomly divided into cages on the day of arrival (-1) and each group (N = 8) was assigned to the treatment groups shown below with the following scheme: Vehicle: Day PO 4, 6, 8, 10 Dexamethasone: lmg / kg IP Days 4, 6, 8, 10 Compound 1-4: 4 mg / kg PO, day 4, 6, 8, 10 Compound 1-4: 7.5 mg / kg PO, day 4, 6 , 10, 10 Compound 1-4: 15 mg / kg PO, day 4, 6, 8, 10
[000363] The health status of the animals was analyzed upon arrival. Only healthy animals were acclimated to laboratory conditions and were used in the study. The animals received ad libitum a diet for rodents, commercial, and free access to drinking water, provided to each cage via polyethylene bottles with stainless steel suction tubes. Automatically controlled environmental conditions were set to maintain the temperature at 20-24 ° C, with relative air humidity (RH) of 30-70%, a dark cycle: 12:12 hour light and 10-30 air changes / h in the study room. The temperature, relative humidity and light cycle were monitored daily by the control computer. The animals received a unique animal identification number and on day 0 of the study each animal received an injection into the tail vein of the antibody cocktail (200 µl of 10 mg / mL). The antibody cocktail was provided by MD Biosciences (Catalog: CIA-MAB-50). On day 3, after the single administration of mAb, all animals were subjected to the administration of LPS (200 µl of 0.5 mg / mL) from a single intraperitoneal (IP) injection. LPS was provided by MD Biosciences (Catalog: MDLPS.5). The mice were examined for signs of arthritogenic responses in peripheral joints, on day 0. Since the onset of the disease, the arthritogenic response will be examined on study days 3-8, 10, and 12. Arthritis reactions are reported for each paw according to a scale of 0-4, in ascending order of severity.

[000364] Animals found in a dying state, animals with cracked skin on a leg with arthritis, or with a greater than 20% reduction in body weight and animals that present intense pain and signs of intense and continued suffering were humanly sacrificed. Severe pain or suffering was assessed on a case-by-case basis by experienced animal technicians. In summary, however, the evaluation looked for abnormal vocalizations, isolation from other animals, unwillingness to use the limbs, abnormal response to handling, tremors and posture. The animals were sacrificed by C02 inhalation followed by cervical dislocation. The evaluation is based mainly on the average values of arthritis marking and measurements of paw thickness. Statistical analysis was also carried out on body weight. Whenever appropriate, ANOVA data analysis with Tukey's post hoc analysis was applied to determine the significance of the treatment effects.
[000365] As part of this model, animals lose weight quickly during the first 5-8 days and slowly start to gain / lose weight, depending on the evolution of the disease. 1-4 increased the rate of weight gain compared to vehicle or dexamethasone treatment groups. FIG. 4 is a graph of the average body weight versus time for days 0 to 12 in male BALB / c mice with arthritis, induced by antibody submitted to the model.
[000366] Furthermore, animals submitted to the CAIA model typically begin to show signs of arthritis around day 4 and as the disease progresses total arthritis markings increase as a function of time. Treatment with compound 1-4 significantly decreased the total label when compared to the vehicle and exhibited a dose dependent effect. FIG. 5 is a graph of the total mean of the clinical arthritic markings of the paw versus time for days 0-12 in male Balb / c mice with arthritis, induced by antibody, submitted to the indicated treatment. EXAMPLE 34: PMA-INDUCED PSORIASIS MODEL
[000367] BALB / c mice were housed in individually ventilated cages in a controlled environment (temperature 22 ± 1 ° C, humidity 70 ± 5%, and 12-hour light / 12-hour dark cycle) in the animal facility. The rats had access to commercially available feed tablets and drinking water treated with UV ad libitum. 4 mice were housed in an individually ventilated cage. Each of the animals in the cage was identified by a tail. 8 mice per group of mice were randomly divided into different treatment groups according to body weight. After randomization, the average body weight for all groups was equivalent. The study design was Group 1: simple, 1% DMSO vehicle (10-30 ul, topical once daily), Group 2: PMA, 1% DMSO vehicle (10-30 ul, topical once daily) ), Group 3: PMA, 1-4 10 mg / kg in PvP / Pluronics (oral, MWF; Day 1-day 3 day-day 5- day 7), Group 4: PMA, 0.1% betamethasone - 25 mg (reference standard) (topical once daily).
[000368] 4 µg of Phorbol 12-myristate 13-acetate (PMA) in 20 µL of acetone was applied daily to the ears of the mice. Beginning on day 2, PMA induction of dermal inflammation / psoriasis manifests with the increase in the clinical activity index of the disease associated with increased ear thickness, ear - skin peeling, and ear - skin fold. The following parameters evaluated were: (i) ear thickness, (ii) peeling of the ear skin. This will be based on the score index - 0, no flaking; 1, mild scaling, 2, moderate scaling, 3, severe scaling, (iii) folding over the ear skin. This will be based on the score index - 0, no fold, 1, light fold, 2, moderate fold, 3, severe fold, (iv) the weight of the ear (on the day of sacrifice).
[000369] FIG. 6 is a bar graph providing marking for ear thickness, ear skin peeling and ear skin fold. The results show that the oral administration of Compound 1-4 at 10 mg / kg, reduced the average ear thickness in a statistically significant way compared to vehicle. The efficacy obtained with 1-4 was comparable with positive control betamethasone. In addition, compound 1-4 was well tolerated. EXAMPLE 35: recognition of new objects
[000370] For the new object recognition test, Zucker rats were placed in a test chamber (dimension 26 "x 18" x 18 "; L x W x H) Food and water were not allowed during the test. The test had three phases: a) Familiarization phase: The rats were individually placed in a test chamber and allowed to explore freely for 60 min.The distance traveled by the animal during this phase was recorded using the tracking software (AnyMaze system). this phase was to familiarize the animals with the test apparatus, which was carried out on day 1. b) sample phase: on day 2, the rats were individually placed in the test chamber for 3 min and allowed to explore freely test arena, which contained two new and identical objects (for example: a metal cube, plastic cylinder) positioned in 2 corners of the test chamber.The distance traveled by the animals during this phase of the sample was automatically recorded, as well as o the time spent by the animal interacting with the new objects, using a tracking and visual observation software system. The interaction with the object was defined as active interaction with the animal's snout in contact or immediate proximity to the object, c) test phase: 1H after the sample phase, the rats were individually returned to the test chamber for 3 min. and allowed to freely explore the test arena which contained two objects, one of which was the object presented during the sample phase, and the second a new object that was exclusive to the test phase. The two objects were positioned in the same two corners of the test chamber as used for the sampling phase. The distance traveled by the animal during the test phase was automatically recorded, as well as the time spent by the animal interacting with familiar and new objects, using a tracking and visual observation software system. Interaction markings of the object both during the test and sample phase were recorded independently by two observers. The final mark represents the difference mark between each reading. Preferences markings for objects presented as Dl (that is, time spent exploring a new object - time spent exploring a familiar object; therefore, positive markings represent a preference for the new object) and D2 (ie Dl / a + b; Dl markings divided by the total object exploration time).
[000371] FIG. 7 provides a set of graphs that show the object preference of untreated and I-4 treated Zucker rats. From fig. 7 it can be seen that Compound 1-4 administered orally in doses of 0.625, 1.25 and 2.5 mg / kg induced trends of improved recognition of the new object in Zucker and I-4 rats was well tolerated.
[000372] EXAMPLE 36: Study of feeding of obese Zucker rats
[000373] Male Zucker rats (fa / fa) and thin male Zucker rats (both from Charles River) at 10 weeks of age - a point in time when Zucker fa / fa rats should show high food intake, body mass and elevated plasma lipid profile in relation to its “lean” copies were individually housed in cages with a plastic bottom and received 14 days of habituation. During this period, the animal's body weight, food and water intakes were recorded daily. All animals received ad-lib access to lab ration. Standard and water throughout the study. After collecting 14-day baseline intake data, obese Zucker rats were assigned to treatment groups based on equivalent baseline data, that is, all obese Zucker rats had daily water / food intakes equivalents and body weights. During this phase the rats also received two vehicle administrations as a familiarization with the dosing procedure. Immediately after the baseline phase, the treatment phase started. The test article and vehicle were administered approximately 1 h before the start of the dark cycle. The dose schedule varied according to the group: 5x a week of dosing was from Monday to Friday. The study design was as follows: group A = lean male Zucker rats, 5x vehicle treatment per week, oral, n = 6, group B = obese male Zucker rats, 5x vehicle treatment per week, oral, n = 6, group C = male obese Zucker rats, I-4 2.5 mg / kg 5x per week, oral, n = 6.
[000374] Body weight, food and water intake, daily, were measured at approximately the same time of day. On day -1 and day 7 of the treatment phase.
[000375] Figure 8A provides cumulative and average food intake in obese and lean Zucker rats (W / O indicates washout period). Administration of compound I-4 at 2.5 mg / kg 5X per week reduced the mean and cumulative food intake in obese Zucker (fa / fa) rats. Compound I-4 was well tolerated.
[000376] Figure 8B provides average and percentage body weight in lean and obese Zucker rats (W / O indicates washout period). Oral administration of I-4 at 2.5 mg / kg 5X per week significantly reduced weight gain compared to the Zucker fa / fa controls. 2 days of washout phase, body weight gain still reduced compared to the Zucker fa / fa controls. I-4 was well tolerated.
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[000406] The relevant teachings of all patents, published applications and references cited here are incorporated by reference in their entirety.
[000407] Although the present invention was particularly mustard and described with reference to the exemplary modalities thereof, it will be understood by those skilled in the art that various changes in shape and details can be made therein without departing from the scope of the invention covered by joined claims.

权利要求:
Claims (36)
[0001]
1. Composed of structural formula I:
[0002]
2. Compound according to claim 1, characterized by the fact that the compound is represented by structural formula II:
[0003]
3. Compound according to claim 2, characterized by the fact that: R1is hydrogen or methyl; and R2 is pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl or pyrimidin-4-yl, wherein R2 is unsubstituted or substituted with a single substituent selected from methyl and chlorine; or R1 and R2 are taken together to form 4-hydroxypiperidin-1-yl.
[0004]
A compound according to any one of claims 1 to 3, characterized by the fact that R3 is hydrogen.
[0005]
Compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the compound is represented by any of the following structural formulas:
[0006]
A compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the compound is represented by any of the following structural formulas:
[0007]
A composition characterized by comprising a compound as defined in any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[0008]
Composition according to claim 7, characterized in that it further comprises a second therapeutic agent useful for treating cancer.
[0009]
A compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized for being for use in the treatment of a disorder associated with CRM1 activity in an individual needing it, wherein the disorder is selected from a proliferative disorder, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, an abnormal tissue growth disorder, a disorder related to food intake, allergies, and a respiratory disorder
[0010]
A compound according to claim 9, or a pharmaceutically acceptable salt thereof, characterized in that it is for administration in conjunction with a second therapeutic agent useful for treating cancer.
[0011]
Use of a compound as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, characterized by being in the manufacture of a medicament for the treatment of a disorder associated with CRM1 activity, wherein the disorder is selected from a proliferative disorder, an inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, an abnormal tissue growth disorder, a disorder related to food intake, allergies, and a respiratory disorder.
[0012]
12. Compound according to claim 9, characterized by the fact that the disorder is a hematological cancer that is a leukemia, a lymphoma or a myeloma or a solid tumor cancer that is head and neck, prostate, breast, lung, ovary, squamous cells, colon or kidney.
[0013]
13. Compound, according to claim 12, characterized by the fact that hematological cancer is multiple myeloma.
[0014]
14. A compound according to any one of claims 8, 12 and 13, characterized by the fact that the use is oral.
[0015]
15. Use according to claim 11, characterized by the fact that the drug is for administration in conjunction with a second therapeutic agent useful for the treatment of cancer.
[0016]
16. Use, according to claim 11, characterized by the fact that the disorder is a hematological cancer that is a leukemia, a lymphoma or a myeloma or a solid tumor cancer that is head and neck, prostate, breast, lung, ovary, squamous cells, colon or kidney.
[0017]
17. Use, according to claim 16, characterized by the fact that cancer is multiple myeloma.
[0018]
18. Use according to any of claims 10 and 15 to 17, characterized by the fact that the drug is for oral release.
[0019]
19. A compound according to claim 1, characterized by being represented by the following structural formula:
[0020]
A compound according to claim 19 or a pharmaceutically acceptable salt thereof, characterized in that it is for use in the treatment of a disorder associated with CRM1 activity in an individual in need of the same, wherein the disorder is a proliferative disorder, a inflammatory disorder, an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, a disorder of abnormal tissue growth, a disorder related to food intake, allergies or respiratory disorder.
[0021]
21. A compound according to claim 20, or a pharmaceutically acceptable salt thereof, characterized in that it is for administration in conjunction with a second therapeutic agent useful for the treatment of cancer.
[0022]
22. Compound according to claim 20, characterized by the fact that the disorder is a hematological cancer that is a leukemia, a lymphoma or a myeloma or a solid tumor cancer that is head and neck, prostate, breast, lung, ovary, squamous cells, colon or kidney.
[0023]
23. Compound according to claim 22, characterized by the fact that cancer is multiple myeloma.
[0024]
24. A compound according to any one of claims 20 to 22, characterized by the fact that the compound is for oral administration.
[0025]
25. Use of the compound as defined in claim 21, or a pharmaceutically acceptable salt thereof, characterized by being in the manufacture of a medicament for the treatment of a disorder associated with CRM1 activity, wherein the disorder is a proliferative disorder, an inflammatory disorder , an autoimmune disorder, a viral infection, an ophthalmic disorder, a neurodegenerative disorder, a disorder of abnormal tissue growth, a disorder related to food intake, allergies or respiratory disorder.
[0026]
26. Use according to claim 25, characterized by the fact that its pharmaceutically acceptable salt is for administration in conjunction with a second therapeutic agent useful for the treatment of cancer.
[0027]
27. Use, according to claim 25, characterized by the fact that the disorder is a hematological cancer that is a leukemia, a lymphoma or a myeloma or a solid tumor cancer that is head and neck, prostate, breast, lung, ovary , squamous cells, colon or kidney.
[0028]
28. Use, according to claim 27, characterized by the fact that hematological cancer is multiple myeloma.
[0029]
29. Use according to any one of claims 25 to 28, characterized by the fact that the drug is for oral administration.
[0030]
30. A compound according to claim 1, characterized by being represented by the following structural formula:
[0031]
31. A compound according to claim 30 or a pharmaceutically acceptable salt thereof, characterized in that it is for use in the treatment of cancer in a veterinary individual in need thereof.
[0032]
32. Compound according to claim 31, characterized by the fact that the veterinarian is a dog.
[0033]
33. A compound according to any one of claims 31 and 32, characterized by the fact that cancer is a lymphoma.
[0034]
34. A compound according to any one of claims 31 and 32, characterized by the fact that the cancer is osteosarcoma, leukemia or myeloma.
[0035]
35. A compound according to any one of claims 31 to 34, characterized by the fact that the treatment is oral, intravenous, intraperitoneal, intramuscular or intradermal.
[0036]
36. Use according to claim 11, characterized by the fact that the individual is a veterinary individual.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-26| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-09-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 26/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161513428P| true| 2011-07-29|2011-07-29|

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US201161513432P| true| 2011-07-29|2011-07-29|

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US61/513,428|2011-07-29|

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US61/513,432|2011-07-29|

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US201261610178P| true| 2012-03-13|2012-03-13|

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US61/610,178|2012-03-13|

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US201261653588P| true| 2012-05-31|2012-05-31|

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US61/653,588|2012-05-31|

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US201261654651P| true| 2012-06-01|2012-06-01|

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US61/654,651|2012-06-01|

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PCT/US2012/048319|WO2013019548A1|2011-07-29|2012-07-26|Hydrazide containing nuclear transport modulators and uses thereof|

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