Hydrazide-containing nuclear transport modulators and uses thereof

Abstract

In general, the present application relates to nuclear transport modulators, such as CRM1 inhibitors, and more particularly to a compound represented by structural formula (I): or a pharmaceutically acceptable salt thereof, wherein the values and substituents for the variables are as defined and described herein. The present application also includes the synthesis and use of a compound having structural formula I or a pharmaceutically acceptable salt or composition thereof, for example, in the treatment, modulation and / or prevention of physiological conditions associated with CRM1 activity.

Description

[0001] This application is a divisional application of two patent applications filed on July 26, 2012, with application number 201280047939.2 and entitled "Nuclear transport modifier containing hydrazide and its use thereof", and filed on July 26, 2012, with application number 201910734865.3 and entitled "Nuclear transport modifier containing hydrazide and its use thereof".

[0002] Related applications

[0003] This application claims the benefits of U.S. Provisional Application No. 61 / 513,428, filed July 29, 2011; U.S. Provisional Application No. 61 / 513,432, filed July 29, 2011; U.S. Provisional Application No. 61 / 610,178, filed March 13, 2012; U.S. Provisional Application No. 61 / 654,651, filed June 1, 2012; and U.S. Provisional Application No. 61 / 653,588, filed May 31, 2012. The contents of these applications are incorporated herein by reference in their entirety. Background of the Invention

[0005] Cells of most major human solid and hematologic malignancies exhibit anomalous cellular localization of various oncogenic proteins, tumor suppressor proteins, and cell cycle regulators (Cronshaw et al., 2004; Falini et al., 2006). For example, certain p53 mutations can lead to matrix localization rather than nuclear localization. This results in the loss of normal growth regulation, although tumor suppressor function remains intact. In other tumors, wild-type p53 is isolated in the cytoplasm or is rapidly degraded, again leading to the loss of its inhibitory function. Restoration of proper nuclear localization of functional p53 protein can normalize some characteristics of tumor cells (Cai et al., 2008; Hoshino et al., 2008; Lain et al., 1999a; Lain et al., 1999b; Smart et al., 1999), restore the sensitivity of cancer cells to DNA-damaging agents (Cai et al., 2008), and lead to the regression of developing tumors (Sharpless & DePinho, 2007; Xue et al., 2007). Similar data have been obtained from other tumor suppressor proteins such as forkhead (Turner and Sullivan, 2008) and c-Abl (Vignari and Wang, 2001). Furthermore, the aberrant localization of some tumor suppressor proteins and growth regulators may be related to the pathogenesis of autoimmune diseases (Davis 2007, Nakahara 2009). CRM1 inhibition can provide particularly meaningful applications in familial cancer syndromes (e.g., Li-Fraumeni syndrome, BRCA1 or BRCA2 cancer syndromes caused by a p53 allele deletion), in which specific tumor suppressor proteins (TSPs) are cleared or dysfunctional, and in which the enhancement of TSP levels achieved through the administration of systemic (or local) CRM1 inhibitors can help restore normal tumor suppressor function.

[0006] Specific proteins and RNA are imported into or exported from the cell nucleus by specific transport molecules. If they import molecules into the nucleus, they are classified as importing proteins; if they export molecules, they are classified as exporting proteins (Terry et al., 2007; Sorokin et al., 2007). Proteins imported into or exported from the nucleus contain nuclear import / localization (NLS) or nuclear export (NES) sequences, enabling them to interact with associated transport factors. Chromosomal region-stabilizing protein 1 (Crm1 or CRM1), also known as exporting protein-1 or Xpo1, is the major exporting protein.

[0007] According to reports, Crm1 is overexpressed 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), liver cancer (Pascale et al., 2005), and osteosarcoma (Yao et al., 2009), and Crm1 overexpression is independently associated with poor clinical outcomes in these tumor types.

[0008] Inhibition of Crm1 blocks the nuclear export 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 forkhead proteins (such as FOXO3a), which are associated with gene expression, cell proliferation, angiogenesis, and phenotypic inheritance. Results have shown that Crm1 inhibitors can induce apoptosis in cancer cells, even in the presence of oncogenic activation signals or growth-stimulating signals, without affecting normal (untransformed) cells. Most studies on Crm1 inhibition have used the natural Crm1 inhibitor lepromycin B (LMB). LMB itself is highly toxic to tumor cells, but its significant gastrointestinal toxicity makes it difficult to be tolerated in animals (Roberts et al., 1986) and humans (Newlands et al., 1996). Derivatizing LMBs to improve drug-like properties can yield compounds that retain antitumor activity while also being better tolerated in animal tumor models (Yang et al., 2007; Yang et al., 2008; Mutka et al., 2009). Therefore, nuclear export inhibitors may have beneficial effects on neoplastic diseases and other proliferative diseases.

[0009] In addition to tumor suppressor proteins, Crm1 also exports several key proteins associated with many inflammatory processes. These proteins include IkB, NF-κB, Cox-2, RXRα, Commd1, HIF1, HMGB1, FOXO, FOXP, and others. The nuclear factor kappa (NF-κB / rel) family of transcriptional activators, named for their ability to induce the expression of the immunoglobulin kappa gene, regulates the expression of mRNAs of various genes related to inflammation, proliferation, immunity, and cell survival. Under normal conditions, an NF-κB protein repressor called IkB binds to NF-κB in the nucleus, and the IkB-NF-κB complex inactivates the transcriptional function of NF-κB. In response to inflammatory stimuli, IkB dissociates from the IkB-NF-κB complex, releasing NF-κB and restoring its potential transcriptional activity. Many signals that activate NF-κB do so by targeting IκB proteolysis (IκB phosphorylation "tags" it for ubiquitination and then proteolysis). The nuclear IκBα-NF-κB complex can be exported to the cytoplasm by Crm1, where it dissociates, thereby reactivating NF-κB. Ubiquitinated IκB can also dissociate from the NF-κB complex, restoring NF-κB transcriptional activity. Inhibition of Crm1-induced export of LMB in human neutrophils and macrophage-like cells (U937) not only leads to the accumulation of transcriptionally inactive nuclear IκBα-NF-κB complexes but also prevents initial NF-κB activation, even under cellular stimulation (Ghosh 2008, Huang 2000). In a separate study, treatment with LMB in lung capillary endothelial cells inhibited IL-1β-induced NF-κB DNA binding (the first step in NF-κB transcriptional activation), IL-8 expression, and intercellular adhesion molecule expression (Walsh 2008). COMMD1 is another inhibitor of the transcriptional activity of both NF-κB and hypoxia-inducible factor 1 (HIF1). Blocking COMMD1 nuclear export by inhibiting Crm1 leads to increased inhibition of the transcriptional activity of both NF-κB and HIF1 (Muller 2009).

[0010] Crm1 also mediates the transport of retinoid X receptor α (RXRα). RXRα is highly expressed in the liver and plays a central role in regulating bile acid, cholesterol, fatty acid, steroid, and xenobiotic metabolism, as well as homeostasis. In hepatitis, nuclear RXRα levels are significantly reduced, primarily due to inflammation-mediated nuclear export of RXRα via Crm1. LMB prevents IL-1β-induced cytoplasmic increase of RXRα levels in human liver-derived cells (Zimmerman 2006).

[0011] The role of Crm1-mediated nuclear output in NF-κB, HIF-1, and RXRα signaling suggests that blocking nuclear output may have potential benefits for many inflammatory processes across multiple tissues and organs, including the vascular system (vasculitis, arteritis, polymyalgia rheumatica, arteriosclerosis), skin diseases (see below), and rheumatic diseases (rheumatoid arthritis and related arthritis, psoriatic arthritis, spondyloarthropathy, crystal arthropathy, systemic lupus erythematosus, mixed connective tissue disease, myositis, dermatomyositis, inclusion body myositis, undifferentiated connective tissue disease, Sjögren's syndrome, overlap syndrome, etc.).

[0012] CRM1 inhibition can affect gene expression by inhibiting / activating a range of transcription factors such as ICP27, E2F4, KLF5, YAP1, and ZAP.

[0013] Crm1 inhibition has potential therapeutic effects on many dermatological syndromes, including inflammatory skin diseases (allergies, allergic dermatitis, chemical dermatitis, psoriasis), sun damage (ultraviolet (UV) damage), and infections. The most well-studied Crm1 inhibition using LMB showed minimal effect on normal keratinocytes and anti-inflammatory activity on keratinocytes stimulated by UV, TNFα, or other inflammations (Kobayashi & Shinkai 2005, Kannan & Jaiswal 2006). Inhibition of Crm1 can also upregulate the activity of NRF2 (nuclear factor erythrocyte-associated factor 2), which protects keratinocytes (Schafer et al., 2010, Kannan & Jaiswal 2006) and other cell types (Wang et al., 2009) from oxidative damage. LMB induces apoptosis in keratinized cells infected with oncogenic human papillomavirus (HPV) strains such as HPV16, but does not induce apoptosis in uninfected keratinized cells (Jolly et al., 2009).

[0014] Crm1 also mediates the transport of key neuroprotective proteins that are useful in neurodegenerative diseases including Parkinson's syndrome (PD), Alzheimer's disease, and amyotrophic lateral sclerosis (ALS). For example, Crm1 inhibition can slow or prevent neuronal death in these diseases by (1) forced nuclear arrest of key neuroprotective regulators such as NRF2 (Wang, 2009) and FOXA2 (Kittappa et al., 2007), anchoring them in neurons, and / or (2) by inhibiting the transcriptional activity of NFkB by isolating IκB in the nucleus of glial cells. There is also evidence that abnormal glial cell proliferation is associated with abnormal CRM1 levels or CRM1 function (Shen, 2008).

[0015] The complete maturation of many viruses also requires complete nuclear export, primarily mediated by CRM1. Viruses involving nuclear export and / or CRM1 itself during their life cycle include human immunodeficiency virus (HIV), adenovirus, simian retrovirus type I, Polner's disease virus, influenza virus (common strains as well as H1N1 and avian H5N1 strains), hepatitis B virus (HBV) and hepatitis C virus (HCV), human papillomavirus (HPV), respiratory syncytial virus (RSV), Dungeee, 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, Whittaker 1998). It is anticipated that more viral infections relying on complete nuclear export will be discovered in the future.

[0016] The HIV-1 Rev protein, which crosses the nucleolus and shuttles between the nucleus and cytoplasm, facilitates the export of unspliced ​​and single-spliced ​​HIV transcripts containing Rev response element (RRE) RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA transport via CRM1 repressors such as LMB or PKF050-638 can prevent HIV-1 transcription, suppress the generation of new HIV-1 viral particles, and thus reduce HIV-1 levels (Pollard 1998, Daelemans 2002).

[0017] Dengue virus (DENV) is the causative agent of dengue fever (DF), a common arthropod-borne viral disease, and its more severe and potentially fatal form, dengue hemorrhagic fever (DHF). DHF appears to be caused by an overactive inflammatory response to DENV. NS5 is the largest and most conserved protein of DENV. CRM1 regulates the transport of NS5 from the nucleus to the cytoplasm, mediating most of NS5's functions. Inhibition of CRM1-mediated NS5 export leads to altered viral dynamics and reduced induction of the inflammatory chemokine interleukin-8 (IL-8), providing a novel avenue for the treatment of DENV and other medically important flaviviruses, including those caused by hepatitis C virus (Rawlinson 2009).

[0018] Other virus-encoded RNA-binding proteins that use CRM1 to export cells to the nucleus include HSV type I mesenchymal proteins (VP13 / 14 or hUL47), human CMV protein pp65, SARS coronavirus ORF 3b protein, and RSV matrix (M) protein (Williams 2008, Sanchez 2007, Freundt 2009, Ghildyal 2009).

[0019] Interestingly, many of these viruses are associated with specific types of human cancers, including liver cancer (HCC) caused by chronic HBV or HCV infection, cervical cancer caused by HPV, and Merkel cell carcinoma associated with MCV. Therefore, the CRM1 inhibitor is beneficial in both the viral infection process and the cancerous transformation process induced by these viruses.

[0020] CRM1 controls nuclear localization, thereby controlling the activity of several DNA metabolic enzymes, including histone deacetylases (HDAC), histone acetyltransferases (HAT), and histone methyltransferases (HMT). It has been demonstrated and is generally accepted that the irreversible inhibition of cardiomyocyte hypertrophy by CRM1 inhibitors is associated with nuclear arrest (and activation) of HDAC5, an enzyme known to inhibit the genetic program of hypertrophy (Monovich et al., 2009). Therefore, CRM1 inhibition may have beneficial effects on hypertrophic syndromes, including certain forms of congestive heart failure and hypertrophic cardiomyopathy.

[0021] CRM1 is also associated with other diseases. Leber's disorder, a genetic disorder characterized by the degeneration of retinal ganglion cells and vision loss, is associated with ineffective CRM1 switching (Gupta N 2008). There is also evidence that neurodegenerative diseases are associated with abnormal nuclear transport.

[0022] However, small-molecule drug-like Crm1 inhibitors are still not commonly used in vitro and in vivo. Invention Overview

[0024] This invention relates to compounds for use as nuclear transport regulators or pharmaceutically acceptable salts thereof. The invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention, and methods for using said compounds and compositions to treat various disorders, such as those associated with abnormal cellular responses triggered by improper nuclear transport.

[0025] In one embodiment of the invention, these compounds are represented by Formula I:

[0026]

[0027] Or a pharmaceutically acceptable salt thereof, wherein the value and alternative values ​​for each variable are defined and described in such a way.

[0028] 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.

[0029] Another embodiment of the present invention is a method for treating a disorder associated with CRM1 activity, the method comprising administering to a subject in need a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0030] Another embodiment of the invention is the use of the compound of the invention to treat a disorder associated with CRM1 activity in a subject.

[0031] Another embodiment of the invention is the use of one of the compounds of the invention in the manufacture of a medicament for treating a disorder in a subject associated with CRM1 activity.

[0032] The nuclear transport modulators of the present invention and their pharmaceutically acceptable salts and / or compositions provide excellent in vivo exposure, as measured by AUC in mice, rats, dogs, and monkeys, while exhibiting low levels of brain penetration. Therefore, the compounds of the present invention and their pharmaceutically acceptable salts and / or compositions are useful for treating a variety of diseases, disorders, or conditions (such as those described herein) associated with anomalous cellular responses triggered by inappropriate nuclear transport. The compounds provided by the present invention are also useful for studying nuclear transport regulation in biological and pathological phenomena; for studying kinase-mediated intracellular signal transduction pathways; and for comparative evaluation of nuclear transport modulators.

[0033] Brief description of the attached figures

[0034] Figure 1 The graph shows tumor volume as a function of time and illustrates the effect of compound I-3 on tumor volume in a mouse xenograft model of triple-negative breast cancer (TNBC).

[0035] Figure 2A These are Western blot images showing the effects of increasing the concentration of compound I-3 on CRM1 and apoptosis marker proteins in MDA-MB-468TNBC cells.

[0036] Figure 2B These are Western blot images showing the effects of increasing compound I-3 concentration on CRM1 and apoptosis marker proteins in DU4475 cavitary BC cells.

[0037] Figure 2C These are Western blot images showing the effects of increasing compound I-3 concentration on CRM1 and apoptosis marker proteins in HS578T TNBC cells.

[0038] Figure 3 These are Western blot images showing the effects of increasing the concentration of compound I-3 on anti-apoptosis and cyclin activity in MDA-MB-468 and HS578T TNBC cell lines.

[0039] Figure 4 This is a graph showing the average body weight versus time from 0 to 12 days in antibody-induced male BALB / c arthritis mice that have undergone the specified treatment.

[0040] Figure 5 This is a graph showing the mean total claw clinical arthritis score over time from 0 to 12 days in antibody-induced male BALB / c arthritis mice that have undergone the specified treatment.

[0041] Figure 6 This is a bar graph showing the scores for mean ear thickness, grime, and folding, determined from days 0 to 7 of PMA-induced male BALB / c psoriasis mice that have undergone the specified treatment.

[0042] Figure 7 It is a series of graphs showing the object preferences of rats treated according to the new object recognition model.

[0043] Figure 8A This is a series of graphs showing the cumulative and mean food intake over time in obese and lean Zucker rats treated according to the specified parameters.

[0044] Figure 8BThis is a series of graphs showing the mean and percentage body weight versus time in obese and lean Juke rats treated according to the specified criteria.

[0045] Detailed description

[0046] Upon examination of the following detailed description of the invention, the novel features of the invention will become clear to those skilled in the art. However, it should be understood that while certain embodiments of the invention have been pointed out, the detailed description of the invention and the specific examples given are provided merely for illustrative purposes, as various changes or modifications within the spirit and scope of the invention will become clear to those skilled in the art from the detailed description and the claims below.

[0047] The compounds of the present invention

[0048] One embodiment of the present invention is a compound represented by formula I:

[0049]

[0050] Or its pharmaceutically acceptable salt, wherein:

[0051] R 1 Selected from hydrogen and methyl;

[0052] R 2 Selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, quinoxalin-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophene-3-yl, and cyclopropyl, wherein R 2 It may optionally be substituted with one or more independent substituents selected from methyl and halogen; or

[0053] R 1 and R 2 Together with the atoms inserted between them, they form 4-hydroxypiperidin-1-yl, pyrrolidine-1-yl, azircycloheptane-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3-hydroxyazircyclobutane-1-yl, or morpholino-4-yl;

[0054] R 3 Selected from hydrogen and halogens; and

[0055] It represents a single bond, wherein the carbon-carbon double bond attached to it is in the (E)- or (Z)- configuration.

[0056] As generally described above, R 1 Selected from hydrogen and methyl. In some embodiments, R 1 It is hydrogen. In some embodiments, R 1 It is a methyl group.

[0057] As generally described above, R 2 Selected from pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrazin-2-yl, quinoxalin-2-yl, pyrimidin-4-yl, 1,1-dioxotetrahydrothiophene-3-yl, and cyclopropyl, wherein R 2 It may optionally be substituted with one or more independent substituents selected from methyl and halogen. In some embodiments of Formula I, R 2 It is pyridin-2-yl. In some embodiments of Formula I, R 2 It is pyridin-3-yl. In some embodiments of Formula I, R 2 It is pyridin-4-yl. In some embodiments of Formula I, R 2 It is pyrazin-2-yl. In some embodiments of Formula I, R 2 It is pyrimidin-4-yl. In some embodiments of Formula I, R 2 It is quinoxalo-2-yl. In some embodiments of Formula I, R 2 Selected from pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl. In some embodiments of Formula I, R 2 It 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, R 2 It is selected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl, and pyrimidin-4-yl.

[0058] In some embodiments, R 2 Selected from:

[0059]

[0060] In some embodiments of Formula I, R 2 It may optionally be substituted with a single substituent selected from methyl and chlorine. In some embodiments of Formula I, R 2 Optionally, it can be substituted with a methyl group. In some embodiments of Formula I, R 2 It may optionally be substituted with a chlorine group. In some embodiments, R 2 Selected from:

[0061]

[0062] In some embodiments, R 2 Selected from:

[0063]

[0064] In some embodiments, R 2 Selected from:

[0065] as well as

[0066] In some embodiments of Formula I, R 1 and R 2 Together with the atoms inserted between them, they form 4-hydroxypiperidin-1-yl, pyrrolidine-1-yl, azircycloheptane-1-yl, 4-benzylpiperazin-1-yl, 4-ethylpiperazin-1-yl, 3-hydroxyazircyclobutane-1-yl, or morpholino-4-yl. In some embodiments of Formula I, R 1 and R 2 Together with the atoms inserted between them, they form 4-hydroxypiperidin-1-yl.

[0067] As generally described above, R 3 Selected from hydrogen and halogens. In some embodiments, R 3 It is hydrogen. In some embodiments, R 3 It is a halogen (e.g., chlorine, bromine, iodine, or fluorine). In some such embodiments, R 3 It is chlorine.

[0068] As generally described above, the carbon-carbon double bond between the triazole moiety and the carbonyl moiety is in either an (E)-configuration or a (Z)-configuration. In some embodiments, the double bond is in an (E)-configuration. In some embodiments, the double bond is in a (Z)-configuration, and the compound is represented by Formula II:

[0069]

[0070] Or a pharmaceutically acceptable salt thereof, wherein R 1 R 2 With R 3 It is as defined above and described here.

[0071] Another embodiment of the invention is a compound represented by Formula II or a pharmaceutically acceptable salt thereof, wherein the values ​​and substitutions for these variables are as defined above for a compound having Formula I.

[0072] In a first aspect of this further embodiment, R 1 It is as defined above; and R 2 Selected from pyridin-2-yl, pyridin-4-yl, pyrazin-2-yl, and pyrimidin-4-yl, wherein R 2 It may optionally be substituted with a single substituent selected from methyl and chlorine; or R 1 and R 2 Together with the atoms inserted between them, they form 4-hydroxypiperidin-1-yl.

[0073] In one specific aspect of this first aspect, R3 It is hydrogen. The values ​​and alternative values ​​for these remaining variables are as defined above for a compound having Formula I, or as defined in other embodiments or in its first aspect.

[0074] Exemplary compounds having Formula I are listed in Table 1.

[0075] Table 1. Exemplary compounds having Formula I.

[0076]

[0077]

[0078] In some embodiments, the compounds of the present invention are selected from any one of compounds I-3 to I-26. In one aspect of these embodiments, the compound is selected from compounds I-3, I-4, I-5, I-7, I-8, I-10, I-12, I-18, I-19, and I-24. In a more specific aspect, the compounds of the present invention are selected from I-3 and I-4.

[0079] Pharmacokinetics (PK) plays an increasingly important role in drug discovery and development. Pharmacokinetics is the quantitative study of the absorption, distribution, metabolism, and / or excretion timelines of a drug. When a drug is administered, it rapidly distributes from its site of administration into the systemic circulation. One measure of the distribution range of a therapeutic agent is the area under the plasma concentration-time curve (AUC), calculated down to the last measured concentration (AUC). t And extrapolate to infinity (AUC) Inf Therefore, AUC is a useful metric for quantifying drug exposure.

[0080] Generally, higher exposure to a therapeutic agent results in a greater effect. However, high exposure to a therapeutic agent can have detrimental effects on certain tissues, such as the brain. Drugs with high AUC can still penetrate the blood-brain barrier (BBB), a protective network of tight junctions between endothelial cells that restricts the diffusion of hydrophilic and / or macromolecules. This type of penetration is often undesirable and can lead to unwanted side effects. Current drug discovery efforts are partly aimed at disrupting the balance between maximizing drug exposure (e.g., AUC) and minimizing brain penetration.

[0081] The brain to plasma (B:P) ratio is a method of quantifying the relative distribution of a therapeutic agent in brain tissue relative to the circulatory system, and thus provides an indication of the brain penetration of a given therapeutic agent. A high B:P ratio is preferred when the targeted disease is located in the central nervous system (CNS) (including the brain and cerebrospinal fluid). However, a lower B:P ratio is generally preferred for non-CNS therapeutic agents to minimize brain penetration and avoid potential side effects caused by unwanted accumulation of the therapeutic agent in brain and CNS tissues.

[0082] As illustrated in more detail in the examples, these compounds of the present invention exhibit higher AUCs and / or lower B:Ps compared to other nuclear transport inhibitors such as those disclosed in common U.S. Patent Application No. 13 / 041,377, filed March 5, 2011 (and published November 10, 2011 as US2009 / 0275607). In some embodiments of the invention, compounds of Formula I, when administered orally to mice at 10 mg / kg po, exhibit nuclear export activity of less than about 1 μM and an AUC greater than about 3300 (e.g., greater than about 3500). Inf And a B:P ratio of less than approximately 2.5.

[0083] Synthesis method of the present invention

[0084] According to the present invention, this provides a method for preparing a (Z)-olefin derivative of a compound having the formula Z, which is useful in the preparation of the compounds of the present invention (e.g., precursors of these compounds of the present invention):

[0085]

[0086] Or its pharmaceutically acceptable salt, wherein:

[0087] Ring A is a ring selected from the following optionally substituted rings: phenyl, 8-10-membered bicyclic aryl ring, 5-6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and 8-10-membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

[0088] Y is a covalent bond or -L-;

[0089] L is a divalent form of C. 1-8Saturated or unsaturated, straight-chain or branched hydrocarbon groups, wherein one or both methylene units of L are optionally replaced 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)-;

[0090] Each R is independently hydrogen or a group selected from the following optionally substituted groups: C 1-6 Aliphatic groups, phenyl, 4-7-membered saturated or partially unsaturated carbon rings, rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 5-6-membered monocyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 8-10-membered bicyclic aryl rings, and 8-10-membered bicyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or

[0091] Two R groups on the same nitrogen atom, together with the nitrogen atom to which they are attached, form a ring of a 4-7-membered saturated or partially unsaturated heterocycle having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5-6-membered heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0092] V 1 V 2 and V 3 Each is independently C(R) y ) or N;

[0093] R x and R y Each 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.

[0094] R 1 and R 2 Each can be independently hydrogen, deuterium, tritium, or a halogen;

[0095] W is -CN, haloalkyl, -NO2, or -C(=Z)R 3 ;

[0096] Z is O, S, or NR;

[0097] R 3Selected from hydrogen, -R, -OR, -SR and -N(R) 4 )2;

[0098] Each R 4 The independent one is -R; or

[0099] Two Rs on the same nitrogen 4 Together with the nitrogen atoms attached to them, they form a 4-7-membered saturated or partially unsaturated heterocycle having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5-6-membered heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the ring thus formed is -(R 5 ) n It can be substituted at will;

[0100] Each R 5 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

[0101] m and n are each an integer independently selected from 0, 1, 2, 3, and 4.

[0102] Compounds having formula Z have been described, for example, in US13 / 041,377, filed March 5, 2011; and in US Provisional Application No. 61 / 513,428, filed July 29, 2011; and in US1 / 653,588, filed June 1, 2012. Compounds having formula Z are typically synthesized as a mixture of (E)- and (Z)-olefin isomers, which must be separated. The separation of these (E)- and (Z)-olefin isomers requires extensive chromatography and results in a 50% loss of the advanced intermediate A, as the undesirable isomer cannot be typically converted to the desired isomer. A 50% yield at any step of the synthesis is inefficient and costly, and such unacceptable yields are even more problematic at the end of a multi-step synthesis. It has been surprisingly found that the use of a sterically hindered base in 1,4-nucleophilic addition can affect the (Z)-selectivity of the reaction, thereby providing cis-olefin isomers as the major or exclusive product. Therefore, the present invention provides the (Z)-selective synthesis of compounds having formula Z, and methods for preparing synthetic intermediates useful for the preparation of compounds having formula Z. A key step in the synthesis of compounds having formula Z is described in Scheme I.

[0103] In some embodiments, these compounds having formula Z are prepared according to scheme I, as listed below:

[0104]

[0105] Where LG is a leaving group, and the rings A, Y, V 1 V 2 V 3 R x R 1 R 2 W and m are each as defined above with respect to compounds having formula Z and as described in the examples herein.

[0106] In some embodiments of step S-1.1, intermediate A is coupled to intermediate B via a 1,4-nucleophilic addition / elimination reaction. In some embodiments of step S-1.1, LG is a suitable leaving group. In some such embodiments of step S-1.1, LG is a halogen. In some embodiments, LG is iodine. In some embodiments of step S-1.1, LG is bromine. In some embodiments of step S-1.1, LG is a sulfonate. In some such embodiments, LG is methanesulfonate (or mesylate).

[0107] In some embodiments of step S-1.1, intermediate A is coupled to intermediate B in the presence of a sterically hindered nucleophilic base. Those skilled in the art will be able to select a suitable sterically hindered base. Suitable sterically hindered nucleophilic bases used in this invention include 1,8-diazabicyclo[5.4.0]undecyl-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, quinone bases, 1,2,2,6,6-pentamethylpiperidine (PMP), 7-methyl-1,5,7-triazabicyclo(4.4.0)decyl-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine, and tricyclohexylphosphine.

[0108] In some embodiments, these compounds having formula Y are prepared according to scheme II, as listed below:

[0109]

[0110] Where LG is a leaving group, and R x R y R 1 R 2W and m are each as defined above with respect to compounds having formula Z and as described in the examples herein.

[0111] In some embodiments of step S-2.1, intermediate C reacts with a thiolate to provide intermediate D. In some embodiments of step S-2.1, the thiolate is sodium thiolate. In some embodiments of step S-2.1, the thiolate is potassium thiolate.

[0112] In step S-2.2, intermediate D reacts with a hydrazine equivalent to provide intermediate E.

[0113] In step S-2.3, intermediate E is coupled to intermediate B to provide a compound having formula Y. In some embodiments of step S-2.3, LG is a suitable leaving group. In some such embodiments of step S-2.3, LG is a halogen. In some embodiments, LG is iodine. In some embodiments of step S-2.3, LG is bromine. In some embodiments of step S-2.3, LG is a sulfonate. In some such embodiments, LG is a methanesulfonate (or mesylate).

[0114] In some embodiments of step S-2.3, intermediate E is coupled to intermediate B in the presence of a sterically hindered nucleophilic base. Those skilled in the art will be able to select a suitable sterically hindered base. Suitable sterically hindered nucleophilic bases used in this invention include 1,8-diazabicyclo[5.4.0]undecyl-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, quinone bases, 1,2,2,6,6-pentamethylpiperidine (PMP), 7-methyl-1,5,7-triazabicyclo(4.4.0)decyl-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine, and tricyclohexylphosphine.

[0115] According to one aspect, the present invention provides a method for providing a compound having the following formula Z:

[0116]

[0117] Or a pharmaceutically acceptable salt thereof, wherein rings A, Y, V 1 V 2 V 3 R x , R, R 1 R 2 W and m are each as defined above for compounds having formula Z.

[0118] Includes the following steps:

[0119] (a) Provide a compound having formula A:

[0120]

[0121] Among them, rings A and R x Y, V 1 V 2 V 3 And m are each as defined above for compounds having formula Z; and

[0122] (b) Reacting the compound having formula A with an olefin having formula B:

[0123]

[0124] in:

[0125] LG is halogen, -OSO2R, or -OSO2CF3; and

[0126] R, W, R 1 and R 2 Each is as defined above for compounds having formula Z;

[0127] A compound with formula Z is formed in the presence of a sterically hindered nucleophilic base.

[0128] As described above, a compound having formula A is coupled to intermediate B via a 1,4-nucleophilic addition / elimination reaction. In some embodiments, a compound having formula A is coupled to intermediate B in the presence of a sterically hindered nucleophilic base. Suitable sterically hindered bases include tertiary amine bases. In some embodiments, a suitable sterically hindered base includes a sterically hindered secondary amine base. In some embodiments, the sterically hindered nucleophilic base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]-5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane (DABCO), N,N-dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinone base, 1,2,2,6,6-pentamethylpiperidine (PMP), 7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine, tri-butanphosphine, 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).

[0129] In some embodiments, the sterically hindered nucleophilic base is a phosphine. In some such embodiments, the sterically hindered nucleophilic base is triphenylphosphine.

[0130] In some embodiments, step (b) above is performed in a temperature range of approximately 0°C to approximately 100°C. In some embodiments, step (b) is performed at approximately 0°C. In some embodiments, step (b) is performed at approximately 25°C. In some embodiments, step (b) is performed at approximately 50°C. In some embodiments, step (b) is performed at approximately 100°C.

[0131] Those skilled in the art will recognize that a 1,4-nucleophilic addition / elimination reaction of a compound having formula A and intermediate B requires the use of a polar, protonic organic solvent. Suitable polar, protonic organic solvents include ethers such as dioxane, tetrahydrofuran, and methyl tert-butyl ether (MTBE), and amides such as dimethylformamide (DMF) and dimethylacetamide (DMA). Those skilled in the art can select an appropriate solvent for the ideal reaction temperature.

[0132] According to another aspect, the present invention provides a method for providing a compound having the following formula Y:

[0133]

[0134] Or a pharmaceutically acceptable salt thereof, wherein R, R x R y R 1 R 2 W and m are each as defined above for a compound having formula Z.

[0135] Includes the following steps:

[0136] (a) Provide a compound having formula E:

[0137]

[0138] Where R x R y And m are each as defined above for compounds having formula Y; and

[0139] (b) Reacting the compound having formula E with an olefin having formula B:

[0140]

[0141] in:

[0142] LG is halogen, -OSO2R, or -OSO2CF3; and

[0143] R, W, R 1 and R 2 Each is as defined above for compounds having formula Y.

[0144] A compound with formula Y is formed in the presence of a sterically hindered nucleophilic base.

[0145] As described above, a compound having formula E is coupled to intermediate B via a 1,4-nucleophilic addition / elimination reaction. In some embodiments, a compound having formula E is coupled to intermediate B in the presence of a sterically hindered nucleophilic base. Suitable sterically hindered bases include tertiary amine bases. In some embodiments, a suitable sterically hindered base includes a sterically hindered secondary amine base. In some embodiments, the sterically hindered nucleophilic base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]nonyl5-ene (DBN), 1,4-diazabicyclo(2.2.2)octane (DABCO), N,N-dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinone base, 1,2,2,6,6-pentamethylpiperidine (PMP), 7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine, tri-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]undecyl-7-ene (DBU).

[0146] In some embodiments, the sterically hindered nucleophilic base is a phosphine. In some such embodiments, the sterically hindered nucleophilic base is triphenylphosphine.

[0147] In some embodiments, step (b) above is performed in a temperature range of approximately 0°C to approximately 100°C. In some embodiments, step (b) is performed at approximately 0°C. In some embodiments, step (b) is performed at approximately 25°C. In some embodiments, step (b) is performed at approximately 50°C. In some embodiments, step (b) is performed at approximately 100°C.

[0148] Those skilled in the art will recognize that a 1,4-nucleophilic addition / elimination reaction of a compound having formula E and intermediate B requires the use of a polar, protonic organic solvent. Suitable polar, protonic organic solvents include ethers such as dioxane, tetrahydrofuran, and methyl tert-butyl ether (MTBE), and amides such as dimethylformamide (DMF) and dimethylacetamide (DMA). Those skilled in the art can select an appropriate solvent for the ideal reaction temperature.

[0149] In some embodiments of a compound having the formula Y, W is -CN. In some embodiments, W is a haloalkyl group. In some such embodiments, W is -CF3. In some embodiments, W is -NO2.

[0150] In some embodiments, W is -C(=Z)R 3 In some such embodiments, Z is O. In some embodiments, W is -C(O)R. 3 , where R 3 Selected from -OR, -SR, or -N(R) 4 2. In some embodiments, W is -C(O)OR. In some embodiments, W is -C(O)OR, wherein 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.

[0151] In some embodiments, W is -C(O)N(R) 4 2. In some embodiments, W is -(O)NH(R) 4 In some embodiments, W is -C(O)NH2. In some embodiments, W is -C(=O)N(R). 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-7-membered saturated heterocycle having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the resulting ring may optionally be modified by -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-7-membered saturated heterocycle having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the resulting ring may optionally be modified by -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-7-membered saturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein the resulting ring may optionally be modified by -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4The groups, together with the nitrogen atoms to which they are attached, form a 4-7-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring may optionally be -(R 5 ) n replace.

[0152] In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-6-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring may optionally be modified by -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-5-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring may optionally be -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring may optionally be -(R 5 ) n Replacement. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring is substituted with at least one fluorine molecule. In some embodiments, W is -C(O)N(R) 4 )2, where two R 4 The groups, together with the nitrogen atoms to which they are attached, form a 4-membered saturated heterocyclic ring having one nitrogen atom, wherein the resulting ring is substituted with at least two fluorine atoms. In some embodiments, W is

[0153] In some embodiments, R1 is hydrogen. In some embodiments, R 1 It is deuterium. In some embodiments, R2 is hydrogen. In some embodiments, R2 is deuterium. In some embodiments, R 1 and R 2 Each is hydrogen.

[0154] In some embodiments, m is 1. In some embodiments, m is 2. In some such embodiments, R x It is a haloalkyl group. In some embodiments, R x It is -CF3.

[0155] In some embodiments, R y It is hydrogen.

[0156] In some embodiments, the present invention provides a method for providing a compound having the following formula E:

[0157]

[0158] Where R x R y And m is as described for compounds having formula Z,

[0159] Includes the following steps:

[0160] (a) Provide a compound having formula D:

[0161]

[0162] Where R x And m are each as defined above for compounds having formula E; and

[0163] (b) React the compound having formula D to form a compound having formula E.

[0164] In some embodiments, the conditions for effectively forming a compound having formula D include a hydrazine equivalent. Therefore, in some embodiments, step (b) of a method for providing a compound having formula E includes reacting the compound having formula D with a hydrazine equivalent to form the compound having formula E. In some embodiments, intermediate D reacts with hydrazine hydrate to provide a compound having formula E. In some embodiments, intermediate D reacts with a protected form of hydrazine, such as tert-butylhydrazine carboxylate, and subsequently deprotects to provide intermediate D.

[0165] Those skilled in the art will recognize that the addition of hydrazine to intermediate D requires a polar, proton-thinning organic solvent. Suitable polar, proton-thinning organic solvents include ethers such as dioxane, tetrahydrofuran, and methyl tert-butyl ether (MTBE), alcohols such as isopropanol, and amides such as dimethylformamide (DMF) and dimethylacetamide (DMA). Those skilled in the art can select an appropriate solvent for the ideal reaction temperature.

[0166] In some embodiments, the present invention provides a method for preparing a compound having the following formula D:

[0167]

[0168] Where R x And m are as defined above for compounds having formula Z.

[0169] Includes the following steps:

[0170] (a) Provide a compound having the formula C:

[0171]

[0172] Where R x And m are each as defined above for compounds having formula D; and

[0173] (b) React the compound having formula C to form a compound having formula D.

[0174] As described above, in some embodiments, intermediate C is treated with a thiolate to provide intermediate D. In some embodiments, the thiolate is sodium thiolate. Those skilled in the art will recognize that reacting intermediate C with a thiolate requires the use of a polar proton-phobic solvent. Suitable polar proton-phobic solvents include ethers such as dioxane, tetrahydrofuran, and methyl tert-butyl ether (MTBE).

[0175] In some embodiments, the present invention provides a method for preparing a compound having the following formula B:

[0176]

[0177] in:

[0178] LG is halogen, -OSO2R, or -OSO2CF3; and

[0179] R, R 1 R 2 W and Z are each as defined above for compounds having formula Z.

[0180] Includes the following steps:

[0181] (a) Provide a compound having the formula F:

[0182]

[0183] Where R 2 Both W and W are as defined above for compounds having formula B; and

[0184] (b) React the compound having formula F to form a compound having formula B.

[0185] As described above, in some embodiments of intermediate B, LG is a halogen. In some such embodiments, a compound having formula F is treated with a halide. In some embodiments, a compound having formula F is treated with a sodium halide. In some such embodiments, a compound having formula F is treated with sodium iodide. In some embodiments, intermediate F is treated with a halide in the presence of an acid. Suitable acids include both inorganic and organic acids. In some embodiments, intermediate F is treated with a halide 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 having formula B.

[0186] Those skilled in the art will recognize that the addition of a halide salt to intermediate F requires a polar, proton-heptane organic solvent. Suitable polar, proton-heptane organic solvents include ethers such as dioxane, tetrahydrofuran, and methyl tert-butyl ether (MTBE).

[0187] According to another aspect, the present invention provides a method for distributing a compound having the following formula X:

[0188]

[0189] Or a pharmaceutically acceptable salt thereof, wherein R, R x R y R 1 R 2 R 4 And m are each as defined above for compounds having formula Z.

[0190] Includes the following steps:

[0191] (a) Provide a compound having formula E:

[0192]

[0193] Where R x R y And m are each as defined above for compounds having formula X; and

[0194] (b) Reacting the compound having formula E with an olefin having formula G:

[0195]

[0196] in:

[0197] LG is halogen, -OSO2R, or -OSO2CF3; and

[0198] R, R 1 R 2 and R 4Each is as defined above for compounds having formula X.

[0199] A compound of formula X is formed in the presence of a sterically hindered nucleophilic base.

[0200] According to another aspect, the present invention provides a method for providing a compound having the following formula W:

[0201]

[0202] Or a pharmaceutically acceptable salt thereof, wherein R, R x R y R 1 R 2 R 5 m and n are each as defined above for compounds having formula Z.

[0203] Includes the following steps:

[0204] (a) Provide a compound having formula E:

[0205]

[0206] Where R x R y And m are each as defined above for compounds having the formula W; and

[0207] (b) Reacting the compound having formula E with an olefin having formula H:

[0208]

[0209] in:

[0210] LG is halogen, -OSO2R, or -OSO2CF3; and

[0211] R, R 1 R 2 R 5 And n are each defined as above for compounds having the formula W.

[0212] A compound with formula W is formed in the presence of a sterically hindered nucleophilic base.

[0213] According to another aspect, the present invention provides a method for distributing a compound having the following formula V:

[0214]

[0215] Or a pharmaceutically acceptable salt thereof, wherein R, R x Ry R 1 R 2 , and m are each as defined above for compounds having formula Z.

[0216] Includes the following steps:

[0217] (a) Provide a compound having formula E:

[0218]

[0219] Where R x R y And m are each as defined above for compounds having formula V; and

[0220] (b) Reacting the compound having formula E with an olefin having formula J:

[0221]

[0222] in:

[0223] LG is halogen, -OSO2R, or -OSO2CF3; and

[0224] R, R 1 and R 2 Each is as defined above for compounds having formula V.

[0225] A compound of formula V is formed in the presence of a sterically hindered nucleophilic base.

[0226] In some embodiments, the present invention provides a method for preparing a compound having the following formula G:

[0227]

[0228] in:

[0229] LG is halogen, -OSO2R, or -OSO2CF3; and

[0230] R, R 1 R 2 and R 4 Each of these is described in the section concerning compounds having formula Z.

[0231] Includes the following steps:

[0232] (a) Provide a compound having the formula K:

[0233]

[0234] Where R 2 and R4 Each is as defined above for compounds having formula G; and

[0235] (b) React the compound having formula K to form a compound having formula G.

[0236] As described above, in some embodiments of intermediate G, LG is a halogen. In some such embodiments, a compound having formula K is treated with a halide. In some embodiments, a compound having formula K is treated with a sodium halide. In some such embodiments, a compound having formula K is treated with sodium iodide. In some embodiments, intermediate K is treated with a halide in the presence of an acid. Suitable acids include both inorganic and organic acids. In some embodiments, intermediate K is treated with a halide 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 having formula G.

[0237] In some embodiments, the present invention provides a method for preparing a compound having the following formula K:

[0238]

[0239] Where R 2 and R 4 Each is as defined above for compounds having formula Z.

[0240] Includes the following steps:

[0241] (a) Provide a compound having the formula L:

[0242]

[0243] Where R 2 It is hydrogen, deuterium, tritium, or a halogen; and

[0244] (b) Reacting the compound having formula L with HN(R) 4 )2 reaction, where each R 4 As defined above regarding compounds having formula K, to form a compound having formula K.

[0245] In some embodiments, in HN(R) 4A compound having formula L is treated with an amide coupling agent in the presence of 2 to form a compound having formula K. Suitable amide coupling agents include HOBt, HOAt, HAMDU, HAMTU, PyBOP, PyBrOP, TBTU, HATU, and T3P. Those skilled in the art will recognize that the use of such amide coupling agents requires the use of a base. Suitable bases include organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylpyridine (DMAP), and the like.

[0246] In some embodiments, a compound having formula L reacts with a chlorinating agent such as thionyl chloride to form an acyl chloride, and then reacts with HN(R) 4 The reaction proceeds to form a compound having the formula K.

[0247] In some embodiments, the present invention provides a method for preparing a compound having the following formula G:

[0248]

[0249] in:

[0250] LG is halogen, -OSO2R, or -OSO2CF3; and

[0251] R, R 1 R 2 and R 4 Each is as defined above for compounds having formula Z.

[0252] Includes the following steps:

[0253] (a) Provides a propynic acid having the formula L:

[0254]

[0255] Where R 2 It is as defined above for compounds having formula G;

[0256] (b) Reacting the compound having formula L with an alcohol having formula HO-R to form a propyne ester having formula M:

[0257]

[0258] Among them, R and R 2 Each is as defined above for compounds having formula G;

[0259] (c) Reacting the propyne ester having formula M to form a compound having formula N:

[0260]

[0261] Among them, R, R 1 R 2 LG and G are each defined as above for compounds having the formula G;

[0262] (d) Hydrolyzing the compound having formula N to form a compound having formula Q:

[0263]

[0264] Among them, R, R 1 R 2 LG and G are each as defined above for compounds having formula G; and

[0265] (e) React the compound having formula Q with HN(R) 4 )2 reaction, where each R 4 As defined above for compounds having formula G, to form a compound having formula G.

[0266] In some embodiments, a propynic acid having formula L is treated with an alcohol to form a propynyl ester having formula M. Suitable alcohols include methanol, ethanol, and isopropanol. Those skilled in the art will recognize that the esterification of a propynic acid having formula L can be achieved by a catalytic acid. Therefore, in some embodiments, a propynic acid having formula L is treated with methanol or ethanol in the presence of a catalytic sulfuric acid to provide a propynyl ester having formula M.

[0267] Those skilled in the art will recognize that such esterification can be carried out at temperatures ranging from about 25°C to about 100°C, or at the boiling point of the alcohol. In some embodiments, the esterification of a propynic acid having formula L is performed by heating to reflux (the boiling point of the alcohol).

[0268] As described above, in some embodiments of a compound having formula N, LG is a halogen. In some such embodiments, a compound having formula M is treated with a halide. In some embodiments, a compound having formula M is treated with a sodium halide. In some such embodiments, a compound having formula M is treated with sodium iodide. In some embodiments, a compound having formula M is treated with a halide in the presence of an acid. Suitable acids include both inorganic and organic acids. In some embodiments, a compound having formula M is treated with a halide and an organic acid such as acetic acid. In some embodiments, a compound having formula M is treated with sodium iodide in the presence of acetic acid to provide a compound having formula N.

[0269] In some embodiments, an ester of a compound having formula N is hydrolyzed to acrylic acid. Suitable hydrolysis conditions are known to those skilled in the art and include hydroxides such as lithium hydroxide, sodium hydroxide, potassium hydroxide, and cesium hydroxide in the presence of water. Those skilled in the art will recognize that such hydrolysis can be carried out at temperatures from about 25°C to about 100°C. In some embodiments, the hydrolysis of an acrylate having formula N is performed by heating to reflux.

[0270] In some embodiments, an acrylic acid having the formula Q reacts with HN(R) 4 The reaction )2 forms a compound having the formula G. In some embodiments, in HN(R) 4 In the presence of 2, an acrylic acid having the formula Q is treated with an amide coupling agent to form a compound having the formula G. Suitable amide coupling agents include HOBt, HOAt, HAMDU, HAMTU, PyBOP, PyBrOP, TBTU, HATU, and T3P. Those skilled in the art will recognize that the use of such amide coupling agents requires the use of a base. Suitable bases include organic bases such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylpyridine (DMAP), and the like.

[0271] In some embodiments, a compound having formula Q reacts with a chlorinating agent such as thionyl chloride to form an acyl chloride, and then reacts with HN(R) 4 The reaction proceeds to form a compound having the formula G.

[0272] In some embodiments, the present invention provides a method for providing a compound having the following formula V:

[0273]

[0274] Or a pharmaceutically acceptable salt thereof, wherein R, R x R y R 1 R 2 And m are each as defined above for compounds having formula Z.

[0275] Includes the following steps:

[0276] (a) Provide a compound having the formula L:

[0277]

[0278] Where R 2 It is as defined above for compounds having formula V;

[0279] (b) Mixing the compound having formula L with... The reaction forms a compound having the formula R:

[0280]

[0281] Where R 2 It is as defined above for compounds having formula V;

[0282] (c) Reacting the compound having formula R to provide a compound having formula J:

[0283]

[0284] in:

[0285] LG is halogen, -OSO2R, or -OSO2CF3; and

[0286] R, R 1 and R 2 Each is as defined above for compounds having formula V; and

[0287] (d) React the compound having formula J with a compound having formula E:

[0288]

[0289] Where R x R y And m are each defined as above for compounds having formula V.

[0290] To provide a compound having formula V in the presence of a sterically hindered nucleophilic base.

[0291] In some embodiments, the present invention provides a method for providing a compound having the following formula V:

[0292]

[0293] Or a pharmaceutically acceptable salt thereof, wherein R, R x R y R 1 R 2 And m are each as defined above for compounds having formula Z.

[0294] Includes the following steps:

[0295] (a) Provide a compound having the formula L:

[0296]

[0297] Where R 2 It is as defined above for compounds having formula V;

[0298] (b) Reacting the compound having formula L with an alcohol having formula HO-R to form a compound having formula M:

[0299]

[0300] Among them, R and R 2 Each is as defined above for compounds having formula V.

[0301] (c) Reacting the compound having formula M to provide a compound having formula N:

[0302]

[0303] in:

[0304] LG is halogen, -OSO2R, or -OSO2CF3; and

[0305] R, R 1 and R 2 Each is as defined above for compounds having formula V;

[0306] (d) Hydrolyzing the compound having formula N to form a compound having formula Q:

[0307]

[0308] Among them, R 1 R 2 LG and V are each defined as above for compounds having formula V;

[0309] (e) to mix the compound having formula Q with The reaction forms a compound having the formula J:

[0310]

[0311] in:

[0312] LG is halogen, -OSO2R, or -OSO2CF3; and

[0313] R, R 1 and R 2 Each is as defined above for compounds having formula V; and

[0314] (f) Reacting the compound having formula J with a compound having formula E:

[0315]

[0316] Where R x Ry And m are each defined as above for compounds having formula V.

[0317] To provide a compound having formula V in the presence of a sterically hindered nucleophilic base.

[0318] definition

[0319] The compounds of this invention include those generally described above, and are further described by the categories, subcategories and species disclosed herein. Unless otherwise specified, the following definitions shall apply as used herein. For all purposes of this invention, these chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Furthermore, the general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5th ed., Smith, MB and March, J., John Wiley & Sons, New York, 2001, the entire contents of which are incorporated herein by reference.

[0320] Unless otherwise stated, the nomenclature used herein generally follows the examples and rules described in Nomenclature of Organic Chemistry, Chapters A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, for which exemplary chemical structure nomenclature and rules concerning chemical structure nomenclature are incorporated herein by reference. Optionally, the names of compounds may be generated using a chemical nomenclature program (ACD / ChemSketch, version 5.09 / September 2001, Advanced Chemistry Development, Inc., Toronto, Canada).

[0321] The compounds of the present invention may have an asymmetry center, a chiral axis, and a chiral plane (e.g., as described in ELEliel and S.Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pp. 1119-1190), and may exist as racemates, racemic mixtures, and individual diastereomers or enantiomers, wherein all possible isomers and mixtures thereof (including optical isomers) are included in the present invention.

[0322] As used herein, the term "aliphatic" or "aliphatic group" refers to a monovalent hydrocarbon group that is straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridged, and spirofused polycyclic). An aliphatic group may be saturated or contain one or more unsaturated but non-aromatic units. Unless otherwise stated, an aliphatic group contains 1-6 carbon atoms. However, in some embodiments, an aliphatic group contains 1-10 or 2-8 carbon atoms. In some embodiments, an aliphatic group contains 1-4 carbon atoms, and in other embodiments, an aliphatic group contains 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched alkyl, alkenyl, and alkynyl groups, and their hybrids such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0323] As used herein, the term "alkyl" means a saturated, straight-chain or branched aliphatic group. In one aspect, an alkyl group comprises 1-10 or 2-8 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and the like.

[0324] As used herein, the term "alkenyl" means a straight-chain or branched 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, but not limited to, vinyl, 1-propenyl, 1-butenyl, and the like. The term "alkenyl" encompasses groups having carbon-carbon double bonds in "cis" and "trans" conformations, or alternatively in "E" and "Z" conformations. If an alkenyl group contains more than one carbon-carbon double bond, each carbon-carbon double bond is independently a cis or trans double bond, or a mixture thereof.

[0325] As used herein, the term "alkynyl" means a straight-chain or branched aliphatic group 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, but not limited to, 1-propynyl (propynyl), 1-butynyl, and the like.

[0326] The terms “alicyclic,” “carbocyclic,” “carbocyclic,” and “carbocyclic” used alone or as part of a larger body refer to a saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring system having from 3 to 10 members, as illustrated herein, wherein the aliphatic ring system as defined above and illustrated herein may optionally be substituted. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. The terms “alicyclic,” “carbocyclic,” “carbocyclic,” and “carbocyclic” also include aliphatic rings fused to one or more aromatic or non-aromatic rings, such as decaaminonaphthyl, tetrahydronaphthyl, decahydronaphthyl, or bicyclic [2.2.2]octane.

[0327] As used herein, the term "cycloalkyl" means a saturated cyclic aliphatic monocyclic or bicyclic ring system having 3 to 10 members. A cycloalkyl group may optionally be substituted, as illustrated herein. In some embodiments, a cycloalkyl group has 3 to 6 carbons.

[0328] As used herein, the term "heterocyclic alkyl" means a saturated or unsaturated aliphatic ring system in which at least one carbon atom is replaced by a heteroatom selected from N, S, and O. A heterocyclic alkyl group may comprise one or more rings that can be attached together in a suspended manner or may be fused. In one aspect, a heterocyclic alkyl group is a tri- to seven-membered ring system and comprises, for example, but not limited to, piperidinyl, piperazine, pyrrolidinyl, tetrahydrofuranyl, and the like.

[0329] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon, and includes any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any basic quaternized form of nitrogen; and a heterocyclic substituted nitrogen, such as N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolealkyl), or NR. + (e.g., in N-substituted pyrroleyl groups).

[0330] The term “unsaturated” as used herein refers to a portion having one or more unsaturated units.

[0331] The term “halo” or “halogen” as used herein means halogens in radioactive and non-radioactive forms, and includes, for example, but not limited to, fluorine, chlorine, bromine, iodine, etc.

[0332] As used herein, the term "haloalkyl" refers to an aliphatic group substituted with one or more halogen atoms. In some embodiments, a haloalkyl refers to a fully halogenated aliphatic group. In some embodiments, a haloalkyl refers to an alkyl group substituted with one or more halogen atoms. Exemplary haloalkyl groups include -CF3, -CCl3, -CF2CH3, -CH2CF3, -CH2(CF3)2, -CF2(CF3)2, and the like.

[0333] The term "aryl," used alone or in combination herein, refers to a carbocyclic aromatic system comprising one or more rings that can be attached together in a suspended manner or may be fused. In specific embodiments, the aryl group is one, two, or three rings. On one hand, the aryl group has 5 to 12 ring atoms. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, biphenyl, phenanthryl, anthraceneyl, and acenaphthyl. An "aryl" group may have 1 to 4 substituents, such as lower alkyl, hydroxyl, halogen, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, etc.

[0334] As used herein, the term "heteroaryl," whether alone or in combination, means an aromatic system in which at least one carbon atom is replaced by a heteroatom selected from N, S, and O. A heteroaryl group may comprise one or more rings that can be attached together in a suspended manner or may be fused. In specific embodiments, the heteroaryl group has one, two, or three rings. On one hand, the heteroaryl group has 5 to 12 ring atoms. The term "heteroaryl" includes heteroaryl groups such as triazolyl, imidazolyl, pyrroleyl, pyrazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazinyl, indolyl, furanyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, oxazolyl, oxadiazolyl, isoxazolyl, and the like. A "heteroaryl" group may have 1 to 4 substituents, such as lower alkyl, hydroxyl, halogen, haloalkyl, nitro, cyano, alkoxy, lower alkylamino, etc.

[0335] It should be understood that the substituents and substitution patterns on the compounds of the present invention can be selected by a person skilled in the art, thereby providing compounds that are chemically stable and readily synthesized by techniques known in the art and those methods listed below. Generally, whether or not preceded by "optionally," the term "substituted" refers to the substitution of one or more hydrogen atoms of a specified moiety by a suitable substituent. Unless otherwise specified, a "optionally substituted" group may have a suitable substituent at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure can be substituted by more than one substituent selected from a specified group. Alternatively, a "optionally substituted" group may be unsubstituted.

[0336] The combinations of substituents considered in this invention are preferably those that result in the formation of stable or chemically viable compounds. If a substituent is itself replaced by more than one group, it should be understood that these multiple groups may be on the same carbon atom or on different carbon atoms, as long as the result is a stable structure. The term "stable" as used herein means that such compounds are substantially unchanged when subjected to conditions permissible for their production, testing, and, in some embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0337] Suitable monovalent substituents on the substituted carbon atom of the "optionally substituted" group are independently: halogen; -(CH2). 0-4 R ο ;-(CH2) 0-4 OR ο ;-O(CH2) 0-4 R ο -O-(CH2) 0-4 C(O)OR ο ;-(CH2) 0-4 CH(OR ο )2;-(CH2) 0- 4SR ο ;-(CH2) 0-4 Ph, which can be R ο Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be R ο Substitution; -CH=CHPh, which can be replaced by R ο Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl group, which can be R ο Substitution; -NO2; -CN; -N3; ​​-(CH2)0-4 N(R ο )2;-(CH2) 0-4 N(R ο )C(O)R ο ;-N(R ο )C(S)R ο ;-(CH2) 0-4 N(R ο )C(O)NR ο 2;-N(R ο )C(S)NR ο 2;-(CH2) 0-4 N(R ο )C(O)OR ο ;-N(R ο )N(R ο )C(O)R ο ;-N(R ο )N(R ο )C(O)NR ο 2;-N(R ο )N(R ο )C(O)OR ο ;-(CH2) 0-4 C(O)R ο ;-C(S)R ο ;-(CH2) 0-4 C(O)OR ο ;-(CH2) 0-4 C(O)SR ο ;-(CH2) 0-4 C(O)OSiR ο 3;-(CH2) 0-4 OC(O)R ο ;-OC(O)(CH2) 0-4 SR-,SC(S)SR ο ;-(CH2) 0-4 SC(O)R ο ;-(CH2) 0-4 C(O)NR ο 2;-C(S)NR ο 2;-C(S)SR ο ;-SC(S)SR ο ,-(CH2) 0-4 OC(O)NR ο 2;-C(O)N(OR ο )R ο ;-C(O)C(O)R ο ;-C(O)CH2C(O)R ο ;-C(NOR ο )Rο ;-(CH2) 0- 4SSR ο (CH2) 0-4 S(O)2R ο ;-(CH2) 0-4 S(O)2OR ο ;-(CH2) 0-4 OS(O)2R ο ;-S(O)2NR ο 2;-(CH2) 0-4 S(O)R ο ;-N(R ο )S(O)2NR ο 2; -N(R) ο )S(O)2R ο ;-N(OR) ο )R ο ;-C(NH)NR ο 2; -P(O)2R ο ;-P(O)R ο 2; -OP(O)R ο 2; -OP(O)(OR ο )2; SiR ο 3; -(C 1-4 (linear or branched alkylene) ON(R) ο )2; or -(C 1-4 (straight-chain or branched alkylene)C(O)ON(R ο )2, where each R ο It can be substituted and independently be hydrogen, C, as defined below. 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms from nitrogen, oxygen, and sulfur, or, despite the above definition, two independently occurring R ο Together with one or more of their inserted atoms, they form a 3-12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, and sulfur, which may be substituted as defined below.

[0338] R ο (or two independently occurring R) ο The suitable monovalent substituents on the ring (formed together with their inserted atoms) are independently halogens, -(CH2). 0-2 R · , -(halogenated R · ), -(CH2)0-2 OH, -(CH2) 0-2 OR · ,-(CH2) 0-2 CH(OR · )2;-O(halogenated R · -CN, -N3, -(CH2) 0-2 C(O)R · ,-(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR · ,-(CH2) 0-2 SR · ,-(CH2) 0-2 SH, -(CH2) 0-2 NH2,-(CH2) 0-2 NHR · ,-(CH2) 0-2 NR · 2,-NO2,-SiR · 3, -OSiR · 3,-C(O)SR · , -(C 1-4 (linear or branched alkylene)C(O)OR · , or -SSR · Each R · The case where it is not substituted or is preceded by "halo" is when it is substituted by only one or more halogens and is independently selected from C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring with 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on the saturated carbon atom at R° include =O and =S.

[0339] Suitable divalent substituents on the saturated carbon atom of the "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)) 2-3 O-, and -S(C(R) * 2)) 2-3 S-, where each independently existing R * Selected from hydrogen, and can be substituted C as defined below.1-6 Aliphatic, or a saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, and sulfur. Suitable divalent substituents on the substituted carbon atom adjacent to the "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently existing R * Selected from hydrogen, and can be substituted C as defined below. 1-6 Aliphatic, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0340] In R * Suitable substituents on the aliphatic group include halogens, -R · , -(halogenated R · ), -OH, -OR · -O(halogenated R) · -CN, -C(O)OH, -C(O)OR · -NH2, -NHR · -NR · 2, and -NO2, where each R · The case where it is unsubstituted or preceded by "halogenation" is when it is substituted by only one or more halogens and is independently C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0341] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include as well as Each of them Hydrogen alone can be substituted by C as defined below. 1-6 Aliphatic, unsubstituted -OPh, or having 0-4 unsubstituted 5-6-membered saturated, partially unsaturated, or aryl rings independently selected from nitrogen, oxygen, and sulfur, or, despite the above definition, two independently occurring... Together with one or more of their inserted atoms, they form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring with 0-4 independent heteroatoms selected from nitrogen, oxygen, and sulfur.

[0342] exist The appropriate substituent on the aliphatic group is independently -R · , -(halogenated R · ), -OH, -OR · -O(halogenated R) · -CN, -C(O)OH, -C(O)OR · -NH2, -NHR · -NR · 2, or -NO2, where each R · It is either unsubstituted or, in the case of being preceded by a "halogen," substituted by only one or more halogens, and is independently C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0343] As used herein, the term "hydrazine equivalent" means a chemical reagent that can be used to introduce an N- moiety into a molecule. Hydrazine equivalents include hydrazine hydrate along with its protected form, such as tert-butylhydrazine carboxylate.

[0344] As used herein, the term "leaving group" refers to a functional group that is transferred from a molecule during a chemical reaction. Leaving groups include halogens and sulfonate groups, such as toluenesulfonate and methanesulfonate.

[0345] As used herein, the term "pharmaceutically acceptable salt" means, within the bounds of reasonable medical judgment, salts suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Such pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. described such 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 suitable inorganic and organic acids and bases, which are compatible with the treatment of the patient.

[0346] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or an organic acid such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-p-methyl, glycerol phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pyruvate, pectin salt, persulfate, 3-phenylpropionate, phosphate, p-pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0347] In some embodiments, exemplary inorganic acids that form suitable salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as acid metal salts such as disodium hydrogen phosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids. Illustrative examples of such acids are, for example, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid, and other sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Salts of mono- or di-acids can be formed, and such salts can exist in hydrated, solvated, or substantially anhydrous forms. Generally, the acid addition salts of these compounds are more soluble in water and various hydrophilic organic solvents compared to their free base forms, and typically exhibit higher melting points.

[0348] In some embodiments, acid addition salts of compounds having Formula I are best suited for formation from pharmaceutically acceptable acids, including, for example, those formed with inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, and organic acids such as succinic acid, maleic acid, acetic acid, or fumaric acid.

[0349] Other non-pharmaceuticalally acceptable salts, such as oxalates, can be used, for example, for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt, for the separation of compounds of Formula I. Base addition salts (such as sodium, potassium, and ammonium salts), solvates of the compounds of the present invention, and hydrates are also included within the scope of the present invention. The conversion from a salt of a given compound to a salt of a desired compound can be accomplished by applying standard techniques well known to those skilled in the art.

[0350] A "pharmaceutically acceptable base addition salt" is any non-toxic, organic, or inorganic base addition salt of the acidic compounds represented by Formula I, or any intermediate thereof. Exemplary inorganic bases forming suitable salts include, but are not limited to, hydroxides of lithium, sodium, potassium, calcium, magnesium, or barium. Exemplary organic bases forming suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and methylpyridine or ammonia. The selection of a suitable salt may be important such that the ester functionality (if present) is not hydrolyzed elsewhere in the molecule. Criteria for selecting a suitable salt are well known to those skilled in the art.

[0351] Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Alkyl salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0352] Unless otherwise stated, the structures described herein also mean all isomers of that structure (e.g., enantiomers, diastereomers, and geometric (or conformational) forms); for example, R and S configurations for each asymmetry center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, individual stereochemical isomers of these compounds, along with mixtures of enantiomers, diastereomers, and geometric (or conformational) isomers, are within the scope of this invention. Unless otherwise stated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0353] Furthermore, unless otherwise stated, the structures described herein also mean those compounds that differ only in the presence of one or more isotopically enriched atoms. For example, by replacing hydrogen with deuterium or tritium, or by using a... 13 C- or 14Compounds derived from carbon substitution by C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or therapeutic agents according to this invention.

[0354] The term "stereoisomer" is a general term used for all isomers of a single molecule that differ only in the spatial orientation of their atoms. It includes mirror isomers (enantiomers), geometric (cis / trans) isomers, and isomers of compounds having more than one chiral center that is not a mirror image of each other (diastereomers).

[0355] The term “treat” or “treating” refers to relieving one or more symptoms, eliminating the cause of one or more symptoms on a temporary or permanent basis, or preventing or delaying the onset of one or more symptoms associated with a disorder or condition.

[0356] The term "therapeutic effective dose" refers to a quantity of a compound that is effective in treating or alleviating the severity of one or more symptoms of a disease or condition.

[0357] The term "pharmaceutically acceptable carrier" refers to a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with the active ingredient to allow for the formation of a pharmaceutical composition (i.e., a dosage form suitable for patient administration). An example of such a carrier is pharmaceutically acceptable oil, typically used for parenteral administration. These pharmaceutically acceptable carriers are well known in the art.

[0358] When describing the elements disclosed herein, the articles “a / an,” “the,” and “said” are intended to indicate that there is one or more of such elements. The terms “comprising,” “having,” and “including” are intended to be open-ended, meaning that there may be other elements besides those listed.

[0359] Preparations and administration

[0360] Pharmaceutically acceptable compositions

[0361] 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 delivery vehicle. The amount of the compound in a composition of the invention is an amount that measurably inhibits CRM1 in a biological sample or patient. In some embodiments, the compositions of the invention are formulated for administration to a patient in need of the composition. As used herein, the term "patient" refers to an animal. In some embodiments, the animal is a mammal. In some embodiments, the patient is a veterinary patient (i.e., a non-human mammalian patient). In some embodiments, the patient is a dog. In other embodiments, the patient is a human.

[0362] The phrase "pharmaceutically acceptable carrier, adjuvant, or transporter" refers to a non-toxic carrier, adjuvant, or transporter that does not impair the pharmacological activity of the compound formulated with it. Pharmaceutically acceptable carriers, adjuvants, or transporters that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partially saturated mixtures of glycerides of the compositional fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0363] The compositions of the present invention can be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), via inhalation spray, topically, rectally, nasally, sublingually, vaginally, or via an implantable reservoir. In some embodiments, the provided compounds or compositions can be administered intravenously and / or intraperitoneally.

[0364] As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraocular, intravitreal, intra-articular, intrasynovial, intrasternal, intramembranous, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. Preferably, the composition is administered orally, subcutaneously, intraperitoneally, or intravenously. The aseptic injection of the composition of the present invention can be in the form of an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The aseptic injection can also be a aseptic injection solution or suspension in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, aseptic fixative oils are conventionally used as solvents or suspension media.

[0365] The pharmaceutically acceptable compositions of the present invention can be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, and solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are typically also added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension for oral use is required, the active ingredient is combined with an emulsifier and a suspending agent. Sweeteners, flavorings, or colorings may also be added if desired. In some embodiments, an oral formulation provided is formulated for immediate or sustained / delayed release. In some embodiments, compositions suitable for sublingual or oral administration include tablets, lozenges, and soft lozenges. An provided compound may also be in a microencapsulated form.

[0366] Alternatively, for rectal administration, the pharmaceutically acceptable compositions of the present invention can be administered in suppository form. The pharmaceutically acceptable compositions of the present invention can also be administered topically, particularly when the therapeutic target includes a region or organ easily accessible for local application, including diseases of the eyes, skin, or lower intestine. Suitable local formulations for each of these regions or organs are readily prepared.

[0367] For local application to the lower intestine, it can be accomplished with rectal suppository formulations (see above) or appropriate enema formulations. Topical transdermal patches may also be used.

[0368] For ophthalmic use, pharmaceutically acceptable compositions can be formulated as micronized suspensions or in an ointment such as petrolatum.

[0369] The pharmaceutically acceptable compositions of the present invention can also be administered via nasal aerosol or inhaler.

[0370] In some embodiments, the pharmaceutically acceptable compositions of the present invention are formulated for intraperitoneal administration.

[0371] The amount of the compounds of the present invention, which can be combined with a carrier material in a single dosage form to produce a composition, will vary depending on the host being treated and the specific route of administration. In one embodiment, a composition is formulated such that a dose of the inhibitor between 0.01 and 100 mg / kg body weight / day can be administered to the patient receiving the composition. In another embodiment, the dose is from about 0.5 to about 100 mg / kg body weight every 4 to 120 hours, or between 1 mg and 1000 mg / dose, or as required by specific pharmaceutical needs. Typically, the pharmaceutical compositions of the present invention will be administered from about once to about six times daily.

[0372] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health, sex, diet, timing of administration, excretion rate, drug combination, the judgment of the treating physician, and the severity of the specific disease to be treated. The amount of the compound of the invention in the composition will also depend on the specific compound in the composition.

[0373] In some embodiments, the composition further comprises one or more additional therapeutic or preventative agents. When the composition of the present invention comprises a compound having the formula described herein combined with one or more additional therapeutic or preventative agents, both the compound and the additional agents should be at a dose level between about 1% and 100% of the normal dose in a single therapy regimen, and more preferably at a dose level between about 5% and 95%. These additional agents may be administered alone, as part of a multi-dose regimen, along with the compounds of the present invention. Alternatively, these additional agents may be part of a single dose form and may be mixed with a compound of the present invention in a single composition.

[0374] Depending on the improvement of the patient's condition, a maintenance dose of the compounds, compositions, or combinations of the present invention may be administered if necessary. Subsequently, once the symptoms have been relieved to a desired level, the dosage or frequency of administration, or both, as a function of these symptoms, may be reduced to a level where the improved condition can be maintained. However, the patient may request long-term intermittent therapy based on any recurrence of disease symptoms.

[0375] Use of compounds and pharmaceutically acceptable compositions

[0376] The compounds and compositions described herein are generally useful for inhibiting CRM1, and therefore useful for treating one or more disorders associated with CRM1 activity. Accordingly, in some embodiments, the present invention provides a method for treating CRM1-mediated disorders, comprising the step of administering a compound of the present invention, or a pharmaceutically acceptable salt or composition thereof, to a patient in need of it. The compounds and compositions described herein may also be administered to cells in a culture system, such as in vivo or in vitro, or to a subject, such as in vivo, to treat, prevent, and / or diagnose a variety of disorders, including those described herein.

[0377] The activity of the compounds used in this invention as inhibitors of CRM1 can be determined in vivo, in vitro, or in cell lines. Detailed conditions for determining the compounds used in this invention as inhibitors of CRM1 are listed in the examples.

[0378] As used herein, the terms "CRM1-mediated disorder or condition" or "disorder or condition associated with CRM1 activity" mean any disease or other harmful condition in which CRM1 plays a role. Therefore, another embodiment of the invention relates to treating or reducing the severity of one or more diseases in which CRM1 plays a role. In some embodiments, the invention provides a method of treating a disease in a subject associated with the expression or activity of p53, p73, p21, pRB, p27, IκB, NFκB, c-Abl, FOXO protein, or COX-2, the method comprising administering to the patient a therapeutically effective amount of the compound described herein. In another embodiment, the invention relates to a method of treating or reducing the severity of a disease or condition selected from proliferative disorders (e.g., cancer), inflammatory disorders, autoimmune disorders, viral infections, ophthalmic disorders, and neurodegenerative disorders, the method comprising administering a compound or composition according to the invention to a patient in need of it. In a more specific embodiment, the invention relates to a method of treating or reducing the severity of cancer. Specific examples of the above disorders are set forth in detail below.

[0379] The cancers treatable by these compounds of the present invention include, but are not limited to: hematologic malignancies (leukemia, lymphoma, myeloma, myelodysplastic syndromes) and solid tumors (cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, and soft tissue and osteosarcoma, and stromal tumors). Breast cancer (BC) can include basal-like breast cancer (BLBC), triple-negative breast cancer (TNBC), and breast cancer that is both BLBC and TNBC. Additionally, breast cancer can include invasive or non-invasive ductal carcinoma or lobular carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma of the breast, male breast cancer, recurrent or metastatic breast cancer, phyllodes tumor of the breast, and Paget's disease of the nipple.

[0380] The inflammatory disorders treatable by these compounds of the present invention include, but are not limited to, multiple sclerosis, rheumatoid arthritis, degenerative arthritis, systemic lupus erythematosus, systemic sclerosis, vasculitis syndromes (small, medium, and large vessels), atherosclerosis, inflammatory bowel disease, irritable bowel syndrome, Crohn's disease, mucinous colitis, ulcerative colitis, gastritis, sepsis, psoriasis, and other inflammatory skin disorders (such as eczema, atopic dermatitis, contact dermatitis, urticaria, scleroderma, psoriasis, and skin diseases with acute inflammatory components, pemphigus, bullous pemphigoid, allergic dermatitis), and urticaria syndrome. In some embodiments, the disorder or condition associated with CRM1 activity is multiple sclerosis, irritable bowel syndrome, rheumatoid arthritis, psoriasis, or other inflammatory skin disorders.

[0381] The viral diseases treatable by these compounds of the present invention include, but are not limited to, acute febrile pharyngitis, pharyngoconjunctival fever, viral keratoconjunctivitis, infantile gastroenteritis, Coxsackie virus infection, infectious mononucleosis, Burkitt lymphoma, acute hepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivostomatitis in children, tonsillitis in adults, and pharyngitis, keratoconjunctivitis), and latent HSV-1 infection. HSV-1 infection (e.g., cold sores and herpes labialis), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, giant cell inclusion body disease, Kaposi's malignant tumor, multicentric giant lymphadenopathy, primary exudative lymphoma, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, proliferative epithelial lesions (e.g., common, flat, plantar and anogenital warts, laryngeal papilloma, verrucous epidermal dysplasia), cervical cancer, squamous cell carcinoma, membranous laryngitis, pneumonia, bronchiolitis, the common cold, poliomyelitis, rabies, influenza-like syndrome, severe bronchiolitis and pneumonia, rubella, congenital rubella, varicella, and herpes zoster. Viral diseases treatable by these compounds of the present invention also include chronic viral infections, including hepatitis B and hepatitis C.

[0382] Exemplary ophthalmic disorders include, but are not limited to, macular edema (diabetic and non-diabetic macular edema), age-related wet and dry macular degeneration, age-related discoid macular degeneration, cystoid macular edema, eyelid edema, retinal edema, diabetic retinopathy, choroidal retinopathy, neovascular macular degeneration, neovascular glaucoma, uveitis, iritis, retinal vasculitis, endophthalmitis, panophthalmitis, metastatic ophthalmitis, choroiditis, retinal pigment epitheliitis, conjunctivitis, cyclitis, scleritis, episcleritis, optic neuritis, retrobulbar optic neuritis, keratitis, blepharitis, and exudative retinal detachment. Corneal ulcer, conjunctival ulcer, nummular keratitis, ophthalmic diseases related to tissue hypoxia or local ischemia, retinopathy of prematurity, diabetic proliferative retinopathy, polypoid choroidal angiopathy, retinal angiomatous hyperplasia, retinal artery occlusion, retinal vein occlusion, Coront's disease, familial exudative vitreoretinopathy, veinless disease (Gau's disease), retinal periphlebitis, antiphospholipid antibody syndrome, leukemic retinopathy, hyperviscosity syndrome, macroglobulinemia, interferon-associated retinopathy, hypertensive retinopathy, radiation retinopathy, corneal epithelial stem cell deficiency or cataract.

[0383] Neurodegenerative diseases treatable by the compounds of this invention include, but are not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS / Lou Gehrig's disease). In some embodiments, the disorder or condition associated with CRM1 activity is ALS.

[0384] The compounds and compositions described herein can also be used to treat disorders of abnormal tissue growth and fibrosis, including dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pulmonary fibrosis, liver fibrosis, glomerulonephritis, and other kidney diseases.

[0385] The compounds and compositions described herein can also be used to treat food intake-related disorders, such as obesity and overeating. In some embodiments, the disorder or condition associated with CRM1 activity is obesity.

[0386] In some embodiments, disorders or conditions associated with CRM1 activity include muscle atrophy, arthritis (e.g., osteoarthritis and rheumatoid arthritis), ankylosing spondylitis, traumatic brain injury, spinal cord injury, sepsis, rheumatoid arthritis, carcinomatous atherosclerosis, type 1 diabetes, type 2 diabetes, leptospirosis-related renal dysfunction, glaucoma, retinal disease, aging, headache, pain, complex regional pain syndrome, cardiac hypertrophy, muscle wasting, catabolism disorders, obesity, fetal growth retardation, hypercholesterolemia, heart disease, chronic heart failure, ischemia / reperfusion, stroke, cerebral aneurysm, angina pectoris, lung disease, cystic fibrosis, acidogenic lung injury, pulmonary hypertension, asthma, chronic obstructive pulmonary disease, Sjögren's syndrome, hyaline membrane disease, kidney disease, glomerular disease, alcoholic liver disease, intestinal disease, and abdominal pain. Membranous endometriosis, skin diseases, sinuses, mesothelioma, anhidrotic ectodermal dysplasia-ID, Bechtel's disease, pigmentary disorders, tuberculosis, asthma, Crohn's disease, colitis, eye allergies, appendicitis, Paget's disease, pancreatitis, periostitis of the dental root, endometriosis, inflammatory bowel disease, inflammatory lung disease, silicosis, sleep apnea, AIDS, HIV-1, autoimmune diseases, antiphospholipid syndrome, lupus, lupus nephritis, familial Mediterranean fever, hereditary periodic fever syndrome, psychosocial stress disorders, 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.

[0387] In other embodiments, disorders or conditions associated with CRM1 activity include head injury, uveitis, inflammatory pain, allergen-induced asthma, non-allergen-induced asthma, glomerulonephritis, ulcerative colitis, necrotizing enterocolitis, hyperimmunoglobulinemia D with recurrent fever (HIDS), TNF receptor-associated periodic syndrome (TRAPS), cryptothermal protein-associated periodic syndrome, Mue-Wei syndrome (urticaria, deafness, amyloidosis), familial cold urticaria, neonatal multisystem inflammatory disease (NOMID), periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFA). PA syndrome, Blau syndrome, suppurative aseptic arthritis, pyoderma gangrenosa, acne (PAPA), interleukin-1 receptor antagonist deficiency (DIRA), subarachnoid hemorrhage, polycystic kidney disease, transplantation, organ transplantation, tissue transplantation, myelodysplastic syndrome, irritant-induced inflammation, phytoirritant-induced inflammation, inflammation caused by kudzu / urushiol crude oil, chemical irritant-induced inflammation, bee sting-induced inflammation, insect bite-induced inflammation, sunburn, burns, dermatitis, endotoxemia, lung injury, acute respiratory distress syndrome, alcoholic hepatitis, or kidney injury caused by parasitic infection.

[0388] In another embodiment, a compound or composition described herein may be used to treat or prevent allergies and respiratory disorders, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any chronic obstructive pulmonary disease (COPD).

[0389] Another embodiment of the invention is the use of a compound having Formula I in the manufacture of a medicament for treating a disorder or condition associated with CRM1 activity. In another aspect, the invention provides the use of a compound having Formula I in the manufacture of a medicament for treating in a subject a disease associated with the expression or activity of p53, p73, p21, pRB, p27, IκB, NFκB, c-Abl, FOXO protein, or COX-2. In some embodiments, the invention provides the use of a compound having Formula I in the manufacture of a medicament for treating any of the following: cancer and / or neoplastic disorders, angiogenesis, autoimmune disorders, inflammatory disorders and / or diseases, epigenetic, hormonal disorders and / or diseases, viral diseases, neurodegenerative disorders and / or diseases, and ophthalmic disorders.

[0390] In some embodiments, the present invention provides a method for inhibiting CRM1 in a biological sample or a patient, the method comprising contacting the biological sample with a pharmaceutically acceptable salt of a compound having Formula I or a pharmaceutically acceptable composition thereof, or administering the biological sample to the patient.

[0391] tumor disorders

[0392] The compounds or compositions described herein can be used to treat neoplastic disorders. A "neoplastic disorder" is a disease or disorder characterized by cells with autonomous growth or replication capabilities, such as an abnormal state or symptom characterized by proliferative cell growth (benign or malignant). Exemplary neoplastic disorders include: cancer, sarcoma (e.g., soft tissue), osteosarcoma, metastatic disorders (e.g., tumors originating from the prostate, brain, bone, gastrointestinal tract, lung, breast, ovary, cervix, pancreas, kidney, head and neck, and liver), hematopoietic neoplastic disorders (e.g., leukemia, lymphoma, myeloma, and other malignant plasma cell disorders), and metastatic tumors. In one embodiment, the cancer to be treated is selected from breast cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, brain cancer, liver cancer, and pancreatic cancer. Treatment using the compound can be at an effective level to improve the symptoms of at least one neoplastic disorder, for example, by reducing cell proliferation, reducing tumor mass, etc.

[0393] In one embodiment, the neoplastic disorder is a basal-like breast cancer (BLBC). BLBCs account for up to 15% of breast cancers (BCs) and are typically triple-negative breast cancer (TNBC), characterized by the absence of ER, progesterone receptor PR, and HER-2 amplification. In one specific embodiment, this breast cancer is TNBC. Additionally, a large proportion of BRCA1-associated BCs are BLBCs and TNBCs, expressing basal keratin and EGFR. BLBCs are characterized by an aggressive phenotype, high histological grade, and poor clinical outcomes with high recurrence and metastasis rates.

[0394] Combination therapy

[0395] In some embodiments, the compound described herein is administered co-administered with an additional "second" therapeutic agent or treatment. The second therapeutic agent can be selected from any agent typically used in a single therapy for treating a specified disease or condition. As used herein, the term "co-administered" and related terms refer to simultaneous or sequential administration of the therapeutic agents according to the invention. For example, a compound of the invention can be administered simultaneously with another therapeutic agent, or sequentially in separate unit dosage forms, or together in a single unit dosage form. Therefore, the invention provides a single unit dosage form comprising a compound having Formula I, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or delivery vehicle.

[0396] In one embodiment of the invention, a second therapeutic agent is administered to a subject in an effective amount less than the effective amount that would be present if the second therapeutic agent were not administered. In another embodiment, the effective amount of the second therapeutic agent is less than the effective amount that would be present if the compound of the invention were not administered. In this manner, undesirable side effects associated with high doses of either of the two agents can be minimized. Other potential advantages (including, but not limited to, improved dosing regimens and / or reduced drug costs) should be apparent to those skilled in the art.

[0397] Other exemplary cancer treatments include, for example, chemotherapy, targeted therapies such as antibody therapy, kinase inhibitors, immunotherapy, and hormone therapy, epigenetic therapy, proteasome inhibitors, and anti-angiogenic therapy. Examples of each of these treatments are provided below.

[0398] Examples of chemotherapeutic agents used in cancer therapy include, for example, antimetabolites (e.g., folic acid, purine and pyrimidine derivatives) and alkylating agents (e.g., nitrogen mustard, nitrosourea, platinum, alkyl sulfonates, hydrazine, triazine, aziridine, spindle inhibitors, cytotoxic agents, topoisomerase inhibitors, and others). Exemplary reagents include arubicin, actinomycin, alitretinoin, hexamethylmelamine, aminopterin, aminolevulinic acid, arubicin, acridine, anagrelide, arsenic trioxide, asparaginase, atrasentan, belotecone, bexarotin, bendamustine, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, carboplatin, carpoquinone, carmoflurane, carmustine, celecoxib, chlorambucil, nitrogen mustard, cisplatin, cladribine, chlorofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, and lincomycin. Narcissusamine, Docetaxel, Doxorubicin, Eproxiro, Ilismo, Exalucin, Enoxabine, Epirubicin, Estrostine, Etoglucoside, Etoposide, Azouracil, Fludarabine, Fluorouracil (5FU), Formosine, Gemcitabine, Carburexa, Hydroxycarbamide, Idarubicin, Ifosfamide, Irinotecan, Irovvin, Ixaspirone, Larotaxel, Leucovorin, Doxorubicin Liposomes, Daunorubicin Liposomes, Clonidamine, Cyclohexanonitrosourea, Thianthrone, Mannosuphan, Masrophenone L-carnitine, mercaptopurine, mesna, methotrexate, methylaminolevulinate, dibromomannitol, mitoxantrone, mitoxantrone, mitomycin, mitoxantrone, nedaplatin, pyrimidine nitrosourea, oblimersen, omacetaxine, oxaliplatin, paclitaxel, polyvinylglycol conjugate, pemetrexed, pentostatin, pirarubicin, pisacotrol, procainamide, porphyrin sodium, prednimustine, procarbazine, raltitrexed, ramustine, rubotecan, sapacitabine, semustine, adenovirus vector coding gene, szostatin Strataplatin, strazocine, tarapofen, tegafur uracil, temopofen, temozolomide, teniposide, tesetaxel, testosterone, tetranitrate, thiotepa, thiazolinone, thioguanine, tepifenabil, topotecan, trabectedin, triaminoquinone, triethylene melamine, trimolybdenum, retinoic acid, butyltetrafluoroethylene sulfonate, chlorocyclophosphamide, uramustine, pentorubicin, vertepofen, vincristine, vinblastine, vindesine, vinflunine, vinorelbine, vorinostat, zorubicin, and other cell growth inhibitors or cytotoxic agents described herein.

[0399] Because the combined effect of some drugs is greater than that of their individual use, two or more drugs are often prescribed simultaneously. Often, two or more chemotherapeutic agents are used as combination chemotherapy. In some embodiments, these chemotherapeutic agents (including combination chemotherapy) may be used in combination with a compound described herein.

[0400] Targeted therapy involves using agents that specifically downregulate proteins in cancer cells. Small molecule targeted therapy drugs are typically inhibitors of the enzyme domains of mutated, overexpressed, or other key proteins within cancer cells. Notable examples include tyrosine kinase inhibitors such as axitinib, bosutinib, cediranib, desatinib, erolotinib, imatinib, gefitinib, lapatinib, letatinib, nilotinib, semasanib, sorafenib, sunitinib, and vandetanib, and cyclin-dependent kinase inhibitors such as alvocidib and 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 antibody trastuzumab, typically used in breast cancer. Including anti-CD20 antibodies rituximab and tosimomab, typically used in various B-cell malignancies. Other exemplary antibodies include cetuximab, panitumumab, trastuzumab, alemtuzumab, bevacizumab, ezetizumab, and gimenumab. Exemplary fusion proteins include aflibercept and deniella interleukin-toxin conjugates. In some embodiments, targeted therapy may be used in combination with a compound described herein (2001), for example, Gleevec (Vignari and Wang).

[0401] Targeted therapy can also include small molecule peptides as "homing devices," which can bind to cell surface receptors or affect the extracellular matrix surrounding the tumor. If the radionuclide decays in neighboring cells, the radionuclide attached to these peptides (e.g., RGD) ultimately kills the cancer cells. An example of such therapy includes...

[0402] Anti-angiogenic therapies may include kinase inhibitors targeting vascular endothelial growth factor (VEGF) such as sunitinib and sorafenib, or monoclonal antibodies or receptor "decoys" targeting VEGF or VEGF receptors, including bevacizumab or VEGF-Trap, or thalidomide or its analogues (lenalidomide, pomalidomide), or agents targeting non-VEGF angiogenic targets such as fibroblast growth factor (FGF), angiopoietin, or statins or endostatins.

[0403] Epigenetic therapies include inhibitors of enzymes that control epigenetic modifications, particularly DNA methyltransferases and histone deacetylases, which have shown good antitumor effects against some malignant tumors, along with antisense oligonucleotides and siRNA.

[0404] Tumor immunotherapy refers to a group of different treatment strategies designed to induce a patient's own immune system to fight tumors. Modern methods for generating an immune response to fight tumors include intravascular BCG immunotherapy for superficial bladder cancer, the prostate cancer vaccine Provenge, and the use of interferon and other cytokines to induce immune responses in patients with renal cell carcinoma and melanoma.

[0405] Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy because, in the transplant-on-tumor effect, the donor's immune cells will frequently attack the tumor. In some embodiments, these immunotherapeutic agents may be used in combination with a compound described herein.

[0406] Hormone therapy agents include those that administer hormone agonists or hormone antagonists, and those that contain retinoids / retinoic acid, compounds that inhibit estrogen or testosterone, and those that administer progestins.

[0407] The foregoing disclosure generally describes the invention. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the invention. Variations in form and equivalent substitutions may be considered as suggestions or for the purpose of convenience. Although specific terminology has been used herein, it is intended in an illustrative sense and is not for limiting purposes.

[0408] illustration

[0409] abbreviation

[0410] atm atmosphere

[0411] aq. water-based

[0412] BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

[0413] Boc tert-butoxycarbonyl

[0414] CDI N,N'-carbonyldiimidazole

[0415] CH2Cl2 Dichloromethane

[0416] DCC N,N-dicyclohexylcarbodiimide

[0417] DCM dichloromethane

[0418] DBU diaza(1,3)bicyclo[5.4.0]undecane

[0419] DIC N,N'-Diisopropylcarbodiimide

[0420] DIPEA N,N-Diisopropylethylamine

[0421] DMAP N,N-dimethyl-4-aminopyridine

[0422] DMF N,N-dimethylformamide

[0423] DMSO (dimethyl sulfoxide)

[0424] DPPF diphenylphosphine ferrocene

[0425] EA (ethyl acetate)

[0426] EDCI N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride

[0427] EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0428] eq. equivalent

[0429] Et2O diethyl ether

[0430] EtOAc (ethyl acetate)

[0431] EtOH (ethanol)

[0432] EtI iodoethane

[0433] Et Ethyl

[0434] Fmoc 9-fluorenyloxycarbonyl

[0435] GC gas chromatography

[0436] h hours

[0437] HetAr heteroaryl

[0438] HOBt N-hydroxybenzotriazole

[0439] HBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate

[0440] HPLC (High Performance Liquid Chromatography)

[0441] LAH Lithium Aluminum Hydrogen

[0442] LCMS (Liquid Chromatography-Mass Spectrometry)

[0443] MCPBA (m-chloroperbenzoic acid)

[0444] MeCN Acetonitrile

[0445] MeOH (methanol)

[0446] min minutes

[0447] MeI iodomethane

[0448] MeMgCl (methyl magnesium chloride)

[0449] Memethyl

[0450] NaOAc Sodium acetate

[0451] NMR nuclear magnetic resonance

[0452] NMP N-methylpyrrolidone

[0453] overnight

[0454] RT (Room temperature or residence time)

[0455] T3P Propionic Anhydride

[0456] TEA Triethylamine

[0457] THF Tetrahydrofuran

[0458] TLC (Thin Layer Chromatography)

[0459] From the following description of the process, it should be understood that, where appropriate, suitable protecting groups will be added and subsequently removed from various reactants and intermediates in a manner readily understood by those skilled in the art of organic synthesis. Conventional procedures for using such protecting groups, along with examples of suitable protecting groups, are described, for example, in "Protective Groups in Organic Synthesis," TW Green, PGM Watts, Wiley-Interscience, New York, (1999). It should also be understood that the conversion of one group or substituent into another group or substituent by chemical manipulation can be carried out on any intermediate or end product within the synthetic pathway that forms the end product, wherein the possible types of conversion are limited only by the inherent incompatibility between the functionality of the molecule at that stage and the conditions or reagents used in the conversion. Such inherent incompatibilities, and the methods by which they are avoided by performing appropriate conversions and synthetic steps in an appropriate order, will be readily understood by those skilled in the art of organic synthesis. Examples of transformations are given below, and it should be understood that the transformations described are not limited to the general group or substituent used in the illustrative transformations. References and descriptions of other suitable transformations are given in “Comprehensive Organic Transformations - A Guide to Functional Group Preparations”, RC Larock, VHC Publishing (1989). References and descriptions of other suitable reactions are given in organic chemistry textbooks such as “Advanced Organic Chemistry”, March, 4th edition, McGraw Hill (1992) or “Organic Synthesis”, Smith, McGraw Hill (1994). Techniques for purifying intermediates and end products include, for example, normal and reversed-phase chromatography on a column or rotating plate, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, which will be readily understood by those skilled in the art. Unless otherwise defined, substituents and groups are defined as described with respect to Formula I. The terms "room temperature" and "ambient temperature" shall mean, unless otherwise stated, a temperature between 16°C and 25°C. The term "reflux" shall mean, unless otherwise stated, with respect to a solvent, a temperature at or above the boiling point of that solvent.

[0460] Example 1: Synthesis of intermediate (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid.

[0461]

[0462] Synthesis of 3,5-bis(trifluoromethyl)benzothioamide:

[0463]

[0464] A solution of 200 g of 3,5-bis(trifluoromethyl)benzonitrile in DMF (1 L) was loaded into a 2-L 3-necked round-bottom flask. The solution was then treated with NaSH (123.7 g, 2.0 eq.) and MgCl2 (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 slurry (10 L) and the compound was extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated 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 following steps.

[0465] Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole:

[0466]

[0467] A solution of 205.65 g of 3,5-bis(trifluoromethyl)benzothioamide in 1.03 L of DMF was collected in a 5-L 3-necked round-bottom flask. 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 h. After being allowed to cool to room temperature, the reaction mixture was poured into a saturated aqueous solution of sodium bicarbonate (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 Na₂SO₄, filtered, and concentrated under reduced pressure (35 °C, 20 mmHg) to obtain 180 g of crude compound. The crude material was stirred with petroleum ether (3 x 500 mL), filtered and dried to obtain 160 g of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole, which was obtained as a light yellow solid (yield: 75%).

[0468] Synthesis of (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0469]

[0470] A solution of 160 g of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole in 960 mL of DMF was transferred to a 2-L, 3-necked round-bottom flask. The solution was treated with DABCO (127.74 g, 2 eq.) and stirred for 30 minutes before the dropwise addition of (Z)-isopropyl 3-iodoacrylate (150.32 g, 1.1 eq.). After approximately 1 hour, the reaction mixture was poured into an ice-water slurry (5 L) and extracted with EtOAc (3 x 1 L). The combined organic layers were washed with aqueous saturated 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. This crude compound was purified by column chromatography (60 / 120 silica gel) using an ethyl acetate / n-hexane gradient (the column was packed with hexane, and the desired compound was eluted starting from 2% EtOAC / n-hexane). The fractions containing the desired compound were combined to obtain 138 g of pure desired compound (yield: 61%).

[0471] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid:

[0472]

[0473] In a 5-L 3-necked round-bottom flask, (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-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 this solution, and the reaction mixture was stirred at room temperature for 4 h before quenching with 400 mL of ice-water slurry and acidifying with dilute aqueous HCl (pH = 2-3). 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 obtain 110 g of the desired carboxylic acid (yield: 94%) (cis content = 90.0%, trans content = 8.2% by LCMS).

[0474] Example 2: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylhydrazide (I-3).

[0475]

[0476] A suspension of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.200 g) in a 1:1 CH2Cl2:AcOEt (25 mL) mixture was placed into a 50 mL three-necked round-bottom flask. 2-Hydroxypyridine (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 at -40 °C for 30 min before concentration under reduced pressure (35 °C, 20 mmHg). The crude oil was purified by preparative TLC (using 5% MeOH in CH2Cl2 as the mobile phase) (under an ammonia atmosphere) to obtain 40 mg (yield: 16%) of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylhydrazide. 1 H NMR(400MHz,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=2.8Hz,1H),7.51-7.53(d,J=10.4Hz,1H),6.07-6.10(d,J=10.4Hz,1H); 17 H 12 The LCMS [M+H]+ prediction for F6N7O was 444.31, and the result was 444.49 (RT 2.70 min, purity: 95.78%).

[0477] Example 3: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-2-yl)acrylhydrazine hydrochloride (I-4).

[0478]

[0479] A suspension of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (10 g, 1.0 eq.) in a 1:1 CH2Cl2:AcOEt (200 mL) was loaded into a 500 mL three-necked round-bottom flask. 2-Hydroxypyridine (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). The reaction mixture was stirred at -40 °C for 30 min before concentration under reduced pressure (35 °C, 20 mmHg) to obtain a crude brown oil, which was purified by column chromatography (eluting with 1.3% MeOH in CH2Cl2). The portions containing the desired compound were combined to obtain 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. 1 H NMR(400MHz,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=5.2Hz,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=8.4Hz,1H),6.07-6.09(d,J=10.4,1H). C 18 H 12 The LCMS [M+H]+ prediction for F6N6O was 443.33, and the result was 443.44 (RT 2.45 min, purity: 100%).

[0480] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-2-yl)acrylhydrazine hydrochloride:

[0481]

[0482] A solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-2-yl)acrylhydrazide (5.5 g) in Et₂O (250 mL) was collected into a 500 mL three-necked round-bottom flask. The solution was cooled to 5 °C, treated with HCl in 1,4-dioxane, allowed to heat to room temperature and stirred until complete (as shown by TLC analysis (approximately 1 h)). The solids were filtered through a Büchner funnel, washed with Et₂O and dried under vacuum to obtain 5.5 g (yield: 92%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-2-yl)acrylhydrazide hydrochloride. 1 H NMR(400MHz,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=10.4Hz,1H),7.21-7.24(m,1H),7.05-7.09(m,1H),6.16-6.19(d,J=10.4Hz,1H), C 18 H 13 The LCMS [M+H]+ of F6N6O was 443.33; the value was 443.44 (RT 3.54 min, purity: 99.0%).

[0483] Example 4: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(4-hydroxypiperidin-1-yl)prop-2-en-1-one (I-5).

[0484]

[0485] A solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.20 g) in CH₂Cl₂ (10 mL) was transferred to a 50 mL three-necked round-bottom flask. Piperidin-4-ol (0.07 g, 1.2 eq.) was added, and the solution was cooled to -60 °C to add T₃P (propylphosphonic 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 aqueous saturated brine (50 mL), dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg). Purification was performed by column chromatography (using silicon 60 / 120 and MeOH:CH2Cl2 as the mobile phase). (The desired compound was eluted with 3.0% MeOH / CH2Cl2) to give 0.025 g (yield: 10%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(4-hydroxypiperidin-1-yl)prop-2-en-1-one. 1 ¹H NMR (400MHz, CDCl₃) δ, 8.75(s, 1H), 8.58(s, 2H), 7.93(s, 1H), 7.08–7.11(d, J = 10.4 Hz, 1H), 6.01–6.04(d, J = 10.4 Hz, 1H), 4.02–4.14(m, 1H), 3.98–4.01(m, 1H), 3.78–3.85(m, 1H), 3.47–3.52(s, 1H), 3.32–3.38(s, 1H), 1.96(s, 1H), 1.83(s, 1H), 1.27(s, 1H), 0.90(s, 1H), Chemical formula: C 18 H 17 The LCMS [M+H]+ concentration of F6N4O2 was 435.34; the concentration found was 435.24 (RT 2.408 min, purity: 89.6%).

[0486] Example 5: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyrrolidone-1-yl)acrylamide (I-6).

[0487]

[0488] A cold (-40°C) solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.35 g) in a 1:1 CH₂Cl₂:EtOAc (200 mL) mixture 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 the slow addition of DIPEA (0.51 mL, 3.0 eq.). The reaction mixture was stirred at -40°C for 30 min before quenching with ice-water and extraction with EtOAc (3 x 20 mL). The combined organic layers were washed with aqueous saturated brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure (35°C, 20 mmHg) to obtain 0.275 g of crude solids. Purification was performed by silica gel column chromatography (60-120 mesh size) (using MeOH in CH2Cl2 as the mobile phase) to obtain pure (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(pyrrolidone-1-yl)acrylamide (yield: 1.7%). 1 H NMR(400MHz,DMSO-d6)δ,9.49(s,1H),8.95(s,1H),8.53(s,2H),8.28(s,1H),7.4-7.38 (d,J=7.6Hz,1H),5.87-5.84(d,J=10.4Hz,1H),2.86-2.81(m,4H),1.74-1.73(m,4H); C 17 H 16 The LCMS [M+H]+ of F6N5O was 420.33; the value found was 420.13 (RT 7.76 min, purity: 92.4%).

[0489] 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 (I-7).

[0490]

[0491] Synthesis of 2-(1-methylhydrazyl)pyridine:

[0492]

[0493] Under a nitrogen atmosphere, 0.31 g of 2-bromopyridine and 5.09 g (34.2 eq.) of methylhydrazine were charged into a 25 mL three-necked round-bottom flask, and the mixture was stirred and heated to reflux at 80-85 °C for 1 hour. The reaction mixture was concentrated under reduced pressure (40 °C, 20 mmHg) to obtain a yellow oil, which was treated with 10% w / v aqueous Na₂CO₃ and extracted with EtOAc. The organic layer was washed with aqueous saturated brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure (40 °C, 20 mmHg) to obtain a yellow oil (0.40 g), which was used in the following steps.

[0494] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.43 g), 2-(1-methylhydrazyl)pyridine (0.15 g, 1.0 eq.) in EtOAc (10 mL) were transferred into a 50 mL three-necked round-bottom flask. T3P (50% in EtOAc; 1.1 g, 1.5 eq.) and DIPEA (0.40 g, 2.5 eq.) were added under a nitrogen atmosphere at -60 °C, and the progress of the reaction was monitored by TLC (using 10% MeOH:CH2Cl2 as the mobile phase and visualized under UV light). The reaction mixture was concentrated under reduced pressure (25 °C, 20 mmHg) to give 0.65 g of crude solids. Purification was performed using Combi-fast column chromatography with CH2Cl2 and MeOH (the desired compound began to elute at 3.0% MeOH in CH2Cl2). The fractions containing the desired compound were combined and concentrated under reduced pressure (35°C, 20 mm Hg) to obtain 90.0 mg (yield: 18%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-methyl-N'-(pyridin-2-yl)acrylhydrazide. 1 H NMR(400MHz,DMSO-d6)δ9.89(s,1H),9.79(brs,1H),8.57-8.62(d,2H),7.92-7.94(d,J=11.2Hz,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=10.8Hz,1H),3.46(d,3H);C 19 H 15 The LCMS [M+H]+ of F6N6O was 457.35; the value was 456.26 (RT 2.52 min, purity: 100.0%).

[0495] 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)acrylhydrazide (I-8).

[0496]

[0497] Synthesis of 2-(1-methylhydrazyl)pyrazine:

[0498]

[0499] In a 25-mL three-necked round-bottom flask under a nitrogen atmosphere at room temperature, 0.5 g of 2-chloropyrazine was dissolved in methylhydrazine (0.5 g, 1.5 eq.). Solid K₂CO₃ (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 concentrated under reduced pressure (40 °C, 20 mmHg) to obtain a yellow oily residue, which was treated with 10% w / v aqueous Na₂CO₃ and extracted with EtOAc. The organic extract was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure (40 °C, 20 mmHg) to obtain 0.43 g of yellow oil, which was used in the following steps.

[0500] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.3 g), 2-(1-methylhydrazyl)pyrazine (0.12 g, 1.1 eq.), and CH2Cl2 (10 mL) were placed in a 50 mL three-necked round-bottom flask. 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, and the progress of the reaction was monitored by TLC (using 10% MeOH:CH2Cl2 as the mobile phase and visualized under UV light). The reaction mixture was concentrated under reduced pressure (25 °C, 20 mmHg) to give 0.265 g of crude solids. Purification was performed using Combi-fast column chromatography (using CH2Cl2:MeOH as the eluent) (the desired compound began to elute at 1.5% MeOH in CH2Cl2), yielding 75.0 mg of the pure compound (yield: 23%); (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-methyl-N'-(pyrazin-2-yl)acryloylhydrazine: 1H NMR(400MHz,DMSO-d6)δ10.77(s,1H),9.40-9.36(br 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=10.4Hz,1H),6.13-6.10(d,J=10.4Hz,1H),3.43(d,3H);C 18 H 14 The LCMS [M+H]+ of F6N7O was 458.34; the concentration was 458.24 (RT 2.83 min; purity: 96.31%).

[0501] 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)acryloylhydrazine (I-9).

[0502]

[0503] 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) was transferred into a 50 mL three-necked round-bottom flask. The solution was cooled to -70 °C and treated sequentially with 3-methyl-2-(1-methylhydrazyl)pyridine (0.135 g, 1.0 eq.), T3P (50% in EtOAc; 1.4 mL, 4 eq.), and DIPEA (0.6 mL, 6 eq.). The clarified reaction mixture was stirred at -60 °C for 4 hr. The progress of the reaction was tracked by TLC analysis using 2.5% MeOH in CH2Cl2 as the mobile phase and visualized under UV light. The reaction mixture was concentrated under reduced pressure (25°C, 20 mmHg) to obtain a crude compound, which was purified by column chromatography (60 / 120 mesh SiO2 with a MeOH:CH2Cl2 gradient elution). The desired compound was eluted with 0.3%–0.4% MeOH in dichloromethane. The fractions containing the desired material were combined to obtain 0.21 g (yield: 40%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-methyl-N'-(3-methylpyridin-2-yl)acrylhydrazide. 1H NMR (400MHz, DMSO-d6) δ = 10.73 (s, 1H), 9.32 (s, 1H), 8.52 (s, 2H), 8.45-8.46 (d, J = 4.4Hz, 1H), 8.29 (s, 1H), 7.9 7-7.99(d,J=8Hz,1H),7.48-7.50(d,J=10Hz,1H),7.01-7.05(m,1H),5.86-5.88(d,J=10Hz,1H),3.26(s,3H); 20 H 14 The LCMS [M+H]+ of F9N6O was 525.35; the value found was 525.19 (RT 3.31 min, purity: 99.40%).

[0504] Example 9: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(5-methylpyridin-2-yl)acrylhydrazide (I-10).

[0505]

[0506] A 50-mL three-necked round-bottom flask containing 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 (10 mL) was treated with 2-hydrazino-5-methylpyridine (0.97 g, 1.1 eq.). The mixture was cooled to -60 °C and treated with T3P (propylphosphonic 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 anhydrous MgSO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain a crude compound, which was purified by column chromatography (SiO2, 60 / 120 mesh, MeOH:CH2Cl2 as mobile phase). The desired compound was eluted with 2.5% MeOH:CH2Cl2. The 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 (400MHz, CDCl3) δ, 10.38 (s, exchangeable, 1H), 9.65 (s, 1H), 8.54 (s, 2H), 8.40 (s, exchangeable, 1H), 8.29 (s, 1H), 7.90 (s, 1H), 7.48–7.51 (d, J = 10.4 Hz, 1H), 7.33–7.36 (dd, J = 2 Hz, J = 6 Hz, 1H), 6.61–6.63 (d, J = 8.4 Hz, 1H), 6.20–6.23 (d, J = 10.4 Hz, 1H), 2.15 (s, 3H); C 19 H 15 The LCMS [M+H]+ of F6N6O was 457.35; the value was 457.24 (RT 2.61 min, purity: 99.13%).

[0507] Example 10: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-methyl-N'-(pyridin-3-yl)acrylhydrazide (I-11).

[0508]

[0509] A 50-mL three-necked round-bottom flask containing a solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.25 g) in CH2Cl2 (12 mL) was treated with 3-(1-methylhydrazyl)pyridine (0.105 g, 1.2 eq.). The mixture was cooled to -60 °C and treated with T3P (propylphosphonic 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 tracked 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 anhydrous MgSO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain a crude compound, which was purified by column chromatography (SiO2, 60 / 120 mesh, MeOH:CH2Cl2 as mobile phase). The desired compound began elution with 3.0% MeOH:CH2Cl2. The fraction containing this compound was collected and concentrated under reduced pressure to obtain 140 mg (yield: 43%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-methyl-N'-(pyridin-3-yl)acrylhydrazide. 1H NMR(400MHz,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),7.51-7.54(d,J=10.4Hz,1H),7.18-7.22(m,2H),6.05-6.07(d,J=10.4Hz,1H),3.20(s,3H);C 19 H 15 The LCMS [M+H]+ of F6N6O was 457.35; the value found was 457.19 (RT 2.43 min, purity: 83.48%).

[0510] Example 11: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(6-chloropyrimidin-4-yl)acrylhydrazide (I-12).

[0511]

[0512] 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) was loaded into a 25 mL three-necked round-bottom flask. 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 eluent) showed that the starting material was depleted after 30 min. The reaction mixture was then diluted with CH2Cl2, washed with water, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain a crude material, which was purified by preparative TLC (using 5% MeOH-CH2Cl2 as the mobile phase). This yielded 250 mg (yield: 36.74%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(6-chloropyrimidin-4-yl)acrylhydrazide. 1 ¹H NMR (400MHz, 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 = 10.4Hz), 6.69 (s, ¹H), 6.05–6.08 (d, ¹H, J = 10.4Hz); LCMS: C17 H 11 ClF6N7O(M+H) + The calculated value was 478.76; the observed value was 478.09 (RT 2.79 min, purity: 97.51%).

[0513] 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).

[0514]

[0515] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.25 g) and 3-hydrazinopyridine (0.077 g, 1.0 eq.) in EtOAc (10 mL) were transferred into a 50 mL three-necked round-bottom flask. 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 °C to -60 °C. The progress of the reaction was tracked 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 a crude solid of 0.475 g. Purification was performed using Combi-fast column chromatography (with MeOH:CH2Cl2). The desired compound was initially eluted at 2.3% MeOH in CH2Cl2. The fraction containing this compound was 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)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-3-yl)acryloylhydrazide. 1 H NMR(400MHz,DMSO-d6)δ10.35(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=10.4Hz,1H),7.09 -7.15(m,2H),6.04-6.07(d,J=10.4Hz,1H),C 18 H 13 LCMS[M+H] of F6N6O + The value was 443.33; the value was 443.19 (RT 2.19 min, purity: 99.60%).

[0516] Example 13: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(quinoxalo-2-yl)acrylhydrazide (I-14).

[0517]

[0518] Synthesis of 2-hydrazinoquinoxaline:

[0519]

[0520] In a nitrogen atmosphere at room temperature, 1.0 g of 2-chloroquinoxaline was dissolved in 8 mL of ethanol, and 8 mL of hydrazine hydrate was added. The mixture was stirred and heated to reflux temperature (80 °C) for 1 hour. The reaction was tracked by TLC analysis using 10% MeOH:CH2Cl2 as the mobile phase and visualized 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 then used in the following steps.

[0521] A solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.25 g) and 2-hydrazinoquinoxaline (0.14 g, 1.2 eq.) in EtOAc was collected into a 50 mL three-necked round-bottom flask. 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°C to -60°C, and the reaction mixture was stirred for 2 hours before concentration under reduced pressure (25°C, 20 mmHg) to obtain a crude solid of 0.150 g. Purification was performed using Combi-fast column chromatography (eluting with MeOH:CH2Cl2; the desired compound begins to elute at 5% MeOH in CH2Cl2) to obtain 60 mg (yield: 20%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(quinoxalo-2-yl)acrylhydrazide. 1 H NMR (400MHz, 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=8Hz,1H),7.45-7.66(m,4H),6.17-6.20(d,J=10.4Hz,1H); C 21 H 14LCMS[M+H] of F6N7O + The value was 494.37; the value found was 494.19 (RT 2.88 min, purity: 100%).

[0522] Example 14: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(1,1-dioxotetrahydrothiophen-3-yl)acryloylhydrazide (I-15).

[0523]

[0524] A 50-mL three-necked round-bottom flask containing a solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.5 g) in EtOAc (20.0 mL) was treated with 2-(1,1-dioxotetrahydrothiophene-3-yl)hydrazine (0.3 g, 1.2 eq.). The mixture was cooled to -60 °C and simultaneously treated with T3P (50% in EtOAc; 2.0 mL, 2 eq.) and DIPEA (1 mL, 4 eq.). The reaction mixture was stirred at -60 °C for 30 min prior to concentration under reduced pressure (35 °C, 20 mmHg) to obtain a solid residue of 0.60 g. Purification was performed by Combi-fast column chromatography (SiO2; elution with MeOH:CH2Cl2; elution of the desired compound at 5% MeOH in CH2Cl2) to obtain 100 mg (yield = 15%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(tetrahydrothiophene-1-1-dioxide-3-yl)acrylhydrazide. 1 H NMR (400MHz, CD3OD) δ=9.57(s,1H),8.64(s,2H),8.10(s,1H),7.34-7.36(d,J=10.4Hz ,1H),5.89-5.92(d,J=10.8Hz,1H),4.01(m,1H),3.04-3.26(m,4H),2.27-2.34(m,2H). C 17 H 15 The LCMS [M+H]+ of F6N5O3S was 484.40; the concentration of the observed concentration was 483.39 (RT 2.63 min, purity: 66.39%).

[0525] Example 15: Synthesis of (Z)-N-(azacyclohepta-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide (I-16).

[0526]

[0527] A solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.3 g) in CH2Cl2:EtOAc (1:1, 200 mL) was loaded into a 500 mL three-necked round-bottom flask and treated with azirmoni-1-heptane (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 at -60 °C for 30 min before quenching with ice-cold water and extraction with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure (35 °C, 20 mmHg) to obtain 0.57 g of solid. Purification was performed by Combi-fast column chromatography (SiO2; MeOH:CH2Cl2 as the mobile phase; the compound was eluted with 0.1% MeOH in CH2Cl2) to obtain 90 mg (yield: 24%) of (Z)-N-(azacyclohepten-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide. 1 H NMR(400MHz,DMSO-d6)δ,9.61(s,1H),9.49(s,1H),9.14(s,1H),8.52(s,2H),8.28(s,1H),7.39-7.97(d,J= 10Hz,1H),6.52-6.49(d,J=10.4Hz,1H),5.86-5.83(d,J=10.4Hz,1H),3.00-2.97(m,4H),1.58-1.54(m,8H)C 19 H 19 The LCMS [M+H]+ of F6N5O was 448.39; the concentration was found to be 448.30 (at RT 3.22 min, purity (96.48%)).

[0528] Example 16: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(2,6-dimethylpyrimidin-4-yl)acrylhydrazide (I-17).

[0529]

[0530] 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) was transferred into a 50 mL three-necked round-bottom flask. The solution was cooled to -40 °C and treated with 4-hydrazino-2,6-dimethylpyrimidine (0.078 g, 1 eq.). Then, 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 at -40 °C for 30 min. The reaction mixture was then allowed to be heated to room temperature and concentrated under reduced pressure (35°C, 20 mmHg) to obtain 0.340 g of an oily crude compound, which was purified by combi-fast (using MeOH:CH2Cl2 as the mobile phase) (eluting the desired compound with 7%-8% MeOH in CH2Cl2) to obtain 50 mg (yield: 18%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(2,6-dimethylpyrimidin-4-yl)acrylhydrazide. 1 H NMR(400MHz,DMSO-d6)δ,10.54(s,1H),9.19(b,1H),8.54(s,2H),8.30(s,1H),7.52-7. 55(d,J=10.4,1H),6.29(s,1H),6.06-6.08(d,J=10.4,1H),2.33(s,3H),2.13(s,3H),C 19 H 15 LCMS[M+H]+[M+H] of F6N7O + The value was 472.37; the value was 472.24 (RT 2.88 min, purity: 99.59%).

[0531] Example 17: Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylhydrazide

[0532]

[0533] Synthesis of 3,5-bis(trifluoromethyl)benzothioamide:

[0534]

[0535] A 2-L three-necked round-bottom flask containing a solution of 200 g of 3,5-bis(trifluoromethyl)benzonitrile in 1 L of DMF was treated with NaSH (123.7 g, 2.0 eq.) and MgCl2 (186.7 g, 1 eq.). The reaction mixture was stirred at room temperature for 3 h before being poured onto an ice-water slurry (10 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 (25 °C, 20 mmHg) to obtain 205 g of crude compound (yield: 90%), which was used in the following steps without further purification.

[0536] Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole:

[0537]

[0538] A 5-L 3-necked round-bottom flask containing a solution of 205.65 g of 3,5-bis(trifluoromethyl)benzothioamide in 1.03 L of DMF was treated dropwise with hydrazine hydrate (73.16 mL, 2.0 eq.). The reaction mixture was stirred at room temperature for 1 h before being treated with dropwise HCOOH (1.028 L). The reaction mixture was refluxed at 90 °C for 3 h, then cooled to room temperature and poured into a saturated aqueous solution of NaHCO3 (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 Na2SO4, 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 obtain the desired triazole, which was a light yellow solid (160 g, yield: 75%).

[0539] Synthesis of (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate and (E)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0540]

[0541] A 2-L 3-necked round-bottom flask containing a solution of 160 g of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole in DMF (0.96 L, 6 V) 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 the mixture was stirred for 1 h before being poured onto an ice-water slurry (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 a crude compound. The pure cis ester (138 g, yield: 61.6%) and the pure trans ester (11.6 g, yield: 5.2%) were obtained by column chromatography (SiO2, 60 / 120 mesh, eluted with EtOAc:hexane gradient; the desired compound was eluted at 2%-2.5% EtOAc in hexane).

[0542] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid:

[0543]

[0544] A solution of (E)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate (5.0 g) in THF (50 mL) was loaded into a 500-mL three-necked round-bottom flask. 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 dilution with 40 mL of water, acidification with dilute aqueous HCl (pH = 2-3), and extraction with EtOAc (3 x 100 mL). The organic extract was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 2.75 g of the desired unsaturated carboxylic acid (yield: 61.6%, purity: 99.0%, by LCMS).

[0545] Synthesis of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylhydrazide:

[0546]

[0547] At room temperature, a solution of 2-hydrazinopyrazine (0.23 g) in 12 mL of THF was added to one of a solution of (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.75 g) in EtOAc (25 mL) and THF (12.5 mL). T3P (50% in ethyl acetate, 1.52 mL) and DIPEA (1.46 mL) were added dropwise and simultaneously, and the reaction mixture was stirred at room temperature for 30 min before quenching with ice-cold water and extraction with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure (35 °C, 20 mmHg) to give 0.698 g of a crude solid. First, it was ground with petroleum ether and then with Et2O to obtain 275 mg (yield: 29%) (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acrylhydrazide. 1 HNMR(400MHz,DMSO-d6)δ,10.3(s,1H),9.15(s,2H),8.59(s,2H),8.30-8.26(d ,J=14.8Hz,1H),8.13(s,1H),8.06-8.07(m,1H),6.98-6.95(d,J=13.4Hz,1H);C 17 H 12 The LCMS [M+H]+ of F6N7O was 443.31; the result was 444.19 (RT 2.625 min, purity: 99.06%).

[0548] Example 18: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-4-yl)acrylhydrazine hydrochloride (I-19).

[0549]

[0550] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.25 g) and EtOAc (10.0 mL) were transferred into a 50-mL three-necked round-bottom flask. 4-Hydroxypyridine 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 at -40 °C for 30 min before concentration under reduced pressure (35 °C, 20 mmHg) to obtain 0.35 g of crude material. The compound was purified by column chromatography (using MeOH:CH2Cl2 as the mobile phase) (eluting the compound with 4% MeOH in CH2Cl2) to obtain 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. 1 ¹H NMR (400MHz, DMSO-d⁶) δ, 10.53 (br s, NH exchangeable, ¹H), 9.58 (s, ¹H), 8.88 (br s, NH exchangeable, 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), 6.66–6.69 (m, 2H), 6.06–6.10 (d, J = 14.4 Hz, H); C 18 H 13 The LCMS [M+H]+ of F6N6O was 443.33; the value was 443.24 (RT 2.241 min, purity: 90.17%).

[0551] A cold (0°C) solution of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-4-yl)acrylhydrazide (0.08 g) in CH2Cl2 (5.0 mL) was transferred into a 25 mL three-necked round-bottom flask and treated with 4N HCl in dioxane (0.5 mL). The reaction mixture was allowed to be heated to room temperature and stirred for 4 h before concentration under reduced pressure (35°C, 20 mmHg) to obtain 0.05 g (yield: 40.81%) of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyridin-4-yl)acrylhydrazide-HCl salt. 1¹H NMR (400MHz, 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); C 18 H 13 LCMS[M+H] of F6N6O + The value was 443.33; the value was 443.24 (RT 3.00 min, purity: 90.97%).

[0552] Example 19: Synthesis of (Z)-N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide (I-20).

[0553]

[0554] Synthesis of 4-benzylpiperazine-1-amine.

[0555]

[0556] Concentrated HCl and water were transferred to a 50-mL three-necked round-bottom flask and cooled to 0-5°C under a nitrogen atmosphere before adding NaNO2 and benzylpiperazine (5.0 g). The reaction mixture was stirred at 0-5°C for 2.5 h before dilution with water and extraction with EtOAc (3 x 100 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure (40°C, 20 mmHg) to obtain 4.40 g of a colorless solid. Purification was performed using combi-rapid chromatography (eluting with 25.5% EtOAc:hexane) to give 2.0 g of the desired compound (yield: 34.3%).

[0557] A cold (-70°C) solution of 1-benzyl-4-nitroso-4-piperazine (0.8 g) in THF was treated with excess LAH under a nitrogen atmosphere. The reaction mixture was allowed to be heated to ambient temperature and stirred for 1.0 h before quenching with water and extraction with EtOAc (3 x 10 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure (40°C, 20 mmHg) to obtain 0.70 g of 4-benzylpiperazine-1-amine as a colorless solid.

[0558] Synthesis of (Z)-N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide.

[0559] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.220 g, 1.2 eq.), 4-benzylpiperazin-1-amine (0.10 g, 1.0 eq.), and EtOAc (15 mL) were placed in a 50-mL 3-necked round-bottom flask. T3P (50% in EtOAc; 0.99 g, 3.0 eq.) and DIPEA (0.27 mg, 4.0 eq.) were added to this cold (-60 °C) solution under a nitrogen atmosphere. The progress of the reaction was tracked by TLC analysis (SiO2, 15% MeOH:CH2Cl2 as the mobile phase, visualized 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 Na₂SO₄, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain 0.35 g of crude solid. Purification was performed using Combi-fast (eluting with 10% MeOH / CH₂Cl₂) to give 20 mg (yield: 6%) (Z)-N-(4-benzylpiperazin-1-yl)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylamide. 1 H NMR (400MHz, DMSO-d6) δ9.44-9.48(t,3H),9.10(s,1H),8.51(s,2H),7.23-7.41(m,6H),6.46-6. 49(d,J=10.4Hz,1H),5.83-5.86(d,J=10.4Hz,1H),3.47(s,2H),2.81(s,4H),2.23-2.33(d,2H)C 24 H 23 The LCMS [M+H]+ of F6N6O was 525.47; the concentration of the observed concentration was 525.20 (RT 9.87 min, purity: 100%).

[0560] Example 20: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(4-ethylpiperazin-1-yl)acrylamide (I-21).

[0561]

[0562] A cold (-40°C) 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) 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 at -40°C for 30 min prior to quenching with ice-cold water and extraction with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure (35°C, 20 mmHg) to obtain 0.280 g of crude compound. Purification was performed by Combi-Rapid Column Chromatography (eluting with 2% MeOH in CH2Cl2), followed by purification on a preparative TLC plate (eluting with 10% MeOH in CH2Cl2) to obtain 60 mg of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(4-ethylpiperazin-1-yl)acrylamide. 1 H NMR (400MHz, CF3COOD) δ: 10.75 (s, 1H), 8.31-8.29 (d, J = 10.2H), 7.98 (s, 1H), 7.21-7.23 (d ,1H),6.08-6.10(d,1H),3.52-3.54(m,3H),3.36(s,1H),3.11(m,8H),1.19-1.22(m,3H);C 19 H 21 The LCMS [M+H]+ of F6N6O was 463.40; the concentration found was 463.23 (RT 2.43 min, purity: 98.63%).

[0563] Example 21: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-morpholinylacrylamide (I-22).

[0564]

[0565] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.250 g), morpholino-4-amine (0.072 g, 1.0 eq.), and EtOAc (10 mL) were placed in a 50 mL three-necked round-bottom flask. The solution was cooled to -60 °C under a nitrogen atmosphere and treated with T3P (50% in EtOAc; 0.63 mL, 1.5 eq.) and DIPEA (0.24 mL, 2.0 eq.). The progress of the reaction was tracked by TLC analysis (using 10% MeOH:CH2Cl2 as the mobile phase) and visualization under UV light. After completion, the reaction mixture was quenched with water and extracted with EtOAc (3 x 15 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain 0.35 g of a crude solid. Purification (Combi-rapid, eluting with 3% MeOH:CH₂Cl₂) yielded 100 mg (yield: 33%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-morpholinylacrylamide. 1 ¹H NMR (400MHz, DMSO-d⁶) δ=9.52(s, NH exchange, 1H), 8.51(s, 2H), 8.28(s, 1H), 7.38-7.42(m, 1H), 6.50-6.53(d, J=10.4Hz, 1H), 5.84-5.86(d, J=10.4Hz, 1H), 3.63(s, 4H), 2.87(s, 4H); C 17 H 16 The LCMS [M+H]+ concentration of F6N5O2 was 436.33; the concentration found was 436.18 (RT 2.64 min, purity: 100%).

[0566] Example 22: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrimidin-4-yl)acrylhydrazide (I-23).

[0567]

[0568] Synthesis of 4-hydrazinopyrimidine:

[0569]

[0570] A solution of 2,4-dichloropyrimidine (2.0 g) in EtOH (25 mL) was cooled to 0 °C–20 °C and treated with hydrazine (2.8 mL). The progress of the reaction was tracked by TLC (using 10% MeOH:CH₂Cl₂ as the mobile phase) and visualized under UV light. The mixture was concentrated under reduced pressure to obtain 3.1 g of crude 2-chloro-4-hydrazinopyrimidine (yield = 94.8%).

[0571] 10% Pd / C (200 mg) was added to a solution of 2-chloro-4-hydrazino-pyrimidine (200 mg) dissolved in MeOH (10 mL), and the suspension was stirred under a hydrogen atmosphere until complete as shown by TLC analysis (using 10% MeOH:CH2Cl2 as the mobile phase and visualized under UV light). The mixture was then subjected to... The sample was filtered and concentrated under reduced pressure to obtain 250 mg of 4-hydrazinopyrimidine.

[0572] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrimidin-4-yl)acrylhydrazide.

[0573] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (250 mg, 1.0 eq.) and EtOAc (20.0 mL) were placed in a 50-mL 3-necked round-bottom flask. 4-Hydroxypyrimidine (231 mg, 3 eq.) was added at -60 °C, followed simultaneously by T3P (50% in EtOAc, 0.84 mL, 2.0 eq.) and DIPEA (0.24 mL, 2.0 eq.). The reaction mixture was stirred at -60 °C for 30 min before concentration under reduced pressure (35 °C, 20 mmHg) to obtain 0.20 g of a solid. Purification by column chromatography (eluting with 5% MeOH in CH2Cl2) yielded 75 mg of material, which was then purified by preparative TLC (using MeOH:CH2Cl2 as the mobile phase) to yield 13 mg (yield: 5%) of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrimidin-4-yl)acrylhydrazide. 1¹H NMR (400MHz, DMSO-d⁶) δ=10.59(s, ¹H), 9.68(s, NH exchange, ¹H), 9.47(s, NH exchange, ¹H), 8.53–8.59(t, ²H), 8.30(s, ¹H), 8.19–8.20(d, ¹H), 7.53–7.56(d, J=11.2Hz, ¹H), 6.66–6.67(d, ¹H), 6.06–6.09(d, J=10.4Hz, ¹H); C 17 H 12 The LCMS [M+H]+ of F6N7O was 444.31; the result was 444.19 (RT 2.39 min, purity: 94.97%).

[0574] 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)acrylhydrazide (I-24).

[0575]

[0576] Synthesis of 4-chloro-3,5-bis(trifluoromethyl)benzamide:

[0577]

[0578] A solution of 4-chloro-3,5-bis(trifluoromethyl)benzonitrile (1.0 g) in DMSO (10 mL) was treated with solid K₂CO₃ (0.55 g, 1.1 eq.) and H₂O₂ (30% v / v, 1.0 mL). The reaction mixture was stirred at room temperature for 3 h before being poured into ice-cold water (20 mL). The precipitate was filtered and washed with petroleum ether to obtain 1.0 g of crude desired primary amine (yield: 90%).

[0579] Synthesis of 4-chloro-3,5-bis(trifluoromethyl)benzothioamide:

[0580]

[0581] Lawesson's reagent (3.32 g, 2.0 eq.) was added to a solution of 1.2 g of 4-chloro-3,5-bis(trifluoromethyl)benzamide in 20 mL of toluene. The reaction mixture was stirred at 90 °C for 8 h before cooling to room temperature and filtering. The filtrate was decanted 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 Na₂SO₄, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain 2 g of a crude compound. The crude compound was purified by combi-fast chromatography (eluting with 7% EtOAc:hexane) to obtain 1.0 g of the desired compound (yield: 79%).

[0582] Synthesis of 3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole:

[0583]

[0584] 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 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 aqueous saturated NaHCO3 (slowly, maintaining the temperature at 25 °C–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 Na2SO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain 1.5 g of a crude compound. Purification was performed by column chromatography (eluting with 40% EtOAc in hexane) to obtain 0.50 g of the desired compound (yield: 36%).

[0585] Synthesis of (Z)-isopropyl 3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0586]

[0587] A solution of 2.1 g of 3-(chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole 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-cold 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 Na2SO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain 3.0 g of a crude compound. The desired unsaturated ester (1.33 g, yield: 52%) was obtained by purification by column chromatography (60 / 120 mesh SiO2, eluted with 1%-1.2% MeOH in CH2Cl2).

[0588] Synthesis of (Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid:

[0589]

[0590] A solution of (Z)-isopropyl 3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate (1.33 g) in a 1:1 THF:water (26 mL) mixture was loaded into a 25 mL three-necked round-bottom flask. The solution was treated with solid LiOH (0.53 g, 4 eq.) and stirred at room temperature for 4 h before dilution with 400 mL of water, acidification to pH 2-3 with dilute aqueous HCl, and extraction with EtOAc (3 x 100 mL). The combined organic extracts were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 0.8 g of crude compound (yield: 66%).

[0591] Synthesis of (Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-(pyrazin-2-yl)acryloylhydrazine:

[0592]

[0593] A solution of (Z)-3-(3-(4-chloro-3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.8 g) in a 1:1 EtOAc:THF (20 mL) was loaded into a 50 mL three-necked round-bottom flask. The solution was cooled to -70 °C and continuously treated dropwise 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.). The clarified reaction mixture was stirred at -60 °C for 1 h before concentration under reduced pressure (25 °C, 20 mmHg) to obtain the crude compound. The product was purified by column chromatography (60 / 120 mesh SiO2, eluted with 3%-4% MeOH in CH2Cl2) to obtain 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)acryloylhydrazide. 1 H NMR (400MHz, DMSO-d6) δ = 10.53 (s, 1H), 9.58 (s, 1H), 9.11 (s, 1H), 8.47 (s, 1H), 8.32 (s, 1H), 8.1 3(s,1H),8.06(s,1H),7.97(s,1H),7.52-7.55(d,J=10.4Hz,1H),6.08-6.11(d,J=10.4Hz,1H);C 17 H 11 LCMS[M+H] of ClF6N7O + The value was 478.76; the value found was 478.1 (RT 2.64 min, purity: 100%).

[0594] Example 24: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-cyclopropylacrylhydrazine (I-25).

[0595]

[0596] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (0.50 g) and CH2Cl2 (25 mL) were placed in a 100-mL three-necked round-bottom flask. DCC (0.29 g, 1.0 eq.) was added and the mixture was cooled to 0 °C to continuously add cyclopropylhydrazine hydrochloride (0.15 g, 1.0 eq.) and DIPEA (0.24 mL, 1.0 eq.). The reaction mixture was stirred for 1 h before being poured into water (50 mL) and extracted with CH2Cl2 (2 x 50 mL). The combined organic extracts were washed with brine (50 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure (25 °C, 20 mmHg) to obtain the crude compound. Purification was performed by Combi-Rapid Column Chromatography (eluting with 1.5%-2.5% MeOH in CH2Cl2), followed by purification on a preparative TLC plate (eluting with 70% EtOAc in hexane) to obtain 15 mg (yield: 2.6%) (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N'-cyclopropylacrylhydrazine. 1 ¹H NMR (400MHz, 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), 0.83–0.87(m, 2H), 0.65–0.69(m, 2H); Chemical formula: C 16 H 14 The LCMS [M+H]+ of F6N5O was 406.31; the result was 406.19 (RT 2.74 min, purity: 98.85%).

[0597] Example 25: Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyazacyclobut-1-yl)acrylamide (I-26).

[0598]

[0599] Synthesis of 1-aminoazacyclobut-3-ol:

[0600]

[0601] A cold (15°C–20°C) solution of aziridine-3-ol hydrochloride (2.0 g) in water (20 mL) was treated with NaOH (0.8 g in 10 mL of water), and the mixture was stirred at 15°C–20°C for 1 h. The reaction mixture was then cooled to 0°C and treated successively with a NaNO2 solution (1.89 g in 10 mL of water) and acetic acid (1.3 mL). After stirring at 0°C–5°C for 2 h, 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 Na2SO4, and concentrated under reduced pressure to obtain 0.26 g of the desired crude compound, which was used in the following steps (LCMS purity: 59.84%).

[0602] A solution of 0.25 g of 1-nitrosoazonicyclobutan-3-ol 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 passed through… The reaction mixture was stirred at approximately -70°C for 3 hours before filtration and concentration under reduced pressure to obtain 90 mg of 1-aminoazacyclobut-3-ol, which was then used in the following steps.

[0603] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyzazocyclobut-1-yl)acrylamide.

[0604] (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid (200 mg) and THF (20.0 mL) were transferred into a 50 mL three-necked round-bottom flask. The solution was cooled to -60 °C and treated with a solution of 1-aminoazacyclobut-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 at -60 °C for 30 min before allowing it to warm to room temperature. The reaction mixture was then concentrated under reduced pressure (35 °C, 20 mmHg) to give 100 mg of solid. Purification was performed by column chromatography (eluting with 3% MeOH in CH2Cl2) to obtain 20 mg of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N-(3-hydroxyzazocyclobut-1-yl)acrylamide. 1¹H NMR (400MHz, DMSO-d⁶) δ 9.60 (s, 1H), 6.38 (s, 1H), 8.52 (s, 2H), 8.26 (s, 1H), 7.32–7.35 (d, J = 10.8 Hz, 1H), 6.40 (d, exchangeable, 1H), 5.78–5.81 (d, J = 10.8 Hz, 1H), 4.14–4.15 (d, 1H), 3.82 (m, 2H), 3.71 (m, 2H); Chemical formula: C 16 H 14 The LCMS [M+H]+ concentration of F6N5O2 was 422.31; the concentration found was 422.19 (RT 2.46 min, purity: 91.49%).

[0605] Examples 26-31: Examples 26-31 describe novel synthetic methods useful in preparing compounds of the present invention (e.g., as precursors of compounds of the present invention, by means of such a compound as described above in formula Z).

[0606] Example 26.

[0607]

[0608] Synthesis of isopropyl propargyl ester:

[0609]

[0610] Propylene acid (1000 g, 1 equivalent) and IPA (8 L, 8 Vol.) were charged into a 20 L four-necked round-bottom flask equipped with a feeding funnel, thermometer holder, and a mechanical stirrer. BF3-ether compound (4.54 kg, 2.0 equivalent) was slowly added from the feeding funnel at 25 °C over 30-minute intervals. The temperature of the reaction mixture was gradually increased to 90 °C and maintained at this temperature for 3 hours. GC monitoring showed completion of the reaction after 3 hours. The reaction mixture was cooled to room temperature, quenched with 20 L of ice-cold DM water, and stirred for 30 minutes. 10 L of dichloromethane was added to the reaction mixture, and the mixture 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 under vacuum at 35°C to 40°C (the product is volatile) to produce the product, a brown liquid (1.32 kg, 81.25%). Purity 89.67% (GC). 1 H NMR (300MHz, CDCl3) δ: 1.22 (d, 6H, J = 6.6Hz), 2.85 (s, 1H), 4.98-5.05 (m, 1H).

[0611] Synthesis of (Z)-isopropyl 3-iodoacrylate:

[0612]

[0613] Isopropyl propyne ester (1000 g, 1 equivalent) and acetic acid (3.7 L, 3.7 Vol.) were loaded into a 20-L four-necked round-bottom flask equipped with a feeding funnel, thermometer holder, and a mechanical stirrer at 25 °C, and the reaction mixture was stirred for 10 min. 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 increased to 110 °C, and the reaction was maintained at this temperature for 1.5 hr. GC monitoring showed that the reaction was complete after 1.5 hr. The reaction mixture was cooled to room temperature, quenched with ice-cold DM water (18.75 L, 18.75 V), and stirred for 30 min. MTBE (5 L) was added to the reaction mixture, and it was stirred for another 30 min. The organic layer was separated, and the aqueous layer was re-extracted with MTBE (5 L). The combined organic layers were washed with NaHCO3 (2 x 10 L), NaHSO3 (2 x 5 L), and saturated brine (5.2 L, 5.2 V), dried over sodium sulfate, and concentrated under vacuum at 35 °C to produce (Z)-isopropyl 3-iodoacrylate, a brown liquid (1.49 kg, 70%). Purity: 87.34% (GC). 1 H NMR (300MHz, CDCl3) δ: 1.28 (d, 6H, J = 6.3Hz), 5.08-5.131 (m, 1H), 6.83 (d, 1H, J = 8.7Hz), 7.38 (d, 1H, J = 8.7Hz).

[0614] Synthesis of 3,5-bis(trifluoromethyl)benzothioamide:

[0615]

[0616] Bis(trifluoromethyl)benzonitrile (1.25 kg, 1.0 equivalent) and DMF (6.25 L, 5V) were loaded into a 20-L multi-necked flask equipped with a top stirrer and thermometer holder, 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·H₂O (2 equivalent) was added over 10 min intervals. After stirring for 15 min, MgCl₂·6H₂O (1.169 kg, 1.1 equivalent) was added fractionally over 15 min intervals, and the reaction was stirred for another 35 min. The progress of the reaction was monitored by HPLC (green solution), showing 99.6% product and 0.03% benzonitrile. The reaction mixture was cooled to 0 °C–5 °C and 30% dilute HCl (3.75 L) was added dropwise to adjust the pH to 2–3. The product 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 then extracted again with MTBE (2.5 L x 1). The combined organic layers were washed with brine (4.5 L x 3), dried, and concentrated under vacuum. Hexane was added to the obtained solid to displace it, and the product was separated into a yellow solid (1.400 kg, 98.0%). Purity: 99.28% (HPLC). 1 H NMR (300MHz, CDCl3) δ: 8.27 (s, 1H), 8.53 (s, 2H), 10.0 (s, 1H), 10.38 (s, 1H).

[0617] Synthesis of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole:

[0618]

[0619] Thioamide (1378 g, 1 equivalent) and DMF (6.89 L, 5 V) were placed in a 20-L multi-necked flask equipped with a top stirrer and thermometer holder, and the mixture was stirred under nitrogen at room temperature (28 °C). The reaction mixture was cooled to 10 °C and hydrazine hydrate (505.4 g, 2 equivalent) was added dropwise over 2 hours with stirring. The reaction mixture was cooled to 0 °C to 5 °C and formic acid (6.89 L, 5 V) was added over a period of 1 hour (exothermic reaction was observed, and the temperature increased to 20 °C). The reaction mixture was then heated to 95 °C to 100 °C for another 12 hours. The progress of the reaction was monitored by HPLC, which showed the formation of 99.5% product. The reaction mixture was cooled to 35 °C to 40 °C, added to 20.6 L of pre-cooled DM water (10 °C to 15 °C), and stirred for 30 minutes. The reaction mixture 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) and brine (6.89 L x 3), dried with sodium sulfate, and concentrated under vacuum. Dichloromethane (2V) was added to the obtained yellow solid, and the mixture was stirred at 0-5°C for 1 hour. The mixture was filtered to give a product as a yellow solid (1156 g, 82.2%). Purity: 99.7% (HPLC). 1 H NMR (300MHz, DMSO) δ: 8.15 (s, 1H), 8.55 (s, 2H), 8.79 (s, 1H), 14.5 (s, 1H, NH).

[0620] Synthesis of (Z)-isopropyl 3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0621]

[0622] 600 g (1.0 eq.) of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole, 480 g (2.0 eq.) of DABCO, and 3.0 L of DMF were placed into a 10 L four-necked round-bottom flask equipped with a feeding funnel, thermometer holder, mechanical stirrer, and brake. The reaction mixture was stirred for 30 minutes. After 30 minutes, an iodoester (1024.8 g, 2.0 eq.) in 1200 mL of DMF was added dropwise over a 1-hour period. The progress of the reaction was monitored by HPLC, showing (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 another hour, one equivalent of DABCO (258 g) was added and the reaction was maintained for another hour. HPLC analysis showed a conversion to 75.63% (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 min, and extracted with ethyl acetate (2 x 6 L). The combined organic layers were washed with saturated brine (30%, 2 x 3 L), dried over anhydrous sodium sulfate (100 g), and concentrated. The crude material (840 g) was transferred to a 10 L round-bottom flask and methanol (1200 mL) was added. The solution was maintained at 0-5 °C and stirred for 30 min. The obtained solid was filtered and washed with methanol (200 mL), yielding a white solid (550 g, 65.0%). Purity: 87.34% (HPLC). 1 HNMR (300MHz, CDCl3) δ: 1.30 (d, 6H, J = 6.0Hz), 5.12 (m, 1H), 5.73 (d, 1H, J = 10.8Hz), 7.24 (d, 1H, J = 10.8Hz), 7.91 (s, 1H), 8.58 (s, 2H), 9.70 (s, 1H). The ratio of cis isomer to trans isomer is 83:8.

[0623] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid:

[0624]

[0625] 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.) were charged into a 5-L four-necked round-bottom flask equipped with a feeding funnel, thermometer holder, mechanical stirrer, and brake. The reaction mixture was cooled to 0°C. A chilled lithium hydroxide solution (66.58 g in 1.25 L of water) was added to the stirred solution over 30-minute intervals via a feeding funnel. The reaction temperature was slowly increased to 25°C and maintained at this temperature for 2 hours. HPLC monitoring showed the following states: (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-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)-1H-1,2,4-triazol-1-yl)acrylate: 2%. The reaction was continued for another 30 minutes and monitored by HPLC ((Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid: 88.20%; (E)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylic acid: 11.03%). After the reaction was complete, the reaction mixture was quenched with ice-cold water (385 mL) and stirred for 30 minutes. The pH was adjusted to 1–2 with olefinic hydrochloric acid (30%, 400 mL) and the reactants were extracted with ethyl acetate (3 x 625 mL). The combined organic layers were washed with saturated brine (30%, 650 mL), dried with anhydrous sodium sulfate (12.5 g), and concentrated under reduced pressure at 30–35 °C. Hexane was added to the crude material and the mixture was stirred for 30 minutes. The obtained solids were filtered through a Buchner funnel and washed with hexane (250 mL). The solids were dried under vacuum for 30 minutes and then at room temperature for 3–4 hours. The product was separated into a white powder (92.8 g, 84.36%). Purity: 93% (HPLC). 1 H NMR (300MHz, DMSO-d6) δ: 5.98 (d, 1H, J = 10.2Hz), 7.48 (d, 1H, J = 10.2Hz), 8.2 (s, 1H), 8.50-8.54 (m, 2H), 9.39 (s, 1H).

[0626] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazacyclobut-1-yl)prop-2-en-1-one:

[0627]

[0628] Add (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) to a 3-L four-necked round-bottom flask equipped with a nitrogen inlet, feeding funnel, thermometer holder, and mechanical stirrer. Cool the reaction mixture to -10 °C. Add HOBT (4.4 g, 0.1 eq.), EDC·HCl (80.6 g, 1.5 eq.), and 3,3-difluoroazacyclobutane hydrochloride (44 g, 1.2 eq.) to the cooled solution. Add DIPEA (72 mL, 1.5 eq.) dropwise to the resulting mixture at -10 °C over a period of 1.5 hours. Monitor the progress of the reaction by HPLC analysis, which showed that the reaction was complete when the addition of DIPEA was terminated. Slowly increase the reaction temperature to 15 °C to 20 °C (~2 h). The reaction mixture was quenched with 1 L of ice-water slurry. The organic layer was separated, and the aqueous layer was extracted with DCM (400 mL x 2). These organic layers were washed with saturated brine (2 x 500 mL), dried with anhydrous Na₂SO₄ (10 g), and concentrated under reduced pressure (~35 °C) to obtain the crude compound. The crude compound thus obtained was dissolved in 5 vol. DIPE and stirred at room temperature for 30 min, and then filtered. The crude weight was 100 g (yield = 82.39%) [cis-85.07% by HPLC, trans-14.36% by HPLC].

[0629] The crude compound thus obtained was further purified by recrystallization using ethyl acetate according to the following procedure. 100 g of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazacyclobut-1-yl)prop-2-en-1-one was added to a 500-mL four-necked round-bottom flask equipped with a mechanical stirrer, thermometer holder, and brake. Ethyl acetate (7 volumes) was added to the compound at room temperature with stirring. However, the compound did not completely dissolve. Therefore, the resulting solution was heated to 60 °C to obtain a clear solution and then 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 3-4 h to produce a product as a white solid. (by HPLC trans-98.9%); (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazacyclobut-1-yl)prop-2-en-1-one. 1 HNMR (300MHz, CDCl3) δ9.57 (s, 1H), 8.56 (s, 2H), 7.90 (s, 1H), 7.18-7.21 (d, J = 10.8Hz, 1H), 5.61-5.65 (d, J = 10.8Hz, 1H), 4.39-4.45 (m, 4H).

[0630] Example 27.

[0631] Synthesis of (Z)-3-iodoacrylic acid:

[0632]

[0633] Add propionic acid (7.0 g, 1.0 eq) and sodium iodide (29.96 g, 2.0 eq) dissolved in acetic acid (70 mL, 10 V) to a 250 mL three-necked round-bottom flask equipped with a nitrogen inlet. Reflux the reaction mixture at 1000°C for 2–3 h. Track the progress of the reaction by silica gel TLC analysis (using 10% MeOH:DCM as the mobile phase). SM Rf = 0.3 and product Rf = 0.5. Decant the reaction mixture into ice water (700 mL) and neutralize with saturated sodium bicarbonate solution. Extract the reaction mixture with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (3 x 100 mL), dried over MgSO4, filtered, and concentrated by rotary evaporation (25 °C, 20 mmHg) to obtain 12.0 g of crude compound, which was purified by column chromatography (using silica 60 / 120, with MeOH:DCM as the mobile phase). The column (5 x 10 cm) was packed with DCM and eluted in a gradient manner, starting with MeOH and collecting fractions (50 mL fractions) from 2% to 5% MeOH (in DCM). The compound eluted starting with 2% MeOH (in DCM). The fractions containing this TLC characteristic curve were combined to obtain 8.0 mg of the desired compound (yield 40.44%).

[0634] Synthesis of (Z)-1-(3,3-difluoroazacyclobut-1-yl)-3-iodoprop-2-en-1-one:

[0635]

[0636] In a 25-mL three-necked round-bottom flask equipped with a 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-difluoroazacyclobutane hydrochloride (0.179 g, 1.1 eq.) were added. The reaction mixture was stirred at 0 °C for 2–3 h. The progress of the reaction was tracked by silica gel TLC analysis (using 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 the mobile phase). The column (5 x 10 cm) was packed with 5% ethyl acetate in hexane and eluted in a gradient manner, starting with ethyl acetate and collecting fractions (50 mL fractions) from 20% to 30% ethyl acetate (in hexane). The compound eluted starting with 20% ethyl acetate (in hexane). The fractions containing this TLC characteristic curve were combined to obtain 0.18 g of the desired compound (yield 52.33%). Mass: [M+H] + :273.8.

[0637] Synthesis of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-1-(3,3-difluoroazacyclobut-1-yl)prop-2-en-1-one:

[0638]

[0639] In a 25-mL three-necked round-bottom flask equipped with a nitrogen inlet, 0.18 g (1.0 eq.) of 3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazole was dissolved in DMF (5.0 mL, 27.0 V), and DABCO (0.143 g, 2.0 eq.) and (Z)-1-(3,3-difluoroazacyclobut-1-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 tracked by silica gel TLC analysis using 80% ethyl acetate-hexane as the mobile phase, with 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 (3 x 25 mL), dried over MgSO4, 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, with ethyl acetate:hexane as the mobile phase). The column (5 x 10 cm) was packed with hexane and eluted in a gradient manner, starting with ethyl acetate and collecting fractions (50 mL fractions) from 40% to 45% ethyl acetate (in hexane). The compound eluted with 40% ethyl acetate (in hexane). The fractions containing this TLC characteristic curve were combined to obtain 70 mg of the desired compound (yield 25.64%).

[0640] Example 28. Synthesis of (Z)-isopropyl 3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0641]

[0642] Synthesis of isopropyl propargyl acid. A BF3 ether compound (2015 g, 14.2 mol) was added to a mixture of propargyl acid (500 g, 7.1 mol) in isopropanol (4000 mL) at 10 °C. After stirring for 10 min, the reaction mixture was heated to 90 °C and stirred for 2 h. The reaction was monitored for completion by TLC. The reaction mixture was brought to 25-30 °C and quenched with crushed ice, followed by extraction with dichloromethane. The organic layer was washed with water and then with a brine solution. The organic layer was dried with sodium sulfate and concentrated under vacuum to give isopropyl propargyl acid (440 g; 55%). 1 The product was confirmed by H NMR.

[0643] Synthesis of (Z)-Isopropyl 3-iodoacrylate. NaI (930 g, 6.2 mol) was added to a mixture of isopropyl propynate (350 g, 3.1 mol) in AcOH (1300 mL) at 25 °C. The reaction mixture was heated to 115 °C and stirred for 1.5 hr. The reaction mixture was cooled to 25-30 °C and quenched with water, followed by extraction with MTBE. The organic layer was washed with saturated solutions of bicarbonate, bisulfite, and brine. The organic layer was dried with sodium sulfate and concentrated under vacuum to give the product (Z)-isopropyl 3-iodoacrylate (626 g; 83.5%). 1 The product was confirmed by H NMR.

[0644] Synthesis of 3-isopropoxy-5-(trifluoromethyl)benzonitrile:

[0645]

[0646] NaH (122 g, 5.08 mol) was added to a mixture of propan-2-ol (102.96 g, 1.76 mol) in DMF (3200 mL, 8 V) at 5 °C. The mixture was stirred for 2 hours. 400 g (2.1 mol) of 3-fluoro-5-(trifluoromethyl)benzonitrile was added dropwise to the reaction mixture. The temperature was increased to 25-30 °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-cold water and extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried with sodium sulfate, and then concentrated under vacuum to give 530 g (2.31 mol; 110%) of 3-isopropoxy-5-(trifluoromethyl)benzonitrile, which was used directly in the next step without further purification. HPLC purity - 96.5% by area (a / a).

[0647] Synthesis of 3-isopropoxy-5-(trifluoromethyl)benzothioamide:

[0648]

[0649] 3-Isopropoxy-5-(trifluoromethyl)benzonitrile (1000 g, 4.3 mol) was dissolved in DMF (4000 mL) and sodium hydrosulfide hydrate (636 g; 8.6 mol) was added, followed by magnesium chloride hexahydrate (960.2 g, 4.7 mol). The reaction mixture was stirred at 25-30 °C for 1 hr. 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 slurry (250 mL) and the pH was adjusted to 5 by adding 10% aqueous HCl. The reaction mixture was extracted with MTBE and washed with 20% brine. The organic layer was concentrated under vacuum to give 1136 g (4.3 mol; 100%) of the title compound, which was then used in the next step. HPLC purity -97.37% a / a.

[0650] Synthesis of 3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole:

[0651]

[0652] 3-Isopropoxy-5-(trifluoromethyl)benzothioamide (646 g, 2.74 mol) was combined with hydrazine hydrate (140 g; 4.4 mol) and DMF (3200 mL; 5V). The mixture was stirred for 30 minutes and cooled to 10°C. Formic acid (3200 mL) was added dropwise to this reaction mixture. The reaction mixture was heated to 90°C to 100°C and maintained for 12 hours. After the reaction was complete (by HPLC), the reactants were cooled to 25°C to 30°C and quenched with ice-cold water. The mixture was extracted in MTBE. The organic layer was washed with brine, followed by washing with aqueous sodium bicarbonate, and concentrated under vacuum. The residue was ejected with hexane, and the resulting residue was pulped at 10°C for 1 hour. The obtained solid was filtered and dried at 25°C to 30°C for 12 hours to produce 550 g (2.26 mol: 82%) of the product 3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole. HPLC purity: 95.24% a / a.

[0653] Synthesis of (Z)-isopropyl 3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0654]

[0655] A mixture of 500 g (1.8 mol) of 3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole and DABCO (417.6 g; 3.6 mol) in 1200 mL of DMF was stirred for 30 minutes. At 25-30°C, (Z)-isopropyl 3-iodoacrylate (864 g; 3.6 mol) in 1200 mL of DMF was added to the mixture, 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 that 3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole: 9.59%, (Z)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate: 73.76%, and (E)-isopropyl3-(3-(3-isopropoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate: 6.66%. The reactants were quenched with water, extracted with dichloromethane, and concentrated under vacuum to give the crude product. Chromatography of the crude product using an ethyl acetate-hexane system in silica gel at 60-120°C yielded 310 g (0.8 mol; 44%). HPLC purity: 99% a / a.

[0656] Example 29. Synthesis of (Z)-isopropyl 3-(3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0657]

[0658] DABCO (2 equivalents) was added to a solution of 0.50 g (prepared according to Example 3) of 3-(3-methoxy-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazole in 1.5 mL of DMF. The resulting reaction mixture was stirred for 30 min at room temperature, and then (Z)-isopropyl 3-iodoacrylate (2.0 equivalents; prepared according to Example 3) was added. The resulting mixture was stirred for 3 hr at room temperature. The reaction mixture was quenched with ice-cold water and extracted with ethyl acetate (3 times). The organic layers were separated, and the combined organic layers were dried over anhydrous sodium sulfate. LC-MS and HPLC analysis showed 62% cis-isomer and 36% trans-isomer. 1H NMR(400MHz, CDCl3)δ:9.72(s,1H),8.02(s,1H),7.86(s,1H),7.30(s,1H),7.28(d,J=8 .8Hz,1H),5.71-5.73(d,J=10.8Hz,1H),5.12-5.18(m,1H),3.94(s,3H),1.34(d,6H):C 16 H 16 F3N3O3[M+1] + The LCMS was 355.31, and at 4.317 min it was found to be 355.92 (LCMS 99.82%).

[0659] Example 30. Synthesis of (Z)-isopropyl 3-(3-(2-chloro-6-isopropoxypyridin-4-yl)-1H-1,2,4-triazol-1-yl)acrylate:

[0660]

[0661] DABCO (0.467 g, 2 equivalents) was added to 0.5 g of 2-chloro-6-isopropoxy-4-(1H-1,2,4-triazol-3-yl)pyridine (as prepared in Example 3) in 3 mL of DMF, and the resulting mixture was stirred for 30 min. A solution of (Z)-isopropyl 3-iodoacrylate (0.990 g, 2 equivalents) (as prepared in Example 3) was added to the reaction mixture, and the resulting mixture was stirred at room temperature for 3 h. As in Example 3, the reaction mixture was exhausted to obtain 53% cis-isomer and 34% trans-isomer.

[0662] Example 31. Synthesis of (Z)-isopropyl 3-(3-(3-(cyclobutylamino)-5-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)acrylate:

[0663]

[0664] DABCO (0.188 g) was added to N-cyclobutyl-3-(1H-1,2,4-triazol-3-yl)-5-(trifluoromethyl)aniline (0.5 g) (as prepared in Example 3) in 1.5 mL of DMF, and the resulting mixture was stirred for 30 min. A solution of (Z)-isopropyl 3-iodoacrylate (0.404 g) (as prepared in Example 3) was added to the reaction mixture, and the resulting mixture was stirred at room temperature for 3 h. As in Example 3, the reaction mixture was exhausted to obtain 44% cis-isomer and 20% trans-isomer.

[0665] Example 32: Determination. The exemplary compounds of the present invention were tested in parallel with compounds X-1, X2, and X-3 in different determinations. These results are listed in Table 2 below.

[0666] Suppression of nuclear output

[0667] The ability of these exemplary compounds of the present invention to inhibit CRM1-mediated nuclear export was evaluated in a RevGFP assay. Rev is a protein derived from human immunodeficiency virus 1 (HIV-1) 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 the Rev protein depends on the classical NES / CRM1 pathway (Neville et al., 1997). Nuclear accumulation of Rev can be observed in cells treated with a specific inhibitor of CRM1 (such as LMB) (Kau et al., 2003).

[0668] In this assay, U2OS-RevGFP cells were seeded in clear-bottomed, black 384-well plates prior to the day of the experiment. Compounds were serially diluted 1:2 in DMEM in a septated 384-well plate, starting at 40 μM, and then transferred to the cells. The cells were incubated with the compounds for approximately 1 hour before fixation with 3.7% formaldehyde and nuclear staining with Hoechst 33258. The amount of GFP in the nuclei was measured, and the IC50 of each compound was determined. 50 (Kau et al., 2003). If the compound of the present invention has an IC50 concentration below approximately 10 μM... 50 In this case, they were considered to be active in the RevGFP assay described above, and the most preferred compound had an IC50 value of less than approximately 1 μM. 50 Table 2 shows the results of this RevGFP assay.

[0669] Cell proliferation assay

[0670] CellTiter The AQueous One Solution cell proliferation assay (Promega) was used on the MM.1S multiple myeloma cell line to investigate the cytotoxic and inhibitory properties of these compounds. The assay is based on the cleavage of tetrazolium salt, MTS, in the presence of an electron-coupling agent, PES (phenazine ethyl sulfate). This MTS tetrazolium compound is then reduced by cell bioreduction to a colored formazanine product, which is soluble in tissue culture medium. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenases in metabolically active cells. The assay was performed by adding small amounts of CellTiter directly to the culture wells. AQueous Onesolution reagent was used for incubation for 1–4 hours, and absorbance at 490 nm was recorded using a 96-well plate reader. The absorbance results showed a direct correlation with cell number and their metabolic activity.

[0671] With a 96-well plate, each well is 5x10 cm. 3 Up to 1.5x10 4 Cells were seeded in 100 μL of fresh culture medium and allowed to adhere overnight. Stock solutions of the compounds were diluted in cell culture medium to obtain eight concentrations of each drug, ranging from 1 nM to 30 μM, with less than 1% v / v DMSO used as a negative control. The resulting drug solutions were transferred to the cells. After 72 h of treatment, 20 μL of CellTiter was added to each well of the 96-well assay plate. Aqueous reagent was used, and the plate was incubated at 37°C in a humid 5% CO2 atmosphere for 1–4 hours. The absorbance of each well was then recorded at 490 nm using a 96-well plate reader. In most cases, the determination was performed in triplicate, and these results are presented as half-maximal inhibition concentrations (IC50). 50 The optical density ratio was plotted against compound concentration, and analyzed using a nonlinear regression equation (IDBS XLfit) to calculate the IC50 for each compound. 50 .

[0672] Drug pharmacokinetic (PK) assays and brain plasma ratio determination

[0673] AUC. Blood was collected from mice (N=3) to contribute to all 10 time points (before administration, 5 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration). Mice were induced to bleed on a rotating basis, with each mouse contributing blood to 3 time points. At the designated time points, animals were anesthetized with isoflurane, and approximately 110 μL of blood was collected from each time point via posterior orbital puncture into pre-chilled K2EDTA (anticoagulant) tubes. Blood samples were placed on wet ice and centrifuged (2000 g, 4°C for 5 min) to obtain plasma within 30 minutes of sample collection. All samples were cryopreserved at approximately -80°C until analysis. Prior to analysis, samples were mixed with an internal standard (dexamethasone) in acetonitrile, vortexed, centrifuged, and the supernatant was injected for analysis. The concentrations of compounds in plasma were determined using LC-MS-MS (API 4000, Triple Quadruple with electro-electro-ionization; Acuity Ultra Performance Liquid Chromatography C18 column, MeOH and formic acid as organic solvents). AUC values ​​were calculated using the WinNonlin Professional Edition 6.2 software package and the non-compartmental pharmacokinetic model NCA200.

[0674] Brain to plasma B:P ratio. Mice in a separate group (N=3) were administered the drug (PO at 10 mg / kg) and then at the time of maximum plasma concentration (estimated T at 2 hours post-administration). max (Drug administration) euthanasia was performed, and plasma and brain tissue were collected at the end of the time interval. After collection, brain tissue was rinsed with cold saline, dried on filter paper, weighed, and flash-frozen on dry ice. All samples were frozen and stored at approximately -80°C until analysis. For analysis, brain tissue was homogenized (using PBS, pH 7.4), mixed with an internal standard (dexamethasone) in acetonitrile, vortexed, centrifuged, and the supernatant was injected for compound concentration analysis using LC-MS-MS (API 4000, Triple Quadruple with electrospray ionization; Acuity Ultra Performance Liquid Chromatography column C18, using MeOH and formic acid as organic solvents). Plasma samples were processed using the same method (except for the homogenization step), and the concentration of compounds in each matrix was calculated based on the generated standard curve. These results for PK determination and B:P ratio determination are presented in Table 2.

[0675] Table 2. Compounds with Formula I and their comparator determination results (A = IC) 50 Value <= 1 μM; B = IC from 1 μM to 10 μM 50 Value; C = IC 50 Value >10μM; NT = Untested.

[0676]

[0677]

[0678]

[0679]

[0680] * Mice were administered the drug at a dose of 10 mg / kg po.

[0681] ** Compound 26 from US2009 / 0275607.

[0682] *** Compound 44 from US2009 / 0275607.

[0683] AUC of compound X-1 administered orally to mice at 10 mg / kg Inf The values ​​were below the quantification limit. Data for 5 mg / kg IV were reported.

[0684] Rats were administered the drug at a dose of 10 mg / kg po.

[0685] When mice were administered 10 mg / kg orally, the AUC of compound X-1 was... Inf Below the detection limit. When administered at 5 mg / kg IV, compound X-1 showed minimal exposure, as indicated by a low AUC of 209 hr·ng / mL. Inf As noted, the brain-to-plasma ratio of compound X-1 was not detected, attributed to its negligible exposure levels when administered orally.

[0686] When administered to rats at a dose of 10 mg / kg po, the AUC of compound X-2 was... Inf The calculated value was 68.3 hr·ng / mL. This level of exposure is very low compared to compound X-3 and the compounds of formula I of this invention. However, compound X-2 exhibited a moderate brain-to-plasma ratio. Low AUC Inf The addition of a non-negligible brain-to-plasma ratio suggests that compound X-2 can cross the BBB despite low exposure levels. This is assuming its AUC...Inf With increased levels, compound X-2 exhibits a significantly higher brain-to-plasma ratio.

[0687] When administered to rats at a dose of 10 mg / kg po, the AUC of compound X-3 was... Inf The calculated value was 12300 hr·ng / mL, indicating good exposure. However, compound X-3 exhibited a high B:P ratio of 5.0.

[0688] These compounds of Formula I are characterized by an AUC greater than about 3300 hr·ng / mL, and in most cases greater than about 3500 hr·ng / mL. Inf And a relatively low B:P ratio (<2.5). Overall, a higher exposure level of a therapeutic agent increases the likelihood of brain penetration. Therefore, it is surprising and unexpected that compounds with formula I exhibit high AUC. Inf Levels and a relatively low brain-to-plasma ratio.

[0689] The in vivo and in vitro activity of the compounds of this invention against breast cancer

[0690] Basal-like breast cancer (BLBC) accounts for up to 15% of breast cancers (BC) and is typically triple-negative breast cancer (TNBC), characterized by the lack of ER, progesterone receptor PR, and HER-2 amplification. Additionally, a large proportion of BRCA1-associated BCs are BLBCs and TNBCs, expressing basal cell keratin and EGFR. BLBCs are characterized by an aggressive phenotype, high histological grade, and poor clinical outcomes with high recurrence and metastasis rates. Additional therapies are required. The in vivo and in vitro activities of these compounds of the present invention, such as compound I-3, in different breast cancer cell lines were evaluated.

[0691] Suppression of TNBC (triple-negative breast cancer) in vivo xenograft

[0692] MDA-MB-468 (ATCC#HTB-132) triple-negative breast cancer cells were obtained from ATCC. These cells were grown in Leibovitz L-15 matrix supplemented with 10% fetal bovine serum (FCS), 1% penicillin and streptomycin, and 2 ml glutamine. These cells were subcultured by dilution at a ratio of 1:3. Fifty (50) female SCID mice (Charles River Labs) aged 5 to 6 weeks with an average pretreated weight of 19.2 g were used. 6 MDA-MB-468 cells were inoculated into the left rib area of ​​SCID mice. When these tumors reached 100 and 200 mm... 3When the average size was between, the mice were randomly and predictably divided into a vector control group of ten (10) mice and five treatment groups of eight (8) mice each. These groups were as follows:

[0693] Carrier (1% PLP in distilled water)

[0694] 5-FU 50mg / kg

[0695] Compound I - 35 mg / kg Monday (M), Wednesday (W), Friday (F)

[0696] Compound I-315 mg / kg, M, W, F

[0697] Compound I-325 mg / kg M, W, F

[0698] Compound 1 - 325 mg / kg M, Thursday (Th).

[0699] All medications are administered orally. Using sterile... Animals were fed 5053 (pre-sterilized) rodent food and provided with sterile water at will. Tumors were measured every two days using a micrometer, and tumor volume was calculated as (length x width x width) / 2. All animals were weighed daily to assess weight differences between treatment groups and to monitor animal health. Any animal exhibiting a loss greater than 20% of its starting weight during the study was euthanized. Animals with a tumor diameter exceeding 1500 mm... 3 Animals with tumors of any size were euthanized. Survival rates were recorded daily. Dosage solutions were freshly prepared daily. Compound I-3 was provided as a lyophilized powder containing 67.8% of the drug product, with the remainder consisting of Prönnicke F-68 and PVP K29 / 32. This was prepared by dissolving the lyophilized powder in sterile water at a ratio of 6.64 mg / 90 μL, and, if necessary, diluting it in sterile water with the carrier (1% Prönnicke F-68 and 1% PVP K29 / 32). All dosage solutions of Compound I-3 were administered at a rate of 0.1 mL / 10 g. Statistical differences between treatment groups were determined using the Mann-Whitney Rank Sum test or ANOVA with a critical value of 0.05.

[0700] These tumors were removed 33 days after vaccination. Figure 1 The graph shows tumor volume as a function of time and demonstrates that compound I-3 exhibits dose-dependent efficacy compared to vector-treated animals, inhibiting tumor growth by approximately 60% (5 mg / kg Monday, Wednesday, Friday) to almost 100% (for the 25 mg / kg Monday, Thursday regimen). Furthermore, compound I-3 is well tolerated.

[0701] After resection, these tumors were stained for tumor suppressor proteins (TSPs) FOXO3a, IκB, and p27, and the nuclear localization of these TSPs was confirmed by immunohistochemistry.

[0702] Proliferation inhibition and cytotoxicity in TNBC and coelenterate BC cell lines

[0703] Using CellTiter The AQueous One Solution cell proliferation assay (Promega) was used to study the cytotoxic and inhibitory properties of compound I-3 in different TNBC and coelomic BC cell lines.

[0704] With a 96-well plate, each well is 5x10 cm. 3 Up to 1.5x10 4 Cells (depending on cell type) were seeded in 100 μL of fresh culture medium and allowed to adhere overnight. Stock solutions of the compounds were diluted in cell culture medium to obtain eight concentrations of each drug, ranging from 1 nM to 30 μM, with less than 1% v / v DMSO used as a negative control. The resulting drug solutions were transferred to the cells. After 72 h of treatment, 20 μL of CellTiter was added to each well of the 96-well assay plate. Aqueous reagent was used, and the plate was incubated at 37°C in a humid 5% CO2 atmosphere for 1–4 hours. The absorbance of each well was then recorded at 490 nm using a 96-well plate reader. In most cases, the determination was performed in triplicate, and these results are presented as half-maximal inhibition concentrations (IC50). 50 The optical density was plotted against the compound concentration, and analyzed using a nonlinear regression equation (Excel Fit). The IC50 of compound I-3 for each cell line was calculated. 50 .

[0705] Table 3 shows the results of this cell proliferation assay. These results demonstrate that compound I-3 is effective at cytotoxicity to nine of the fifteen BC cell lines tested. If it has an IC50 concentration of less than approximately 1.0 μM... 50 The compound was considered effective in a certain cell line based on its IC50 value. Compound I-3 in this cell line had an IC50 value of less than 1.0 μM. 50 The cell lines in which the value is indicated are considered sensitive cell lines, and in which compound I-3 has an IC50 greater than 1.0 μM. 50 Cell lines with the specified values ​​were considered resistant. Seven of these nine sensitive cell lines were TNBC. Genomic analysis of all BC lines showed that p53, PI3K / AKT, and BRCA1 or 2 status did not affect cytotoxicity.

[0706] Table 3. IC50 of compound I-3 in different breast cancer cell lines 50 value.

[0707]

[0708] Compounds 1-3 induce apoptosis and inhibit long-term BC growth.

[0709] The ability of compound I-3 to induce apoptosis and inhibit the long-term growth of selected BC cell lines was evaluated.

[0710] MDA-MB-468 TNBC, DU4475, and HS578T TNBC cells were exposed to compound I-3 at concentrations ranging from 0 to 10 μM for 24 hours. After 24 hours, whole protein cell extracts were run on Western blotting and exposed to antimicrobial agents. Figure 2A-2C Antibodies against the proteins mentioned in the text.

[0711] Figure 2A-2C These are immunoblot images obtained from some of the most tolerant and sensitive breast cancer cell lines mentioned above, including MDA-MB-468TNBC, DU4475, and HS578T TNBC. The study showed that after 24 hours, compound I-3 induced apoptosis in sensitive TNBC and coelenterate BC cell lines (MDA-MB-468 and DU4475, respectively), as indicated by decreased PARP and caspase 3, two apoptosis markers, and increased lysed PARP and caspase 3. In contrast, when a tolerant cell line, HS578T, was treated with compound I-3, only a negligible increase in lysed PARP and caspase 3 was observed.

[0712] Long-term growth assays were also performed, in which MDA-MB-468, MDA-MB-231, and HS578T cells were treated with 1 μM compound I-3 and incubated for 7 days (HS578T) or 10 days (MDA-MB-468 and MDA-MB-231). At the end of the assay, the medium was removed from these cells, and the remaining cells were stained with gentian violet. This study showed that compound I-3 inhibited the long-term growth of all three cell lines, including the sensitive (MDA-MB-468 and MDA-MB-231) and tolerant (HS578T) BC cell lines.

[0713] Compound I-3 increases nuclear FOX03a and IKB in the TNBC cell line.

[0714] MDA-MB-468TNBC basal A cells and BT-20TNBC basal B cells were exposed to DMSO or 1 μM compound I-3 for 24 hours, and then stained with FOXO3a or IκB with or without DAPI nuclear staining. Nuclear localization was examined in these stained cells. After treatment with compound I-3, both FOXO3a and IκB were localized in the nucleus, while in DMSO-treated cells, they were localized in the cytoplasm.

[0715] Effects of compound I-3 on anti-apoptosis and cyclin activity in two TNBC cell lines

[0716] The effects of increasing compound I-3 concentration on MDA-MB-468 and HS578T cells were examined. MDA-MB-468 and HS578T cells were exposed to increased concentrations of compound I-3 for 24 hours, and were treated with anti-inflammatory drugs. Figure 3 The antibody detection method for the proteins mentioned in the text is used to detect the total cellular protein level of different proteins.

[0717] Figure 3 The results showed that, although the IC50 of compound I-3 decreased after 72 hours, 50 There was approximately a 100-fold difference between the two cell lines (10 nM vs. 1.5 μM), and a decrease in MCL-1 was observed in both cell lines in response to an increase in the concentration of compound I-3.

[0718] The experiments described in Example 32 indicate that the inhibition of CRM1-mediated nuclear export by these compounds of the present invention (including compounds I-3) induces nuclear localization and activation of tumor suppressor gene proteins, which leads to selective apoptosis, cancer cell cytotoxicity, and inhibition of tumor growth.

[0719] Example 33: Monoclonal antibody-induced arthritis (CAIA)

[0720] On the day of arrival (-1), BalbC mice were randomly assigned to cages, and each group (n=8) was assigned to a treatment group with the following protocol as shown below:

[0721] Transport vehicle: Days 4, 6, 8, and 10 of PO

[0722] Dexamethasone: 1 mg / kg IP on days 4, 6, 8, and 10.

[0723] Compound I-4: 4 mg / kg PO, on days 4, 6, 8, and 10.

[0724] Compound I-4: 7.5 mg / kg PO, on days 4, 6, 8, and 10.

[0725] Compound I-4: 15 mg / kg PO, on days 4, 6, 8, and 10.

[0726] Upon arrival, the animals' health status was assessed. Only well-healthy animals were acclimatized to laboratory conditions and used in this study. Each animal was provided with any commercial rodent diet and free access to drinking water, supplied to each cage via polyethylene bottles with stainless steel pipettes. Automated environmental conditions were set up to maintain the laboratory temperature at 20°C–24°C, relative humidity (RH) at 30%–70%, a 12:12 light:dark cycle, and 10–30 air changes / hr. Temperature, RH, and photocycle were monitored daily via a control computer. Each animal was given a unique animal identification number, and on Day 0 of the study, each animal received a tail vein injection of an antibody mixture (10 mg / mL in 200 μL). This antibody mixture was supplied by MD Biosciences (catalog number: CIA-MAB-50). On Day 3, following the administration of mAb alone, all animals received LPS (0.5 mg / mL in 200 μL) via a single intraperitoneal (IP) injection. LPS was supplied by MD Biosciences (catalog #: MDLPS.5). Signals of arthritic responses in the peripheral joints were examined on day 0. From the onset of disease, arthritic responses were examined on study days 3–8, 10, and 12. Arthritic responses in each paw were reported in ascending order of severity on a 0–4 scale.

[0727]

[0728] Animals in a near-death state, with broken skin on their arthritic paws, or with a weight loss greater than 20%, or showing signs of severe pain and tolerability, were euthanized. Severe pain or tolerability was assessed by an experienced animal technician on a case-by-case basis. In short, the assessment was directed at abnormal vocalizations, isolation from other animals, reluctance to use limbs, and abnormal responses to handling, vibration, and posture. Animals were euthanized by CO2 inhalation followed by cervical dislocation. Evaluation was initially based on the mean of arthritis scores and paw thickness measurements. Weight was also statistically analyzed. Where appropriate, data analysis was performed using ANOVA and Tukey post-hoc comparative analysis to determine the significance of the treatment effect.

[0729] As part of this model, animals experienced rapid weight loss during the initial 5–8 days followed by a slow increase / loss of weight (depending on disease progression). I-4 increased the rate of weight gain compared to the carrier or dexamethasone treatment groups. Figure 4This is a graph showing the average body weight of male BALB / c arthritis mice subjected to antibody-induced arthritis of this model over time from 0 to 12 days.

[0730] Furthermore, animals subjected to the CAIA model typically began to show signs of arthritis around day 4, and the total arthritis score increased as the disease progressed as a function of time. Treatment with compound I-4 significantly reduced this total score and demonstrated a dose-dependent effect compared to the vector. Figure 5 This is a graph showing the mean total claw clinical arthritis score against time 0–12 days in antibody-induced male BALB / c arthritis mice that have undergone the specified treatment.

[0731] Example 34: PMA-induced psoriasis model

[0732] BALB / c mice were housed in single, ventilated cages in a controlled environment at an animal facility (temperature 22℃±1℃, humidity 70%±5%, and a 12h light / 12h dark cycle). The mice had free access to commercially available feed pellets and UV-treated drinking water. Each ventilated cage contained four mice. Each animal in the cage was identified by its tail. Eight mice from each group were randomly assigned to different treatment groups based on their weight. Randomization was followed to ensure that the mean weight was equal across all groups. The experimental design was as follows: Group 1: original... Group 1: 1% DMSO carrier (10-30 μL, topical once daily); Group 2: PMA, 1% DMSO carrier (10-30 μL, topical once daily); Group 3: PMA, I-4 10 mg / kg in PVP / Pranic (oral, MWF; Day 1-3-5-7); Group 4: PMA, 0.1% betamethasone - 25 mg (reference standard) (topical once daily).

[0733] Mice were administered 4 μg phorbol 12-myristate-13-acetate (PMA) in 20 μL of acetone daily into their ears. From day 2 onwards, an increase in the clinical disease activity index, associated with increased ear thickness, ear-skin crusting, and ear-skin folding, indicated PMA-induced skin inflammation / psoriasis. The following parameters were evaluated: (i) ear thickness; (ii) ear skin crusting, which was based on a score index of -0, no crusting; 1, slight crusting; 2, moderate crusting; 3, severe crusting; (iii) ear skin folding, which was based on a score index of -0, no folding; 1, slight folding; 2, moderate folding; 3, severe folding; and (iv) ear weight (on the day of sacrifice).

[0734] Figure 6The results are bar graphs providing scores for ear thickness, ear crusting, and ear skin folds. These results show that administration of compound I-4 at 10 mg / kg significantly reduced mean ear thickness compared to the carrier. The potency obtained with I-4 was comparable to that of the positive control betamethasone. Furthermore, compound I-4 was well tolerated.

[0735] Example 35: New Object Recognition

[0736] For the new object recognition test, Zucker rats were placed in a test chamber (26" x 18" x 18"; L x W x H). Food and water were not allowed during this test. The test had three phases: a) Familiarization phase: Rats were placed one by one in the test chamber and allowed free exposure for 60 minutes. The distance traveled by the animal during this phase was recorded using tracking software (AnyMaze system). The purpose of this phase was to familiarize the animals with the testing equipment. This phase was conducted on the first day. b) Sampling phase: On the second day, the rats were placed one by one in the test chamber for 3 minutes and allowed free exposure on a testing platform containing two identical new objects (e.g., metal cubes, plastic cylinders) located in two corners of the test chamber. The distance traveled by the animal and the time spent interacting with these new objects were automatically recorded during this sampling phase using the tracking software system and visual observation. Interaction with the object was defined as active nose-to-nose contact or immediate approach to the object. c) Measurement Trial Phase: One hour after the sampling phase, each rat was placed in a test chamber for three minutes and allowed free exposure to the test platform, which contained two objects: one of the objects present in the sampling phase and the second a novel object unique to this test phase. These two objects were located in the same two corners of the test chamber as those used in the sampling phase. Using a tracking software system and visual observation, the distance the animal traveled and the time spent interacting with both the novel and familiar objects were automatically recorded during this test phase. Object interaction scores were independently recorded by two observers during both the sampling and test phases. The final score represents the difference between each reading. Object preference scores are presented as D1 (time spent exploring a new object minus time spent exploring a familiar object; therefore, a positive score indicates a preference for the new object) and D2 (D1 / a+b; D1 score divided by the total object exploration time).

[0737] Figure 7 A series of figures are provided, illustrating object preferences in untreated and I-4 treated Jooker rats. Figure 7As can be seen, oral administration of compound I-4 at doses of 0.625, 1.25, and 2.5 mg / kg induced a trend of increased recognition of novel objects in Jukes rats, and compound I-4 was well tolerated.

[0738] Example 36: Feeding Study of Obese Zhuke Rats

[0739] Male Juck (fa / fa) rats and lean male Juck rats (both from Charles River) (10 weeks old – at this time, Juck fa / fa rats should show increased food intake, body weight, and elevated plasma lipid profiles relative to their "lean" counterparts) were individually housed in plastic-bottomed cages and given 14 days of acclimatization. During this period, animal body weight, food, and water intake were recorded daily. All animals had free access to standard laboratory food and water throughout the study. Once baseline intake data for 14 days was collected, these obese Juck rats were assigned to treatment groups based on equal baseline data, i.e., all obese Juck rats had equal daily food / water intake and body weight. During this phase, the rats also received two administrations of the carrier as familiarization with the dosing protocol. The treatment phase began immediately after this baseline phase. Test items and the carrier were administered approximately 1 hour before the start of the dark cycle. The dosing regimen varied by group: 5x weekly administration was administered Monday through Friday. The study design was as follows: Group A = male rats with juvenile leukemia, treated with the vector for 5x weeks orally, n=6; Group B = male rats with juvenile leukemia, treated with the vector for 5x weeks orally, n=6; Group C = male rats with juvenile leukemia, treated with I-4 2.5mg / kg for 5x weeks orally, n=6.

[0740] Daily weight, food, and water intake were measured at almost the same time each day, on days 1 and 7 of the treatment phase.

[0741] Figure 8A Cumulative and mean food intake (W / O) were provided for obese and lean Juck rats. Oral administration of I-4 at 2.5 mg / kg 5X weekly reduced mean and cumulative food intake in obese (fa / fa) Juck rats. Compound I-4 was well tolerated.

[0742] Figure 8B Mean and percentage body weights (W / O) are provided for obese and lean Zuck rats. Oral administration of I-4 at 2.5 mg / kg 5X weekly significantly reduced weight gain compared to the Zuck fa / fa control. Weight gain remained reduced over a 2-day clearance period compared to the Zuck fa / fa control. I-4 was well tolerated.

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[0772] All relevant teachings in patents, published applications, and references are incorporated in their entirety through citation.

[0773] While the invention has been specifically shown and described with reference to its exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A therapeutically effective amount of a compound represented by the following structural formula. The use of its pharmaceutically acceptable salts in the preparation of a medicament for the treatment of a malignant hematologic disorder in subjects in need, wherein the malignant hematologic disorder is myeloproliferative syndrome, but not leukemia.

2. The use according to claim 1, wherein the drug is formulated for oral administration.

3. The use according to claim 1 or 2, wherein the subject is a human.

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