Imidazopyridine and oxazolopyridine derivatives and analogs thereof, methods for their preparation, methods for inhibiting the HIF-1 / 2a pathway, and induction of ferroptosis

Abstract

Inhibitors of HIF-1α and HIF-2α due to fluctuations in iron metabolism, and novel substituted imidazopyridine and oxazolopyridine compounds useful as inducers of ferroptosis, synthetic methods for preparing the above compounds, pharmaceutical compositions containing the compounds, and methods of using the compounds and compositions for treating disorders associated with HIF-1 / 2α or dysfunction of iron metabolism.

Description

Technical Field

[0001] Statement Regarding Federal Government Funds The present invention was funded in part by grants from the National Institutes of Health, the National Institute on Drug Abuse 1R03DA033980, and the National Cancer Institute's Small Business Innovation Research Program R43CA217385. The government has certain rights in the invention.

[0002] 1. Technical Field The present disclosure relates to novel compounds, more specifically, imidazopyridine and oxazolopyridine derivatives and analogs thereof, and methods of making and using such compounds.

Background Art

[0003] 2. Related Art Hypoxia provides extracellular stimuli necessary for proper embryonic development and wound healing and maintains the pluripotency of stem cells. Apart from these cellular processes, pathological hypoxia can be caused by a decrease in oxygen supply, such as at high altitudes, or by local ischemia due to disruption of blood flow to a given area. Furthermore, most solid tumors contain hypoxic regions because the tumor vasculature has profound structural abnormalities and the rapid proliferation of the tumor cells themselves often exceeds the available oxygen levels.

[0004] The transcription factor hypoxia-inducible factor (HIF) is a central mediator of the response to hypoxia or hypoxemia.

[0005] HIF is a heterodimer that includes one of three major oxygen-labile HIF-α subunits (HIF-1α, HIF-2α, and HIF-3α) and the constitutive HIF-1β subunit (also known as the aryl hydrocarbon receptor nuclear translocator or ARNT), and these subunits together form HIF-1, HIF-2, and HIF-3 transcription complexes, respectively. Of the three α-subunits, HIF-1α and HIF-2α have been the most studied.

[0006] In the presence of oxygen, HIF-α is hydroxylated at two conserved proline residues (P402 / P564 and P405 / P531 for human HIF-1α and HIF-2α, respectively) located within the oxygen-dependent degradation domain (ODD) by specific prolyl hydroxylases (PHDs) in a reaction that requires oxygen, 2-oxoglutarate, ascorbate, and iron (Fe 2+ ). The hydroxylation of HIF-α promotes the binding of the von Hippel-Lindau protein (pVHL) to the HIF-α ODD. pVHL forms the substrate recognition molecule of the E3 ubiquitin ligase complex, which directs HIF-1 / 2α poly-ubiquitination and proteasomal degradation. In the hypoxic state, PHD activity is inhibited, pVHL disappears, HIF-α is stabilized and translocates into the nucleus, where it heterodimerizes with HIF-1β and binds to a conserved DNA sequence known as the hypoxia-responsive element (HRE), transactivating various hypoxia-responsive genes.

[0007] HIF activates the transcription of numerous genes that are critical for adaptation to hypoxia and tumor progression, such as those promoting aerobic glycolysis, angiogenesis, and metastasis. While sharing many transcriptional targets, HIF also plays non-redundant roles. For example, anaerobic glycolysis appears to be mainly controlled by HIF-1, whereas erythropoietin (EPO) synthesis and iron metabolism appear as processes controlled by HIF-2. Furthermore, in addition to canonical HRE-mediated transcription that requires heterodimerization with HIF-1β, the HIF-1α and HIF-2α subunits independently regulate cell signaling pathways through interaction with proteins that do not contain a PAS domain, including the tumor suppressor protein p53, the c-MYC proto-oncogene, β-catenin, and the Notch intracellular domain.

[0008] HIF-1α is ubiquitously expressed in hypoxic tissues, while HIF-2α is detected in a more restricted set of cell types, including vascular endothelial cells and macrophages, where it is frequently expressed in both hypoxic and normoxic states. In particular, most healthy adult tissues do not experience hypoxia, and as a result, the HIF family of transcription factors generally cannot be detected in normal, non-inflammatory tissues.

[0009] Tumor hypoxia is of great clinical significance as it promotes both tumor progression and resistance to therapy. By shifting cells towards anaerobic metabolism, angiogenesis, and resistance to apoptosis, hypoxia promotes the survival of tumor cells and also elicits other responses that contribute to tumor aggressiveness, such as increased genetic instability, invasion, metastasis, and dedifferentiation, mainly through the activation of HIF.

[0010] Elevated levels of tumor HIF-1α are associated with poor patient prognosis in multiple tumor types. Elevated HIF-2α is also associated with poor prognosis in specific tumor types, such as neuroblastoma, glioblastoma (GBM), and non-small cell lung cancer.

[0011] ccRCC most commonly begins with the loss of pVHL, leading to the pseudohypoxic activation of both HIF-1 and HIF-2. The upregulation of HIF as a result of pVHL loss in ccRCC is associated with mitochondrial dysfunction, including decreased mitochondrial respiration and suppression of fatty acid metabolism, which enables a metabolic shift towards glycolysis and thereby promotes tumor progression.

[0012] The need for new treatments for ccRcc is unmet. ccRcc is highly refractory to standard chemotherapy and radiation, and the 5-year survival rate for patients with progressive or metastatic tumors is only 13%. Furthermore, many ccRccs remain asymptomatic, and approximately 30% of patients with ccRcc present with metastatic disease. Current treatments include various anti-angiogenic agents (mainly kinase inhibitors), which do not induce a sustained response in the majority of patients, the inevitable development of resistance, immune checkpoint inhibitors, and are limited by combinations of these two.

[0013] In addition to its role in promoting tumor progression, activation mutations within EPAS1 (the gene encoding HIF-2α), or overproduction of HIF-2α caused by inactivating mutations in pVHL or PHD2, can lead to overproduction of red blood cells or erythrocytosis. This is mainly mediated by an increase in HIF-2α-dependent production of erythropoietin (EPO), a cytokine that promotes the production of red blood cells. Mutations in EPAS1 are also said to cause tumorigenesis, particularly paraganglioma. Consistent with its unique role in regulating erythropoiesis, inactivating mutations in EPAS1 are associated with adaptation to high altitude and reduce increased erythropoiesis and high blood viscosity associated with populations not adapted to high altitude. Thus, inhibition of HIF-2α can provide benefits for erythrocytosis associated with pVHL, PHD2, or EPAS1 mutations, or due to overproduction of EPO. Furthermore, HIF-2α may be beneficial for the treatment of paraganglioma associated with EPAS1 mutations. Finally, inhibition of HIF-2α can provide benefits for the treatment of altitude sickness associated with increased blood viscosity. Due to its widespread expression in multiple tumor types associated with poor patient prognosis, HIF-1α is also a promising therapeutic target for cancer. Furthermore, both acquired resistance to anti-angiogenic therapies and innate resistance to immune checkpoint therapies are associated with upregulation of various HIF target genes, suggesting that targeting HIF-1α and HIF-2α may be beneficial for cancer treatment.

[0014] Belzutifan, a novel selective HIF-2α antagonist, demonstrates promising single-agent activity in VHL disease-related non-metastatic ccRCC and was approved for the treatment of cancers associated with VHL disease in August 2021, supporting the efficacy of HIF-2α inhibition in ccRCC. However, this approach to inhibiting HIF-2 transcriptional activity does not address the non-transcriptional targets of HIF-2α, such as c-Myc, EGFR, and β-catenin, which are activated by protein-protein interactions with HIF-2α and are also associated with tumor progression and resistance to therapies.

[0015] Since oxygen transport is closely related to iron availability, depletion of both oxygen and iron has very similar molecular consequences. Consistent with the central role of HIF-2α in the regulation of iron homeostasis, HIF-2α is also regulated by iron through the presence of an RNA stem-loop element known as an iron-responsive element (IRE) in the 5’ untranslated region (UTR) of the HIF-2α transcript. Under iron-depleted conditions, iron-responsive proteins (IRP1 and IRP2) bind to the IRE within the 5’ or 3’ UTR of the transcript, resulting in translational repression and transcript stabilization, respectively.

[0016] IRPs coordinate the cellular response to iron depletion by reducing iron storage and increasing iron uptake through downregulation of ferritin (both heavy and light chains, 5’ IRE), a central iron storage molecule, and upregulation of transferrin receptor (TfR1, 3’ IRE), a major mediator of cellular iron uptake, respectively.

[0017] Under iron-depleted conditions, IRP1 binds to the IRE within the 5’UTR of HIF-2α and suppresses the translation of HIF-2α. Similarly, under iron-depleted conditions, IRP2 is stabilized, binds to the 5’IRE of ferritin, and suppresses the translation of ferritin to reduce iron storage. Conversely, IRP2 also binds to the 3’IRE of TfR1 and promotes iron uptake. Consequently, cellular iron-depleted conditions can be indicated by an increase in the levels of IRP2 and TfR1, and a decrease in the levels of ferritin (both heavy and light chains, FTH1, FTL).

[0018] The IRE-binding activities of IRP1 and IRP2 are induced by different stimuli. IRP1 is induced by the disruption of its [4Fe-4S] cluster (such as oxidative stress or nitric oxide), and IRP2 is induced by iron or oxygen depletion. These different regulatory mechanisms can promote the specific induction of IRP1 IRE binding by the disruption of its [4Fe-4S] cluster.

[0019] Together with its binding partners ISCA1 and IBA57, ISCA2 is required for the maturation of a subset of mitochondrial [4Fe-4S] proteins and may play a role in the assembly of [2Fe-2S] proteins in both mitochondria and the cytoplasm.

[0020] Iron is critically required by tumor cells to enable the function of key proteins involved in DNA replication, maintenance of genomic integrity (including DNA repair), and progression of the cell cycle, which are frequently upregulated in cancer. Furthermore, many signaling pathways known to drive cancer, such as Wnt, PI-3K / AKT / mTor, and Ras / Raf / MEK / ERK, require iron and are inhibited by iron depletion.

[0021] Increased iron demand by tumor cells, as well as changes in the pathways of iron uptake and utilization, are particularly key metabolic changes characteristic of cancer. This change includes increases in both TfR1 and circulating ferritin in many cancer types associated with tumor progression. Therefore, antibodies targeting TfR1 are currently being developed as an anticancer strategy for the functional neutralization or internalization of conjugated toxic moieties. Furthermore, the ability of tumor-associated macrophages (TAMs) to promote tumor growth is associated with the ability of TAMs to release iron into the local microenvironment as part of the wound healing response.

[0022] Despite the well-established association between iron and cancer, current treatment strategies for iron depletion are limited to iron chelation, which is not specific and has significant side effects, thus limiting its practicality.

[0023] Ferroptosis is a form of necrotic cell death associated with iron-dependent oxidation of phospholipid membranes, which results in the loss of selective permeability of the plasma membrane and defects in the mitochondrial membrane. Since the avoidance of apoptosis-mediated cell death is a characteristic trait in human cancers, therapeutic approaches that mediate non-apoptotic mechanisms of cell death have become attractive treatment strategies for cancer. Ferroptosis itself promotes immune activation through the release of damage-associated molecular patterns (DAMPs), which can also contribute to the efficacy of immune checkpoint inhibitors. Furthermore, the abnormally increased amount of iron in many cancer types makes ferroptosis prone to occur and provides an indicator of selectivity to help normal tissues. In addition to iron, other transition metals such as zinc also promote ferroptosis.

[0024] Typical features of apoptosis, such as mitochondrial cytochrome c release, caspase activation, and chromatin fragmentation, were shown in initial studies that characterized ferroptosis to not be observed in ferroptotic cells. However, ferroptosis is associated with increased levels of intracellular reactive oxygen species (ROS) and is prevented by iron chelation or genetic inhibition of cellular iron uptake. In recent systematic studies of various lethality compounds with unique mechanisms, prevention of cell death by iron chelation is a rare phenomenon, suggesting that most triggers cannot access an iron-dependent lethality mechanism.

[0025] The standard pathway of ferroptosis induction involves inactivation of the central protective mechanism of the membrane against peroxidative damage, including those that control cysteine availability and glutathione biosynthesis. Glutathione peroxidase 4 (GPX4), a selenoprotein enzyme, has been shown to be the only enzyme capable of directly reducing complex hydroperoxides and thus protecting cells from ferroptosis and can be inactivated by direct or indirect targeting mechanisms such as depletion of intracellular glutathione. A number of potent ferroptosis inducers that trigger ferroptosis in vitro have been described, such as by depleting intracellular glutathione or GPX4, but many of these are inappropriate as clinical candidates because they target nodes that can be bypassed in vivo or require large amounts of inducer or additional delivery vehicles for activity.

[0026] In general, there is a compelling theoretical basis for inducing ferroptosis for the treatment of cancer, and in particular, clear cell renal carcinoma. First, loss of pVHL, an early event in ccRCC, promotes metabolic reprogramming that increases lipid storage and impairs fatty acid oxidation, and ccRCC cells sense ferroptosis. In this regard, HIF-2α, which increases as a result of pVHL deficiency, selectively concentrates polyunsaturated lipids, which are rate-limiting substrates for lipid peroxidation associated with ferroptosis. Thus, ccRCC cells are highly sensitive to ferroptosis induction, particularly to the oxidation of GPX4. Second, ccRCC is an iron-concentrating tumor and also increases sensitivity to ferroptosis. Third, the wild-type state associated with the mesenchymal phenotype and resistance to standard therapies are associated with ferroptosis sensitivity, suggesting the potential utility of ferroptosis inducers in drug-resistant tumors. Finally, the ferroptosis-susceptible state is also associated with an immunosuppressive phenotype, suggesting that cells resistant to immune checkpoint inhibitors may exhibit increased sensitivity to ferroptosis.

[0027] Strategies that selectively induce ferroptosis are attractive potential anti-tumor strategies for cancer because dysregulated iron metabolism and iron accumulation are frequently observed across both solid tumors and hematological malignancies. SUMMARY OF THE INVENTION

[0028] The present disclosure relates to novel compounds, more specifically, imidazopyridine and oxazolopyridine derivatives and analogs thereof, and methods of making and using such compounds. The present disclosure further relates to the use of these compounds as medicaments. In certain embodiments, the treatment of disorders associated with HIF-1α or HIF-2α upregulation or activation, and / or dysfunction of iron or lipid metabolism, which can be addressed by induction of ferroptosis, is contemplated. Such disorders can include certain cancer types such as clear cell renal cell carcinoma, breast cancer, liver cancer, pancreatic cancer, and glioblastoma. The present disclosure also relates to the use of the compounds for the manufacture of medicaments useful for treating such disorders. The present disclosure further relates to pharmaceutical compositions comprising the novel compounds, and methods for the preparation of the pharmaceutical compositions.

[0029] The present disclosure provides novel compounds that reduce HIF-1α and HIF-2α proteins by targeting protein iron-sulfur cluster assembly 2 (ISCA2). Without being bound to any particular theory, inhibition of ISCA2 disrupts cellular iron homeostasis, leading to an increase in cellular iron content. This can result in the loss of the [4Fe-4S] cluster within IRP1, which in turn promotes the functional switch from aconitase to IRE binding within IRP1 and inhibits the translation of HIF-2α mRNA. Since the production of HIF-2α is reduced or eliminated, these novel compounds block both the transcriptional and non-transcriptional targets of HIF-2α. Although the specific mechanism is unclear, the synthesis of HIF-1α is also reduced. Furthermore, these compounds interfere with cellular iron metabolism, triggering the iron deficiency response (brought about by an increase in IRP2 and a decrease in FTH1), thereby promoting the accumulation of iron and other transition metals that trigger ferroptosis. The present disclosure provides compounds useful for preventing or treating HIF-1 / 2α-related disorders and / or disorders associated with the accumulation of iron or lipids, where the induction of ferroptosis can be beneficial, inter alia, in solid tumors such as ccRCC, breast cancer, liver cancer, pancreatic cancer, and glioblastoma. The present disclosure demonstrates that these compounds efficiently reduce HIF-1α and HIF-2α proteins and induce ferroptosis. Accordingly, these compounds constitute a class of useful compounds that can be used in the treatment of HIF-1 / 2α, and / or iron-related disorders, including types of tumors driven by HIF-1 / 2α, and types of tumors and disorders associated with the accumulation of iron or lipids.

[0030] Embodiments of the present disclosure (or, in one aspect of the invention) include (or relate to) a compound of formula I, or a pharmaceutically acceptable salt thereof.

[0031]

Chemical formula

[0032] In some embodiments, each of X1 and X2 is independently CH2, O, S, or NH. In some embodiments, each of X3 and X4 is independently CH or N.

[0033] In some embodiments, Z is CH2 or O or S or NR A wherein, R A is H or C1-4 alkyl.

[0034] In some embodiments, each of C1, C2, C3, and C4 (C 1-4 ) is independently C, S, O, N, or sulfur dioxide.

[0035] In some embodiments, each of C1, C2, C3, and C4 (C 1-4 ) is independently C or N.

[0036] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently CH, CH2, O, or N.

[0037] In some embodiments, "a" (e.g., within a cyclic structure) represents an option of a single bond or a double bond. In some embodiments, each "a" is independently a single bond or a double bond.

[0038] In some embodiments, R1, R2, R3, and R4 (R 1-4Each of ( ), and each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently hydrogen, halo, CN, nitro, hydroxy, dioxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, di-C1-4 alkylaminosulfonylamino, and is selected from the absence. In some embodiments, (the above R 1-4 or of Ra-e) hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, or di-C1-4 alkylaminosulfonylamino is optionally substituted with 1, 2, or 3 groups independently selected from halo, CN, hydroxy, C1-3, alkoxy, amino, C1-3 alkylamino, di-C1-3-alkylamino, and none.

[0039] In some embodiments, each of R 1-4 or Ra-e, when present, together with one of R 1-4 or Ra-e, and, when present, the above R 1-4 or Ra-e to which each is attached, C 1-4Optionally form a 3- to 7-membered carbocyclic or 4- to 6-membered heterocyclic ring, each optionally substituted with 1, 2, 3, or 4 C1-3 alkyl groups, together with Ca-e.

[0040] In some embodiments, X1 is O or S or NH.

[0041] In some embodiments, X2 is O.

[0042] In some embodiments, Z is NH.

[0043] In some embodiments, each of C1, C2, C3, C4 is independently CH or N.

[0044] In some embodiments, Ca, Cb, Cc, Cd, and Ce are each CH, N.

[0045] In some embodiments, R1 is H, CH3,

[0046]

Chemical formula

[0047] is.

[0048] In some embodiments, R2 is H, CH3, Cl, CF3, OCH3,

[0049]

Chemical formula

[0050] is.

[0051] In some embodiments, R3 is H, OCH3, CF3.

[0052] In some embodiments, R4 is H, OCH3.

[0053] In some embodiments, Ra is H or OCH3.

[0054] In some embodiments, Rb is H, F, Cl, CH3CN, OCF3, OCH3, OCD3, or together with Rc, forms methylenedioxy, ethylenedioxy, furan, or hydrofuran.

[0055] In some embodiments, Rc is H, F, Cl, CH3, OCH3, CN, OCF3, OCD3, SCH3, N(CH3)2,

[0056]

Chemical formula

[0057] or together with Rb, forms methylenedioxy, ethylenedioxy, furan, or hydrofuran.

[0058] In some embodiments, Rd and Re are each independently H.

[0059] In some embodiments, R1 is not H. In some embodiments, R2 is not H. In some embodiments, R3 is not H. In some embodiments, R4 is not OCH3. In some embodiments, Ra is not H. In some embodiments, Rb is not H. In some embodiments, Rc is not F. In some embodiments, Rd is not H. In some embodiments, Re is not H. In some embodiments, any permutation or combination of the foregoing.

[0060] In some embodiments, the compound of formula I is not 5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazole-2-amine. In some embodiments, the following compound of formula I is not 5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazole-2-amine.

[0061] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ia, or a pharmaceutically acceptable salt thereof.

[0062]

Chemical formula

[0063] In some embodiments, X is S, O, or NH.

[0064] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently CH or N.

[0065] In some embodiments, R1 is H, CH3,

[0066]

Chemical formula

[0067] and so on.

[0068] In some embodiments, R2 is H, CH3, Cl, CF3, OCH3,

[0069]

Chemical formula

[0070] and so on.

[0071] In some embodiments, R3 is H or OCH3.

[0072] In some embodiments, Rb and Rc are independently H, F, Cl, CH3, OCH3, CN, OCF3, OCD3, SCH3, N(CH3)2,

[0073]

Chemical formula

[0074] Selected from. Alternatively, both Rb and Rc form methylenedioxy or ethylenedioxy.

[0075] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ib or a pharmaceutically acceptable salt thereof.

[0076]

Chemical formula

[0077] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N.

[0078] In some embodiments, R1 is H.

[0079] In some embodiments, R2 is H.

[0080] In some embodiments, R3 is H or OCH3.

[0081] In some embodiments, each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from H, F, and OCH3.

[0082] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ic or a pharmaceutically acceptable salt thereof.

[0083]

Chemical formula

[0084] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N.

[0085] In some embodiments, R1 is H.

[0086] In some embodiments, R2 is H or Cl.

[0087] In some embodiments, each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from H and F.

[0088] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Id or a pharmaceutically acceptable salt thereof.

[0089]

Chemical formula

[0090] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N.

[0091] In some embodiments, R1 is H.

[0092] In some embodiments, R2 is H, CH3, or Cl.

[0093] In some embodiments, R3 and R4 are H or OCH3.

[0094] In some embodiments, Ra, Rb, and Re are H.

[0095] In some embodiments, Rb is H, F, OCH3, OCD3, or together with Rc forms methylenedioxy, ethylenedioxy, furan, or hydrofuran.

[0096] In some embodiments, Rc is H, F, CH3, OCH3, OCD3, OC2H5, * , or

[0097]

Chemical formula

[0098] is, or together with Rb, forms methylenedioxy, ethylenedioxy, furan, or hydrofuran.

[0099] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ie, or a pharmaceutically acceptable salt thereof.

[0100]

Chemical formula

[0101] In some embodiments, each of C1, C2, C3, and C4 (C 1-4 ) is independently C or N.

[0102] In some embodiments, each of R1, R2, R3, and R4 (R 1-4 ), and each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from hydrogen, halo, CN, nitro, hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, di-C1-4 alkylaminosulfonylamino, and absent. In some embodiments, (the above R 1-4、or a hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, or di-C1-4 alkylaminosulfonylamino of Ra-e is optionally substituted with 1, 2, or 3 groups independently selected from halo, CN, hydroxy, C1-3, alkoxy, amino, C1-3 alkylamino, and di-C1-3-alkylamino.

[0103] In some embodiments, each of Ra-e is independently, if present, together with one of Ra-e, and if present, together with Ca-e to which the respective Ra-e is attached, each optionally forms a 3- to 7-membered carbocyclic or 4- to 6-membered heterocyclic ring optionally substituted with 1, 2, 3, or 4 C1-3 alkyl groups.

[0104] Non-limiting examples or embodiments of the compounds provided include the following:

[0105]

Chemical formula

[0106]

Chemical formula

[0107]

Chemical formula

[0108]

Chemical formula

[0109] In some embodiments, the compound is not 5-(4-fluorophenyl)-N-(4-methoxybenzothiazol-2-yl)-1,3,4-oxadiazol-2-amine:

[0110]

Chemical formula

[0111] Compositions described herein include, but are not limited to, hydrates, solvates, polymorphs, isomers, tautomers, pharmaceutically acceptable salts of the compounds, and pharmaceutically acceptable salts of the tautomers.

[0112] Embodiments include pharmaceutical formulations, medicaments containing the compound, pharmaceutical formulations, medicaments, methods for preparing the compound, and methods for treating a patient using the provided pharmaceutical formulations and compound.

[0113] Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of the disclosed compound, with or without a pharmaceutically acceptable carrier.

[0114] Also disclosed are synthetic methods for making the disclosed compounds. Accordingly, embodiments of the present disclosure include methods for making the disclosed compounds according to the disclosed schemes. In a further aspect, the products of the disclosed synthetic methods are disclosed.

[0115] Embodiments include methods for inhibiting HIF-1α or HIF-2α (HIF-1 / 2α) activity and methods for inducing ferroptosis. Such methods can include contacting HIF-1 / 2α with an effective amount of one or more of the compounds disclosed herein and / or inducing ferroptosis with one or more of the compounds disclosed herein. By way of example and not limitation, one or more of the compounds can be contacted with HIF-1 / 2α such that they bind to or interact with HIF-1 / 2α, HIF-1 / 2α-encoding mRNA, the gene encoding HIF-1 / 2α, or a protein that regulates the HIF-1 / 2α gene, protein, or mRNA. Alternatively, one or more of the compounds can effect ferroptosis and / or induce a process that decreases HIF-1 / 2α protein or mRNA by contacting a component of the ISC complex such as ISCA2.

[0116] A method of treating a disorder in a mammal associated with HIF-1 / 2α activity and / or iron dysfunction, the method comprising administering to the mammal a therapeutically effective amount of one or more of the disclosed compounds or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof is also disclosed.

[0117] A method of inhibiting HIF-1 / 2α activity and inducing ferroptosis in a mammal, the method comprising administering to the mammal a therapeutically effective amount of at least one of the disclosed compounds or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof is also disclosed.

[0118] A method of inhibiting HIF-1 / 2α activity and inducing ferroptosis in at least one cell, the method comprising contacting the at least one cell with an effective amount of at least one of the disclosed compounds or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof is also disclosed.

[0119] A method for treating a disorder associated with HIF-1 / 2α activity and / or iron or lipid dysfunction in a mammal by inducing an immune response in the mammal, the method comprising administering to the mammal a therapeutically effective amount of a disclosed compound or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof, the compound being capable of eliciting a beneficial immune response in the treatment of a disorder associated with HIF-1 / 2α and / or iron or lipid dysfunction is also disclosed. Such disorders can be, but are not limited to, any type of cancer in which HIF-1 / 2α activity and / or iron or lipid is involved, or any disease caused by bacteria and / or viruses.

[0120] A method for inducing cell death by lipid peroxidation is also disclosed. The method can comprise administering to the cell one or more of the disclosed compounds.

[0121] A method for inducing the accumulation of iron in a cell is also disclosed. The method can comprise administering to the cell one or more of the disclosed compounds.

[0122] A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a disclosed compound or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof is also disclosed.

[0123] A kit comprising at least one disclosed compound or a pharmaceutically acceptable salt, tautomer, isomer, hydrate, solvate, or polymorph thereof is also disclosed.

[0124] Also disclosed is a method for manufacturing a medicament, which comprises combining at least one of the disclosed compounds or at least one of the disclosed products with a pharmaceutically acceptable carrier or diluent. In a further aspect, the present disclosure relates to the use of the disclosed compounds in the manufacture of a medicament for treating HIF-1 / 2α activity dysfunction and / or lipid / iron regulation abnormalities. In a further aspect, the present disclosure relates to the use of the disclosed compounds in the use of a medicament for treating disorders of uncontrolled cell proliferation.

[0125] Also disclosed is the use of the disclosed compounds or the disclosed products in the manufacture of a medicament for treating disorders in mammals associated with HIF-1 / 2α and / or iron or lipid dysfunction.

[0126] Aspects of the present disclosure may be described and claimed in certain statutory classes, such as system statutory classes, but this is for convenience only, and those skilled in the art will understand that each aspect of the present disclosure may be described and claimed in any statutory class. Unless otherwise explicitly specified, none of the methods or aspects shown in this specification are intended to be construed as requiring their steps to be performed in a particular order. Accordingly, when a method claim does not specifically define in the claim or description that the steps should be limited to a particular order, in no way is it intended to define an order in any aspect. This is the same for any possible implicit interpretation criteria, including logical matters regarding the context of steps or workflows, general meanings derived from grammatical composition or punctuation, or the number or type of aspects described in this specification.

[0127] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention will be better understood by reference to these one or more drawings in combination with the detailed description of the specific embodiments presented herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0128]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 8E

Figure 8F

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 12A

Figure 12B

Figure 12C

Figure 12D

Figure 12E

Figure 12F

Mode for Carrying Out the Invention

[0129] Before detailing various embodiments of the present disclosure, it should be understood that the present disclosure is not limited to specific parameters and descriptions of the specifically illustrated systems, methods, and / or products that may vary between one embodiment and the next. Thus, while certain embodiments of the present disclosure are detailed with reference to specific features (such as structures, parameters, properties, steps, components, ingredients, members, elements, parts, and / or portions, etc.), the description is illustrative and should not be construed as limiting the scope of the present disclosure and / or the claimed invention. Further, the terms used herein are for the purpose of describing embodiments and do not necessarily have the intention of limiting the scope of the present disclosure and / or the claimed invention.

[0130] Although the detailed description is divided into chapters, the chapter headings and the content of each chapter are not intended to be self - contained descriptions and embodiments. Rather, the content of each chapter in the detailed description is intended to be read and understood as a whole, where elements of one chapter may relate to, and / or may inform, other chapters. Thus, embodiments specifically disclosed in one chapter may also relate to, and / or may serve a role in, additional and / or alternative embodiments in another chapter having the same and / or similar systems, devices, methods, and / or terms.

[0131] To assist in the understanding of the scope and content of the foregoing and the following descriptions, as well as the appended claims, some terms are explained and directly defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0132] As used herein, including in the claims, the terms "comprising", "comprise", "comprises" and the like shall be inclusive and / or non-limiting and shall not exclude additional, unrecited elements or method steps. Further, as used herein, including in the claims, the terms "including", "having", "involving", "containing", "characterized by", variations thereof (e.g., "includes", "have", and "involves", "contains"), and like terms shall be inclusive and / or non-limiting and shall have the same meaning as the term "comprising" and its variations (e.g., "comprise" and "comprises") and shall not exclude additional, unrecited elements or method steps.

[0133] As used herein, the transitional phrases "consisting of", "consist of", and the like shall be limiting and shall exclude additional, unrecited elements or method steps.

[0134] As used herein, the transitional phrase "consisting essentially of" shall be construed to mean that a claim's scope is to include the specific materials or steps recited in the claim's scope, and those that do not materially affect the "basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in original), and also see MPEP § 2111.03. Accordingly, the term "consisting essentially of" as used in the claims of the present disclosure is not intended to be construed as equivalent to "comprising".

[0135] As used herein, the term "composition" includes products, formulations, and mixtures, as well as devices, apparatuses, assemblies, kits, etc. Similarly, the term "method" includes processes, procedures, steps, etc. The terms "formulation" and "composition" can be used interchangeably herein, unless the context clearly indicates otherwise.

[0136] As used herein, the term "method" also encompasses processes, procedures, steps, etc. Any step recited in the methods described herein and / or recited in the claims can be performed in any suitable order, unless otherwise described (either explicitly or implicitly), and is not necessarily limited to the order described and / or recited. Further, the term "product" also contemplates systems, compositions, kits, etc.

[0137] Various aspects of the present disclosure, including systems, methods, and / or products, can be described with reference to one or more embodiments or implementations, which are essentially exemplary. As used herein, the terms "embodiment" and "implementation" mean "serving as an example, as an instance, or as an illustration", and should not necessarily be construed as being more preferred or advantageous than other aspects disclosed herein. Further, references to "implementations" of the present disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to illustrate examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the description of the present invention.

[0138] It should be noted that embodiments of the present disclosure can include one or more of any combination of two or more of the features described herein. As used herein, "feature(s)" and similar terms can include, for example, one or more compositions, components, constituents, elements, members, parts, portions, systems, methods, steps, configurations, parameters, properties, or other readily available aspects of the subject matter. Embodiments can include any of the features, options, and / or possibilities listed elsewhere in the present disclosure, including those in other aspects or embodiments of the present disclosure. Each of the foregoing, following, and / or other features described herein represents different embodiments of the present disclosure, but the features can be combined, and / or combinable, in any suitable combination and / or order, with one or more other features, with one or more additional features included in or implemented between them, or without them, to form unique embodiments contemplated by the present disclosure. Any two or more such combinations of such features represent different embodiments of the present disclosure. Accordingly, the present disclosure is not limited to the specific combinations of exemplary embodiments detailed herein, and the disclosure of a particular feature with respect to a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of the feature with respect to that particular embodiment.

[0139] Furthermore, unless a feature is required in a particular embodiment, the features described in the various embodiments can be optional and may not be included in other embodiments of the present disclosure. Further, unless a feature is described as requiring another feature in combination with that feature, any feature herein can be combined with any other feature, whether the same or different, of the embodiments disclosed herein. Similarly, any steps recited in any method described herein and / or recited in the claims can be performed in any suitable order, unless otherwise stated (either explicitly or implicitly), and are not necessarily limited to the order described and / or recited. However, it is also possible that such steps may be required to be performed in a particular order in certain embodiments of the present disclosure.

[0140] As used throughout this application, the words "can" and "may" are used in a permissive sense (i.e., having the possibility) rather than a mandatory sense (i.e., an obligatory sense).

[0141] The word "or" as used herein means any one of the particular list of elements and also includes any combination of the elements of that list.

[0142] As used in this specification and the appended claims, the singular forms "a", "an", and "the" also contemplate reference to the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a layer" includes one, two, or more layers. Similarly, references to a plurality of referents must be construed as including a single referent and / or a plurality of referents unless the content and / or context clearly indicates otherwise. Thus, references to "layers" do not necessarily require a plurality of such layers. Instead, one or more layers are understood to be contemplated herein, regardless of combination. Similarly, for example, reference to "a functional group", "an alkyl", or "a residue" includes mixtures such as two or more such functional groups, alkyls, or residues.

[0143] As used herein, the nomenclature of compounds containing organic compounds can be given using the common names, IUPAC, IUBMB, or CAS recommendations regarding nomenclature. If one or more stereochemical features are present, the Cahn-Ingold-Prelog rules regarding stereochemistry are used to represent stereochemical priorities, E / Z specifications, etc. One of ordinary skill in the art, given a name, can systematically reduce the structure of a compound using the naming rules or can readily confirm the structure of the compound by any of commercially available software such as TM (Cambridgesoft Corporation, U.S.A.).

[0144] Ranges may be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, further aspects include from one particular value and / or to another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about” it will be understood that the particular value forms further aspects. Further, it will be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. It will also be understood that there are several values disclosed herein, and that each value, in addition to the value itself, is also disclosed herein as “about” that particular value. For example, if the value “10” is disclosed, “about 10” is also disclosed (e.g., 10 ± 10%, or 10 ± 5%). It will also be understood that each unit between two particular units is also disclosed. For example, if 10 - 15 is disclosed, 11, 12, 13, and 14 are also disclosed.

[0145] References in the specification and concluding claims to the weight parts of a particular element or component in a composition represent the weight relationship between the element or component and any other element or component in the composition or article in which the weight parts are expressed. Thus, in a compound containing 2 weight parts of component X and 5 weight parts of component Y, X and Y are present in a weight ratio of 2:5 and are present in such ratio regardless of whether additional components are contained in the compound.

[0146] The weight percent (wt.%) of a component is based on the total weight of the formulation or composition in which the component is included, unless otherwise specifically stated.

[0147] As used herein, the term "HIF-1α" means hypoxia-inducible factor 1-α, which is well-known in the art. Non-limiting examples of HIF-1α are encoded by the gene HIF1A. HIF-1α is a transcription factor that activates gene transcription in response to hypoxia or hypoxemia. Homologs, paralogs, orthologs, etc. of HIF-1α, as well as genes encoding these proteins, are well-known in the art and can be readily retrieved from commonly available databases. Such additional homologs, paralogs, orthologs, etc. of HIF-1α can be considered as described herein.

[0148] As used herein, the term "HIF-2α" means hypoxia-inducible factor 2-α, which is well-known in the art. Non-limiting examples of HIF-2α are encoded by the gene EPAS1. HIF-2α is a transcription factor that activates gene transcription in response to hypoxia or hypoxemia. Homologs, paralogs, orthologs, etc. of HIF-2α, as well as genes encoding these proteins, are well-known in the art and can be readily retrieved from commonly available databases. Such additional homologs, paralogs, orthologs, etc. of HIF-2α can be considered as described herein.

[0149] As used herein, hypoxemia is defined as an oxygen threshold below what is required for normal physiological function of a cell or tissue and is typically defined as an oxygen percentage of <5%.

[0150] As used herein, the term "IRP1" means iron-responsive element-binding protein 1, which is well-known in the art. Non-limiting examples of IRP1 are encoded by the gene ACO1. Homologs, paralogs, orthologs, etc. of IRP1, as well as genes encoding these proteins, are well-known in the art and can be readily retrieved from commonly available databases. Such additional homologs, paralogs, orthologs, etc. of IRP1 can be considered as described herein.

[0151] As used herein, the term "IRP2" means iron-responsive element-binding protein 2, which is well-known in the art. Non-limiting examples of IRP2 are encoded by the gene IREB2. Homologs, paralogs, orthologs, etc. of IRP2, in addition to the genes encoding these proteins, are well-known in the art and can be easily retrieved in commonly available databases. Such additional homologs, paralogs, orthologs, etc. of IRP2 can be considered as described herein.

[0152] As used herein, the term "FTH1" means ferritin heavy chain or the heavy subunit of ferritin, which is a major intracellular iron storage protein in the cell and is well-known in the art. Homologs, paralogs, orthologs, etc. of FTH1, in addition to the genes encoding these proteins, are well-known in the art and can be easily retrieved in commonly available databases. Such additional homologs, paralogs, orthologs, etc. of FTH1 can be considered as described herein.

[0153] As used herein, the term "ISCA2" means iron-sulfur cluster assembly 2, which is a mitochondrial protein involved in the synthesis of iron-sulfur clusters. ISCA2 interacts with protein ISCA1, which means iron-sulfur cluster assembly 1, and IBA57, which means an iron-sulfur cluster assembly factor for biotin synthase and aconitase-like proteins, and is involved in the mitochondrial iron-sulfur cluster assembly pathway. Homologs, paralogs, orthologs, etc. of ISCA1, ISCA2, and IBA57, in addition to the genes encoding these proteins, are well-known in the art and can be easily retrieved in commonly available databases. Such additional homologs, paralogs, and orthologs can be considered as described herein.

[0154] As used herein, the term "inhibiting iron metabolism" means the ability to interfere with a cell's ability to sense, utilize, absorb, accumulate, and / or otherwise track iron, whether in its free form or as a complex with a carrier such as, by way of non-limiting example, transferrin, lipocalin, or ferritin.

[0155] As used herein, the term "ferroptosis" means a regulated cell death mechanism that is morphologically, biochemically, and genetically distinct from apoptosis, various forms of necrosis, and autophagy. Ferroptosis is characterized by the overwhelming accumulation of lethal lipid-derived reactive oxygen species in an iron-dependent manner. The increased iron levels observed in many solid tumor types, including ccRCC and breast cancer, tend to drive these tumors towards ferroptotic death. Other transition metals with oxidizing capabilities, such as copper or zinc, may also contribute to ferroptosis.

[0156] As used herein, the term "subject" can be a vertebrate such as a mammal, fish, bird, reptile, or amphibian. Thus, subjects for the methods disclosed herein can be, by way of non-limiting example, humans, non-human primates, domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, flies, zebrafish, etc.). This term does not denote a particular age or sex. Thus, both male and female subjects, as well as adult and neonatal subjects, and fetuses are intended to be included. In one aspect, the subject is a mammal. A patient means a subject suffering from a disease or disorder. The term "patient" includes human and non-human subjects. In some aspects of the disclosed methods, the subject is diagnosed, prior to the administration step, as in need of treatment for a disorder associated with a dysfunction of HIF-1 / 2α and / or iron or lipid metabolism, including, but not limited to, a disorder of uncontrolled cell proliferation. In a further aspect, the subject is determined by one of ordinary skill in the art, e.g., a physician, prior to the administration step, to have a high likelihood of benefiting from an effect that alleviates or reduces the symptoms or other negative effects of a disease or disorder associated with a dysfunction of HIF-1 / 2α and / or iron or lipid metabolism, including, but not limited to, a disorder of uncontrolled cell proliferation.

[0157] As used herein, the term "treatment" means medically managing a patient with the intent to cure, relieve, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment directed particularly towards improvement of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed towards removal of the cause of the relevant disease, pathological condition, or disorder. Further, the term includes palliative treatment, i.e., treatment aimed at relief of symptoms rather than treatment of the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the progression of a relevant disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy for the improvement of a relevant disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), (i) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease, (ii) inhibiting a disease, i.e., inhibiting its onset, (iii) alleviating a disease, i.e., causing regression of the disease, and (iv) reducing the symptoms of an underlying disease and / or reducing and / or eliminating one of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms. In one aspect, the subject is a mammal such as a primate, and in a further aspect, the subject is a human.

[0158] As used herein, the term "prevent" or "preventing" means, among other things, precluding, averting, forestalling, preempting, stopping, or inhibiting something from occurring by prior action. When the words "reduce," "weaken," "inhibit," or "prevent" are used herein, unless otherwise indicated, the use of the other three words is also to be understood as being explicitly disclosed.

[0159] As used herein, the term "diagnosed" means that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, "diagnosed with a disorder of uncontrolled cell proliferation" means that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has a condition that can be diagnosed or treated by a compound or composition that inhibits HIF-1 / 2α and / or inhibits iron or lipid metabolism. As a further non-limiting example, "diagnosed as in need of inhibition of HIF-1 / 2α" means that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has a condition characterized by HIF-1 / 2α and / or iron or lipid dysfunction. Such a diagnosis can refer to disorders such as disorders of uncontrolled cell proliferation, cancer, etc., as discussed herein. "Diagnosed as in need of treatment of one or more diseases of uncontrolled cell proliferation associated with HIF-1 / 2α and / or iron or lipid dysfunction" means, as used herein, that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has one or more diseases of uncontrolled cell proliferation associated with HIF-1 / 2α and / or iron or lipid dysfunction.

[0160] Furthermore, "diagnosed as in need of inhibition of iron accumulation" means that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has a condition characterized by iron accumulation or iron metabolism dysfunction. Such a diagnosis can refer to disorders such as disorders of uncontrolled cell proliferation, cancer, hemochromatosis, etc., as discussed herein. "Diagnosed as in need of treatment of one or more diseases of uncontrolled cell proliferation associated with iron accumulation" means, as used herein, that a person skilled in the art, such as a physician, has conducted a physical examination and has found that the person has one or more diseases of uncontrolled cell proliferation associated with iron accumulation dysfunction.

[0161] Alternatively, the term "diagnosed" in the foregoing examples can also mean recognizing or determining a disease or condition from its signs and / or symptoms, which can occur independently of a physical examination and has been found to be likely to provide benefits through diagnosis, treatment, or other intervention.

[0162] As used herein, phrases such as "identified as in need of treatment of a disorder" mean selecting a subject based on the need for treatment of the disorder or on the likelihood of a benefit in alleviating or reducing the negative impact of a disease or disorder. For example, a subject can be identified as in need of treatment of a disorder (e.g., a disorder associated with a dysfunction of HIF-1 / 2α, or a disorder associated with a dysfunction of iron or lipid metabolism) based on an early diagnosis or measurement by one of ordinary skill in the art, and then can be subjected to treatment of the disorder. In this example, "in need of" also means "likely to provide a benefit in alleviating or reducing the negative impact of a disease or disorder." The identification can, in one aspect, be performed by a person different from the person making the diagnosis. In a further aspect, the administration can also be performed by a person other than the person who subsequently administers it.

[0163] As used herein, "a disorder associated with HIF-1 / 2α activity dysfunction" or "a disorder associated with iron or lipid metabolism dysfunction" refers to any disorder in which HIF-1 / 2α activity or iron or lipid metabolism is abnormal and / or exceeds the normal physiological range. By way of non-limiting example, HIF-1 / 2α activity may exceed the activity expected in a normal cell, tissue, subject, or sample derived from a subject. Similarly, the levels of iron or lipids, or proteins and tissues known to be associated with iron or lipids, including but not limited to ferritin, transferrin, hematocrit, hemoglobin, IRP1, or IRP2, may be less than or greater than the normal physiological range in a cell, tissue, subject, or sample derived from a subject. Examples of such disorders associated with HIF-1 / 2α and / or iron or lipid metabolism dysfunction include clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer, other subtypes of kidney cancer (e.g., the chromophobe papillary subtype), ovarian cancer, liver cancer (hepatocellular carcinoma), pancreatic cancer, breast cancer, neuroblastoma, glioblastoma (GBM), non-small cell lung cancer, altitude sickness associated with increased blood viscosity, and excessive EPO, as well as various hematological disorders, but are not limited thereto. Measurement of HIF-1 / 2α activity and / or iron or lipid metabolism that is abnormal or exceeds the normal physiological range can be performed by comparing the above activities or levels to normal (disease-free) cells, tissues, subjects, or samples from a subject.

[0164] As used herein, the terms "administering" and "administration" mean any method of providing a pharmaceutical to a subject. Such methods are well known to those of skill in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, buccal administration, intracerebral administration, rectal administration, sublingual administration, intraoral administration, intraurethral administration, and parenteral administration including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, i.e., administered to treat an existing disease or condition. Further, in various embodiments, the preparation can be administered prophylactically, i.e., administered to prevent a disease or condition.

[0165] As used herein, the term "contacting" means bringing into contact the disclosed compound with a cell, target receptor, or other biological component in a manner such that the compound affects the activity of the target (e.g., receptor, cell, etc.) either directly, i.e., by interacting with the target itself, or indirectly, i.e., by interacting with another molecule, cofactor, factor, or protein upon which the activity of the target depends.

[0166] As used herein, the terms "effective amount" and "amount effective to" mean an amount sufficient to achieve a desired result or to affect an undesirable condition. For example, a "therapeutically effective amount" means an amount sufficient to achieve a desired therapeutic result or to affect an undesirable symptom. A "therapeutically effective amount" may be insufficient to cause adverse side effects. The specific therapeutically effective dosage level for any particular patient will depend upon a variety of factors including the disorder being treated, the severity of the disorder; the specific composition being utilized; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound being utilized; the duration of the treatment; agents used in combination with, or concurrently with, the specific compound employed, and like factors well known to the medical practitioner. For example, it is within the skill of the art to initiate administration of a compound at a level below that required to achieve the desired therapeutic effect and to increase the dosage gradually until the desired effect is achieved. If desired, the effective daily dosage may be divided into multiple dosages for purposes of administration. Consequently, single dose compositions may contain such amounts, or an approximation thereof, which constitute the effective daily dosage. Dosages may be adjusted by the individual physician if so indicated by the exigencies of the case. Dosages may vary and may be administered in one or more dose administrations daily, for one day or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, the preparation may be administered in a "prophylactically effective amount", i.e., an amount effective to prevent a disease or condition.

[0167] As used herein, "EC 50 " is intended to mean the concentration of a substance (e.g., a compound or agent) required for 50% agonism or activation of a biological process, or a component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. In one aspect, EC 50 means the concentration of a substance required for 50% agonism or activation in vivo, as further defined elsewhere herein. In a further aspect, EC 50 means the concentration of an agonist or activator that elicits a response midway between baseline and maximum response.

[0168] As used herein, "IC 50 " is intended to mean the concentration of a substance (e.g., a compound or agent) required for 50% inhibition of a biological process, or a component of a process, including proteins, subunits, organelles, ribonucleoproteins, etc. For example, IC 50 means the concentration required for 50% inhibition in vivo, or the concentration of a substance whose inhibition is measured in vitro, as further defined elsewhere herein. Alternatively, IC 50 means the maximum half-maximal (50%) inhibitory concentration (IC) of a substance. Inhibition is measured in cell lines including, but not limited to, 786-0, ACHN, RCC4, A498, Caki, HT29, AN3 CA, BT-20, BT-549, HCT 116, HER218, MCF7, MDA-MB-231, MDA-MB-235, MDA-MB-435S, MDA-MB-468, PANC-1, PC-3, SK-N-MC, T-47D, and U-87 MG.

[0169] The term "pharmaceutically acceptable" describes a substance that is not biologically or otherwise undesirable, i.e., a substance that does not cause unacceptable levels of undesirable biological effects or that does not interact harmfully.

[0170] The term "stable" means a compound that does not substantially change when subjected to conditions for production, detection, and / or recovery, purification, and use for one or more of the purposes disclosed herein.

[0171] As used herein, the term "derivative" has a structure derived from the structure of a parent compound (e.g., a compound disclosed herein), the structure of which is sufficiently similar to those disclosed herein such that, based on that similarity, it is predicted by one of ordinary skill in the art to exhibit the same or similar activity and utility as the claimed compound, or, as a precursor, to induce the same or similar activity as the claimed compound. Non-limiting examples of derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0172] As used herein, the term "pharmaceutically acceptable carrier" means a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powders. Such powders can be used immediately before use for reconstitution into a sterile injectable solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose, and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, antibacterial and antifungal agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be achieved by containing various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents such as sugars, sodium chloride, etc. may also be included. The duration of absorption of injectable pharmaceutical forms can be effected by including agents such as aluminum monostearate and gelatin that delay absorption. Injectable depot forms are prepared by forming a microencapsulation matrix of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The rate of drug release can be controlled depending on the ratio of the drug to the polymer and the nature of the specific polymer used. Depot injectable formulations can also be prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers include, but are not limited to, sugars such as lactose. In certain embodiments, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0173] The residues of chemical species used in this specification and in the claims that state the conclusion mean a part that is a product obtained from a chemical species, or a subsequent formulation or chemical product, in a particular reaction scheme, regardless of whether the part is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester means one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester means one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether sebacic acid or its ester was reacted to obtain the polyester.

[0174] As used herein, the term "substituted" is intended to encompass all possible substituents of an organic compound. In one broad aspect, possible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of the organic compound. Exemplary substituents include, but are not limited to, those described hereinafter. The acceptable substituents may be one or more and may be the same or different for a suitable organic compound. As used herein, a heteroatom such as nitrogen may have a hydrogen substituent and / or any acceptable substituent of the organic compounds described herein that satisfies the valence of the heteroatom. The present disclosure is not intended to be limited in any way by the acceptable substituents of the organic compounds. Also, the terms "substituted" or "substituted with" imply that such substitution follows the acceptable valences of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not undergo spontaneous transformations such as rearrangement, cyclization, elimination, etc. In certain embodiments, unless explicitly indicated otherwise, individual substituents may be further optionally substituted (i.e., may be further substituted or unsubstituted). Unless otherwise explicitly indicated in the context, the term "substituted" when used in connection with a substituent, functional group, or (organic or inorganic) compound generally does not include replacement (i.e., substitution) of such substituent, functional group, or conjugate.

[0175] In the definitions of various terms, "A" 1 ", "A" 2 ", "A" 3 ", and "A" 4」 etc. are used herein as general symbols for representing various specific substituents. These symbols can be any substituents and are not limited to those disclosed herein. When defined as a particular substituent in one example, they can be defined as several other substituents (plural) in another example.

[0176] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. By way of non-limiting example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (e.g., 1 to 4) carbon atoms.

[0177] As a non-limiting example, the "C1-C3 alkyl" group can be selected from methyl, ethyl, n-propyl, i-propyl, and cyclopropyl, or a subset thereof. In certain embodiments, the "C1-C3 alkyl" group may optionally be further substituted. As a non-limiting example, the "C1-C4 alkyl" group can be selected from methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, and cyclobutyl, or a subset thereof. In certain embodiments, the "C1-C4 alkyl" group may optionally be further substituted. As a further non-limiting example, the "C1-C6 alkyl" group can be selected from methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, and cyclohexane, or a subset thereof. In certain embodiments, the "C1-C6 alkyl" group may optionally be further substituted. As a non-limiting example, the "C1-C8 alkyl" group can be selected from methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane, cycloheptane, octane, and cyclooctane, or a subset thereof. In certain embodiments, the "C1-C8 alkyl" group may optionally be further substituted.As a non-limiting example, the "C1-C12 alkyl" group can be selected from methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane, cycloheptane, octane, cyclooctane, nonane, cyclononane, decane, cyclodecane, undecane, cycloundecane, dodecane, and cyclododecane, or a subset thereof. In certain embodiments, the "C1-C12 alkyl" group may optionally be further substituted.

[0178] Throughout this specification, the term "alkyl" is generally used to mean both unsubstituted alkyl groups and substituted alkyl groups, although substituted alkyl groups are also generally referred to herein by identifying specific substituent(s) on the alkyl group. As a non-limiting example, the term "halogenated alkyl" or "haloalkyl" specifically means an alkyl group substituted with one or more halides, such as fluorine, chlorine, bromine, or iodine. The term "alkoxyalkyl" specifically means an alkyl group substituted with one or more alkoxy groups, as described hereinafter. The term "alkylamino" specifically means an alkyl group substituted with one or more amino groups, etc., as described hereinafter. When "alkyl" is used in one instance and a specific term such as "alkyl alcohol" is used in another instance, this does not mean that the term "alkyl" does not also refer to specific terms such as "alkyl alcohol".

[0179] This convention is also used with respect to other groups described herein. That is, terms such as "cycloalkyl" mean both unsubstituted and substituted cycloalkyl moieties, where the substituted moieties are further specifically identified herein. For example, a particular substituted cycloalkyl may be referred to as, for example, "alkylcycloalkyl". Similarly, a substituted alkoxy may be specifically referred to as, for example, "halogenated alkoxy", a particular substituted alkenyl may be specifically referred to as, for example, "alkenyl alcohol", and so on for others. Again, the convention of using general terms such as "cycloalkyl" and specific terms such as "alkylcycloalkyl" does not mean that the general term does not also include the specific term.

[0180] As used herein, the term "cycloalkyl" is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, etc. The term "heterocycloalkyl" is, as described above, a type of cycloalkyl group and is included in the meaning of the term "cycloalkyl", where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl groups and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl groups and heterocycloalkyl groups can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, nitrile, sulfonamide, or thiol, as described herein.

[0181] As used herein, the term "aryl" refers to a group containing any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, etc. The term "aryl" also includes "heteroaryl", which is defined as a group containing an aromatic group in which at least one heteroatom is incorporated into the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Similarly, the term "non-heteroaryl", which is also included in the term "aryl", defines a group containing an aromatic group that does not contain a heteroatom. An aryl group can be substituted or unsubstituted. An aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, sulfo-oxo, nitrile, sulfonamide, or thiol, as described herein. The term "biaryl" is a particular type of aryl group and is included in the definition of "aryl". Biaryl means two aryl groups bonded to each other via a fused ring structure, such as in naphthalene, or bonded via one or more carbon-carbon bonds, such as in biphenyl.

[0182] As used herein, the terms "halogen", "halide", and "halo" mean fluorine, chlorine, bromine, and iodine, which are halogens. In various embodiments, it is also contemplated that the halogen can be selected from fluoro, chloro, bromo, and iodo, or any combination thereof. Further, and / or alternatively, the halogen can be any one of fluoro, chloro, bromo, or iodo. By way of non-limiting example, the halogen can be selected from fluoro, chloro, and bromo. As a further non-limiting example, the halogen can be selected from fluoro and chloro. As a further non-limiting example, the halogen can be selected from chloro and bromo. As a further non-limiting example, the halogen can be selected from bromo and iodo. As a further non-limiting example, the halogen can be selected from chloro, bromo, and iodo. In one embodiment, the halogen can be fluoro. In a further embodiment, the halogen can be chloro. In still a further embodiment, the halogen is bromo. In yet a further embodiment, the halogen is iodo.

[0183] In certain embodiments, it is also contemplated that pseudohalogens (e.g., triflate, mesylate, tosylate, brosylate, etc.) can be used in place of the halogen. For example, in certain embodiments, the halogen can be replaced by a pseudohalogen. As a further non-limiting example, the pseudohalogen can be selected from triflate, mesylate, tosylate, and brosylate. In one embodiment, the pseudohalogen is triflate. In a further embodiment, the pseudohalogen is mesylate. In a further embodiment, the pseudohalogen is tosylate. In a further embodiment, the pseudohalogen is brosylate.

[0184] As used herein, the term "heterocyclic ring" means a monocyclic or polycyclic aromatic or non-aromatic ring system in which at least one of the ring members is other than carbon. Examples of heterocyclic rings include, but are not limited to, azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxadiazole (including 1,2,3-oxadiazole, 1,2,5-oxadiazole, and 1,3,4-oxadiazole), piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine (including 1,2,4,5-tetrazine), tetrazole (including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole), thiadiazole (including 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole), thiazole, thiophene, triazine (including 1,3,5-triazine and 1,2,4-triazine), triazole (including 1,2,3-triazole and 1,3,4-triazole), etc.

[0185] As used herein, the term "hydroxyl" is represented by the formula -OH (or, R-OH). When shown, the hydroxyl group (or, "hydroxy" substituent) can be "substituted" or "optionally substituted", thereby forming, for example, an ether represented by the formula R-O-R'.

[0186] As used herein, "R 1 ", "R 2 ", "R 3 ", "R n " (where n is an integer) can independently have one or more of the above groups. For example, R 1When it is a linear alkyl group, one of the hydrogen atoms of the alkyl group may be optionally substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, or the like. Depending on the selected group, the first group may be incorporated into the second group, or the first group may be pendant (i.e., attached) to the second group. As a non-limiting example, in the phrase "alkyl group containing an amino group", the amino group may be incorporated into the main chain of the alkyl group. Alternatively, the amino group may be bonded to the main chain of the alkyl group. The nature of the selected group(s) determines whether the first group is embedded in or attached to the second group.

[0187] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each position where substitution is possible for that group, and when more than one position in any given structure may be substituted by more than one substituent selected from a particular group, the substituents may be the same or different at all positions. Combinations of substituents contemplated by the present invention preferably result in the formation of stable or chemically suitable compounds. Also, in certain embodiments, unless expressly indicated otherwise, individual substituents may themselves be further optionally substituted (i.e., may be further substituted or unsubstituted).

[0188] The compounds described herein may contain one or more double bonds and, therefore, may optionally give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless otherwise specified, the present invention includes all such possible isomers, as well as mixtures of such isomers.

[0189] Unless otherwise indicated, formulas having chemical bonds represented only by solid lines and not by wedge-shaped or dashed lines contemplate each possible isomer, e.g., each enantiomer, and diastereomers, as well as mixtures of isomers (racemic or scalemic mixtures). Compounds described herein may contain one or more chiral centers and, thus, in some cases, diastereomers and optical isomers may result. Unless otherwise indicated, the present invention includes such possible diastereomers and their racemic mixtures, their substantially purely resolved enantiomers, any possible geometric isomers, and their pharmaceutically acceptable salts. Also included are mixtures of stereoisomers and isolated specific stereoisomers. During the process of synthetic procedures used to prepare such compounds, or in the use of racemization or epimerization procedures well known to those skilled in the art, the products of such procedures can be mixtures of stereoisomers.

[0190] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule around its chiral center(s). If desired, chiral carbons can be denoted by an asterisk (*). The prefixes d and l, or (+) and (-), are used to indicate the sign of rotation of plane-polarized light by the compound, and (-) or l indicates that the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are mirror images that cannot be superimposed on one another. Specific stereoisomers are also referred to as enantiomers, and mixtures of such isomers may be called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture.

[0191] Many of the compounds described herein may have one or more chiral centers and, therefore, may exist in different enantiomeric forms. If desired, chiral carbons may be indicated by an asterisk (*). In the disclosed formulas, when bonds to chiral carbons are drawn as straight lines, both the (R) and (S) configurations of the chiral carbon, and, therefore, both enantiomers, and mixtures thereof, are understood to be included in the formula. As used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be drawn as a wedge (bond to an atom above the plane), and the other bonds can be drawn as a series of short parallel lines or wedges (bonds to atoms below the plane). The (R) or (S) configuration can be assigned to the chiral carbon using the Cahn-Ingold-Prelog priority rules.

[0192] The compounds described herein include atoms in both their natural isotopic abundances and their non-natural abundances. The disclosed compounds can be the same, isotopically labeled, or isotopically substituted compounds as those described, except for the fact that one or more atoms are replaced with atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include, respectively, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, and 36 isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, Cl. The compounds further include their prodrugs, and pharmaceutically acceptable salts of the foregoing compounds or the foregoing prodrugs containing the aforementioned isotopes and / or other isotopes of other atoms are also within the scope of the present disclosure. Non-limiting examples of certain isotopically labeled compounds of the present disclosure include 3 H and 14Those incorporating radioisotopes such as C are useful in drug and / or substrate tissue distribution assays. Due to the ease of preparation and detectability, tritium labeling, i.e., 3 H, and carbon-14, i.e., 14 C isotopes can be used. Further, substitution with heavier isotopes such as deuterium (i.e., 2 H) etc. leads to higher metabolic stability, for example, a longer in vivo half-life or a lower required dosage, and as a result, specific therapeutic advantages can be obtained, and thus, it can be used. The isotope-labeled compounds and their prodrugs of the present disclosure can generally be prepared by implementing the following procedure and using readily available isotope-labeled reagents in place of non-isotope-labeled reagents.

[0193] The compounds described herein can exist as solvates. In some cases, the solvent used to prepare the solvate is an aqueous solution, and in that case, the solvate is often called a hydrate. The compound can exist as a hydrate, which can be obtained, by way of non-limiting example, by crystallization from a solvent or from an aqueous solution. In this regard, 1, 2, 3, or any number of solvates or water molecules can be combined with the compound according to the present invention to form solvates and hydrates. Unless otherwise stated, the composition includes all such possible solvates.

[0194] It is also understood that certain compounds described herein can exist as an equilibrium of tautomers. For example, a ketone having an α-hydrogen can exist in an equilibrium between the keto form and the enol form.

[0195]

Chemical Formula

[0196] Similarly, an amide having an N-hydrogen can exist in an equilibrium between the amide form and the imidic acid form. Unless otherwise stated, the compounds described herein can include all such possible tautomers.

[0197] Chemical substances are known to form solids that exist in different ordered forms, called polymorphic forms or modifications. Different modifications of polymorphic substances may vary significantly in their physical properties. Thus, compounds described herein may exist in different polymorphic forms, and certain modifications may be metastable. Unless otherwise stated, compounds include all such possible polymorphic forms.

[0198] In some embodiments, the structure of the compound can be represented by the following formula:

[0199] [ka]

[0200] This is understood to be equivalent to the following formula:

[0201] [ka]

[0202] In the formula, n is usually an integer. That is, Rn is understood to represent five independent substituents Rn(a), Rn(b), Rn(c), Rn(d), and Rn(e). "Independent substituent" means that each R substituent can be defined independently. For example, if in one example Rn(a) is halogen, Rn(b) is not necessarily halogen in that example.

[0203] Certain materials, compounds, compositions, and components disclosed herein are commercially available or can be readily synthesized using techniques generally known to those of ordinary skill in the art. By way of non-limiting example, starting materials and reagents used in the preparation of the disclosed compounds and compositions are available from commercial suppliers or can be prepared by methods known to those of ordinary skill in the art following procedures described in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0204] Unless otherwise expressly specified, none of the methods shown in this specification are intended to be construed as requiring that their steps be performed in a particular order. Accordingly, where a method claim does not actually recite a particular order for following its steps or, in some other way, the steps are not specifically limited to a particular order in the claims or specification, no order is intended in any instance. This applies to all possible implicit criteria for interpretation, including matters of logic regarding the sequence or flow of steps, general meanings derived from grammatical construction or punctuation, and the number or type of embodiments described herein.

[0205] The components used to prepare the compositions of the present disclosure, and the compositions themselves as used within the methods disclosed herein are disclosed. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to the various, individual and collective, combinations and permutations of these compounds may not be explicitly disclosed, but it is understood that each is specifically contemplated and described herein. By way of non-limiting example, if a particular compound is disclosed and discussed, and some modifications that can be made to several molecules containing the compound are discussed, then every combination and permutation of the compound, as well as possible modifications, are clearly contemplated unless specifically indicated otherwise. Thus, not only classes of molecules A, B, and C, but also classes of molecules D, E, and F, and as an example of a combination molecule, A-D are disclosed, then each is individually and collectively contemplated, i.e., A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, by way of non-limiting example, sub-groups of A-E, B-F, and C-E would be considered to be disclosed. This concept applies to all aspects of the present application, including but not limited to steps in the methods of making and using the compositions of the present invention. Thus, if there are various additional steps that can be performed, it should be understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the method of the present invention.

[0206] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are specific structural requirements for performing the disclosed functions, and there are various structures that can perform the same functions associated with the disclosed structures, and it is understood that these structures will generally achieve the same results.

[0207] In the formula, n is usually an integer. That is, Rn is understood to represent five independent substituents Rn(a), Rn(b), Rn(c), Rn(d), and Rn(e). The "independent substituents" means that each R substituent can be defined independently. For example, in one example, when Rn(a) is a halogen, Rn(b) is not necessarily a halogen in that example.

[0208] The components used to prepare the compositions of the present disclosure, and the compositions themselves used within the methods disclosed herein are disclosed. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to the various, individual and collective, combinations and permutations of these compounds may not be explicitly disclosed, but it is understood that each is specifically contemplated and described herein. By way of non-limiting example, if a particular compound is disclosed, discussed, and some modifications that can be made to several molecules containing the compound are discussed, then all combinations and permutations of the compound, and possible modifications, are clearly contemplated, unless specifically indicated otherwise. Thus, not only the classes of molecules A, B, and C, but also the classes of molecules D, E, and F, and as an example of a combined molecule, A-D is disclosed, then each is individually and collectively contemplated, that is, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered to be disclosed, even if each is not individually listed. Similarly, any subset or combination of these is also disclosed. Thus, by way of non-limiting example, subgroups of A-E, B-F, and C-E would be considered to be disclosed.

[0209] In one aspect, the present disclosure provides a specific compound. In certain aspects, such a compound may be useful as an inhibitor of HIF-2α. Further, in one aspect, the compound is useful for the treatment of disorders of uncontrolled cell proliferation. In a further aspect, the disorder of uncontrolled cell proliferation is cancer or a tumor. In still further aspects, the disorder of uncontrolled cell proliferation is HIF-2α dysfunction as further described herein. In a further aspect, there is provided a method for treating a disorder of uncontrolled cell proliferation, comprising administering to a subject a therapeutically effective amount of a compound of the present invention.

[0210] In another aspect, the compound is useful in the treatment of diseases derived from bacteria or viruses. Accordingly, in one aspect, there is provided a method for treating a disease caused by bacteria or viruses, comprising administering to a subject a therapeutically effective amount of a compound of the present invention.

[0211] It is contemplated that each of the disclosed derivatives may optionally be further substituted. It is also contemplated that any one or more of the derivatives may optionally be excluded from any claim. It is understood that the disclosed compounds may be provided by the disclosed methods. It is also understood that the disclosed compounds can be used in the disclosed methods of use or treatment.

[0212] Embodiments of the present disclosure (or, in one aspect of the present invention) include (or relate to) a compound of formula I, or a pharmaceutically acceptable salt thereof.

[0213]

Chemical formula

[0214] In some embodiments, each of X1 and X2 is independently CH2, O, S, or NH. In some embodiments, each of X3 and X4 is independently CH or N. In some embodiments, Z is CH2 or O or S or NR A wherein, R Ais H or C1-4 alkyl. In some embodiments, each of C1, C2, C3, and C4 (C 1-4 ) is independently C, S, O, N, or sulfur dioxide, preferably C or N.

[0215] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently CH, CH2, O, or N.

[0216] In some embodiments, "a" (e.g., within a cyclic structure) represents an option of a single bond or a double bond.

[0217] In some embodiments, each of R1, R2, R3, and R4 (R 1-4 ) and each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from hydrogen, halo, CN, nitro, hydroxy, dioxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, di-C1-4 alkylaminosulfonylamino, and absent. In some embodiments, (the above R 1-4、or a hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, or di-C1-4 alkylaminosulfonylamino of Ra-e is optionally substituted with 1, 2, or 3 groups independently selected from halo, CN, hydroxy, C1-3, alkoxy, amino, C1-3 alkylamino, di-C1-3-alkylamino, and none.

[0218] In some embodiments, R 1-4 or each of Ra-e is independently, when present, R 1-4 or together with one of Ra-e and, when present, the above R 1-4 or Ra-e is each attached to, C 1-4 or together with Ca-e, each optionally forms a 3- to 7-membered carbocyclic or 4- to 6-membered heterocyclic ring optionally substituted with 1, 2, 3, or 4 C1-3 alkyl groups.

[0219] In some embodiments, X1 is O or S or NH. In some embodiments, X2 is O. In some embodiments, Z is NH. In some embodiments, each of C1, C2, C3, C4 is independently CH or N. In some embodiments, Ca, Cb, Cc, Cd, and Ce are each, CH, N.

[0220] In some embodiments, R1 is H, CH3,

[0221]

Chemical formula

[0222] is.

[0223] In some embodiments, R2 is H, CH3, Cl, CF3, OCH3,

[0224]

Chem.

[0225] is.

[0226] In some embodiments, R3 is H, OCH3, CF3. In some embodiments, R4 is H, OCH3. In some embodiments, Ra is H or OCH3. In some embodiments, Rb is H, F, Cl, CH3CN, OCF3, OCH3, OCD3, or together with Rc, forms methylenedioxy, ethylenedioxy, furan, or hydrofuran. In some embodiments, Rc is H, F, Cl, CH3, OCH3, CN, OCF3, OCD3, SCH3, N(CH3)2,

[0227]

Chem.

[0228] is, or together with Rb, forms methylenedioxy, ethylenedioxy, furan, or hydrofuran.

[0229] In some embodiments, Rd and Re are each independently H. In some embodiments, R1 is not H. In some embodiments, R2 is not H. In some embodiments, R3 is not H. In some embodiments, R4 is not OCH3. In some embodiments, Ra is not H. In some embodiments, Rb is not H. In some embodiments, Rc is not F. In some embodiments, Rd is not H. In some embodiments, Re is not H. In some embodiments, any permutation or combination of the foregoing.

[0230] In some embodiments, the compound of formula I is not 5-(4-fluorophenyl)-N-(4-methoxybenzothiazol-2-yl)-1,3,4-oxadiazol-2-amine.

[0231] In some embodiments, the (compound of formula I) compound is (more specifically) a compound of formula Ia, or a pharmaceutically acceptable salt thereof.

[0232]

Chemical Structure

[0233] In some embodiments, X is S, O, or NH.

[0234] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently CH or N.

[0235] In some embodiments, R1 is H, CH3,

[0236]

Chemical Structure

[0237] and so on.

[0238] In some embodiments, R2 is H, CH3, Cl, CF3, OCH3,

[0239]

Chemical Structure

[0240] and so on.

[0241] In some embodiments, R3 is H, OCH3.

[0242] In some embodiments, Rb and Rc are independently H, F, Cl, CH3, OCH3, CN, OCF3, OCD3, SCH3, N(CH3)2,

[0243]

Chem.

[0244] and are selected from the group consisting of. Alternatively, Rb and Rc together form methylenedioxy or ethylenedioxy.

[0245] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ib, or a pharmaceutically acceptable salt thereof.

[0246]

Chem.

[0247] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N. In some embodiments, R1 is H. In some embodiments, R2 is H. In some embodiments, R3 is H or OCH3. In some embodiments, each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from H, F, OCH3.

[0248] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ic, or a pharmaceutically acceptable salt thereof.

[0249]

Chem.

[0250] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N. In some embodiments, R1 is H. In some embodiments, R2 is H or Cl. In some embodiments, each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently selected from H, F.

[0251] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Id, or a pharmaceutically acceptable salt thereof.

[0252]

Chemical formula

[0253] In some embodiments, each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C or N. In some embodiments, R1 is H. In some embodiments, R2 is H, CH3, or Cl. In some embodiments, R3, R4 are H or OCH3. In some embodiments, Ra, Rd, Re are H. In some embodiments, Rb is H, F, OCH3, OCD3, or together with Rc, forms methylenedioxy, ethylenedioxy, furan, hydrofuran.

[0254] In some embodiments, Rc is H, F, CH3, OCH3, OCD3, OC2H5, *, or

[0255]

Chemical formula

[0256] or together with Rb, forms methylenedioxy, ethylenedioxy, furan, hydrofuran. In some embodiments, the compound of formula Ic is not 5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine.

[0257] In some embodiments, the compound (of formula I) is (more specifically) a compound of formula Ie, or a pharmaceutically acceptable salt thereof.

[0258]

Chemical formula

[0259] In some embodiments, each of C1, C2, C3, and C4 (C 1-4 ) is independently C or N.

[0260] In some embodiments, each of R1, R2, R3, and R4 (R 1-4 ), and each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently hydrogen, halo, CN, nitro, hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, di-C1-4 alkylaminosulfonylamino. In some embodiments, (the above R 1-4、 or Ra-e) hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, or di-C1-4 alkylaminosulfonylamino is optionally substituted with 1, 2, or 3 groups independently selected from halo, CN, hydroxy, C1-3, alkoxy, amino, C1-3 alkylamino, and di-C1-3-alkylamino.

[0261] In some embodiments, each of Ra-e, independently, when present, together with one of Ra-e, and when present, together with Ca-e to which each of the above Ra-e is attached, each optionally forms a 3- to 7-membered carbocyclic or 4- to 6-membered heterocyclic ring optionally substituted with 1, 2, 3, or 4 C1-3 alkyl groups.

[0262] Non-limiting examples or embodiments of the provided compounds include the following:

[0263]

Chemical formula

[0264]

Chemical formula

[0265]

Chemical formula

[0266]

Chemical formula

[0267] In certain embodiments, the compound is not 5-(4-fluorophenyl)-N-(4-methoxybenzothiazol-2-yl)-1,3,4-oxadiazol-2-amine:

[0268] [Chemical formula]

[0269] Also provided are pharmaceutical compositions comprising a therapeutically effective amount of any of the compounds described herein and a pharmaceutically acceptable carrier.

[0270] A further embodiment is a method of treating a disorder of uncontrolled cell proliferation in a mammal, the method comprising administering to the mammal an effective amount of any of the compounds described herein.

[0271] A further embodiment includes a method of reducing HIF-1 / 2α activity. In certain embodiments, the method comprises administering to a subject an effective amount of any of the compounds described herein.

[0272] Yet a further aspect includes a method of inhibiting HIF-1 / 2α activity. In certain embodiments, the method comprises administering to a subject an effective amount of any of the compounds of the present invention.

[0273] In one aspect, provided is a method of making a compound useful as an inhibitor of HIF-1 / 2α, such as a compound disclosed herein. In a further aspect, the product of the disclosed method of making is a modulator of HIF-1 / 2α activity.

[0274] The reactions used to produce the compounds described in this specification can be prepared by using the reactions shown in the following reaction schemes, in addition to those described in the literature or other standard procedures well known to those skilled in the art. To enable a more complete understanding of the present invention, the following examples are provided so that the present invention is for illustrative purposes only and should not be construed as limiting. For clarity, when multiple substituents are permitted under the definitions disclosed herein, examples with a single substituent are shown.

[0275] In one aspect, the disclosed compounds include the products of the synthetic methods described herein. In a further aspect, the disclosed compounds include the compounds produced by the synthetic methods described herein. In still a further aspect, a pharmaceutical composition is described that comprises a therapeutically effective amount of the product of the disclosed method and a pharmaceutically acceptable carrier. In still a further aspect, a method for manufacturing a medicament is provided that comprises combining at least one compound of any of the disclosed compounds, or at least one product of the disclosed method, with a pharmaceutically acceptable carrier or diluent.

[0276] When the reaction conditions and amounts of the components are not described, it is considered to be within the skill of the art to determine these. It is contemplated that each of the disclosed methods may further include additional steps, operations, and / or components. It is also contemplated that any one or more of the steps, operations, and / or components can optionally be removed. The disclosed methods are understood to be capable of being used to provide the disclosed compounds. It is also understood that the products of the disclosed methods can be used in the disclosed methods of use.

[0277] In a further aspect, a pharmaceutical composition is provided that comprises a pharmaceutically acceptable carrier and an effective amount of the product of the disclosed synthetic method. In a further aspect, the effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount. In a further aspect, the compound is the disclosed compound.

[0278] In certain embodiments, the disclosed pharmaceutical compositions comprise, as an active ingredient, one or more of the disclosed compounds (including its pharmaceutically acceptable salt(s)), a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients or adjuvants. The compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although in any given case, the most suitable route will depend on the particular subject and on the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy.

[0279] As used herein, the term "pharmaceutically acceptable salt" means a salt prepared from a pharmaceutically acceptable non-toxic base or acid. When the compounds described herein are acidic, the corresponding salts can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include salts of aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc, etc. Particularly preferred are ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines, and substituted amines such as naturally occurring and synthetic substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include, for example, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and ion exchange resins such as these.

[0280] As used herein, the term "pharmaceutically acceptable non-toxic base" includes inorganic acids, organic acids, and salts prepared therefrom, such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.

[0281] In practice, the compounds described in this specification, or pharmaceutically acceptable salts thereof, can be formulated as an active ingredient in intimate mixture with a pharmaceutical carrier in accordance with conventional pharmaceutical compounding techniques. Depending on the desired form of administration, for example oral or parenteral (including intravenous), the carrier can take a variety of forms. Thus, the pharmaceutical composition can be presented as individual units suitable for oral administration, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient. Additionally, the composition can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a water-in-oil liquid emulsion. In addition to the above general dosage forms, the compounds described in this specification, and / or pharmaceutically acceptable salts (plural) thereof, can also be delivered by controlled release means and / or delivery devices. The composition can be prepared by any of the methods of pharmacy. Generally, such methods include the step of associating the active ingredient with a carrier that constitutes one or more required ingredients. Generally, the composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier, or both. The product can then be conveniently shaped into the desired form.

[0282] Accordingly, the pharmaceutical compositions of the present disclosure can comprise a pharmaceutically acceptable carrier and a compound, or a pharmaceutically acceptable salt of a compound of the present invention. The compounds described in this specification, or pharmaceutically acceptable salts thereof, can also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds.

[0283] The pharmaceutical carriers used can be, for example, solids, liquids, or gases. Examples of solid carriers include, but are not limited to, lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include, but are not limited to, sugar syrup, peanut oil, olive oil, and water. Examples of gas carriers include, but are not limited to, carbon dioxide and nitrogen.

[0284] In the preparation of compositions for oral dosage forms, any convenient pharmaceutical vehicle can be used. As non-limiting examples, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. can be used to form oral liquid preparations such as suspensions, elixirs, and solutions. On the other hand, carriers such as starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used to form oral solid preparations such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units when solid pharmaceutical carriers are used because they are easy to administer. Optionally, tablets can be coated by standard aqueous or non-aqueous techniques.

[0285] Tablets containing the compositions described herein can be prepared, optionally, by compression or molding with one or more auxiliary components or adjuvants. Compressed tablets can be prepared in a suitable machine by compressing the active ingredient in free-flowing form as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersant. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0286] The pharmaceutical compositions described herein can contain, as an active ingredient, the compounds described herein (or pharmaceutically acceptable salts thereof), with or without a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The compositions can include, but are not limited to, compositions suitable for oral, rectal, topical, and parenteral (subcutaneous, intramuscular, and intravenous) administration. In any given case, the most suitable route will depend on the particular host, as well as the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy.

[0287] The pharmaceutical compositions containing the compounds described herein can be suitable for parenteral administration and can be prepared as an aqueous solution or suspension of the active compound. Suitable surfactants can include, for example, hydroxypropylcellulose. The dispersion can be prepared in an oil, as glycerol, liquid polyethylene glycol, and mixtures thereof. Further, preservatives can be included to prevent the harmful growth of microorganisms.

[0288] The pharmaceutical compositions containing the compounds described herein can potentially be suitable for injectable use, including a sterile aqueous solution or dispersion. Further, the composition can be in the form of a sterile powder for the immediate preparation of such a sterile injectable solution or dispersion. In all cases, the final injectable form must be sterile and must be substantially fluid for easy injectability. In embodiments where the pharmaceutical composition must be stable under the manufacturing and storage conditions, it is therefore preferably protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0289] The pharmaceutical composition containing the compounds described in this specification can be in a form suitable for topical use, such as, for example, aerosol, cream, ointment, lotion, powder, mouthwash, gargle, etc. Further, the composition can be in a form suitable for use in a transdermal device. These formulations can be prepared using the compounds of the present invention, or pharmaceutically acceptable salts thereof, by conventional processing methods. As a non-limiting example, a cream or ointment is prepared by mixing a hydrophilic material and water together with about 5 wt% to about 10 wt% of the compound to produce a cream or ointment having the desired consistency.

[0290] When the carrier is solid, the pharmaceutical composition containing the compounds described in this specification can take a form suitable for rectal administration. In certain embodiments, the pharmaceutical composition forms a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. First, the composition can be easily formed into a suppository by mixing it with the softened or melted carrier(s) and then cooling and shaping it in a mold.

[0291] In addition to the above carrier components, the above pharmaceutical formulations can optionally contain one or more additional carrier components such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Further, other adjuvants can be included so that the formulation is isotonic with the blood of the intended recipient.

[0292] For treatment conditions that require inhibition or negative control of HIF-1 / 2α protein activity, appropriate dosage levels are generally about 0.01 to 500 milligrams (mg) per 1 kg of the patient's body weight per day and can be administered as single or multiple doses. By way of non-limiting example, dosage levels can be about 0.1 to about 250 mg / kg / day, or 0.5 to 100 mg / kg / day. Suitable dosage levels can be about 0.01 to 250 mg / kg / day, about 0.05 to 100 mg / kg / day, or about 0.1 to about 50 mg / kg / day. Within this range, the dosage can be 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50 mg / kg / day. For oral administration, the composition can be provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, in particular 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient, to adjust the dosage for the subject being treated according to the symptoms. The compound can be administered in a regimen of 1 to 4 times / day, preferably 1 or 2 times / day. This dosing regimen can be adjusted to provide an optimal therapeutic response.

[0293] However, it is understood that the specific dosage level for any particular patient will depend on a variety of factors. Such factors include the patient's age, weight, general health, gender, and diet. Other factors include the time and route of administration, rate of excretion, combination of drugs, and the type and severity of the particular disease being treated.

[0294] A method for manufacturing a medicament for inhibiting or negatively regulating HIF-1 / 2α protein activity and inducing ferroptosis (e.g., treating one or more neurodegenerative diseases associated with uncontrolled cell growth disorders, or HIF-1 / 2α dysfunction, and / or iron or lipid accumulation) in a subject (e.g., a human), which comprises combining one or more disclosed compounds, products, or compositions with a pharmaceutically acceptable carrier or diluent, is further provided. Thus, in one aspect, the present disclosure provides the manufacture of a medicament comprising combining at least one disclosed compound, or at least one disclosed product, with a pharmaceutically acceptable carrier or diluent.

[0295] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be used in the disclosed methods of use.

[0296] The disclosed compounds can be used as a single agent or in combination with one or more other agents in treating, preventing, controlling, alleviating, or reducing the risk of the above-mentioned diseases, disorders, and conditions, where combinations of the agents are safer or more effective than either agent alone, and the compounds of Formula I or other agents have utility. The other agent(s) can be administered by generally used routes and amounts simultaneously with or sequentially to the disclosed compound. When the disclosed compound is used simultaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such an agent and the disclosed compound is preferred. However, combination therapy can also be administered on overlapping schedules. It is also contemplated that a combination of one or more active ingredients and the disclosed compound will be more effective than either single agent.

[0297] The pharmaceutical compositions and methods of the present invention can further comprise other therapeutically active compounds described herein, which are typically applied to the treatment of the above-mentioned medical conditions.

[0298] The compounds disclosed herein are useful for treating, preventing, alleviating, controlling, or reducing the risk of various disorders in which a patient or subject would benefit from inhibition or negative regulation of HIF-2α, or induction of ferroptosis. In one aspect, provided is a method of treating or preventing a disorder in a subject, the method comprising administering to the subject at least one disclosed compound, at least one disclosed pharmaceutical composition, and / or at least one disclosed product, in an effective dosage and amount to treat the disorder in the subject.

[0299] Also provided is a method of treating one or more disorders in a subject in which HIF-2α inhibition or ferroptosis induction is expected to be beneficial, the method comprising administering to the subject at least one disclosed compound, at least one disclosed pharmaceutical composition, and / or at least one disclosed product, in an effective dosage and amount to treat the disorder in the subject.

[0300] In one aspect, provided is a method of treating a disorder of uncontrolled cell growth, comprising administering to the subject at least one disclosed compound, at least one disclosed pharmaceutical composition, and / or at least one disclosed product, in an effective dosage and amount to treat the disorder in the subject. In a further aspect, provided is a method of treating or preventing a neurodegenerative disease, comprising administering to the subject at least one disclosed compound, at least one disclosed pharmaceutical composition, and / or at least one disclosed product, in an effective dosage and amount to treat the disorder in the subject. Also provided is a method of treating a disorder in a mammal, the method comprising administering to the mammal at least one disclosed compound, composition, or agent.

[0301] The use of one or more of the disclosed compounds or products for treating a disease or disorder in a patient (e.g., a human) where HIF-1 / 2α inhibition or ferroptosis induction is predicted to have a therapeutic effect, such as an impairment of uncontrolled cell proliferation (e.g., cancer), as well as neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, and / or diseases caused by bacteria and / or viruses, is provided herein.

[0302] The compounds described herein can also be used in immunotherapy. In one embodiment, the disclosed compounds treat diseases of uncontrolled cell proliferation and / or diseases caused by bacteria and / or viruses by immunotherapy, which means that the compounds induce an immunotherapeutic response that results in the treatment of these diseases.

[0303] The compounds disclosed herein are useful for treating, preventing, alleviating, controlling, or reducing the risk of various disorders of uncontrolled cell proliferation.

[0304] Methods of using the disclosed compounds, compositions, or agents are also provided. In one aspect, the method of use is directed to treating a disorder. In a further aspect, the disclosed compounds, or other agents, are useful for treating, preventing, controlling, alleviating, or reducing the risk of the above-described diseases, disorders, and conditions, either as a single agent or in combination with one or more other agents, when the combination of agents is safer or more effective than either agent alone.

[0305] Examples of disorders treatable by the compounds provided include disorders of uncontrolled cell proliferation. In a further aspect, the disorder of uncontrolled cell proliferation is cancer. In a still further aspect, the cancer is leukemia, sarcoma, solid tumor, and / or lymphoma. In certain embodiments, compounds for use in treating HIF dysregulation diseases with inflammatory elements, such as cancer, stroke, and rheumatoid arthritis, are disclosed herein.

[0306] Cancer is generally understood to mean or describe a physiological state in mammals characterized by uncontrolled cell proliferation. Cancer can be multi-drug resistant (MDR) or drug sensitive. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include breast cancer, prostate cancer, colorectal cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, peritoneal cancer, liver cancer, e.g., hepatocarcinoma, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, and thyroid cancer.

[0307] In a further aspect, the cancer is a brain cancer. In a still further aspect, the brain cancer is selected from glioma, medulloblastoma, primitive neuroectodermal tumor (PNET), acoustic neuroma, meningioma, pituitary adenoma, schwannoma, CNS lymphoma, craniopharyngioma, chordoma, cerebral neuroblastoma, central neurocytoma, pineocytoma, pineoblastoma, atypical teratoid rhabdoid tumor, chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexus papilloma, craniopharyngioma, dysplastic neuroepithelial tumor, gangliocytoma, germ cell tumor, hemangioblastoma, angioepithelial cell tumor, and metastatic brain tumor. In a still further aspect, the glioma is selected from ependymoma, astrocytoma, oligodendroglioma, and oligoastrocytoma. In a still further aspect, the glioma is selected from juvenile pilocytic astrocytoma, subependymal giant cell astrocytoma, ganglioglioma, subependymal tumor, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, glioblastoma multiforme, brainstem glioma, oligodendroglioma, ependymoma, oligoastrocytoma, cerebellar astrocytoma, fibrillary infantile astrocytoma, subependymal giant cell astrocytoma, diffuse astrocytoma, mixed glioma, optic nerve glioma, gliomatosis cerebri, multicentric gliomatous tumor, multicentric glioblastoma multiforme, paraganglioma, and ganglioglioma.

[0308] In one embodiment, the cancer can be a cancer selected from cancers of the blood, brain, urogenital organs, gastrointestinal tract, colon, rectum, chest, liver, kidney, lymphatic system, stomach, lung, pancreas, and skin. In a further embodiment, the cancer is selected from prostate cancer, glioblastoma multiforme, endometrial cancer, breast cancer, and colorectal cancer. In a further embodiment, the cancer is selected from cancers of the chest, ovary, prostate, head, neck, and kidney. In yet a further embodiment, the cancer is selected from cancers of the lung and liver. In yet a further embodiment, the cancer is selected from cancers of the chest, ovary, testis, and prostate. In yet a further embodiment, the cancer is breast cancer. In yet a further embodiment, the cancer is ovarian cancer. In yet a further embodiment, the cancer is prostate cancer. In yet a further embodiment, the cancer is testicular cancer.

[0309] In a further embodiment, the cancer is a hematological cancer. In yet a further embodiment, the hematological cancer is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and plasmacytoma. In yet a further embodiment, the cancer is selected from chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell non-Hodgkin lymphoma, and large cell type B-cell lymphoma.

[0310] In certain embodiments, compounds for use in the treatment of HIF dysregulation cardiovascular diseases such as cardiac arrhythmia and heart failure are disclosed herein. In certain embodiments, compounds for use in a treatment to prevent or reduce resistance to radiation therapy and chemotherapy are disclosed herein. In certain embodiments, compounds for use in the prevention or reduction of tumor invasion and tumor metastasis are disclosed herein.

[0311] In certain embodiments, compounds for use in angiogenesis and for the prevention or mitigation of disorders associated with angiogenesis are disclosed herein. In certain embodiments, compounds for use in the treatment of hematological diseases with HIF dysregulation, particularly polycythemia, such as Chuvash polycythemia, are disclosed herein.

[0312] In various aspects, disorders associated with HIF-1 / 2α dysfunction and / or iron or lipid dysfunction include neurodegenerative diseases. In a further aspect, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0313] The compounds are further useful in methods for the prevention, treatment, control, alleviation, or reduction of the risk of the diseases, disorders, and conditions described herein. The compounds are further useful in methods for the prevention, treatment, control, alleviation, or reduction of the risk of the above-described diseases, disorders, and conditions in combination with other agents.

[0314] A further aspect relates to the administration of HIF-1 / 2α inhibitors and ferroptosis inducers to improve treatment outcomes in the context of disorders of uncontrolled cell proliferation, including cancer. That is, in one aspect, the method relates to a co-treatment method that includes administering to a mammal an effective amount and dosage of at least one compound of the invention in connection with a cancer treatment method.

[0315] In a further aspect, administration improves treatment outcomes in relation to a cancer treatment method. In relation to a cancer treatment method, the administration can be continuous or intermittent. The administration need not be concurrent with the treatment method and can, therefore, be before, during, and / or after the treatment method. For example, a cancer treatment method can be provided within 1, 2, 3, 4, 5, 6, or 7 days before and after administration of the compound. In a further example, a cancer treatment method can be provided within 1, 2, 3, or 4 weeks before and after administration of the compound. As yet a further example, a cognitive or behavioral therapy can be provided within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 half-lives of the administered compound before and after administration.

[0316] In one aspect, when a combination of agents is safer or more effective than either agent alone, the disclosed compounds can be used in combination with one or more other agents in the treatment, prevention, control, alleviation, or risk reduction of a disease or condition for which the disclosed compound or other drug may have utility. Such other agent(s) may be administered simultaneously with or sequentially to the compounds of the invention, by generally used routes and amounts. When the described compounds are used simultaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such other agent(s) and the disclosed compound can be used. However, combination therapy may also include therapies in which the disclosed compound and one or more other agents are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the disclosed compounds and the other active ingredients may be used in lower dosages than when each is used separately.

[0317] Accordingly, pharmaceutical compositions include pharmaceutical compositions containing one or more other active ingredients in addition to the compounds of the invention.

[0318] Examples of such combinations include combinations of the disclosed compounds with not only one other active compound but also two or more other active compounds. Similarly, the disclosed compounds can be used in combination with other agents used in the prevention, treatment, control, alleviation, or risk reduction of a disease or condition for which the disclosed compound is useful. Such other agents may be administered simultaneously with or sequentially to the compounds of the invention, by generally used routes and amounts. When the disclosed compounds are used simultaneously with one or more other agents, a pharmaceutical composition containing such other agents in addition to the disclosed compound can be prepared and / or used. Accordingly, pharmaceutical compositions include pharmaceutical compositions containing one or more other active ingredients in addition to the compounds of the invention.

[0319] The weight ratio of the disclosed compound to the second active ingredient may vary and will depend on the effective amounts of the respective components. Generally, the effective amounts of each will be used. Thus, for example, when the compounds of the present invention are combined with another agent, the weight ratio of the disclosed compound to the other agent will generally range from about 1000:1 to about 1:1000, preferably from about 200:1 to about 1:200. Combinations of one or more of the disclosed compounds with other active ingredients will also generally be within the above ranges, but in any case, the effective dosage of each active ingredient must be used.

[0320] In such combinations, the disclosed compound and the other active agent can be administered individually or in combination. Further, the administration of one element can be before, at the same time as, or after the administration of the other agent(s). The present compound and the other agent can be co-administered either in a combination therapy or in a fixed combination.

[0321] Accordingly, the present compound can be used alone or in combination with other agents known to be beneficial for the indication of the subject, or with other agents that either enhance efficacy, safety, convenience, or reduce the undesirable side effects or toxicity of the disclosed compound, and that affect receptors or enzymes. The present compound and the other agent can be co-administered either in a combination therapy or in a fixed combination.

[0322] The term "co-administration" and like terms mean the simultaneous, sequential, and / or combined administration of two or more components. For example, two components can be co-administered by administering each component in an integrated dosage. Alternatively, or further, two components can be co-administered by administering each component in individual dosages simultaneously, concurrently, or sequentially (e.g., separate administrations separated by a period). The period can be very short (e.g., substantially immediately following the first administration), or long (e.g., 1 to 60 seconds, 1 to 60 minutes, 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, etc., or any value, or range of values, therebetween). Concurrent or co-administration can include the overlapping dosing time frames of two or more components, or the administration of a combination product that includes a mixture of two or more components.

[0323] In one aspect, the compound can be used in combination with an anti-cancer therapeutic agent, or other well-known therapeutic agents.

[0324] In the treatment of conditions that require inhibition or negative regulation of HIF-1 / 2α and / or induction of ferroptosis, suitable dosage levels are generally about 0.01 to 1000 mg per 1 kg of patient body weight per day, which can be administered in single or multiple doses. In non-limiting examples, the dosage level can be about 0.1 to about 250 mg / kg / day, or about 0.5 to about 100 mg / kg / day. Suitable dosage levels can be about 0.01 to 250 mg / kg / day, about 0.05 to 100 mg / kg / day, or about 0.1 to about 50 mg / kg / day. Within this range, the dosage can be 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg / day. For oral administration, the composition is preferably provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, in particular 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 mg of the active ingredient, to adjust the dosage for the patient being treated according to the symptoms. The compound can be administered in a regimen of 1 to 4 times / day, preferably 1 or 2 times / day. This dosing regimen can be adjusted to provide an optimal therapeutic response. However, the specific dosage level and frequency of administration for any particular patient can vary and will depend on various factors including the activity of the specific compound being used, the metabolic stability and duration of action of the compound, age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, combination of drugs, severity of the particular condition, and the host receiving the treatment.

[0325] Accordingly, in one aspect, a method of inhibiting or negatively regulating HIF-2α and / or inducing ferroptosis in at least one cell, the method comprising contacting the at least one cell with at least one of the described compounds, for example, in an amount effective to control or activate HIF-2α activity response and ferroptosis intracellularly in at least one cell, is provided. In a further aspect, the cell is a mammal (e.g., human). In a further aspect, the cell is isolated from a subject prior to the contacting step. In a further aspect, the contacting is effected by administration to the subject.

[0326] In one aspect, a method of treating a disorder of uncontrolled cell proliferation in a mammal, the method comprising treating the disorder of uncontrolled cell proliferation in the mammal by administering to the mammal an effective amount of at least one compound, or a product of a method of making the disclosed compounds, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, is provided.

[0327] In yet a further aspect, the effective amount is a therapeutically effective amount. Still yet in a further aspect, the effective amount is a prophylactically effective amount.

[0328] In a further aspect, the mammal is a human. In yet a further aspect, the method further comprises identifying a mammal in need of treatment of a disorder of uncontrolled cell proliferation. Still yet in a further aspect, the mammal has been diagnosed as being in need of treatment of a disorder of uncontrolled cell proliferation prior to the administering step.

Examples

[0329] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described in the claims of this specification are made and evaluated, and are intended as a pure illustration of the invention and not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or ambient temperature, and pressure is at or near atmospheric pressure.

[0330] Several methods for preparing the disclosed compounds are described. Starting materials and necessary intermediates may be commercially available or can be prepared according to literature procedures or as described herein.

[0331] The following exemplary compounds of the invention were synthesized. Exemplary compounds are usually described in basic form according to the IUPAC naming rules. Some of the exemplary compounds were obtained or isolated in salt form.

[0332] Some of the exemplary compounds were obtained as racemic mixtures of one or more enantiomers or diastereomers. The compounds can be separated by those skilled in the art to isolate the individual enantiomers. Separation can be carried out by combining the racemic mixture of the compound with an enantiomerically pure compound to form a mixture of diastereomers, followed by separating the individual diastereomers by standard methods such as fractional crystallization or chromatography. The racemic or diastereomeric mixtures of the compounds can also be separated directly by chromatographic methods using a chiral stationary phase.

[0333] Example 1: Synthetic Scheme, Method, and Procedure: Scheme 1

[0334]

Chemical formula

[0335] Dimethyl(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (S1).

[0336] To a 100 mL round-bottom flask equipped with a magnetic stir bar and dried by heating, 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (1.00 g, 5.58 mmol) and DMF (10 mL) were added. The reaction solution was cooled to 0 °C, and an aqueous NaOH solution (20 M, 0.31 mL) was added dropwise. After the addition was complete, the reaction solution was stirred for 10 minutes, and then CS2 (0.62 mL) was added dropwise. The resulting solution was warmed to room temperature (RT) over 30 minutes. Again, the flask was cooled to 0 °C, and iodomethane (1.6 g) was added dropwise. A yellow precipitate formed during the addition. The reaction was allowed to proceed for 30 minutes and completion was confirmed by LC-MS. The reaction contents were then poured into a Erlenmeyer flask containing 50 mL of H2O, and the resulting precipitate was collected by vacuum filtration. The yellow precipitate was recrystallized from hot MeOH (70 mL) to give off-white crystals (0.83 g), which were used without further purification. 1 1H NMR (500 MHz, DMSO-d6) δ 8.02 (dd, 2H), 7.41 (t, 2H), 2.66 (s, 6H). LCMS [M + H] 284.2.

[0337] As used herein, "room temperature" (or, RT) means any temperature above freezing (0 °C, or other (equivalent) freezing temperature that varies depending on the presence of a freezing point adjusting component) and below normal human body temperature (37 °C, or other (equivalent) boiling point that varies depending on the presence of a boiling point adjusting component), preferably greater than about 4 °C to less than about 35 °C, more preferably about 5 °C to about 32 °C, about 8 °C to about 30 °C, about 10 °C to about 30 °C, about 10 °C to about 25 °C, about 10 °C to about 20 °C, about 10 °C to about 15 °C, about 15 °C to about 30 °C, about 15 °C to about 25 °C, about 15 °C to about 20 °C, about 20 °C to about 30 °C, about 20 °C to about 25 °C, about 20 °C to about 22 °C, or any value or range of values between these.

[0338] [Chemical formula]

[0339] 5-(4-Fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine (1). To a 40 mL vial equipped with a magnetic stir bar, dried in an oven, was added 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (0.1 g, 0.35 mmol), 2-amino-3-methoxybenzenethiol hydrochloride (0.075 g, 0.39 mmol), K2CO3 (0.107 g, 0.77 mmol), and DMF (3 mL). The reaction mixture was flushed with nitrogen, sealed, and placed in a heating block at 120 °C and stirred overnight (16 h). The reaction mixture was cooled to RT and the solvent was reduced to approximately 1 mL. Cold water (25 mL) was added and 1N HCl was added to neutralize the pH to approximately 7. The solid was collected and washed with water. Recrystallization from MeOH / acetone followed by HPLC purification (C18 0 - 90% 0.1% TFA in water / 0.1% TFA in CH3CN, 254 nM) gave the TFA salt upon removal of the solvent. Addition of 2.0 M HCl in 1 mL of MeOH and removal of the solvent gave 5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine hydrochloride. 1 H NMR (400 MHz, DMSO-d6) δ 7.90 (t, 2H), 7.36 (t, 12H), 7.22 (d, 1H), 6.89 (t, 1H), 6.77 (d, 1H), 3.87 (s, 3H). LCMS [M + H] 343.1.

[0340] [Chemical formula]

[0341] 5-(4-Fluorophenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (3). To a 40 mL vial equipped with a magnetic stir bar, dried in an oven, was added 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (0.1 g, 0.35 mmol), 2-methoxypyridine-3,4-diamine (0.055 g, 0.39 mmol), K2CO3 (0.107 g, 0.77 mmol), and DMF (3 mL). The reaction mixture was flushed with nitrogen, sealed, and placed in a heating block at 120 °C and stirred overnight (16 h). The reaction mixture was cooled to rt and the solvent was reduced to approximately 1 mL. Cold water (25 mL) was added and 1N HCl was added to neutralize the pH to approximately 7. The solid was collected and washed with water. Recrystallization from MeOH gave 5-(4-fluorophenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine. 1 H NMR (500 MHz, DMSO-d6) δ 7.95 (dd, 2H), 7.85 (s, 1H), 7.39 (t, 2H), 7.16 (s, 1H), 3.99 (s, 3H).LCMS [M + H] 327.1.

[0342]

Chemical Structure

[0343] 5-(4-Fluorophenyl)-N-(5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (4). The target compound was prepared in the same manner as in Scheme 1 from 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine and 6-methoxypyridine-2,3-diamine. 1 H NMR (500 MHz, DMSO-d6) δ 7.86 (dd, 2H), 7.41 (d, 1H), 7.33 (t, 2H), 6.16 (d, 1H), 3.78 (s, 3H).LCMS [M + H] 327.1.

[0344]

Chem.

[0345] N-(6-chloro-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (5). 1 H NMR (500 MHz, DMSO d6) δ 8.31 (s, 1H), 7.97 (t, 2H), 7.40 (t, 2H), 7.35 (s, 1H). LCMS [M + H] 331.1

[0346]

Chem.

[0347] 5-(4-fluorophenyl)-N-(6-(trifluoromethyl)-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (6).

[0348] 1 H NMR (500 MHz, DMSO d6) δ 8.66 (s, 1H), 7.97 (dd, 2H), 7.76 (s, 1H), 7.40 (t, 2H). LCMS [M + H] 365.1.

[0349]

Chem.

[0350] 5-(4-fluorophenyl)-N-(7-methyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (7). 11H NMR (500 MHz, DMSO d6) δ 8.61 (s, 1H), 8.42 (s, 1H), 7.99 (dd, 2H), 7.42 (t, 2H), 2.49 (s, 3H). LCMS [M + H] 311.2.

[0351]

Chem.

[0352] N-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)oxazolo[4,5-b]pyridin-2-amine (8). 1 1H NMR (500 MHz, DMSO-d6) δ 8.04-7.88 (m, 4H), 7.49-7.35 (m, 3H), 6.55 (s, 1H). LCMS [M + H] 298.2.

[0353] Scheme 2

[0354]

Chem.

[0355] 6-Morpholinopyridine-3,4-diamine (S2). To a 20 mL pressure vial equipped with a magnetic stir bar was added 2-chloro-5-nitropyridin-4-amine (0.20 g, 1.15 mmol), morpholine (0.69 mL, 11.5 mmol), and isopropanol (5 mL). The flask was heated to 90 °C and the reaction was allowed to proceed for 18 h. The reaction mixture was then partitioned between dichloromethane (20 mL) and saturated NaHCO3 (20 mL). The organic material was recovered and the aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic material was recovered, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting yellow solid was taken up in freshly distilled methanol (10 mL), heated to dryness, and transferred to a 50 mL round bottom flask equipped with a magnetic stir bar. The atmosphere was replaced with N2 and Pd / C (20 mg, 10% w / w) was added. The atmosphere was purged with H2 and the reaction was allowed to proceed for 16 h under an atmosphere of H2. The resulting suspension was filtered through diatomaceous earth, concentrated to give a yellow solid which was used without further purification. 1 1H NMR (500 MHz, DMSO-d6) 7.31 (s, 1H), 5.92 (s, 1H), 5.21, (s, 2H), 3.92 (s, 2H), 3.63 (t, 4H), 3.09 (t, 4H). LCMS [M + H] 195.4.

[0356] 5-(4-Fluorophenyl)-N-(6-morpholino-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (10). In a 20 mL vial equipped with a magnetic stir bar, dried in an oven, 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (0.066 g, 0.26 mmol), 6-morpholinopyridine-3,4-diamine (0.050 g, 0.26 mmol), K2CO3 (0.072 g, 0.52 mmol), 4 Å molecular sieve (100 mg, powder), and DMF (2 mL) were added. The reaction solution was flushed with nitrogen, sealed, placed in a heating block at 110 °C, and stirred overnight for 16 h. The reaction solution was cooled to RT and filtered. The solid was washed with hot MeOH (15 mL), and the resulting filtrate was evaporated under reduced pressure. The resulting mixture was diluted with water (5 mL), and 1N HCl was added to neutralize the pH to about 7. The resulting precipitate was collected and washed with water. Recrystallization from MeOH / acetone, followed by HPLC purification (C18 0-90 0.1% TFA in water / 0.1% TFA in CH3CN, 254 nM) gave the TFA salt upon removal of the solvent. 2.0 M HCl in 1 mL of MeOH was added, and the solvent was removed to obtain 5-(4-fluorophenyl)-N-(6-morpholino-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine hydrochloride. 1 1H NMR (500 MHz, DMSO d6) δ 7.97 (s, 1H), 7.97 (dd, 2H), 7.39 (t, 2H), 6.89 (s, 1H), 3.75 (t, 4H), 3.58 (t, 4H). LCMS [M + H] 381.2.

[0357]

Chemical Structure

[0358] 6-(4-Methylpiperazin-1-yl)pyridine-3,4-diamine (S3) was prepared from 2-chloro-5-nitropyridin-4-amine and N-methylpiperazine in the same manner as in Scheme 2. LCMS [M + H] 208.3.

[0359]

Chemical Structure

[0360] 5-(4-Fluorophenyl)-N-(6-(4-methylpiperazin-1-yl)-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazole-2-amine (11) was prepared from 6-(4-methylpiperazin-1-yl)pyridine-3,4-diamine and 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 2. 1 H NMR (500 MHz, DMSO d6) δ 8.14 (s, 1H), 7.95 (dd, 2H), 7.37 (t, 2H), 6.63 (s, 1H), 3.37 (t, 4H), 2.48 (s, 3H), 2.41 (t, 4H). LCMS [M + H] 395.2.

[0361]

Chemical Structure

[0362] N-(2-Chloro-9H-purin-8-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazole-2-amine (14). 1 H NMR (500 MHz, DMSO d6) δ 8.53 (s, 1H), 7.99 (dd, 2H), 7.42 (t, 2H). LCMS [M + H] 332.1.

[0363]

Chemical Structure

[0364] 6-(4-(Methylsulfonyl)piperidin-1-yl)pyridine-3,4-diamine (S4) was prepared from 2-chloro-5-nitropyridin-4-amine and 4-(methylsulfonyl)piperidine in the same manner as in Scheme 2. 1 H NMR (500 MHz, DMSO-d6) δ 7.30 (s, 1H), 5.99 (s, 1H), 5.23 (s, 2H), 4.13 - 3.98 (m, 2H), 3.20 (ddt, 1H), 2.90 (s, 3H), 2.60 (td, 2H), 2.01 - 1.88 (m, 2H), 1.55 (qd, 2H).LCMS [M + H] 271.2.

[0365]

Chemical Structure

[0366] 5-(4-Fluorophenyl)-N-(6-(4-(methylsulfonyl)piperidin-1-yl)-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazole-2-amine (16) was prepared from 6-(4-(methylsulfonyl)piperidin-1-yl)pyridine-3,4-diamine and 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 2. 1 H NMR (500 MHz, DMSO d6) δ 7.99 (dd, 2H), 7.97 (s, 1H), 7.40 (t, 2H), 6.94 (s, 1H), 4.29 (d, 2H), 3.45 (t, 1H), 3.18 (t, 2H), 2.96 (s, 3H), 2.14 (d, 2H), 1.74 - 1.67 (m, 2H).LCMS [M + H] 458.2.

[0367]

Chemical Structure

[0368] 2-Morpholinopyridine-3,4-diamine (S5) was prepared from 2-chloro-3-nitropyridin-4-amine and morpholine in the same manner as in Scheme 2. 1 H NMR (500 MHz, DMSO-d6) δ 7.30 (d, 1H), 6.27 (d, 1H), 5.26 (s, 2H), 4.07 (s, 2H), 3.75 - 3.67 (m, 4H), 2.90 - 2.83 (m, 4H). LCMS [M + H] 195.3.

[0369]

Chem.

[0370] 5-(4-Fluorophenyl)-N-(4-morpholino-3H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazole-2-amine (17) was prepared from 2-morpholinopyridine-3,4-diamine and 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 2. 1 H NMR (500 MHz, DMSO-d6) δ 7.94 (dd, 2H), 7.68 (d, 1H), 7.43 (t, 2H), 7.20 (d, 1H), 4.17 (t, 4H), 3.81 (t, 4H). LCMS [M + H] 382.1.

[0371]

Chem.

[0372] 5-Morpholinopyridine-3,4-diamine (S6). To a 20 mL vial equipped with a magnetic stir bar, 3-bromo-5-nitropyridin-4-amine (0.20 g, 0.92 mmol) and morpholine (5 mL) were added. The reaction mixture was flushed with nitrogen, sealed, placed in a heating block at 110 °C, and stirred for 48 h. The reaction mixture was extracted with dichloromethane (20 mL) and saturated NaHCO3 (20 mL). The organic layer was recovered, and the aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic matter was recovered, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting yellow solid was purified by column chromatography (2.5% MeOH / CH2Cl2). The resulting solid was recrystallized from hot MeOH to give fine yellow needles, which were then taken up in freshly distilled methanol (10 mL), heated to dryness, and transferred to a 50 mL round-bottom flask equipped with a magnetic stir bar. The atmosphere was replaced with N2, and Pd / C (20 mg, 10% w / w) was added. The flask was purged with H2, and the reaction mixture was stirred under an atmosphere of H2 for 16 h. The resulting suspension was filtered through celite and concentrated to give a yellow solid. LCMS [M + H] 195.3.

[0373] [Chemical formula]

[0374] 5-(4-Fluorophenyl)-N-(7-morpholino-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (18) was prepared from 5-morpholinopyridine-3,4-diamine and 5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine in a similar manner to Scheme 2. 1 H NMR (500 MHz, DMSO d6) δ 8.16 (s, 1H), 7.94 (t, 2H), 7.37 (t, 2H), 6.64 (s, 1H), 3.70 (t, 4H), 3.33 (t, 4H). LCMS [M + H] 382.1

[0375] [Chemical formula]

[0376] 2-(3-Methoxyphenyl)pyridine-3,4-diamine. Under N2 atmosphere, into a 50 mL round-bottom flask equipped with a magnetic stir bar that was heated and dried, 2-chloro-3-nitropyridin-4-amine (285 mg, 1.64 mmol), (3-methoxyphenyl)boronic acid (375 mg, 2.47 mmol), K2CO3 (566 mg, 4.1 mmol), 1,4-dioxane (15 mL), and H2O (1.5 mL) were added. The solution was degassed by bubbling nitrogen through it for 30 min. Next, Pd(PPh3)4 (185 mg, 0.16 mmol) was added all at once, the reaction solution was sealed, and the reaction was allowed to proceed at 90 °C for 16 h. The reaction material was concentrated, loaded onto silica gel, and purified by flash chromatography (1:1 hexane / EtOAc) to obtain a yellowish-brown solid. Next, the material was dissolved in freshly distilled MeOH (15 mL) and transferred to a 50 mL round-bottom flask equipped with a magnetic stir bar. The atmosphere was replaced with N2, and Pd / C (10% w / w, 15 mg) was added all at once. The reaction solution was flushed with H2(g), and the reaction mixture was stirred under H2(g) for 16 h. The material was filtered through celite, concentrated to obtain a yellowish-brown solid (64 mg), which was used without further purification. 1 H NMR (500 MHz, Chloroform-d) δ 8.26 (d, 1H), 7.36 - 7.28 (m, 1H), 7.09 - 7.06 (m, 1H), 7.04 (dd, 1H), 7.00 - 6.94 (m, 1H), 6.66 (d, 1H), 5.51 (s, 2H), 3.84 (s, 3H).

[0377]

Chemical Structure

[0378] 5-(4-Fluorophenyl)-N-(4-(3-methoxyphenyl)-3H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazole-2-amine (19). The title compound was prepared from 2-(3-methoxyphenyl)pyridine-3,4-diamine and dimethyl (5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. 1 H NMR (500 MHz, DMSO-d6) δ 88.41-8.38 (m, 1H), 7.99-7.94 (m, 2H), 7.58-7.41 (m, 3H), 7.41 (t, 2H), 7.13-7.08 (M, 2H), 3.86 (t, 3H). LCMS [M+H] 403.2.

[0379] Scheme 3

[0380]

Chem.

[0381] 5-(4-Fluorophenyl)-N-(5-methoxythiazolo[5,4-b]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (9). To a 50 mL flask equipped with a magnetic stir bar, dried by heating, were added 5-methoxythiazolo[5,4-b]pyridin-2-amine (0.208 g, 1.14 mmol), iPr2NEt (0.389 mL, 2.29 mmol), and acetonitrile (10 mL). 1,1-Thiocarbonyldiimidazole (204 mg, 1.14 mmol) was added in one portion, and the reaction mixture was heated to 80 °C and the reaction was allowed to proceed until consumption of the starting material was observed by LC-MS (6 h). Next, 4-fluorobenzohydrazide (0.154 g, 1.00 mmol) was added in one portion, and the reaction mixture was stirred overnight for 16 h. Saturated aqueous NaHCO3 was added, and the mixture was extracted with CH2Cl2 (3 × 25 mL). The organic material was collected, dried over Na2SO4, filtered, and concentrated under reduced pressure. Next, the obtained off-white solid was dissolved in DMSO (4 mL) and transferred to a 25 mL round-bottom flask. Next, EDC (0.167 g, 0.86 mmol) was added in one portion, and the reaction mixture was heated to 60 °C. After the reaction mixture was stirred for 6 h, the contents were poured into H2O. The obtained precipitate was collected by vacuum filtration and washed with EtOAc. The obtained solid was recrystallized from hot MeOH to give 5-(4-fluorophenyl)-N-(5-methoxythiazolo[5,4-b]pyridin-2-yl)-1,3,4-oxadiazol-2-amine as a yellow solid. LCMS [M + H] 344.0.

[0382] Scheme 4

[0383] [Chemical formula]

[0384] Dimethyl (5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (S7) was prepared from 5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine in the same manner as S1 in Scheme 1. 11H NMR (500 MHz, DMSO-d6) δ 7.90 (d, 2H), 7.11 (d, 2H), 3.83 (s, 3H), 2.65 (s, 6H). LCMS [M + H] 296.2.

[0385] From S6, in the same manner as compound 1 in Scheme 1, N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine (22) was prepared. 1 1H NMR (500 MHz, DMSO-d6) δ 7.88 - 7.79 (m, 3H), 7.15 (d, 1H), 7.09 (d, 2H), 3.99 (s, 3H), 3.82 (s, 3H). LCMS [M + H] 339.2.

[0386] Scheme 5

[0387]

Chemical Structure

[0388] From 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine, in the same manner as S1 in Scheme 1, dimethyl (5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (S8) was prepared. 1 1H NMR (500 MHz, DMSO-d6) δ 7.97 (d, 2H), 7.64 (d, 2H), 2.66 (s, 6H). LCMS [M + H] 300.1.

[0389] From S7, in the same manner as compound 1 in Scheme 1, N-(4-chloro-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine (23) was prepared. 11H NMR (500 MHz, DMSO-d6) δ 7.91 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 5.5 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 5.5 Hz, 1H), 3.99 (s, 3H). LCMS [M + H] 343.1.

[0390] Scheme 6

[0391]

Chem.

[0392] 5-(4-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (S9). Under a N2 atmosphere, 4-(trifluoromethyl)benzohydrazide (200 mg, 0.98 mmol), methanol (6 mL), and 1,4-dioxane (3 mL) were added to a 25 mL round-bottom flask equipped with a magnetic stir bar. Cyanogen bromide (155 mg, 1.47 mmol) was added in one portion, and the reaction was allowed to proceed for 1 h. Next, sodium bicarbonate (150 mg) was added in one portion, and the resulting mixture was stirred vigorously for 16 h. The resulting precipitate was collected by vacuum filtration and washed with a large amount of water to give a white solid (160 mg). 1 1H NMR (500 MHz, DMSO-d6) δ 7.97 (d, 2H), 7.8 (d, 2H), 7.41 (s, 2H). LCMS [M + H] 230.1.

[0393] Dimethyl (5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (S10) was prepared from 5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (s8) in the same manner as S1 in Scheme 1. 1 1H NMR (500 MHz, DMSO-d6) δ 8.18 (d, 2H), 7.94 (d, 2H), 2.68 (s, 6H). LCMS [M + H] 334.0.

[0394] From S10, in the same manner as 1 in Scheme 1, N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole-2-amine (20) was prepared. 1 H NMR (500 MHz, DMSO-d6) δ 8.11 (d, 2H), 7.91 (d, 2H), 7.87 (d, 1H), 7.18 (d, 1H), 4.00 (s, 3H). LCMS [M + H] 377.0.

[0395] Scheme 7

[0396]

Chemical formula

[0397] From 4-(trifluoromethoxy)benzohydrazide, in the same manner as S8 in Scheme 6, 5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazole-2-amine (S11) was prepared. 1 H NMR (500 MHz, DMSO-d6) δ 7.92 - 7.87 (m, 2H), 7.50 (d, 2H), 7.29 (s, 2H). LCMS [M + H] 246.1.

[0398] From 5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazole-2-amine (s10), in the same manner as S1 in Scheme 1, dimethyl(5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (S12) was prepared. 1 H NMR (500 MHz, DMSO-d6) δ 8.12 - 8.06 (m, 2H), 7.56 (d, 2H), 2.67 (s, 6H). LCMS [M + H] 350.0.

[0399] From S11, in the same manner as 1 in Scheme 1, N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazole-2-amine (21) was prepared. 1 H NMR (500 MHz, DMSO-d6) δ 8.07 - 7.98 (m, 2H), 7.86 (d, 1H), 7.56 - 7.51 (m, 2H), 7.17 (d, 1H), 3.99 (s, 3H). LCMS [M + H] 393.0.

[0400]

Chemical formula

[0401] 5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazole-2-amine. From 3,5-dimethoxybenzohydrazide, in the same manner as Scheme 5, the title compound was prepared. LCMS [M+H] 222.0.

[0402]

Chemical formula

[0403] Dimethyl (5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. From 5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2-amine, in the same manner as Scheme 5, the title compound was prepared. LCMS [M+H] 326.0.

[0404]

Chemical formula

[0405] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-amine (26). The title compound was prepared in the same manner as in Scheme 5 from 2-methoxypyridine-3,4-diamine and dimethyl (5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. 1 H NMR (500 MHz, DMSO-d6) δ 7.55 (d, 1H), 7.39-7.33 (m, 2H), 7.07 (d, 1H), 6.86 (d, 1H), 3.93 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H). LCMS [M+H] 369.1.

[0406]

Chem.

[0407] 5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-amine. The title compound was prepared in the same manner as in Scheme 5 from benzo[d][1,3]dioxole-5-carbohydrazide. LCMS [M+H] 206.0.

[0408]

Chem.

[0409] Dimethyl (5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared in the same manner as in Scheme 5 from 5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-amine.

[0410]

Chem.

[0411] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-amine (27). The title compound was prepared from 2-methoxypyridine-3,4-diamine and dimethyl (5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate in the same manner as in Scheme 5. 1 H NMR (500 MHz, DMSO-d6) δ 7.85 (d, 1H), 7.43 (d, 1H), 7.37 (s, 1H), 7.15 (d, 1H), 7.07 (d, 1H), 6.12 (s, 2H), 3.99 (s, 3H). LCMS [M+H] 353.1.

[0412]

Chem.

[0413] 5-(4-(Dimethylamino)phenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 4-(dimethylamino)benzohydrazide in the same manner as in Scheme 5. LCMS [M+H] 205.1.

[0414]

Chem.

[0415] Dimethyl (4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5. LCMS [M+H] 309.0.

[0416]

Chem.

[0417] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-amine (28). The title compound was prepared from 2-methoxypyridine-3,4-diamine and dimethyl (4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate in the same manner as in Scheme 5. 1 H NMR (500 MHz, DMSO-d6) δ 7.84 (d, 1H), 7.69 (d, 2H), 7.15 (d, 1H), 6.80 (d, 2H), 3.99 (s, 3H), 2.98 (s, 6H). LCMS [M+H] 352.1.

[0418]

Chem.

[0419] 5-(p-Tolyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from p-tolylbenzohydrazide in the same manner as in Scheme 5.

[0420]

Chem.

[0421] Dimethyl (p-tolyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(p-tolyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5. LCMS [M+H] 280.0.

[0422]

Chem.

[0423] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole-2-amine (29). The title compound was prepared in the same manner as in Scheme 5 from 2-methoxypyridine-3,4-diamine and dimethyl (p-tolyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. 1 H NMR (500 MHz, DMSO-d6) δ 7.85 (d, 1H), 7.79 (d, 2H), 7.35 (d, 2H), 7.16 (d, 1H), 3.99 (s, 3H), 2.37 (s, 3H). LCMS [M+H] 323.1.

[0424]

Chemical formula

[0425] N-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)oxazolo[4,5-b]pyridin-2-amine (30). The title compound was prepared in the same manner as in Scheme 4 from 2-aminopyridin-3-ol and dimethyl (5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. 1 H NMR (500 MHz, DMSO-d6) δ 7.90 (dd, 1H), 7.78 (d, 2H), 7.35 (dd, 1H), 7.06 (d, 2H), 6.73 (dd,1H), 3.80 (s, 3H). LCMS [M+H] 310.1.

[0426]

Chemical formula

[0427] 2-(Benzo[d][1,3]dioxol-5-yl)pyridine-3,4-diamine. The title compound was prepared in the same manner as in Scheme 6 from 2-chloro-3-nitropyridin-4-amine and benzo[d][1,3]dioxol-5-ylboronic acid. 11H NMR (400 MHz, chloroform-d) δ 8.20 (d, 1H), 7.00 - 6.96 (m, 2H), δ 6.83 (dd, 1H), 6.61 (d, 1H), 5.99 (s, 2H), 5.48 (s, 3H).

[0428]

Chem.

[0429] N-(4-(benzo[d][1,3]dioxol-5-yl)-3H-imidazo[4,5-c]pyridin-2-yl)-5-(4-fluorophenyl)-1,3,4-oxadiazol-2-amine (31). The title compound was prepared from 2-(benzo[d][1,3]dioxol-5-yl)pyridine-3,4-diamine and dimethyl (5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate in the same manner as in Scheme 6. 1 1H NMR (500 MHz, DMSO-d6) δ 8.34 (d, 1H), 7.97 (dd, 2H), 7.54 - 7.43 (m, 3H), 7.40 (t, 2H), 7.12 - 7.08 (m, 2H). LCMS [M+H] 417.2.

[0430]

Chem.

[0431] 4-(5-((4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)amino)-1,3,4-oxadiazol-2-yl)benzonitrile (32). The title compound was prepared from 2-methoxypyridine-3,4-diamine and dimethyl (5-(4-cyanophenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate in the same manner as in Scheme 5. 11H NMR (500 MHz, DMSO-d6) δ 8.00 (m, 4H), 7.85 (m, 1H), 7.18 (m, 1H), 3.99 (s, 3H). LCMS [M+H] 334.1.

[0432]

Chem.

[0433] 3-Fluoro-4-methoxybenzohydrazide. Under a N2 atmosphere, methyl 3-fluoro-4-methoxybenzoate (500 mg), ethanol (10 mL), and hydrazine monohydrate (1.5 mL) were added to a 40 mL pressure vial equipped with a magnetic stir bar. The flask was then heated at 80 °C for 16 h. A solid white precipitate was observed upon cooling to RT. Next, the contents of the pressure vial were poured into water (50 mL), and the precipitate was collected by vacuum filtration. The resulting hydrazide was used in the next transformation without further purification.

[0434]

Chem.

[0435] 5-(3-Fluoro-4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 3-fluoro-4-methoxybenzohydrazide in the same manner as in Scheme 5.

[0436]

Chem.

[0437] Dimethyl (5-(3-fluoro-4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(p-tolyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5.

[0438]

Chem.

[0439] 5-(3-Fluoro-4-methoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (33). The title compound was prepared from dimethyl (5-(3-fluoro-4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate and 2-methoxypyridine-3,4-diamine in the same manner as in Scheme 5. 1 H NMR (500 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.71 - 7.03 (m, 5H), 3.95 (s,3H), 3.91 (s, 3H). LCMS [M+H] 357.1.

[0440]

Chem.

[0441] 4-Fluoro-3-methoxybenzohydrazide. Under N2 atmosphere, methyl 3-fluoro-4-methoxybenzoate (500 mg), ethanol (10 mL), and hydrazine monohydrate (1.5 mL) were added to a 40 mL pressure vial equipped with a magnetic stir bar. Then, the flask was heated at 80 °C for 16 h. A solid white precipitate was observed when cooling to RT. Next, the contents of the pressure vial were poured into water (50 mL), and the precipitate was collected by vacuum filtration. Next, the obtained hydrazide was used in the next conversion without further purification.

[0442]

Chem.

[0443] 5-(4-Fluoro-3-methoxyphenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 4-fluoro-3-methoxybenzohydrazide in the same manner as in Scheme 5.

[0444]

Chem.

[0445] Dimethyl (5-(4-fluoro-3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(4-fluoro-3-methoxyphenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5.

[0446]

Chem.

[0447] 5-(3-Fluoro-4-methoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (34). The title compound was prepared from dimethyl (5-(4-fluoro-3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate and 2-methoxypyridine-3,4-diamine in the same manner as in Scheme 5. 1 H NMR (500 MHz, DMSO-d6) δ 7.85 (d, 1H), 7.69-7.66 (m, 2H), 7.33 (t, 1H), 7.16 (d, 1H), 3.98 (s, 3H), 3.90 (s, 3H). LCMS [M+H] 357.1.

[0448]

Chem.

[0449] 6-Methoxynicotinohydrazide. The title compound was prepared from methyl 6-methoxynicotinate in the same manner as 3-fluoro-4-methoxybenzohydrazide.

[0450]

Chem.

[0451] 5-(6-Methoxypyridin-3-yl)-1,3,4-oxadiazol-2-amine. The title compound was prepared in the same manner as in Scheme 5 from 6-methoxynicotinohydrazide. LCMS [M+H] 193.1.

[0452]

Chemical formula

[0453] Dimethyl (5-(6-methoxypyridin-3-yl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared in the same manner as in Scheme 5 from 5-(6-methoxypyridin-3-yl)-1,3,4-oxadiazol-2-amine. LCMS [M+H] 297.0.

[0454]

Chemical formula

[0455] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-(6-methoxypyridin-3-yl)-1,3,4-oxadiazol-2-amine (39). The title compound was prepared in the same manner as in Scheme 5 from dimethyl (5-(6-methoxypyridin-3-yl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate and 2-methoxypyridine-3,4-diamine. 1 H NMR (500 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.18 (dd, 1H), 7.86 (d, 1H), 7.16 (d, 1H), 7.00 (s, 1H), 3.99 (s, 3H), 3.92 (s, 3H). LCMS [M+H] 340.1.

[0456]

Chemical formula

[0457] 4-Cyclopropoxybenzohydrazide. The title compound was prepared from methyl 4-cyclopropoxybenzoate in the same manner as 3-fluoro-4-methoxybenzohydrazide.

[0458] [Chemical formula]

[0459] 5-(4-Cyclopropoxyphenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 4-cyclopropoxybenzohydrazide in the same manner as in Scheme 5.

[0460] [Chemical formula]

[0461] Dimethyl (5-(4-cyclopropoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(4-cyclopropoxyphenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5. LCMS [M+H] 322.1.

[0462] [Chemical formula]

[0463] 5-(4-Cyclopropoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (41). The title compound was prepared from dimethyl (5-(4-cyclopropoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate and 2-methoxypyridine-3,4-diamine in the same manner as in Scheme 5. 11H NMR (500 MHz, DMSO-d6) δ 7.87-7.80 (m, 3H), 7.20 (d, 2H), 7.14 (d, 1H), 3.98 (s, 3H), 3.92 (tt, 1H), 0.84-0.76 (m, 2H), 0.73-0.65 (m, 2H). LCMS [M+H] 365.2.

[0464]

Chem.

[0465] 5-(4-Isopropoxyphenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 4-isopropoxybenzohydrazide in the same manner as in Scheme 5.

[0466]

Chem.

[0467] Dimethyl(5-(4-isopropoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(4-isopropoxyphenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5. LCMS [M+H] 324.0.

[0468]

Chem.

[0469] 5-(4-Isopropoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (42). The title compound was prepared from dimethyl(5-(4-isopropoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate and 2-methoxypyridine-3,4-diamine in the same manner as in Scheme 5. 11H NMR (500 MHz, DMSO-d6) δ 8.02-7.87 (m, 3H), 7.16-7.06 (m, 3H), 4.76-4.65 (m, 1H), 3.98 (s, 3H), 1.24 (d, 6H). LCMS [M+H] 367.1.

[0470]

Chem.

[0471] 5-(4-(tert-Butyl)phenyl)-1,3,4-oxadiazol-2-amine. The title compound was prepared from 4-(tert-butyl)benzohydrazide in the same manner as in Scheme 5.

[0472]

Chem.

[0473] Dimethyl(5-(4-(tert-butyl)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate. The title compound was prepared from 5-(4-(tert-butyl)phenyl)-1,3,4-oxadiazol-2-amine in the same manner as in Scheme 5. LCMS [M+H] 322.0.

[0474]

Chem.

[0475] 5-(4-(tert-Butyl)phenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (43). The title compound was prepared from (5-(4-(tert-butyl)phenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate in the same manner as in Scheme 5. LCMS [M+H] 365.2.

[0476]

Chem.

[0477] 5-(2-Bromoacetyl)-2-methoxybenzonitrile (53-02). To a stirred and degassed solution of 5-bromo-2-methoxybenzonitrile (1) (5 g, 23.58 mmol, 1.0 eq) in 1,4-dioxane (50 mL) at RT were added tributyl(1-ethoxyvinyl)stannane (8.53 g, 23.58 mmol, 1 eq), CsF (8.95 g, 58.95 mmol, 2.5 eq), and Pd(PPh3)4 (1.36 g, 1.17 mmol, 0.05 eq). The resulting mixture was stirred at 80 °C for 16 h under N2. After completion of the reaction by TLC, the reaction mixture was diluted with ice-cold water (50 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and 5-(1-ethoxyvinyl)-2-methoxybenzonitrile (53-01) was obtained as a brown rubbery solid (7 g, yield: crude product). TLC system: EtOAc:hexane (5:95), R f value: ~0.2. This crude product was dissolved in THF:H2O (3:1) (80 mL) at RT, to which NBS (6.13 g, 34.46 mmol, 1.5 eq) was added and stirred for 1 h. After completion of the reaction by TLC, the reaction mixture was diluted with ice water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (50 mL), brine solution (50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. The crude compound was purified by silica gel (60 - 120 mesh) column chromatography [(10 - 15% ethyl acetate / hexane gradient elution] to give 5-(2-bromoacetyl)-2-methoxybenzonitrile (53-02) as an off-white solid [4.2 g, yield: 71% (2 steps)]. TLC system: EtOAc:hexane (20:80), R f value: ~0.3, 1HNMR (400 MHz, CDCl3) δ 8.23 - 8.20 (m, 2H), 7.08 (d, J = 8.8 Hz, 1H), 4.36 (s, 2H), 4.04 (s, 3H).

[0478] 5-(2-Aminothiazol-4-yl)-2-methoxybenzonitrile (53-03). To a stirred solution of 5-(2-bromoacetyl)-2-methoxybenzonitrile (53-02) (2.5 g, 9.88 mmol, 1.0 eq) in EtOH (25 mL) at RT was added thiourea (0.75 g, 9.88 mmol, 1 eq), and the mixture was heated to 80 °C and stirred for 2 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure, diluted with ice water and stirred for 1 h. The precipitated solid was filtered, washed with water and n-pentane, and dried under reduced pressure to give 5-(2-aminothiazol-4-yl)-2-methoxybenzonitrile (53-03) as an off-white solid (2 g, yield: 87%). TLC system: EtOAc:hexane (20:80), R f value: ~0.2, LCMS (m / z): 231.9 (M+H) + and 272.9 (ACN adduct); 1 HNMR (400 MHz, CDCl3) δ 8.12 (d, J = 2.4 Hz, 1H), 8.05 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.32 (d, J = 9.2 Hz, 1H), 7.16 (s, 1H), 5.10 - 4.55 (br, 2H), 3.95 (s, 3H).

[0479] Dimethyl (4-(3-cyano-4-methoxyphenyl)thiazol-2-yl)carbodiimidodithioate (53-04). To a stirred solution of 5-(2-aminothiazol-4-yl)-2-methoxybenzonitrile (53-03) (2 g, 8.65 mmol, 1.0 eq) in DMF (20 mL) at 0 °C was added 20 M aqueous NaOH solution (0.64 mL, 12.97 mmol, 1.5 eq), and the mixture was stirred for 10 min. Then CS2 (1.64 g, 21.62 mmol, 2.5 eq) was added. After stirring at RT for 30 min, the mixture was cooled back to 0 °C, and MeI (3.07 g, 21.62 mmol, 2.5 eq) was added, followed by stirring at RT for 4 h. After completion of the reaction by TLC, the volatiles were evaporated under reduced pressure, diluted with ice water, and stirred for 30 min. The precipitated solid was filtered, washed with water, and dried under reduced pressure to give dimethyl (4-(3-cyano-4-methoxyphenyl)thiazol-2-yl)carbodiimidodithioate (53-04) as a brown solid (1.8 g, yield: 62%). TLC system: EtoAc:hexane (20:80), R f value: ~0.6, LCMS (m / z): 335.9 (M+H) + ; 1 H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 2.4 Hz, 1H), 8.05 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.20 (s, 1H), 7.00 (d, J = 9.8 Hz, 1H), 3.97 (s, 3H), 2.59 (s, 6H).

[0480] 2-Methoxy-5-(2-((4-methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)amino)thiazol-4-yl)benzonitrile (53). To a stirred solution of dimethyl (4-(3-cyano-4-methoxyphenyl)thiazol-2-yl)carbodiimidodithioate (53-04) (1 g, 2.98 mmol, 1 eq) and 2-methoxy-6-methylpyridine-3,4-diamine (55-04) (0.45 g, 2.98 mmol, 1 eq) in DMF (10 mL) was added K2CO3 (1.02 g, 7.45 mmol, 2.5 eq) at RT, and the mixture was stirred at 150 °C for 2 h under MW. After completion of the reaction by TLC, the reaction mixture was diluted with ice water and extracted with ethyl acetate (2 × 70 mL). The combined organic layers were washed with water (30 mL) and brine solution (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was triturated with ethyl acetate to enhance the purity, and the resulting material was passed through silica gel combiflash (12 g of general silica) chromatography [gradient elution with 5 - 10% MeOH / DCM] to give 2-methoxy-5-(2-((4-methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)amino)thiazol-4-yl)benzonitrile (53) as an off-white solid (30 mg, yield: 2.5%). TLC system: MeOH:DCM (5:95), R f value: ~0.3, LCMS (m / z): 392.9 (M+H) + ; 1 1H NMR (400 MHz, DMSO-d6) δ 11.90 - 11.64 (br, 2H), 8.30 - 8.23 (m, 2H), 7.55 (brs, 1H), 7.33 (d, J = 9.2 Hz, 1H), 6.93 (brs, 1H), 3.97 (s, 6H), 2.42 (s, 3H).

[0481]

Chemical Structure

[0482] 2,4-Dichloro-6-methyl-3-nitropyridine (55-01). To a stirred solution of 6-methyl-3-nitropyridine-2,4-diol (1) (4 g, 23.55 mmol, 1 eq) in POCl3 (80 mL) was added TEA (3.3 mL, 23.55 mmol, 1 eq) dropwise at 0 °C. The resulting mixture was heated to 110 °C and stirred for 5 h. After completion of the reaction by TLC, the volatiles were evaporated and poured very slowly into saturated aqueous NaHCO3, and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with brine (1000 mL), dried over sodium sulfate, and concentrated. The crude compound was purified by silica gel (60 - 120 mesh) column chromatography [gradient elution with 15 - 20% ethyl acetate / hexane], and 2,4-dichloro-6-methyl-3-nitropyridine (55-01) was obtained as an off-white solid (3.1 g, yield: 64%). TLC system: EtOAc / hexane (10:90), R f value: ~0.7, 1 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H), 2.60 (s, 3H).

[0483] 2-Chloro-6-methyl-3-nitropyridin-4-amine (55-02). To a stirred solution of 2,4-dichloro-6-methyl-3-nitropyridine (55-01 (3.1 g, 15.05 mmol, 1.0 eq) in THF (30 mL) was added 7N MeOH-NH3 (30 mL) at RT, and the mixture was stirred at 80 °C for 24 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure, diluted with ice water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (40 mL), brine solution (40 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude compound was purified by silica gel (60 - 120 mesh) column chromatography [gradient elution with 20 - 30% ethyl acetate / hexane], and 2-chloro-6-methyl-3-nitropyridin-4-amine (55-02) was obtained as a yellow solid (1.2 g, yield: 42%). TLC system: EtOAc:hexane (20:80), R f value: ~0.3, LCMS (m / z): 188.0 (M+H)+ ; 1 HNMR (400 MHz, CDCl3) δ 7.27 (s, 2H), 6.63 (s, 1H), 2.27 (s, 3H).

[0484] 2-Methoxy-6-methyl-3-nitropyridin-4-amine (55-03). To a solution of 2-chloro-6-methyl-3-nitropyridin-4-amine (55-02) (1.2 g, 6.41 mmol, 1.0 eq) in MeOH (15 mL) was added NaOMe (1.38 g, 25.66 mmol, 4 eq) at RT, and the mixture was stirred at 80 °C for 4 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure and diluted with ice water. After stirring for 30 min, the stirred solid was filtered, washed with water and pentane, and dried under reduced pressure to obtain 2-methoxy-6-methyl-3-nitropyridin-4-amine (55-03) as a yellow solid (0.84 g, yield: 72%). TLC system: EtOAc:hexane (20:80), R f value: ~0.4, 1 HNMR (400 MHz, CDCl3) δ 6.13 (s, 1H), 6.00 (s, 2H), 4.00 (s, 3H), 2.30 (s, 3H).

[0485] 2-Methoxy-6-methylpyridine-3,4-diamine (55-04). To a stirred solution of 2-methoxy-6-methyl-3-nitropyridin-4-amine (55-03) (0.84 g, 4.59 mmol, 1.0 eq) in MeOH (10 mL) was added 10% Pd / C (150 mg) at RT. The reaction mixture was stirred at RT for 3 h under a H2 balloon pressure. After completion of the reaction by TLC, the reaction mixture was filtered through a celite bed, washed with MeOH (20 mL) and concentrated to give the crude product, which was purified by pentane trituration to obtain 2-methoxy-6-methylpyridine-3,4-diamine (55-04) as a pale brown solid (600 mg, yield: 86%). TLC system: EtoAc:hexane (30:70) R f value: ~0.2, LCMS (m / z): 154 (M+H) + ; 11H NMR (400 MHz, CDCl3) δ 6.15 (s, 1H), 3.94 (s, 3H), 3.75 (brs, 2H), 3.05 (br, 2H), 2.28 (s, 3H).

[0486] 5-(3,4-Dimethoxyphenyl)-N-(4-methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (55). To a stirred solution of dimethyl(5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)carbodiimidodithioate (1 g, 3.08 mmol, 1.0 eq) and 2-methoxy-6-methylpyridine-3,4-diamine (55-04) (0.42 g, 3.08 mmol, 1 eq) in DMF (10 mL) was added K2CO3 (0.85 g, 6.16 mmol, 2.0 eq) at RT, and the mixture was stirred under microwave irradiation at 150 °C for 2 h. After completion of the reaction by TLC, the reaction mixture was diluted with ice water and extracted with ethyl acetate (2 × 70 mL). The combined organic layers were washed with water (30 mL) and brine solution (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The purity of the crude product was enhanced by ethyl acetate trituration followed by silica gel grace (12 g of general silica) column chromatography [gradient elution with 5-10% MeOH / DCM] to afford 5-(3,4-dimethoxyphenyl)-N-(4-methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (55) as an off-white solid (26 mg, yield: 2.2%). TLC system: MeOH:DCM (5:95), R f value: ~0.4, LCMS (m / z): 383.1 (M+H) + ; 1HNMR (400 MHz, DMSO-d6) δ 12.2 - 11.5 (br, 1H), 7.47 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.99 (s, 1H), 3.98 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 2.42 (s, 3H).

[0487]

Chem.

[0488] 5-(3,4-Dimethoxyphenyl)oxazol-2-amine (59 - 01). Urea (2.89 g, 48.25 mmol, 2.5 eq) was added to a stirred solution of 2-bromo-1-(3,4-dimethoxyphenyl)ethan-1-one (1) (5 × 1 g, 19.30 mmol, 1 eq) in DMF (50 mL) at RT, and the mixture was heated at 140 °C for 10 min under microwave irradiation. After completion of the reaction by TLC, the reaction mixture was diluted with ice-cold water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with ice water (70 mL) and brine solution (50 mL), dried over sodium sulfate, concentrated to give a residue. The crude compound was purified by silica gel (60 - 120 mesh) column chromatography [gradient elution with 30 - 60% ethyl acetate / hexane] to afford 5-(3,4-dimethoxyphenyl)oxazol-2-amine (59 - 01) as an off-white solid (1 g, yield: 23%). TLC system: EtOAc / hexane (50:50), R f value: ~0.2, LCMS (m / z): 221.0 (M + H) + ; 1 HNMR (400 MHz, DMSO-d6) δ 7.78 (s, 1H), 7.21 - 7.16 (m, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.65 (s, 2H), 3.77 (s, 3H), 3.75 (s, 3H).

[0489] 4-Methoxy-1H-imidazo[4,5-c]pyridine-2-thiol (59-02). To a stirred solution of 2-methoxypyridine-3,4-diamine (1 g, 7.19 mmol, 1.0 eq) in EtOH:H2O (3:1) (12 mL) at RT was added KOH (0.443 g, 7.90 mmol, 1.1 eq), and after stirring for 10 min, CS2 (0.601 g, 7.90 mmol, 1.1 eq) was added. The resulting mixture was heated at 130 °C for 20 min under microwave irradiation. After completion of the reaction by TLC, the volatiles were removed under reduced pressure, diluted with ice water, and extracted with ethyl acetate (2 × 70 mL). The combined organic layers were washed with water (20 mL), brine solution (20 mL), dried over anhydrous sodium sulfate, and concentrated to give 4-methoxy-1H-imidazo[4,5-c]pyridine-2-thiol (59-02) as a brown solid (1.1 g, yield: 84%). TLC system: EtOAc:hexane (50:50), R f value: ~0.6, LCMS (m / z): 181.9 (M+H) + ; 1 1H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 12.84 (s, 1H), 7.82 (d, J = 5.6 Hz, 1H), 6.84 (d, J = 5.6 Hz, 1H), 3.96 (s, 3H).

[0490] 4-Methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine (59-03). To a stirred solution of 4-methoxy-1H-imidazo[4,5-c]pyridine-2-thiol (59-02) (1.1 g, 6.07 mmol, 1.0 eq) in acetone (10 mL) were added K2CO3 (1.25 g, 9.10 mmol, 1.5 eq) and MeI (0.861 g, 6.07 mmol, 1 eq) at RT. The resulting mixture was stirred at RT for 3 h. After completion of the reaction by TLC, the volatiles were evaporated under reduced pressure, diluted with ice water, and extracted with ethyl acetate (2×80 mL). The combined organic layers were washed with water (40 mL), brine solution (20 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was purified by trituration with n-pentane to afford 4-methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine (59-03) as a brown rubbery solid (1 g, yield: 84%). TLC system: EtOAc:hexane (30:70), R f value: ~0.4, LCMS (m / z): 196.1 (M+H) + ; 1 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 7.89 (d, J = 5.6 Hz, 1H), 6.99-6.95 (br, 1H), 4.12 (s, 3H), 2.81 (s, 3H).

[0491] tert-Butyl 4-methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-04). To a stirred solution of 4-methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine (59-03) (1 g, 5.12 mmol, 1.0 eq) in DCM (10 mL) was added TEA (0.71 mL, 5.12 mmol, 1.0 eq), DMAP (0.062 g, 0.51 mmol, 0.1 eq), and (Boc)2O (1.35 mL, 6.14 mmol, 1.2 eq) at RT. The resulting mixture was stirred at RT for 16 h. After completion of the reaction by TLC, it was diluted with ice-cold water and extracted with dichloromethane (2 × 50 mL). The combined organic layers were washed with ice-cold water (20 mL), brine solution (20 mL), dried over sodium sulfate, concentrated to give a residue. The residue was purified by silica gel (60 - 120 mesh) column chromatography [gradient elution with 30 - 60% ethyl acetate / hexane] to afford tert-Butyl 4-methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-04) as an off-white solid (1.2 g, yield: 80%). TLC system: EtOAc:hexane (30:70) R f value: ~0.6, LCMS (m / z): 295.9 (M+H) + ; 1 H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 5.6 Hz, 1H), 7.40 (d, J = 5.6 Hz, 1H), 4.15 (s, 3H), 2.75 (s, 3H), 1.72 (s, 9H).

[0492] tert-Butyl 4-methoxy-2-(methylsulfonyl)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-05). To a stirred solution of tert-butyl 4-methoxy-2-(methylthio)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-04) (1.2 g, 4.06 mmol, 1.0 eq) in DCM (15 mL) was added mCPBA (1.4 g, 8.12 mmol, 2 eq) at 0 °C under nitrogen flush, and the mixture was stirred at RT for 4 h. After completion of the reaction by TLC, it was quenched with water (20 mL) and extracted with dichloromethane (2 × 50 mL). The combined organic layers were washed with aqueous sodium bicarbonate solution (2 × 20 mL), brine solution (20 mL), dried over anhydrous sodium sulfate, concentrated, and tert-butyl 4-methoxy-2-(methylsulfonyl)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-05) was obtained as an off-white solid (1 g, purity 65%). TLC system: EtOAc:hexane (50:50), R f value: ~0.5, 227.9 (M+H-Boc) + , purity 65%.

[0493] 5-(3,4-Dimethoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)oxazole-2-amine (59). To a stirred solution of 5-(3,4-dimethoxyphenyl)oxazole-2-amine (59-01) (200 mg, 0.91 mmol, 1 eq) in THF (5 mL) was added 55% NaH (44 mg, 1.83 mmol, 2 eq) at 0 °C under a nitrogen flush and the mixture was stirred for 30 min. Subsequently, a solution of tert-butyl 4-methoxy-2-(methylsulfonyl)-1H-imidazo[4,5-c]pyridine-1-carboxylate (59-05) (300 mg, purity 65%, 0.91 mmol, 1 eq) in THF (1 mL) was added at 0 °C. The resulting solution was stirred at RT for 3 h. After completion of the reaction by TLC, the volatiles were evaporated under reduced pressure, diluted with ice water and extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with water (10 mL), brine solution (10 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was purified by multiple ethyl acetate triturations to afford 5-(3,4-dimethoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)oxazole-2-amine (59) as an off-white solid (15 mg, yield: 4.4%). TLC system: MeOH:DMC (5:95), R f value: ~0.2, LCMS (m / z): 368.4 (M+H) + ; 1 HNMR (400 MHz, DMSO-d6) δ 12.00 (br, 2H), 8.02 (s, 1H), 7.85 (d, J = 5.6 Hz, 1H), 7.49 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.17 (d, J = 5.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H).

[0494]

Chemical Structure

[0495] (1s,4s)-Methyl 4-methoxycyclohexanecarboxylate (62a). To a stirred solution of (1s,4s)-4-hydroxyhexanoic acid (500.0 mg, 3.47 mmol) in DMF (10.0 mL) was added NaH (345.0 mg, 8.67 mmol) at 0 °C, and the mixture was stirred at 0 °C for 30 min. Then CH3I (0.65 mL, 10.05 mmol) was added, and the mixture was stirred at 25 °C for 4 h. The mixture was quenched with ice-cold water and extracted with EA (20.0 mL × 3). The organic phases were combined, dried over Na2SO4, concentrated in vacuo, and the residue was purified by column (PE:EA = 2:1) to give 62a (350.0 mg, 58.6%) as a colorless oil. 1 H-NMR (400 MHz, CDCl3-d): δ(ppm) : 3.67 (s, 3H), 3.35 - 3.39 (m, 1H), 3.31 (s, 3H), 2.34 - 2.41 (m, 1H), 1.79 - 1.88 (m, 4H), 1.61 - 1.65 (m, 2H), 1.47 - 1.58 (m, 2H).δ

[0496] (1s,4s)-4-Methoxycyclohexanecarbohydrazide (62b). To a solution of 62a (350.0 mg, 2.0 mmol) in EtOH (3.0 mL) was added hydrazine hydrate (3.0 mL), and the mixture was stirred at 80 °C for 16 h, concentrated in vacuo, and the residue was purified by column (DCM:MeOH = 20:1) to give 62b (130.0 mg, 37.1%) as a white solid. 1H-NMR (400 MHz, DMSO-d6): δ(ppm) : 8.86 (s, 1H), 4.11 (s, 2H), 3.33 - 3.35 (m, 1H), 3.19 (s, 3H), 2.03 - 2.10 (m, 1H), 1.80 - 1.84 (m, 2H), 1.59 - 1.69 (m, 2H), 1.31 - 1.38 (m, 4H).

[0497] N-(4-Methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-2-((1s,4s)-4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (62c). To a solution of 62b (65.0 mg, 0.38 mmol) in DMF (6.0 mL) were added 2-isothiocyanato-4-methoxy-3H-imidazo[4,5-c]pyridine (63c, 78.0 mg, 0.38 mmol) and DIPEA (98.2 mg, 0.76 mmol). The mixture was stirred at 70 °C for 2 h. 62C was detected by LCMS and the mixture was used directly in the next step. MS: m / z: 379.3 [M+H]+

[0498] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-((1s,4s)-4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (62). To a stirred solution of 62c (143.0 mg, 0.38 mmol) in DMF (6.0 mL) was added EDCI (72.8 mg, 0.38 mmol) at 25 °C. Then the mixture was stirred at 60 °C for 2 h. The mixture was purified by preparative HPLC to give 62 (58.1 mg, 44.4%) as a white solid. MS: m / z: 345.3 [M+H] + , 1 1H-NMR (400 MHz, DMSO-d6): δ(ppm): 7.85 - 7.86 (m, 1H), 7.14 - 7.15 (m, 1H), 4.00 (s, 3H), 3.39 - 3.40 (m, 1H), 3.23 (s, 3H), 2.87 - 2.92 (m, 1H), 1.70 - 1.85 (m, 6H), 1.55 - 1.60 (m, 2H).

[0499]

Chemical Structure

[0500] 2-Methoxypyridine-3,4-diamine (63a). To a solution of 2-methoxy-3-nitropyridin-4-amine (5.0 g, 29.8 mmol) in MeOH (200.0 mL) was added Pd / C (500.0 mg, 10%). The mixture was stirred at RT for 16 h under a H2 balloon. After the reaction was complete, the resulting mixture was filtered through celite and the filtrate was concentrated in vacuo to give 63a (4.14 g, 100%) as a brown solid, which was used in the next step without purification. MS: m / z: 140.1 [M+H] + .

[0501] 4-Methoxy-3H-imidazo[4,5-c]pyridin-2-amine (63b). To a stirred solution of 63a (4.1 g, 29.8 mmol) in EtOH (10.0 mL) was added CNBr (4.7 g, 44.6 mmol) portionwise at 0 °C and the mixture was stirred at 80 °C for 16 h under Ar. The mixture was diluted with water (300.0 mL), basified to pH = 10 with 1 N NaOH and extracted with EA (100.0 mL × 3). The organic phase was extracted twice with brine, dried over Na2SO4 and concentrated in vacuo to give 63b (2.9 g, 59.4%) as a grey solid.

[0502] 2-Isothiocyanato-4-methoxy-3H-imidazo[4,5-c]pyridine (63c). To a solution of 63b (1.0 g, 6.0 mmol) in ACN (30.0 mL) was added thio-CDI (1.4 g, 7.9 mmol). The mixture was stirred at 50 °C for 16 h. The precipitated solid was collected by filtration, washed with ACN (30 mL) and dried under vacuum to give 63c (660.0 mg, 53%) as a yellow solid. MS: m / z: 239.0 [M+CH3OH]+.

[0503] N-(4-Methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-2-((1r,4r)-4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (63d). To a solution of 63c (206 mg, 1.0 mmol) in DMF (5.0 mL) were added (1r,4r)-4-methoxycyclohexanecarbohydrazide (172.0 mg, 1.0 mmol) and DIPEA (258.5 mg, 2.0 mmol). The mixture was stirred at 70 °C for 2 h. 63d was detected by LCMS and the mixture was used directly in the next step. MS: m / z: 379.2 [M+H] + .

[0504] N-(4-Methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-5-((1r,4r)-4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (63). To a solution of 63d (174.0 mg, 0.46 mmol) in DMF (6.0 mL) was added EDCI (88.0 mg, 0.46 mmol) at 25 °C and the mixture was stirred at 60 °C for 2 h. The mixture was purified by preparative HPLC to give 63 (11.1 mg, 7.0%) as a white solid. MS: m / z: 345.1 [M+H] + , 1 H-NMR (400 MHz, DMSO-d6): δ(ppm) : 7.85 - 7.86 (m, 1H), 7.14 - 7.15 (m, 1H), 3.99 (s, 3H), 3.25 (s, 3H), 3.13 - 3.23 (m, 1H), 2.76 - 2.81 (m, 1H), 2.03 - 2.06 (m, 4H), 1.48 - 1.57 (m, 2H), 1.22 - 1.31 (m, 2H).

[0505]

Chemical Structure

[0506] 1-Methylpiperidine-4-carbohydrazide (64-01). In a sealed tube, hydrazine hydrate H2O (31.8 g, 636 mmol, 10 eq) was added to a stirred solution of methyl 1-methylpiperidine-4-carboxylate (10 g, 63.60 mmol, 1 eq) in EtOH (200 mL) at RT, and the mixture was stirred at 120 °C for 16 h. After completion of the reaction by TLC (ninhydrin staining), the reaction mixture was concentrated under reduced pressure, and the resulting material was triturated with diethyl ether (100 mL) and dried under vacuum to obtain 1-methylpiperidine-4-carbohydrazide (64-01) as an off-white solid (9 g, yield: 90%). TLC system: MeOH:DCM (10:90), (ninhydrin staining) R f value: ~0.1 LCMS (m / z): 158.1 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.91 (s,1H), 4.13 (brs, 2H), 2.76-2.72 (m, 2H), 2.11 (s, 3H), 2.00-1.93 (m, 1H), 1.81-1.73 (m, 2H), 1.63-1.55 (m, 4H).

[0507] N-(4-Methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazinecarbothioamide (64-02). To a stirred solution of 4-methoxy-3H-imidazo[4,5-c]pyridin-2-amine (63b) (3 g, 18.2 mmol, 1.0 eq) in DMF (30 mL) was added thioCDI (16.2 g, 91.4 mmol, 5 eq) at 0 °C and the mixture was stirred at RT for 3 h. Then, 1-methylpiperidine-4-carbohydrazide (70-01) (7.14 g, 45.5 mmol, 2.5 eq) was added and the mixture was stirred at RT for 16 h. After observing the product by LCMS, the volatiles were evaporated under reduced pressure to give the crude product. The crude product was purified by reverse phase (C18 column) purification [gradient elution with 10 - 30% ACN in 0.1% FA in H2O] to afford N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazinecarbothioamide (64-02) as an off-white solid (160 mg, yield: 2.4% over 2 steps). TLC system: MeOH:DCM (20:80), R f value: ~0.05, LCMS (m / z): purity 88%, 364.0 (M+H) + .

[0508] N-(4-Methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-amine (64). A solution of N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazinecarbothioamide (64-02) (170 mg, 0.46 mmol, 1.0 eq) and mercury(II) acetate (190 mg, 0.60 mmol, 1.3 eq) in MeOH (3.5 mL) was stirred at 80 °C for 2 h. After completion of the reaction by TLC, the reaction mixture was filtered through a pad of celite and washed with 20% MeOH / DCM (40 mL). The filtrate was concentrated under reduced pressure and subsequently purified by preparative HPLC to give N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazol-2-amine (64) as an off-white solid (20 mg, yield: 13%). TLC system: MeOH:DCM (20:80), R f value: ~0.05, LCMS (m / z): 330.5 (M+H) + ; 1 HNMR (400 MHz, DMSO-d6) δ 12.00 (br, 1H), 7.84 (d, J = 5.6 Hz, 1H), 7.14 (d, J = 5.6 Hz, 1H), 3.99 (s, 3H), 2.86-2.81 (m, 3H), 2.25 (s, 3H), 2.14-2.10 (m, 2H), 1.98-1.95 (m, 2H), 1.75-1.72 (m, 2H).

[0509]

Chemical Structure

[0510] 2,6-Dimethylmorpholine-4-carbohydrazide (70-02). To a solution of 2,6-dimethylmorpholine (10 g, 86.95 mmol, 1 eq) and TEA (13.17 g, 130.42 mmol, 1.5 eq) in DCM (300 mL) was added dropwise a solution of triphosgene (12.87 g, 43.47 mmol, 0.5 eq) in DCM (80 mL) at -10 °C, and the mixture was stirred at -10 °C for 2 h. After completion of the reaction by TLC, the reaction mixture was concentrated to the minimum volume under reduced pressure, the precipitated solid was filtered and washed under reduced pressure with diethyl ether (300 mL). The filtrate was concentrated under reduced pressure to obtain 2,6-dimethylmorpholine-4-carbonyl chloride (70-01) as a brown rubbery solid (14 g, crude product), TLC system: EtOAc (100%), R f value: ~0.7, and it was carried on to the next step without analysis.

[0511] To a stirred solution of 2,6-dimethylmorpholine-4-carbonyl chloride (70-01) (14 g, 79.06 mmol, 1 eq) in dioxane (280 mL) was added hydrazine hydrate (25.29 g, 790 mmol, 10 eq) at RT, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to obtain a residue. The crude product was washed with diethyl ether (100 mL) to obtain 2,6-dimethylmorpholine-4-carbohydrazide (70-02) as an off-white solid (7 g, yield: 46% over 2 steps). TLC system: MeOH:DCM (10:90), R f value: ~0.05, 1 1H NMR (400 MHz, DMSO- d d6) δ 7.67-7.44 (br, 1H), 3.78 (dd, J = 8.8 Hz, 0.8 Hz, 2H), 3.44-3.38 (m, 2H), 2.33-2.27 (m, 2H), 1.05 (d, J = 5.2 Hz, 6H).

[0512] 2-(2,6-Dimethylmorpholine-4-carbonyl)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)hydrazine-1-carbothioamide (70-03). To a stirred solution of 4-methoxy-3H-imidazo[4,5-c]pyridin-2-amine (63b) (4.5 g, 27.43 mmol, 1.0 eq) in DMF (45 mL) was added thioCDI (7.32 g, 41.14 mmol, 1.5 eq) at 0 °C, and the mixture was stirred at RT for 16 h. Then, 2,6-dimethylmorpholine-4-carbohydrazide (70-02) (5.7 g, 32.91 mmol, 1.2 eq) was added, and the mixture was stirred at RT for 4 h. After observation of the conversion by LCMS, the mixture was diluted with ice-cold water and extracted with 10% MeOH in DCM (2 × 100 mL). The combined organic layers were washed with brine solution (10 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase grace purification [gradient elution with 10 - 30% (0.1% HCOOH in H2O) / ACN] to give 2-(2,6-dimethylmorpholine-4-carbonyl)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)hydrazine-1-carbothioamide (70-03) as an off-white rubbery solid (150 mg, yield: 1.4% in 2 steps). TLC system: MeOH:DCM (10:90), R f value: ~0.5, LCMS (m / z): purity 67%, 380.0 (M+H) + .

[0513] 5-(2,6-Dimethylmorpholino)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (70). A stirred solution of 2-(2,6-dimethylmorpholine-4-carbonyl)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)hydrazine-1-carbothioamide (70-03) (100 mg, 0.26 mmol, 1.0 eq) and EDC.HCl (61.3 mg, 0.39 mmol, 1.5 eq) in DMF (2 mL) was stirred at 65 °C for 2 h at RT. After completion of the reaction by TLC, it was diluted with ice water, and the resulting precipitate was filtered and dried, and this was further purified by successive trituration with 2 mL each of IPA, ACN, and ether to give 5-(2,6-dimethylmorpholino)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (70) as an off-white solid (15 mg, yield: 16%). TLC system: MeOH:DCM (10:90), R f value: ~0.5 (The distinction between the SM and the product could not be concluded), LCMS (m / z): 346.4 (M+H) + ; 1 HNMR (400 MHz, DMSO-d6) δ 7.82 (d, J = 5.6 Hz, 1H), 7.11 (d, J = 5.6 Hz, 1H), 3.98 (s, 3H), 3.69-3.65 (m, 2H), 3.61-3.58 (m, 2H), 2.63-2.57 (m, 2H), 1.12 (d, J = 6.4 Hz, 6H).

[0514]

Chem.

[0515] (1H-Imidazol-1-yl)(4-methoxypiperidin-1-yl)methanone (71-01). To a stirred solution of 4-methoxypiperidine (10 g, 87 mmol, 1.0 eq) in THF (200 mL) was added CDI (42 g, 261 mol, 3 eq) at RT, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC (UV and ninhydrin staining), the reaction mixture was evaporated and purified twice by a neutral alumina column (eluted with DCM) to obtain (1H-Imidazol-1-yl)(4-methoxypiperidin-1-yl)methanone (71-01) as a brown liquid (15.4 g, yield: 83%). TLC system: MeOH:DCM (10:90), R f value: ~0.6, LCMS (m / z): 98%; 209.9 (M+H) + ; 1 HNMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.19 (t, J = 1.6 Hz, 1H), 7.09 (t, J = 1.6 Hz, 1H), 3.75-3.71 (m, 2H), 3.55-3.45 (m, 3H), 3.37 (s, 3H), 1.94-1.87 (m, 2H), 1.77-1.70 (m, 2H).

[0516] 4-Methoxypiperidine-1-carbohydrazide (71-02). To a stirred solution of (1H-Imidazol-1-yl)(4-methoxypiperidin-1-yl)methanone (71-01) (15.4 g, 73.7 mmol, 1.0 eq) in dioxane (300 mL) was added NH2NH2·H2O (36.8 g, 737 mmol, 10 eq), and the mixture was stirred at RT for 16 h. After conversion based on TLC (ninhydrin staining), the volatiles were evaporated to obtain the crude material. The crude product was purified by a silica (60~120 mesh) column (eluted with 0~3% MeOH in DCM) to obtain 4-methoxypiperidine-1-carbohydrazide (71-02) as a brown solid (6.2 g, yield: 49%), and 3 g of an impure fraction was removed. TLC system: MeOH:DCM (90:10), R f value: ~0.4, 11H NMR (400 MHz, CDCl3) δ 5.64 (s, 1H), 3.79 (br, 2H), 3.65 - 3.57 (m, 2H), 3.42 - 3.36 (m, 1H), 3.35 (s, 3H), 3.17 - 3.11 (m, 2H), 1.87 - 1.81 (m, 2H), 1.57 - 1.53 (m, 2H).

[0517] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxypiperidine-1-carbonyl)hydrazine-1-carbothioamide (71 - 03). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72 - 03) (0.5 g, 1.83 mmol, 1.0 eq) and 4-methoxypiperidine-1-carbohydrazide (71 - 02) (0.47 g, 2.74 mmol, 1.5 eq) in DMF (5 mL) was added DIPEA (0.59 g, 4.57 mmol, 2.5 eq) at 0 °C, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to give the crude product, which was purified by reverse-phase column (eluting with 30 - 60% ACN and 0.1% FA in water) to afford N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxypiperidine-1-carbonyl)hydrazine-1-carbothioamide (71 - 03) as an off-white solid (155 mg, yield: 22%). TLC system: MeOH:DCM (10:90), R f value: ~0.6, LCMS (m / z): 379.4 (M + H) + ; Purity 80%.

[0518] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxypiperidin-1-yl)-1,3,4-oxadiazol-2-amine (71). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxypiperidine-1-carbonyl)hydrazine-1-carbothioamide (71-03) (0.155 g, 0.41 mmol, 1.0 eq) in DMF (1.5 mL) was added EDC·HCl (0.12 g, 0.61 mmol, 1.5 eq) at RT, and the mixture was heated to 60 °C and stirred for 1 h. After completion of the reaction by LCMS, the reaction mixture was diluted with ice water, and the precipitated solid was filtered and dried to give the crude product. The crude product was purified by trituration with 0.5% MeOH in DCM (2 × 5 mL) to afford N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxypiperidin-1-yl)-1,3,4-oxadiazol-2-amine (71) as a white solid (45 mg, yield: 32%). TLC system: MeOH:DCM (10:90), R f value: ~0.5, LCMS (m / z): 345.4 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ 11.63 (br, 2H), 7.06 - 7.01 (m, 2H), 6.80 - 6.77 (m, 1H), 3.89 (s, 3H), 3.57 - 3.52 (m, 2H), 3.42 - 3.36 (m, 1H), 3.15 - 3.09 (m, 2H), 1.93 - 1.88 (m, 2H), 1.55 - 1.48 (m, 2H).

[0519]

Chemical Structure

[0520] 3-Methoxybenzene-1,2-diamine (72-01). To a stirred solution of 2-methoxy-6-nitroaniline (1) (10 g, 59.5 mmol, 1.0 eq) in EtOH:H2O (2:1, 100 mL) at RT, Fe (16.6 g, 0.3 mol, 5 eq) and NH4Cl (16 g, 0.3 mol, 5 eq) were added and the mixture was stirred at 80 °C for 2 h. After completion of the reaction by TLC, the reaction mixture was filtered through a Celite bed and the filtrate was evaporated to give a residue. The obtained residue was diluted with water and extracted with EtOAc (2 × 200 mL). The organic layer was dried over Na2SO4, concentrated and triturated with n-pentane to give 3-methoxybenzene-1,2-diamine (72-01) as a red rubbery solid (8 g, yield: 99%). TLC system: EtOAc:hexane (50:50), R f value: ~0.2, LCMS (m / z): 89%; 138.9 (M+H) + ; 1 HNMR (400 MHz, CDCl3) δ 6.66 (d, J = 8.0 Hz, 1H), 6.42-6.38 (m, 2H), 3.83 (s, 3H), 3.43 (brs, 4H).

[0521] 7-Methoxy-1H-benzo[d]imidazol-2-amine (72-02). To a stirred solution of 3-methoxybenzene-1,2-diamine (72-01) (8 g, 58 mmol, 1.0 eq) in EtOH (80 mL) at RT, CNBr (12.3 g, 116 mmol, 2 eq) was added and the mixture was stirred at 80 °C for 16 h. After completion of the reaction by TLC, the volatiles were evaporated and the residue was purified by silica gel column chromatography (eluting with 10% MeOH in DCM) to give 7-methoxy-1H-benzo[d]imidazol-2-amine (72-02) as a brown solid (8.2 g, yield: 85%). TLC system: MeOH:DMC (10:90), R f value: ~0.2, LCMS (m / z): 92%; 163.9 (M+H) + ; 11H NMR (400 MHz, DMSO-d6) δ 12.60 (br, 1H), 8.05 (s, 2H), 7.15 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 3.93 (s, 3H).

[0522] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03). To a solution of 7-methoxy-1H-benzo[d]imidazol-2-amine (72-02) (8.2 g, 50.3 mmol, 1.0 eq) in DMF (80 mL) was added thio-CDI (13.4 g, 75.4 mmol, 1.5 eq) at 0 °C, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC, it was diluted with acetonitrile and stirred for 10 min. The precipitated solid was filtered and dried under vacuum to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) as an off-white solid (4.7 g, yield 34%). TLC system: MeOH:DCM (10:90), R f value: ~0.2, LCMS (m / z): 72%; 273.9 (M+H) + ; 1 1H NMR (400 MHz, DMSO-d6) δ 13-12 (br, 1H), 8.54 (t, J = 1.2 Hz, 1H), 7.92 (d, J = 1.2 Hz, 1H), 7.30-7.24 (m, 2H), 7.06 (d, J = 0.8 Hz, 1H), 6.99-6.95 (m, 2H), 3.97 (s, 3H).

[0523] Morpholine-4-carbohydrazide (72-05). To a stirred solution of morpholine (2) (5 g, 57.5 mmol, 1.0 eq) in DCM (100 mL) at -10 °C was added TEA (8.7 g, 86.2 mmol, 1.5 eq), and the mixture was stirred for 10 min. Then, a solution of triphosgene (8.53 g, 28.7 mmol, 0.5 eq) in DCM (100 mL) was added dropwise at -10 °C over 45 min, and the mixture was stirred for an additional 75 min. After completion of the reaction by TLC (ninhydrin staining), the reaction mixture was filtered through a celite bed, the filtrate was evaporated, triturated with diethyl ether, and intermediate 72-04 (5.6 g) was obtained. This material was diluted with dioxane (112 mL), NH2NH2·H2O (28.7 g, 575 mmol, 10 eq) was added, and the mixture was stirred at RT for 16 h. After conversion by TLC (ninhydrin staining), the volatiles were evaporated to a minimum volume under reduced pressure, resulting in a precipitate. The solid was filtered, triturated with diethyl ether, and morpholine-4-carbohydrazide (72-05) was obtained as an off-white solid (3.2 g, yield: 38%). TLC system: MeOH:DCM (10:90), R f value: ~0.1, MS (m / z): 146.1 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 7.69 (s, 1H), 3.88 (br, 2H), 3.51 (t, J = 5.2 Hz, 4H), 3.23 (t, J = 5.2 Hz, 4H).

[0524] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(morpholine-4-carbonyl)hydrazine-1-carbothioamide (72-06). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) (0.5 g, 1.83 mmol, 1.0 eq) and morpholine-4-carbohydrazide (72-05) (0.39 g, 2.74 mmol, 1.5 eq) in DMF (5 mL) was added DIPEA (0.59 g, 4.57 mmol, 2.5 eq) at RT, and the mixture was stirred for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to afford the crude product. The crude product was purified by reverse-phase column (eluting with 0 - 60% ACN and 0.1% FA in water) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(morpholine-4-carbonyl)hydrazine-1-carbothioamide (72-06) as an off-white solid (130 mg, yield: 20%). TLC system: MeOH:DCM (10:90), R f value: ~0.5, LCMS (m / z): 351.1 (M+H) + ; purity 82%.

[0525] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-5-morpholino-1,3,4-oxadiazol-2-amine (72). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(morpholine-4-carbonyl)hydrazine-1-carbothioamide (72-06) (0.13 g, 0.37 mmol, 1.0 eq) in DMF (1.3 mL) was added EDCI HCl (0.086 g, 0.56 mmol, 1.5 eq) at RT, and the mixture was heated to 60 °C and stirred for 1 h. After completion of the reaction by LCMS, the reaction mixture was diluted with ice water and extracted with 10% MeOH / DCM (2 × 30 mL). The combined organic layers were washed with water (10 mL) and brine solution (10 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative HPLC (FA buffer) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-5-morpholino-1,3,4-oxadiazol-2-amine (72) as a white solid (22 mg, yield: 19%). TLC system: MeOH:DCM (10:90), R f value: ~0.5, LCMS (m / z): 317.4 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ 11.63 (br, 2H), 7.08-7.02 (m, 2H), 6.81-6.78 (m, 1H), 3.89 (s, 3H), 3.69 (t, J = 4.8 Hz, 4H), 3.28 (t, J = 4.8 Hz, 4H).

[0526]

Chemical Structure

[0527] 4-Methylpiperazine-1-carbohydrazide (73-01). In a sealed tube, hydrazine monohydrate (19.7 g, 617 mmol, 10 eq) was added to MeOH (100 mL) of 4-methylpiperazine-1-carbonyl chloride (10 g, 61.7 mmol, 1.0 eq) at RT, and the mixture was stirred at 80 °C for 16 h. After completion of the reaction by TLC (ninhydrin staining), the reaction mixture was evaporated and triturated with diethyl ether to obtain 4-methylpiperazine-1-carbohydrazide (73-01) as a brown rubbery liquid (7.4 g, yield: 83%). TLC system: MeOH:DCM (10:90), R f value: ~0.1, LCMS (m / z): 70%; 159.1 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 3.25 (t, J = 5.2 Hz, 4H), 2.21 ( (t, J = 5.2 Hz, 4H), 2.15 (s, 3H). The NH2 hydrogen was not clearly distinguishable in the spectrum.

[0528] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methylpiperazine-1-carbonyl)hydrazinecarbothioamide (73-02). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) (1 g, 3.66 mmol, 1.0 eq) in DMF (15 mL) was added 4-methylpiperazine-1-carbohydrazide (73-01) (0.86 g, 5.49 mmol, 1.5 eq) at 0 °C, and the mixture was stirred at RT for 16 h. After completion of the reaction by LCMS, the reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by a reverse-phase column (eluted with 0-15% ACN in 0.1% FA in water) to obtain N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methylpiperazine-1-carbonyl)hydrazinecarbothioamide (73-02) as an off-white solid (170 mg, yield: 13%). TLC system: MeOH:DCM (20:80), R fValue: ~0.2, LCMS (m / z): 364.4 (M+H) + ; Purity 73%.

[0529] N-(4-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-amine (73). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methylpiperazin-1-carbonyl)hydrazinecarbothioamide (73-02) (0.17 g, 0.46 mmol, 1.0 eq) in MeOH (5 mL) was added Hg(OAc)2 (0.19 g, 0.6 mmol, 1.3 eq) at RT, and the mixture was heated to 85 °C and stirred for 3 h. After completion of the reaction by LCMS, the reaction mixture was diluted with 20% MeOH + DCM, filtered through a celite pad, and the crude product was evaporated. The crude product was purified by preparative HPLC purification (ABC of H2O + ACN method) to give N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methylpiperazin-1-yl)-1,3,4-oxadiazol-2-amine (73) as a white solid (16 mg, yield: 10%). TLC system: MeOH:DCM (20:80), R f Value: ~0.1, LCMS (m / z): 330.5 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ 11.62-11.52 (br, 2H), 7.06-7.01 (m, 2H), 6.80-6.77 (m, 1H), 3.89 (s, 3H), 3.30 (t, J = 4.8 Hz, 4H), 2.40 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H).

[0530]

Chemical Structure

[0531] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazine-1-carbothioamide (74-01). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) (0.5 g, 1.83 mmol, 1.0 eq) and 1-methylpiperidine-4-carbohydrazide (64-01) (0.43 g, 2.74 mmol, 1.5 eq) in DMF (10 mL) was added DIPEA (0.47 g, 3.66 mmol, 2 eq) at 0 °C, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by reverse-phase column (using gradient elution of 0 - 5% ACN and 0.1% FA in water) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazine-1-carbothioamide (74-01) as an off-white solid (155 mg, yield: 23%). TLC system: MeOH:DCM (20:80), R f value: ~0.05, LCMS (m / z): 363.4 (M+H) + ; Purity 95%.

[0532] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazole-2-amine (74). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(1-methylpiperidine-4-carbonyl)hydrazine-1-carbothioamide (74-01) (150 mg, 0.41 mmol, 1.0 eq) in MeOH (3.5 mL) was added mercury(II) acetate (170 mg, 0.53 mmol, 1.3 eq) at RT, and the mixture was stirred at 85 °C for 2 h. After completion of the reaction by TLC, the reaction mixture was filtered through a pad of Celite, washed with 20% MeOH / DCM (40 mL), the filtrate was evaporated, and purified by preparative HPLC (ABC in H2O + ACN) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-5-(1-methylpiperidin-4-yl)-1,3,4-oxadiazole-2-amine (74) as a white solid (20 mg, yield 13%). TLC system: MeOH:DCM (20:80), R f value: ~0.05 (almost the same Rf spot, indistinguishable clearly); LCMS (m / z): 329.5 (M+H) + ; 1 HNMR (400 MHz, DMSO-d6) δ 11.77 (br, 2H), 7.07-7.03 (m, 2H), 6.82-6.79 (m, 1H), 3.90 (s, 3H), 2.78-2.67 (m, 3H), 2.17 (s, 3H), 2.03-1.91 (m, 4H), 1.75-1.66 (m, 2H).

[0533]

Chemical Structure

[0534] tert-Butyl 2-(4-methoxycyclohexanecarbonyl)hydrazinecarboxylate (75-02). To a stirred solution of SOCl2 (100 mL) of cis / trans racemic-4-methoxycyclohexanecarboxylic acid (1) (5 g × 2, 63.3 mmol, 1.0 eq) was added DMF (1 mL) at 0 °C, and the mixture was stirred at 80 °C for 1 h. After completion of the reaction by TLC (KMnO4 staining), the reaction mixture was evaporated under a N2 atmosphere to obtain 10.2 g of the intermediate (75-01) as a brown liquid. This substance was diluted with THF (200 mL), cooled to 0 °C, and triethylamine (26.4 mL, 190 mmol, 3 eq) and Boc-hydrazine (8.36 g, 63.3 mmol, 1.0 eq) were added, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC (ninhydrin staining), the reaction mixture was diluted with water (200 mL) and extracted with EtOAc (2 × 200 mL). The organic layer was dried over Na2SO4 and concentrated to obtain the crude product. The crude product was purified by silica gel (60 - 120 mesh) column (eluted with 1 - 3% MeOH in DCM) to obtain tert-Butyl 2-(4-methoxycyclohexanecarbonyl)hydrazinecarboxylate (75-02) as a brown liquid (7.5 g, yield: 44%) (TLC system: MeOH:DCM(5:95), R f value: ~0.5, 1 1H NMR (400 MHz, CDCl3 / D2O exchange) δ 3.70 - 3.68 (m, 1H), 3.35 (s, 3H), 3.15 - 3.11 (m, 1H), 2.17 - 2.12 (m, 2H), 1.98 - 1.95 (m, 2H), 1.66 - 1.59 (m, 2H), 1.46 (s, 9H), 1.25 - 1.17 (m, 2H).

[0535] 4-Methoxycyclohexanecarbohydrazide (75-03). To a stirred solution of tert-butyl 2-(4-methoxycyclohexanecarbonyl)hydrazinecarboxylate (75-02) (7.5 g, 27.5 mmol, 1.0 eq) in dioxane (15 mL) cooled to 0 °C was added 4 M HCl in dioxane (22.5 mL), and the mixture was stirred at RT for 2 h. After conversion by TLC (ninhydrin staining), the reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was triturated with diethyl ether (100 mL) to give 4-methoxycyclohexanecarbohydrazide (75-03) as an off-white solid (7 g, crude product). TLC system: MeOH:DCM (5:95), R f value: ~0.3, MS (m / z): 173.1 (M+H) + ; 1 H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.40 (br, 2H), 3.23 (s, 3H), 3.11 - 3.06 (m, 1H), 2.33 - 2.23 (m, 1H), 2.05 - 2.02 (m, 2H), 1.80 - 1.77 (m, 2H), 1.47 - 1.37 (m, 2H), 1.14 - 1.04 (m, 2H). Based on 1H NMR, the compound may be in the HCl salt form, and the calculated yield based on the HCl M.W. was >100%. Considering this as the crude product, the next step was proceeded with.

[0536] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (75-04). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) (1 g, 3.65 mmol, 1.0 eq) and 4-methoxycyclohexanecarbohydrazide (75-03) (0.94 g, 5.48 mmol, 1.5 eq) in DMF (20 mL) was added DIPEA (0.94 g, 7.3 mmol, 2 eq) at 0 °C, and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC, the reaction mixture was evaporated and purified by reverse phase column (using gradient elution of 30 - 60% ACN and 0.1% FA in water) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (75-04) as an off-white solid (250 mg, yield: 18%). TLC system: MeOH:DCM (10:90), R f value: ~0.6, LCMS (m / z): 378.4 (M+H) + ; Purity 75%.

[0537] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (75). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (75-04) (0.25 g, 0.66 mmol, 1.0 eq) in DMF (2.5 mL), EDC HCl (0.15 g, 0.99 mmol, 1.5 eq) was added at RT, and the mixture was heated to 65 °C and stirred for 2 h. After completion of the reaction by LCMS, the reaction mixture was poured into ice water, and the precipitated solid was filtered and dried to obtain the crude product. The crude product was purified by preparative HPLC purification (ABC of H2O+ACN method) to give N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (75) as a white solid [(Peak-1: 15 mg) and (Peak-2: 9 mg), yield: 10%]. Based on TLC, the SM and the product could not be clearly distinguished.

[0538] Peak-1: LCMS (m / z): 344.5 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ 11.64 (br, 2H), 7.07-7.05 (m, 2H), 6.82-6.80 (m, 1H), 3.90 (s, 3H), 3.25 (s, 3H), 3.19-3.14 (m, 1H), 2.80-2.74 (m, 1H), 2.07-2.02 (m, 4H), 1.56-1.47 (m, 2H), 1.30-1.25 (m, 2H). 343 / 21.

[0539] Peak-2: LCMS (m / z): 344.5 (M+H) + . 11H NMR (400 MHz, DMSO-d6) δ 11.63 (br, 2H), 7.07-7.05 (m, 2H), 6.82-6.79 (m, 1H), 3.90 (s, 3H), 3.39-3.37 (m, 1H), 3.23 (s, 3H), 2.88-2.85 (m, 1H), 1.82-1.77 (m, 4H), 1.73-1.70 (m, 2H), 1.61-1.54 (m, 2H).

[0540]

Chem.

[0541] Thiomorpholine-4-carbohydrazide-1,1-dioxide (76-02). To a stirred solution of thiomorpholine 1,1-dioxide (1) (10 g, 74.07 mmol, 1.0 eq) in ACN (150 mL) was added triphosgene (26.3 g, 88.88 mmol, 1.2 eq) at 0 °C and the mixture was stirred at RT for 16 h. After completion of the reaction by TLC (ninhydrin staining), the reaction mixture was concentrated under reduced pressure to give 13 g of intermediate 76-01. This material was diluted with dioxane (195 mL) and NH2NH2·H2O (21 g, 575 mmol, 7.7 mmol) was added and the mixture was stirred at RT for 16 h. After conversion was observed by TLC (ninhydrin staining), the volatiles were evaporated under reduced pressure to give the crude product. The crude product was purified by silica gel (60~120 mesh) column (eluting with 5% MeOH in DCM) to give thiomorpholine-4-carbohydrazide-1,1-dioxide (76-02) as a white solid (1.7 g, yield: 13%). TLC system: MeOH:DCM (5:95), R f value: ~0.4, 1 1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 3.91 (s, 2H), 3.72 (t, J = 4.8 Hz, 4H), 3.04 (t, J = 4.8 Hz, 4H).

[0542] 2-(1,1-Dioxidothiomorpholine-4-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbothioamide (76-03). To a stirred solution of N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1H-imidazole-1-carbothioamide (72-03) (0.5 g, 1.83 mmol, 1.0 eq) and thiomorpholine-4-carbohydrazide-1,1-dioxide (76-02) (0.53 g, 2.74 mmol, 1.5 eq) in DMF (10 mL) was added DIPEA (0.47 g, 3.66 mmol, 2 eq) at RT and the mixture was stirred for 16 h. After completion of the reaction by TLC, the reaction mixture was concentrated under reduced pressure to give a residue, which was purified by reverse phase column (using a gradient elution of 0 - 60% ACN and 0.1% FA in water) to afford 2-(1,1-dioxidothiomorpholine-4-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbothioamide (76-03) as an off-white solid (140 mg, yield: 19%). TLC system: MeOH:DCM (10:90), R f value: ~0.5, LCMS (m / z): 399 (M+H) + ; purity 88%.

[0543] 4-(5-((4-Methoxy-1H-benzo[d]imidazol-2-yl)amino)-1,3,4-oxadiazol-2-yl)thiomorpholine 1,1-dioxide (76). To a stirred solution of 2-(1,1-dioxidothiomorpholine-4-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazine-1-carbothioamide (76-03) (0.14 g, 0.35 mmol, 1.0 eq) in DMF (2.8 mL) was added EDC HCl (0.081 g, 0.52 mmol, 1.5 eq) at RT, and the mixture was heated to 65 °C and stirred for 2 h. The reaction mixture was poured into ice water, and the precipitated solid was filtered and dried under vacuum to give a substance with >90% purity. This was triturated with MeOH:ACN (1:1) (5 mL) to give 4-(5-((4-methoxy-1H-benzo[d]imidazol-2-yl)amino)-1,3,4-oxadiazol-2-yl)thiomorpholine 1,1-dioxide (76) as a white solid (13 mg, yield: 10%). TLC system: MeOH:DCM (10:90), R f value: ~0.5 (almost the same Rf spot, indistinguishable clearly), LCMS (m / z): 365.4 (M+H) + . 1 H NMR (400 MHz, DMSO-d6) δ 11.65 (br, 2H), 7.05 - 7.03 (m, 2H), 6.81 - 6.78 (m, 1H), 3.90 (s, 3H), 3.84 - 3.80 (t→br, 4H), 3.29 - 3.26 (t→br, 4H).

[0544]

Chemical Structure

[0545] tert-Butyl 2-(4-methoxybenzoyl)hydrazinecarboxylate (82a). To a stirred solution of 4-methoxybenzoic acid (910 mg, 5 mmol), tert-butyl hydrazinecarboxylate (660 mg, 5 mmol), and HATU (2280 mg, 6 mmol) in DMA (5 mL) was added DIEA (1290 mg, 10 mmol) at 0 °C. The mixture was stirred at RT for 2 h. The resulting mixture was poured into brine (25 mL), and the precipitate was collected by filtration, washed with water, and dried in vacuo to give compound c (1340 mg, 100%) as a white solid. Rt: 1.138 min, MS: m / z: 210.9 [M+H + .

[0546] 4-Methoxybenzohydrazide hydrochloride (82b). A solution of compound 82a (1340 mg, 5 mmol) in HCl / EA (3 M, 5 mL) was stirred at RT for 1 h. The precipitated solid was collected by filtration, washed with EA (10 mL), and dried under vacuum to give compound 82b (1 g, 100%) as a white solid. Rt: 0.734 min, MS: m / z: 167.2 [M+H + .

[0547] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxybenzoyl)hydrazinecarbothioamide (82c). To a stirred solution of 82b (273 mg, 1 mmol), compound 72-03 (200 mg, 1 mmol), and HATU (570 mg, 1.2 mmol) in DMA (5 mL) was added DIEA (258 mg, 2 mmol) at 0 °C. The mixture was stirred at RT for 16 h. The resulting mixture was poured into brine (25 mL), and the precipitate was collected by filtration, washed with water, and dried in vacuo to give compound 82c (370 mg, 100%) as a grey solid. Rt: 1.19 min, MS: m / z: 371.9 [M+H + .

[0548] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine (82). To a solution of compound 82c (370 mg, 1 mmol) in DMA (2 mL) was added EDC (230 mg, 1.2 mmol), and the mixture was stirred at 60 °C for 3 h. Next, the mixture was cooled to RT, poured into brine (10 mL), and the precipitate was collected by filtration, washed with water, purified through a C18 column, and recrystallized from MeOH to give compound 82 (12 mg, 3.5%) as a white solid. MS, m / z: 338.1 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ(ppm) : 11.80 (s, 2 H), 7.85 (s, 4 H), 6.83 (s, 2 H), 4.00 (s, 3 H), 3.92 (s, 3 H).

[0549]

Chem.

[0550] The preparation of 83 - 89 was the same as that described for 82.

[0551]

Chem.

[0552] tert-Butyl 2-(4-methoxy-2-methylbenzoyl)hydrazinecarboxylate (83 - 1). MS: m / z:281.1 [M+H +

[0553]

Chem.

[0554] 4-Methoxy-2-methylbenzohydrazide (83 - 2). MS: m / z:181.1 [M+H +

[0555] ​​​ [Chemistry]

[0556] N-(7-Methoxy-1H-benzo[d]imidazol-2-yl)-2-(4-methoxy-2-methylbenzoyl)hydrazinecarbothioamide (83-3). MS: m / z: 386.0 [M+H +

[0557] [Chemistry]

[0558] N-(4-Methoxy-1H-benzo[d]imidazol-2-yl)-5-(4-methoxy-2-methylphenyl)-1,3,4-oxadiazol-2-amine (83). MS, m / z: 352.2 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ (ppm): 11.91 (s, 2 H), 7.75 (d, J = 7.6 Hz, 4 H), 6.84 - 7.10 (m, 5 H), 3.93 (s, 3 H), 3.82 (s, 3 H), 2.61 (s, 3 H).

[0559] [Chemistry]

[0560] 3,4-Dimethoxybenzohydrazide (84-2). MS: m / z: 196.9 [M+H +

[0561] [Chemistry]

[0562] ​​​2-(3,4-Dimethoxybenzoyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (84-3). MS: m / z: 402.1 [M+H +

[0563]

Chem.

[0564] 5-(3,4-Dimethoxyphenyl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (84). MS, m / z: 368.1 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ (ppm): 12.29 (s, 2 H), 7.55 (dd, J = 8.4 Hz, J = 2.0 Hz, 1 H), 7.45 (d, J = 2.0 Hz, 1 H), 7.14 - 7.16 (m, 3 H), 6.88 (dd, J = 6.8 Hz, J = 2.0 Hz, 1 H), 3.94 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 3 H).

[0565]

Chem.

[0566] tert-Butyl 2-(3-fluoro-4-methoxybenzoyl)hydrazinecarboxylate (85-1). MS: m / z: 569.1 [M+H] +

[0567]

Chem.

[0568] 3-Fluoro-4-methoxybenzohydrazide (85-2). MS: m / z: 185.1 [M+H +

[0569] ​​​ [Chemistry]

[0570] 2-(3-Fluoro-4-methoxybenzoyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (85-3). MS: m / z: 389.9 [M+H +

[0571] [Chemistry]

[0572] 5-(3-Fluoro-4-methoxyphenyl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (85). MS, m / z: 356.1 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ(ppm): 11.95 (s, 2 H), 7.67 - 7.71 (m, 2 H), 7.32 - 7.36 (m, 1 H), 7.09 - 7.11 (m, 2 H), 6.83 - 6.85 (m, 1 H), 3.92 (s, 6 H).

[0573] [Chemistry]

[0574] tert-Butyl 2-(2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)hydrazinecarboxylate (86-1). MS: m / z: 238.9 [M+H +

[0575] [Chemistry]

[0576] ​​​2,3-Dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (86-2). MS: m / z: 195.0 [M+H +

[0577]

Chem.

[0578] 2-(2,3-Dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (86-3). MS: m / z: 400.1 [M+H +

[0579]

Chem.

[0580] 5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (86). MS, m / z: 365.9 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ (ppm): 12.22 (s, 2 H), 7.39 - 7.44 (m, 2 H), 7.11 - 7.14 (m, 2 H), 7.04 (d, J = 8.4 Hz, 1 H), 6.85 - 6.88 (m, 1 H), 4.32 (s, 4 H), 3.91 (s, 3 H).

[0581]

Chem.

[0582] 2,3-Dihydrobenzofuran-5-carbohydrazide (87-2). MS: m / z: 179.0 [M+H +

[0583] ​​​​ [Chemical]

[0584] 2-(2,3-Dihydrobenzofuran-5-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (87-3). MS: m / z: 383.9 [M+H +

[0585] [Chemical]

[0586] 5-(2,3-Dihydrobenzofuran-5-yl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (87). MS, m / z: 368.1 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ(ppm): 12.48 (s, 2 H), 7.85 (s, 1 H), 7.75 (dd, J = 8.0 Hz, J = 1.2 Hz, 1 H), 7.13 - 7.18 (m, 2 H), 6.89 - 6.97 (m, 2 H), 4.62 - 4.66 (m, 2 H), 3.94 (s, 3 H), 3.26 - 3.30 (m, 2 H).

[0587] [Chemical]

[0588] 3,4-Diethoxybenzohydrazide (88-2). MS: m / z: 225.0 [M+H +

[0589] [Chemical]

[0590] ​​​2-(3,4-Diethoxybenzoyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (88-3). MS: m / z: 429.9 [M+H +

[0591]

Chem.

[0592] 5-(3,4-Diethoxyphenyl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (88). MS, m / z: 396.0 [M+H + , 1 H-NMR (400 MHz, DMSO-d6): δ (ppm): 11.80 (s, 2 H), 7.39 - 7.45 (m, 2 H), 7.08 - 7.12 (m, 2 H), 6.82 - 6.85 (m, 1 H), 4.09 - 4.12 (m, 4 H), 3.92 (s, 3 H), 1.34 - 1.39 (m, 6 H).

[0593]

Chem.

[0594] tert-Butyl 2-(benzofuran-5-carbonyl)hydrazinecarboxylate (89-1). MS: m / z: 220.9 [M+H +

[0595]

Chem.

[0596] Benzofuran-5-carbohydrazide (89-2). MS: m / z: 177.0 [M+H +

[0597]

Chem.

[0598] 2-(Benzofuran-5-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (89-3). MS: m / z: 382.0 [M+H +

[0599] [Chemical Formula]

[0600] 5-(Benzofuran-5-yl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (89). MS, m / z: 347.9 [M+H + 1 H-NMR (400 MHz, DMSO-d6): δ (ppm): 12.02 (s, 2 H), 8.24 (s, 1 H), 8.12 (s, 1 H), 7.91 (d, J = 8.8 Hz, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 7.12 - 7.15 (m, 3 H), 6.85 (d, J = 6.8 Hz, 1 H), 3.93 (s, 3 H).

[0601] [Chemical Formula]

[0602] (3R,5S)-3,5-Dimethylpiperidine-1-carbonyl chloride (92a). To a stirred solution of (3R,5S)-3,5-dimethylpiperidine (1.5 g, 13.3 mmol) and pyridine (3.1 mg, 39.8 mmol) in DCM (30 mL) under N2 at 0 °C was added BTC (4.7 g, 15.9 mmol). The mixture was stirred at RT overnight. The resulting mixture was poured into 1N HCl (100 mL), extracted with DCM and concentrated to give 92a (1.5 g, crude product, 100%) as a yellow solid.

[0603] ​​tert-Butyl 2-((3R,5S)-3,5-dimethylpiperidine-1-carbonyl)hydrazinecarboxylate (92b). A solution of compound 92a (1.5 g, crude product), tert-butyl hydrazinecarboxylate (754 mg), and TEA (2.38 mL) in dioxane (10 mL) was stirred overnight at RT under N2. The mixture was concentrated and purified by silica gel column flash chromatography (PE / EA = 1 / 1) to afford 92b (1.0 g) as a yellow solid. RT: 1.622 min, MS: m / z: 272.2 [M+H + .

[0604] (3R,5S)-3,5-Dimethylpiperidine-1-carbohydrazide (92c) A solution of compound 92b (1.0 g, 3.7 mmol) in HCl / EA (3 M, 20 mL) was stirred at RT for 1 h. The precipitated solid was collected by filtration, washed with EA (10 mL), and dried in vacuo to afford compound 92c (700 g, 100%) as a white solid. RT: 0.758 min, MS: m / z: 172.2 [M+H + .

[0605] 2-((3S,5R)-3,5-Dimethylpiperidine-1-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (92d) To a solution of compound 72-03 (500 mg, 1.8 mmol), DIPEA (1.2 g, 9.2 mmol), and compound 92c (340 mg, 1.9 mmol) in DMF (10 mL). The mixture was stirred at 100 °C for 3 h under N2. The resulting mixture was poured into H2O (100 mL). The precipitated solid was collected by filtration, washed with EA (20 mL), and dried in vacuo to afford compound 92d (300 mg, 43.9%) as a yellow solid. RT: 1.673 min, MS: m / z: 377.2 [M+H + .

[0606] 5-((3R,5S)-3,5-Dimethylpiperidin-1-yl)-N-(4-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (92) To a stirred solution of compound 92d (300 mg, 0.8 mmol) in DMF (5 mL) was added EDCI (306 mg, 1.6 mmol). The mixture was stirred at 60 °C for 3 h under N2. The resulting mixture was purified by preparative HPLC to afford compound 92 (10.78 mg, 3.9%) as a gray solid. RT: 1.514 min, MS: m / z: 343.2 [M+H + , 1 1H-NMR (400 MHz, DMSO-d6): δ(ppm): 11.47 (s, 1 H), 7.02 - 7.04 (m, 2 H), 6.77 - 6.80 (m, 1 H), 3.90 (s, 3 H), 3.68 - 3.72 (m, 2 H), 2.40 (t, J = 12.0 Hz, 2 H), 1.75 (d, J = 12.4 Hz, 1 H), 1.63 - 1.69 (m, 2 H), 0.88 - 0.89 (m, 6 H), 0.74 (dd, J = 24.0 Hz, J = 12.0 Hz,1 H).

[0607]

Chemical Structure

[0608] tert-Butyl 2-(4-(tert-butyl)cyclohexanecarbonyl)hydrazinecarboxylate (94a) To a stirred solution of 4-(tert-butyl)cyclohexanecarboxylic acid (2 g, 10.9 mmol) and tert-butyl hydrazinecarboxylate (1.6 g, 11.9 mmol) in DCM (30 mL) were added HATU (5.4 g, 14.1 mmol) and TEA (3.3 g, 32.7 mmol) at RT. The mixture was stirred at RT for 1.5 h. The resulting mixture was poured into H2O (100 mL) and extracted with DCM and concentrated to afford the compound (94a, 2.3 g, 71.8%) as a yellow solid. RT: 1.382 min, MS: m / z: 243.1 [M-(t-Bu)+H + .

[0609] 4-(tert-Butyl)cyclohexanecarbohydrazide (94b) A solution of compound (94a, 2.3 g, 7.7 mmol) in HCl / EA (3 M, 20 mL) was stirred at RT for 1 h. The precipitated solid was collected by filtration, washed with EA (10 mL), and then dried in vacuo to give compound (94b, 1.4 g, 91.5%) as a white solid. RT: 1.136 min, MS: m / z: 199.2 [M+H + .

[0610] 2-(4-(tert-Butyl)cyclohexanecarbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (94c) To a solution of compound 72-03 (300 mg, 1.1 mmol), DIPEA (0.7 g, 5.5 mmol), and compound (94c, 240 mg, 1.2 mmol) in DMF (10 mL). The mixture was stirred at 100 °C for 3 h under N2. The resulting mixture was poured into H2O (100 mL). The precipitated solid was collected by filtration, washed with EA (20 mL), and dried in vacuo to give compound (94c, 270 mg, 61.1%) as a yellow solid. RT: 1.393 min, MS: m / z: 404.0 [M+H + .

[0611] 5-(4-(tert-Butyl)cyclohexyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (94) To a stirred solution of compound (94c, 270 mg, 0.7 mmol) in DMF (5 mL), EDCI (154 mg, 0.8 mmol) was added. The mixture was stirred at 60 °C for 3 h under N2. The resulting mixture was purified by preparative HPLC to give compound 94 (20.41 mg, 8.1%) as a gray solid. Rt: 2.109 min, MS: m / z: 370.2 [M+H + , 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 11.78 (s, 2 H), 7.06 - 7.07 (m, 2 H), 6.81 (t, J = 4.4 Hz, 1 H), 3.90 (s, 3 H), 3.14 (s, 1 H), 2.07 - 2.19 (m, 2 H), 1.59 - 1.64 (m, 2 H), 1.43 (dd, J = 23.6 Hz, J = 12.4 Hz, 1 H), 1.02 - 1.23 (m, 4 H), 0.86 (s, 3 H), 0.80 (s, 6 H).

[0612]

Chem.

[0613] The preparation of 96 was the same as described for 94.

[0614] 2 - ((1R,4S)-Bicyclo[2.2.1]heptane-2-carbonyl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)hydrazinecarbothioamide (96c) MS: m / z: 360.2 [M + H + .

[0615] 5 - ((1R,4S)-Bicyclo[2.2.1]heptan-2-yl)-N-(7-methoxy-1H-benzo[d]imidazol-2-yl)-1,3,4-oxadiazol-2-amine (96) MS: m / z: 326.1 [M + H + , 11H-NMR (400 MHz, DMSO-d6): δ (ppm): 11.79 (s, 2H), 7.06 - 7.07 (m, 2H), 6.81 (t, J = 4.8 Hz, 1H), 3.91 (s, 3H), 3.17 - 3.21 (m, 1H), 2.49 - 2.53 (m, 1H), 2.28 - 2.34 (m, 1H), 1.86 - 1.94 (m, 1H), 1.69 - 1.73 (m, 1H), 1.49 - 1.55 (m, 2H), 1.33 - 1.39 (m, 2H), 1.10 - 1.23 (m, 2H).

[0616] [Chemical formula]

[0617] Methyl 3,4-bis(methoxy-d3)benzoate (97a): To a suspension of methyl 3,4-dihydroxybenzoate (2.0 g, 11.9 mmol) in acetonitrile (10.0 mL) were added potassium carbonate (4.9 g, 35.7 mmol) and iodomethane-d3 (2.4 mL, 38.1 mmol), and then the mixture was stirred at 45 °C for 15 h. The mixture was concentrated under vacuum. The crude residue was diluted with EtOAc (100.0 mL) and water (100.0 mL), and the resulting aqueous layer was further extracted with EtOAc (3 × 20.0 mL). The combined organic layers were washed with Na2CO3 (2 × 20 mL), dried over MgSO4, concentrated under vacuum, and methyl 3,4-bis(methoxy-d3)benzoate (97a, 1.8 g, 75%) was obtained. MS (M + H): 203.2.

[0618] 3,4-Bis(methoxy-d3)hydrazine (97b): A 20 mL ethanol solution of methyl 3,4-bis(methoxy-d3)benzoate (97a, 1.8 g, 8.9 mmol) and hydrazine hydrate (20.0 mL) was perfused for 8 h. The resulting mixture was concentrated to half under vacuum by evaporating the solvent, and the residue was purified by preparative HPLC to obtain 3,4-bis(methoxy-d3)hydrazine (97b, 1.4 g, 77.7%). MS (M+H): 203.2.

[0619] To a DMF (5.0 mL) solution of 2-isothiocyanato-4-methoxy-3H-imidazo[4,5-c]pyridine (63c, 206.0 mg, 1.0 mmol), 97b (202.0 mg, 1.0 mmol) and DIPEA (258.5 mg, 2.0 mmol) were added. The mixture was stirred at 70 °C for 2 h. 2-(3,4-Dimethoxy-d3-benzoyl)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)hydrazinecarbothioamide (97c) was detected by LCMS, and the mixture was used directly in the next step. MS: m / z: 409.2 [M+H] + .

[0620] 5-(3,4-Dimethoxy-d3-phenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (97) To a DMF (6.0 mL) solution of 97c (187.0 mg, 0.46 mmol), EDCI (88.0 mg, 0.46 mmol) was added at 25 °C. The mixture was stirred at 60 °C for 2 h. The mixture was purified by preparative HPLC to obtain 97 (22.1 mg, 12.8%) as a white solid. MS: m / z: 375.1 [M+H] + 1H-NMR (400 MHz, DMSO-d6): δ(ppm) : 7.87 - 7.88 (m, 1H), 7.47 - 7.49 (m, 1H), 7.40 - 7.41 (m, 1H), 7.15 - 7.19 (m, 1H), 7.11 - 7.13 (m, 1H), 4.01 (s, 3 H).

[0621]

Chem.

[0622] 2-(3,4-Dimethoxy-d3-benzoyl)-N-(4-methoxy-3H-imidazo[4,5-c]pyridin-2-yl)hydrazinecarbothioamide (98a) To a solution of 2-isothiocyanato-4-methoxy-3H-imidazo[4,5-c]pyridine (99e) (206.0 mg, 1.0 mmol) in DMF (5.0 mL) were added 97b (202.0 mg, 1.0 mmol) and DIPEA (258.5 mg, 2.0 mmol). The mixture was stirred at 70 °C for 2 h. 98a was detected by LCMS and the mixture was used directly in the next step. MS: m / z: 423.1 [M+H] + .

[0623] 5-(3,4-Dimethoxyphenyl)-N-(4-methoxy-1H-imidazo[4,5-c]pyridin-2-yl)-1,3,4-oxadiazol-2-amine (98) To a solution of compound 98a (194.0 mg, 0.46 mmol) in DMF (6.0 mL) was added EDCI (88.0 mg, 0.46 mmol) at 25 °C. The mixture was stirred at 60 °C for 1 h. The mixture was purified by preparative HPLC to give compound 98 (29.1 mg, 16.3%) as a white solid. MS: m / z: 389.2 [M+H] + 1H-NMR (400 MHz, DMSO-d6): δ(ppm): 11.80 - 12.31 (broad, 1H), 7.46 - 7.53 (multiplet, 1H), 7.40 - 7.41 (multiplet, 1H), 7.11 - 7.15 (multiplet, 1H), 6.98 - 7.02 (multiplet, 1H), 3.99 (singlet, 3H), 2.42 (singlet, 3H).

[0624]

Chemical formula

[0625] 2-Methoxy-6-methyl-3-nitropyridin-4-amine (99b) To a solution of 99a (1.3 g, 6.9 mmol) in MeOH (30.0 mL) was added CH3ONa (740.0 mg, 13.7 mmol), and then this was stirred at 80 °C overnight. The resulting mixture was cooled to 25 °C, concentrated in vacuo, poured into water (40.0 mL), and extracted with DCM (50.0 mL × 3). The combined organic layers were washed with brine (50.0 mL), dried over Na2SO4, filtered, concentrated in vacuo, and 99b (1.2 g, 95%) was obtained as a white solid. MS: m / z: 184.2 [M+H] + .

[0626] 2-Methoxy-6-methylpyridine-3,4-diamine (99c) Pd / C was added to a solution of 99b (2.1 g, 11.5 mmol) in MeOH (70.0 mL). The reaction mixture was stirred under a H2 atmosphere for 16 h, filtered, concentrated in vacuo, and 99c (1.5 g, 87.1%) was obtained as a yellow solid. MS: m / z: 154.1 [M+H] + .

[0627] 4-Methoxy-6-methyl-3H-imidazo[4,5-c]pyridin-2-amine (99d) CNBr (2.1 g, 19.6 mmol) was added to a solution of 99c (1.5 g, 9.8 mmol) in EtOH (40.0 mL), and the reaction solution was stirred at 80 °C overnight. The mixture was diluted with water (50 mL), basified to pH = 10 with 1 N NaOH, and extracted with EA (60.0 mL × 3). The organic phase was extracted with brine (60.0 mL), dried over Na2SO4, filtered, and concentrated, and purified by column (DCM:MeOH = 10:1) to obtain 99d (0.8 g, 46%) as a yellow solid. MS: m / z: 179.0 [M+H] + .

[0628] 2-Isothiocyanato-4-methoxy-6-methyl-3H-imidazo[4,5-c]pyridine (99e): To a solution of 99c (530.0 mg, 3.0 mmol) in ACN (30.0 mL) was added thioCDI (690 g, 3.9 mmol). The mixture was stirred at 50 °C for 16 h. The precipitated solid was collected by filtration, washed with ACN (20 mL), and dried under vacuum to obtain 99e (440.0 mg, 67%) as a yellow solid. MS: m / z: 221.0 [M+H] + .

[0629] N-(4-Methoxy-6-methyl-3H-imidazo[4,5-c]pyridin-2-yl)-2-((1r,4r)-4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (99f): To a solution of 99e (220 mg, 1.0 mmol) in DMF (5.0 mL) were added 99f (172.0 mg, 1.0 mmol) and DIPEA (258.5 mg, 2.0 mmol). The mixture was stirred at 70 °C for 2 h. 99f was detected by LCMS and the mixture was used directly in the next step. MS: m / z: 393.2 [M+H] + .

[0630] N-(4-Methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)-5-((1r,4r)-4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (99): To a solution of 99e (180.0 mg, 0.46 mmol) in DMF (6.0 mL) was added EDCI (88.0 mg, 0.46 mmol) at 25 °C. The mixture was stirred at 60 °C for 2 h. The mixture was purified by preparative HPLC to give 99 (22.7 mg, 16.9%) as a white solid. MS: m / z: 359.2 [M+H] + 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 6.96 (s, 1H), 3.97 (s, 3H), 3.25 (s, 3H), 3.13 - 3.23 (m, 1H), 2.74 - 2.81 (m, 1H), 2.40 (s, 3H), 2.03 - 2.06 (m, 4H), 1.47 - 1.56 (m, 2H), 1.22 - 1.31 (m, 2H).

[0631]

Chemical Structure

[0632] N-(4-Methoxy-6-methyl-3H-imidazo[4,5-c]pyridin-2-yl)-2-((1s,4s)-4-methoxycyclohexanecarbonyl)hydrazinecarbothioamide (100a): To a solution of 2-isothiocyanato-4-methoxy-6-methyl-3H-imidazo[4,5-c]pyridine (99e) (55.0 mg, 0.34 mmol) in DMF (5.0 mL) were added (1s,4s)-4-methoxycyclohexanecarbohydrazide (62b) (74.8 mg, 0.34 mmol) and DIPEA (87.8 mg, 0.68 mmol). The mixture was stirred at 70 °C for 2 h. The crude product 100a was used directly in the next step. MS: m / z: 3933 [M+H] + .

[0633] N-(4-Methoxy-6-methyl-1H-imidazo[4,5-c]pyridin-2-yl)-5-((1s,4s)-4-methoxycyclohexyl)-1,3,4-oxadiazol-2-amine (100): To a stirred solution of 100a (133.4 mg, 0.34 mmol) in DMF (5.0 mL) was added EDCI (65.1 mg, 0.34 mmol) at 25 °C. The mixture was stirred at 60 °C for 2 h. The mixture was purified by preparative HPLC to give compound 100 (37.1 mg, 30.7%) as a white solid. MS: m / z: 359.3 [M+H] + 1 1H-NMR (400 MHz, DMSO-d6): δ(ppm): 6.97 (s, 1H), 3.98 (s, 3H), 3.39 - 3.40 (m, 1H), 3.23 (s, 3H), 2.87 - 2.91 (m, 1H), 2.41 (s, 3H), 1.70 - 1.85 (m, 6H), 1.54 - 1.61 (m, 2H).

[0634] Example 2: Screening of luciferase induced by hypoxia-responsive element to identify inhibitors having HIF-2α transcriptional activity. The screening of the inhibitory activity of the compounds described in this specification was performed using 786-0 ccRCC cells that stably express five copies of the hypoxia-responsive element (HRE) fused to HRE-Luc:pGL3 luciferase reporter (Promega Corp, Madison WI). The 786-0 cells are pVHL-deficient and thus express HIF-2α constitutively, regardless of the oxygen tension of the cells. Since the 786-0 cells lack HIF-1α, the HRE-driven luciferase activity is mainly HIF-2α-driven and has been previously validated. The cells were maintained in Dulbecco's minimum essential medium (DMEM) containing 10% FBS in a humidified incubator at 37 °C and 5% CO2 during logarithmic growth. For the screening assay, the cells were seeded at a density of 4,000 cells / well in 50 μL of complete medium / well in 4 replicates per well in a 96-well plate. After 24 h, 50 μL of a 2-fold concentrated test compound was added to each well. The compounds were diluted from a stock solution of 5 - 10 mM in DMSO, and the final concentration of DMSO in each well of the control and treated cells was kept constant at ≤0.6% DMSO. After 24 h of treatment, luciferase activity was measured using the Steady-Glo luciferase assay system (Promega Corp) according to the manufacturer's procedure. Cell viability was measured in 4 replicate wells using resazurin, as recommended by the manufacturer (R&D systems, Minneapolis, MN). The data for luciferase and resazurin were normalized against the DMSO-treated control (defined as 100%) and wells containing the assay reagents but no cells (defined as 0%), and graphed using GraphPad Prism 9.3.1 software with the log inhibitor and normalized response options. The corresponding data measured using Steady-Glo luciferase and resazurin are shown in Figures 1A and 1B, respectively, and the error bars indicate SD. The filled black diamonds, open squares, and open triangles indicate treatments at 3, 8, and 22, respectively, and the results for HRE-Luc are shown in Figure 1A. Table 1 shows the IC for the hypoxia-responsive element-driven luciferase assay performed as described in this example.50 Value range (HIF-2α IC 50 ) is shown.

[0635]

Table 1-1

[0636]

Table 1-2

[0637] Regarding the data shown in FIG. 1, the ratios of HRE-Luc IC 50 to resazurin IC 50 were 39.05, 3.2, and 2.75 for 3, 8, and 22, respectively. The high ratio of HRE-Luc to resazurin IC 50 indicates that it is unlikely that the decrease in HRE-Luc activity was caused by the decrease in cell viability. The effect of the compound on IRE-mediated translation was investigated using 786-0 ccRCC cells that stably express the HIF-2α URE-Luc reporter using the same method as above, and exemplary results are shown in FIG. 2D. The data show that the compound inhibits IRE-mediated translation of HIF-2α. FIGS. 8A and 8B show exemplary plots and resazurin IC 50 values for 786-0 and RCC4 cells ± pVHL re-expression. Similarly, FIGS. 8D and E show exemplary plots and resazurin IC 50 values for 786-0 and RCC10 cells with empty vector or overexpression of ISCA2. The data in FIG. 8 suggest that an increase in VHL or ISCA2 protects against compound-induced cell death.

[0638] Example 3: Western blot and quantitative real-time polymerase chain reaction (qPCR) to measure the effect of the compound on HIF-2α and the cellular iron sensing mechanism. Western blot was performed to measure the effect of the compound on the levels of HIF-2α and other related proteins, while qPCR was performed to measure the effect of the compound on the transcriptional levels of HIF target genes. 786-0 or RCC10 cells were seeded into 6-well tissue culture plates at 1×10E5 cells / well in 2 mL of DMEM with 10% FBS / well. Cells were allowed to adhere overnight at 37 °C, 5% CO2 in a humidified incubator, after which the appropriate concentration of the compound in DMSO was added. The DMSO concentration was kept constant across all wells. After 24 h of exposure to the compound, the cells were lysed and run on a Western blot according to standard procedures. Antibodies against HIF-1α and HIF-2α, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA), while antibodies against IRP2 and pVHL were purchased from Santa Cruz Biotechnology (Dallas, TX), and the GPX4 antibody was purchased from R&D Systems, Inc (Minneapolis, MN). The exemplary data in Figure 2 shows the dose-dependent effect of the compound in the reduction of HIF-1 / 2α. The compound also affects cellular iron sensing brought about by the increase in IRP2, and together these indicate that the cells sense a decrease in the amount of available iron, which triggers an iron deficiency response initiated by the stabilization of IRP2. The exemplary data shown in Figure 3D shows the dose-dependent effect of the compound on GPX4, which may indicate a decrease in resistance to ferroptosis. The exemplary data shown in Figure 8C shows the effect of the re-expression of pVHL in the reduction of the levels of HIF-1 / 2α, confirming that the function of pVHL, which degrades HIF-1 / 2α in the presence of oxygen, is restored. The data also shows an increase in the expression of ISCA2 accompanying the re-expression of pVHL. The data in Figure 8D shows the overexpression of FLAG-tagged ISCA2 in RCC10 and 786-0 cells, suggesting that overexpression of ISCA2 increases HIF-1α and, to a lesser extent, also HIF-2α in RCC10 cells.

[0639] For qPCR, cells seeded as described above were harvested for RNA isolation using an RNA Clean and Concentrator kit (Zymo Research, Irvine CA) according to the manufacturer's procedure. cDNA was prepared using a High Capacity cDNA-to-RNA kit from Thermo Fisher Scientific (Waltham, MA). qRT-PCR was performed using a QuantStudio 3 real-time PCR system (Thermo Fisher) according to the manufacturer's procedure with pre-designed Taqman gene expression assay primers / probes and master mix (Thermo Fisher Scientific). Relative changes in gene expression were normalized to a control gene, and β2-microglobulin was measured using the ΔΔCt method. The exemplary data shown in Figure 2C indicate that treatment with the compound decreases the transcription of the HIF target genes VEGFA and POU5F1.

[0640] Example 4: Inductively coupled plasma mass spectrometry (ICP-MS) to measure the effect of the compounds on the concentration of iron and other transition metals in cancer cells. These studies were performed to measure the intracellular content of iron and other transition metals after exposure to the compounds described herein. 786-0 cells were seeded at 1.5 million cells / flask in T75 cm 2 flasks with DMEM + 10% FBS and allowed to adhere overnight. Next, the cells were treated for an additional 24 h with the indicated concentration of the compound in DMSO or DMSO alone (vehicle), after which the cells were detached by trypsin treatment, counted, washed twice with phosphate-buffered saline (PBS), and pelleted. The experiment was performed with 3 replicates of T75 cm for each condition 2This was performed using a flask. A 5:1 mixture of nitric acid (OPTIMA grade, 70%, Fisher Scientific) and ultrapure hydrogen peroxide (ULTREX II, 30%, Fisher Scientific) was added to the cell pellet. This mixture was digested overnight, heated until dry, and resuspended in 2% nitric acid for analysis using an Agilent 7900 ICP-MS (Agilent Technologies, Santa Clara, CA). A calibration standard for measuring Fe was prepared from Agilent's multi-element composition standard 2A. Agilent's environmental calibration standard was used as an independent control. Background was measured using a control digestion with PBS only. Metal readings were normalized to the number of cells. Data obtained from these studies for three replicate readings (including SEM) are shown in Figure 3, which shows an increase in the iron (Fe) content of the cells after treatment with the indicated compounds. The content of other transition metals such as zinc (Zn) and copper (Cu) was also measured simultaneously and increased significantly as well.

[0641] Combining the findings of Example 3 (decreased cellular iron availability, as indicated by increased IRP2) and the findings of Example 4 (increased total cellular iron content, as indicated by mass spectrometry), the compounds described herein appear to prevent cells from sensing and / or utilizing iron, triggering an iron-deficiency response, which contributes to the significantly increased cellular iron levels, or alternatively, despite this, the significantly increased cellular iron levels occur.

[0642] Example 5: Confirmation of ferroptosis as a mechanism of compound-mediated cell death. Considering the increase in cellular iron concentration caused by the compounds described herein, the involvement of iron in mediating cell death was measured. The cell viability assay was performed as described in Example 2 using resazurin as a readout of cell viability, in the absence or presence of 100 μM of the iron chelator DFO (D9533, MilliporeSigma, St Louis, MO). Exemplary results are shown in FIGS. 4A and B. The concentration required to reduce cell viability by 50% (cell viability IC 50 ) was 11.36 μM, and this IC 50 increased to 83.63 μM when 22 was added in the presence of DFO. A similar protective effect of DFO was also observed with other compounds 3 and 26HCL shown in FIG. 4B. FIG. 4C shows the effect of co-treatment with DFO, the antioxidant N-acetylcysteine (NAC), the ferroptosis inhibitor liproxstatin (LIP), and the apoptosis / caspase inhibitor ZVAD-FMK on cell death induced by compound 26HCL. The viability IC 50 values are shown in parentheses. The data support a mechanism of cell death that is iron-oxidant dependent and independent of apoptosis / caspase, i.e., ferroptosis.

[0643] Example 6: Quantification of lipid peroxidation using the thiobarbituric acid reactive substrate (TBARS) assay. Malondialdehyde (MDA) is the end product of lipid peroxidation products, and using this, lipid peroxidation, which is a characteristic of ferroptosis, can be shown. Following the manufacturer's procedure, MDA adducts were quantified in cells and tumor tissues using a TBARS-TCA method kit (catalog 700870, Cayman Chemicals, Ann Arbor Michigan). Briefly, 786-0 cells were seeded in T75 cm 2The flask was seeded with 1.75×10E6 cells and allowed to adhere overnight at 37°C and 5% CO2 in a humidified incubator. Subsequently, it was treated with the test compound for 48 h, RSL3 (6 h as a positive control), or DMSO control (the volume of DMSO was kept constant for all flasks) for 6 h. The cells were detached by trypsin treatment, pelleted by centrifugation, washed twice with PBS, resuspended in 200 μL of PBS, and then sonicated in a sonication water bath for 3 min. Exemplary data showing the effects of 26HCL or 62TFA compared to RSL3, a classical ferroptosis inducer, are shown in Figure 5. For the quantification of MDA in tumor masses, 20 - 50 mg of tumor tissue was homogenized in 200 - 500 μL of PBS and then sonicated in a sonication water bath for 5 min. The cell or tumor lysates were passed through a colorimetric detection assay described in the manufacturer's procedure. The MDA concentration in the cells was measured by comparison with a standard curve of the MDA stock solution provided by the manufacturer. The data were plotted using Prism GraphPad 9.3.1 software. Exemplary data showing the effects of oral administration of the compound on the MDA content in tumors are shown in Figures 9B, 10B, and 12C.

[0644] Example 7: Thermal shift assay to verify that iron-sulfur cluster assembly 2 (ISCA2) is the molecular target of the compounds described herein. In the absence or presence of the test compound, the melting temperature (T of the protein was determined using the hydrophobic protein dye SYPRO Orange (S6650, Thermo Fisher Scientific). mBy monitoring the change of ), a thermal shift assay was performed and measured using a LightCycler 480 (Roche Life Sciences, Indianapolis, IN) according to the manufacturer's procedure. Recombinant ISCA2 was produced by expressing amino acid residues 9 - 154 of ISCA2 (ISCA2 lacking its mitochondrial localization sequence) in a pET28 vector containing an N-terminal His6 tag in Rosetta(DE3) competent cells (Novagen, Millipore Sigma). Production of ISCA2 was induced by treating logarithmic phase cells transformed with ISCA2 with 0.25 mM IPTG at 18 °C for 4 hours. According to standard procedures, Ni 2+ affinity purification was used to purify ISCA2 and eluted with 50 nM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM DTT. A thermal shift assay was performed in a 384-well plate using 1 μL of 10-fold concentrated SYPRO Orange, 8 μL of ISCA2 (4 μg of protein), and 1 μL of a stock solution of test compounds 1 - 2 mM per well. The LightCycler was used according to the following settings: LightCycler 480 instrument temperature settings: The first target is 20 °C with a hold time of 15 seconds, the second target is 95 °C with an acquisition mode that is continuous at 10 times / °C, and the third target is 20 °C with a hold time of 15 seconds. T m was measured using Roche protein melting analysis software. Exemplary data using compound 1 and 26HCL at final concentrations of 200 and 100 μM are shown in Figures 6 and 7 respectively. The data show that treatment with 1 or 26HCL results in a significant change in the melting temperature of ISCA2, suggesting that ISCA2 is the molecular target of the described compounds. These findings are supported by studies showing that re-expression of VHL (increasing ISCA2) and overexpression of ISCA2 itself promote cellular resistance to the compounds (shown in Figure 8).

[0645] Example 8: Use of a compound for inhibiting the growth of cancer cells in vivo. If the compounds are based on both the ability to inhibit HIF-1 / 2α and promote ferroptosis, these compounds may be useful for inhibiting tumor growth in animals. This is expected to occur by inhibition of angiogenesis induced by HIF-1 / 2α and also by induction of cell death by ferroptosis, which may also induce immune activation, including increasing the infiltration of cytotoxic CD8+ T cells. To evaluate this use in a mouse model of renal cancer, subcutaneous tumors were generated from 786-0 cells purchased from ATCC. Subcutaneous tumors were established by subcutaneous injection of 10 million cells in a 1:1 ratio of DMEM and growth factor-reduced Matrigel (Corning Life Sciences, Tewksbury MA) (100 μL volume) into the flanks of male immunodeficient NRG mice. When the tumors reached an average tumor size of approximately 150 mm 3 3, the mice were stratified into compound or vehicle treatment groups with equal initial average tumor burdens (8 - 15 mice per group) and treatment was initiated. In the example shown in Figure 9, the mice were orally treated twice daily at 8 am and 4 pm with vehicle, 6.25 mg / kg or 12.5 mg / kg of 26HCL in 0.5% methylcellulose vehicle, and 1% Tween80 in ultrapure distilled water. In a bioavailability study, an F = 27.3% and a half-life of 3.61 hours were shown in Swiss albino mice, justifying the oral route of drug administration. The maximum volume administered was 200 μL in 30 g mice. When the tumors reached approximately 1,500 mm 3The mice were treated until either endpoint was reached, or euthanasia was required (in accordance with the facility's animal care and use procedures). Tumor volumes of the mice were measured twice a week. At the end of the study, the mice were euthanized and tumors were harvested to measure the effect of the treatment on the levels of HIF-2α, GPX4, and cellular iron. To detect the tumor levels of HIF-2α and GPX4, rapidly frozen tumor sections were homogenized in cell lysis buffer and run on a Western blot as described in Example 3. Band intensities of the Western blot for the relevant proteins were measured by densitometry of the gel image, and the data were normalized against a loading control such as GAPDH to measure the relative intensities. These values were expressed as a ratio to the mean value obtained in vehicle-treated mice and are shown in Figure 9B. To measure the cellular iron levels within the tumors, rapidly frozen tumor samples were run on ICP-MS as described in Example 4 and are shown in Figure 9C. The MDA content of the tumors was quantified by the TBAR method described in Example 5 and is shown in Figure 9D.

[0646] A similar study was performed using RENCA cells derived from spontaneous renal tumors in male Balb / c mice. RENCA cells were purchased from ATCC and 2 million cells were transplanted into the flanks of Balb / c mice. 3 When the average tumor size reached approximately 75 - 100 mm 3 the mice were stratified into two groups and the animals were treated PO once daily with either vehicle or 70 mg / kg of 26HCL. Tumor volumes of the mice were measured three times a week until the tumors reached approximately 3000 mm

[0647] Similar studies were conducted using the 786-0 and RENCA tumor models treated with 62TFA. In the bioavailability study, a half-life of F = 55% and 7 hours (at a dose of 100 mg / kg) was shown in Swiss albino mice, justifying the oral route of drug administration. Data from the studies in the 786-0 and RENCA tumor models are shown in Figures 11 and 12, respectively. Immunohistochemistry was performed using standard techniques with CD8 (CST98941) and CD31 (CST77699) purchased from Cell Signaling Technologies.

[0648] Example 9: Use of a compound for inhibiting tumor growth in a mammalian (e.g., human) patient. The above-mentioned compounds are useful in the treatment of solid or liquid tumors that exhibit upregulation of iron, lipids, and / or HIF-1 / 2α. Many studies have shown the identified mechanisms by which upregulation of HIF-1 / 2α, lipids, or iron in tumor tissue, and / or increased HIF-21 / α or iron, promote tumor growth, while inhibition of HIF-1 / 2α, or promotion of ferroptosis, can inhibit tumor growth. Thus, the above-mentioned compounds provide an effect for these patients. As fully described above, in some embodiments, suitable dosage levels of the compounds of the present invention can generally be from about 0.01 to 1000 mg per kg of patient body weight per day, which can be administered in single or multiple doses. By way of non-limiting example, dosage levels can be from about 0.1 to about 250 mg / kg / day, or from about 0.5 to about 100 mg / kg / day. Suitable dosage levels can be from about 0.01 to 250 mg / kg / day, from about 0.05 to 100 mg / kg / day, or from about 0.1 to about 50 mg / kg / day. Within this range, the dosage can be from 0.05 to 0.5, 0.5 to 5, or 5 to 50 mg / kg / day. For oral administration, the composition is preferably provided in the form of tablets containing from 1.0 to 1000 milligrams of the active ingredient, in particular 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient, to adjust the dosage to the patient being treated according to the symptoms. The compounds can be administered in a regimen of 1 to 4 times per day, preferably 1 or 2 times per day. This dosing regimen can be adjusted to provide an optimal therapeutic response. However, the specific dosage level and frequency of administration for any particular patient can vary and will depend on various factors including the activity of the specific compound being used, the metabolic stability and duration of action of the compound, age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, combination of drugs, severity of the particular condition, and the host receiving the treatment.Treatment with such compounds can result in patient benefits brought about by a decrease in tumor burden (shown radiologically or by other means by a person trained in the art), an increase in progression-free survival, a decrease in metastases, or an improvement in overall survival. Monitoring of the drug effect can include measurement of circulating ferritin by biopsy of tumor sections or analysis of the patient's serum or plasma, and / or measurement of HIF-1 / 2α and other target genes or proteins, and other iron regulatory proteins described herein.

Claims

1. A compound of formula I: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein said compound is not 5-(4-fluorophenyl)-N-(4-methoxybenzo[d]thiazol-2-yl)-1,3,4-oxadiazol-2-amine, and wherein, X １ and X ２ each of which is independently CH ２ , O, S, or NH, X ３ and X ４ each is independently CH or N, Z is C or O or S or NRA, wherein RA is H or C １－４ alkyl, C １ 、 C ２ 、 C ３ 、 and C ４ (C １－４ ), respectively, and each of Ca, Cb, Cc, Cd, and Ce (Ca-e) is independently C, S, O, N, or sulfur dioxide, "a" is a single bond or a double bond, R １ 、 R ２ 、 R ３ 、 and R ４ (R １－４ ) respectively, and each of Ra, Rb, Rc, Rd, and Re (Ra-e) is independently, (a) is selected from hydrogen, halo, CN, nitro, hydroxy, dioxide, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, di-C1-4 alkylaminosulfonylamino, and absent, and in some embodiments, (said R1-4, or Ra-e) hydroxy, C1-6 alkyl, aryl, haloalkoxy, amino, C1-6 alkylamino, di-C1-4-alkylamino, carboxy, carbamyl, C1-6 alkylcarbamyl, di(C1-4 alkyl)carbamyl, C1-6 alkylcarbonyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbonyloxy, C1-6 alkylsulfonyl, C1-6 alkylcarbonylamino, C1-6 alkylsulfonylamino, aminosulfonyl, C1-6 alkylaminosulfonyl, di-C1-4 alkylaminosulfonyl, aminosulfonylamino, C1-6 alkylaminosulfonylamino, or di-C1-4 alkylaminosulfonylamino is optionally substituted with 1, 2, or 3 groups independently selected from halo, CN, hydroxy, C1-3, alkoxy, amino, C1-3 alkylamino, di-C1-3-alkylamino, and absent, and / or If present, R １－４ Together with one of Ra-e and, if present, said R １－４ Each of which is attached to C １－４ Together with Ca-e, each optionally forms a 3- to 7-membered carbocyclic or 4- to 6-membered heterocyclic ring, each optionally substituted with 1, 2, 3, or 4 C1-3 alkyl groups a compound or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is a compound of formula Ia: 【Chemical Formula 2】

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is a compound of formula Ib: ​ 【Chemical Formula 3】 ​

4. The compound is a compound of formula Ic: 【Chemical Formula 4】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is as defined above.

5. The compound is a compound of formula Id: 【Chemical Formula 5】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is as defined above.

6. The compound is a compound of formula Ie: 【Chemical Formula 6】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is as defined above.

7. X １ is C, CH, O, or S, or NH, X ２ is O, NH, X ３ is CH, N, X ４ is CH, N, Z is NH, C １ 、 C ２ 、 C ３ 、 C ４ Each of them is independently C or N, Ca, Cc, Cd, and Ce are each C, Cb is C or N, R １ is H, CH ３ , 【Chemical Formula 7】 and is, R ２ is H, Cl, CH ３ , CF ３ , OCH ３ , [Chemical 8] and is, R ３ is H, OCH ３ , CF ３ or, R ４ is H, OCH ３ , or 【Chemical Formula 9】 and is, Ra is H or OCH ３ and Rb is H, F, Cl, CH ３ CN, OCF ３ , OCH ３ , OCD ３ or, together with Rc, forms methylenedioxy, ethylenedioxy, furan, hydrofuran, Rc is H, F, Cl, CH ３ , OCH ３ , CN, OCF ３ , OCD ３ , SCH ３ , N(CH ３ ) ２ , 【Chemical Formula 10】 and is, or together with Rb forms methylenedioxy, ethylenedioxy, furan, or hydrofuran, Rd and Re are each independently H, The compound according to claim 1.

8. The compound is 【Chemical 11】 【Chemical Formula 12】 【Chemical 13】 The compound according to claim 1, which is selected from the group consisting of

9. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier.

10. A method of treating a disorder of uncontrolled cell growth in a mammal, the method comprising administering to the mammal an effective amount of the compound according to claim 1.

11. A method of inducing ferroptosis in a cell, the method comprising administering to the cell the compound according to claim 1.

12. A method of increasing the iron, and optionally zinc and / or copper content in a cell, the method comprising administering to the cell the compound according to claim 1.

13. A method of reducing the amount of HIF-1 / 2α in a cell, the method comprising administering to the cell the compound according to claim 1.

14. A method of binding to ISCA2, the method comprising contacting ISCA2 with the compound according to claim 1.

15. A method of inducing cell death by lipid peroxidation, the method comprising administering to the cell the compound according to claim 1.

16. A method of inducing iron accumulation in a cell, the method comprising administering to the cell the compound according to claim 1.

Images