WO2020210428A1

WO2020210428A1 - Novel inhibitors of flavivirus protease for prevention and treatment of zika, dengue and other flavivirus infections

Info

Publication number: WO2020210428A1
Application number: PCT/US2020/027374
Authority: WO
Inventors: Yongcheng Song; Rebecca Rodea RICO-HESSE; Yuan Yao; Tong HUO; Yi-lun LIN; Fangrui WU; Shenyou NIE; Jing-Yu Wu; Yuanda HUA
Original assignee: Baylor College Of Medicine
Priority date: 2019-04-09
Filing date: 2020-04-09
Publication date: 2020-10-15

Abstract

In some embodiments, the present disclosure pertains to compositions that inhibit flavivirus proteases. In additional embodiments, the present disclosure pertains to methods for treating a viral infection in a subject by administering the compositions of the present disclosure to the subject. In some embodiments, the viral infection is caused by a flavivirus such as, for example, dengue virus, West Nile virus, Zika virus, tick-bome encephalitis virus, yellow fever virus, viruses causing encephalitis, insect- specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof.

Description

TITLE NOVEL INHIBITORS OF FLAVIVIRUS PROTEASE FOR PREVENTION AND TREATMENT OF ZIKA, DENGUE AND OTHER FLAVIVIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 62/831,397, filed on April 9, 2019. The entirety of the aforementioned application is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under W81XWH-18-1-0368 awarded by the Department of Defense. The government has certain rights in the invention. BACKGROUND [0003] Flaviviruses, including dengue, West Nile, and the recently emerged Zika virus are human pathogens. However, there are no drugs to prevent or treat these viral infections. The highly conserved flavivirus NS2B-NS3 protease is used for viral replication and therefore a drug target. The present disclosure yields a series of drug-like, broadly active inhibitors of flavivirus proteases. SUMMARY [0004] In an embodiment, the present disclosure pertains to a composition. In some embodiments, the composition has one or more compounds defined generally as:

. [0005] In some embodiments, R₁, R₂, and R₃ can each independently include, without limitation, hydrogen, aromatic groups, phenyl groups, benzyl, furan groups, phenyl furan groups, pyridine groups, phenyl pyridine groups, biphenyl groups, phenyl piperidine groups, pyrazole groups, amine groups, piperidine groups, amine groups, alkyl amine groups, aniline groups, methyl piperidine groups, benzene groups, cyclohexane groups, methyl benzoate groups, benzyl piperidine groups, imidazole groups, piperidine amine groups, -NHCH₃ groups, - CH(CH₃)CH₂CH₂NH₂ groups, -NH₂ groups, furan, 3-phenylfuran, 2-phenylfuran, 3- phenyloxolane, phenylmethanamine, propan-2-ylbenzene, tert-butylbenzene, phenylpyrrolidine, 1H-1,3-benzodiazole, benzimidazole, 3-phenylthiophene, 4-phenyl-1H-pyrazole, 4-phenyl- 1,2,3,6-tetrahydropyridine, 1-(pyridin-2-yl)piperazine, biphenyl, 3-phenylpyridine, 4- phenylpyridine, pyridine, 4-(1H-pyrazol-1-yl)piperidine, 1-tert-butyl-3-methoxybenzene, 1- phenylethan-1-one, 2-methoxybenzamide, 2-phenylpropan-2-amine, 2-fluorobenzamide, benzoylpiperidine, 4-phenyloxane, 2-(oxan-4-yl)pyridine, phenol, 2-methylpyridine, 3- propylpyridine, 4-methylpyridine, benzylpiperidine, piperidine, benzene, cyclohexane, methylcyclohexane, 3-methylbutan-1-amine, butan-1-amine, NHCH₃, CH(CH₃)CH₂CH₂NH₂, NH₂, aniline, p-toluidine, 4-ethylaniline, methylpiperidine, methyl benzoate, methyl 4- methylbenzoate, 4-benzylpiperidine, imidazole, piperidin-4-amine, 1,4-dimethylpiperidine, acetophenone, N-methylethanamine, 4-isopropylbenzne, 1-(4-methylpiperidin-1-yl)ethan-1-one, 1-(piperidin-1-yl)ethan-1-one, phenyl(piperidin-1-yl)methanone, 3-phenyltetrahydrofuran, 1- ethyl-1H-imidazole, 4-(1-phenyl)piperidine, 3-methylpiperidine, 4-ethylpiperidine, 4- bromobenzene (4-Br-Ph), 4-tertbutyl-benzene (4-t-Bu-Ph), 4-benzylamine (4-(NH₂CH₂)-Ph), 4- benzamide (4-(NH₂CO)-Ph), N-methyl-4-benzylamine (4-(MeNHCH₂)-Ph), 5-indole(Indol-5- yl), 4-(3-phenylfuran) (4-(furan-3-yl)-Ph), (4-(pyrazol-4-yl)-Ph), or combinations thereof. [0006] In some embodiments, X includes, without limitation, O, NH, C=O, CH₂, CONH, or combinations thereof. In some embodiments, Y includes, without limitation, N, CH, or combinations thereof. In some embodiments, Z includes, without limitation, N, CH, or combinations thereof. In some embodiments, m is an integer greater than or equal to 0. [0007] In another aspect, the present disclosure pertains to a method of treating a viral infection in a subject. In some embodiments, the method includes administering a composition of the present disclosure to the subject. In some embodiments, the viral infection is caused by a flavivirus that includes, without limitation, dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof DESCRIPTION OF THE DRAWINGS [0008] FIGURE 1A illustrates a general composition structure according to embodiments of the present disclosure. [0009] FIGURE 1B depicts a method of treating a viral infection in a subject. [0010] FIGURES 2A-F illustrate X-ray structures of the DV2pro-9 complex. FIG. 2A illustrates the overall structure of the DV2pro in complex with compound 9 (ball and stick model with C atoms in brown). NS3 is shown in green and NS2B in purple. FIG.2B illustrates the 2Fo- F_c electron density map of DV2pro-9 at the inhibitor binding site (contoured at 1s) and FIG.2C illustrates the Fo-Fc omit map (at 3s). FIG. 2D shows compound 9 is located in an L-shaped, deep pocket of NS3 (shown as an electrostatic surface) that is mostly hydrophobic. FIG. 2E shows aligned structures of DV2pro-9 with the substrate-bound DV3pro (PDB: 3U1I, NS3 in cyan and NS2B in yellow), showing 9 occupies a different binding site from the substrate (tube model with C atoms in black). FIG.2F shows interactions between compound 9 and DV2pro. [0011] FIGURES 3A-E illustrate cellular and in vivo antiviral activities of compound 9. FIGS. 3A-C show treatment of U87 cells with 9 caused dose-dependent reduction of RNA copies of ZIKV (FLR strain) (FIG. 3A), infectious ZIKV (FLR strain) (FIG. 3B), and infectious ZIKV (HN16 strain) (FIG. 3C). FIG. 3D illustrates treatment with compound 9 significantly reduced ZIKV RNA in plasma (left) and brains (right) of ZIKV-infected mice. FIG. 3E illustrates treatment with compound 9 significantly prolonged the survival of ZIKV-infected mice. DETAILED DESCRIPTION [0012] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word“a” or“an” means“at least one”, and the use of“or” means“and/or”, unless specifically stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as“includes” and “included”, is not limiting. Also, terms such as“element” or“component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise. [0013] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls. [0014] Dengue, West Nile, and Zika viruses belong to the genus Flavivirus in the Flaviviridae family of RNA viruses. These viruses are transmitted primarily by Aedes mosquitos. Four serotypes of dengue infect approximately 400 million people each year with 100 million developing dengue fever. Around 500,000 cases develop serious dengue hemorrhagic fever, causing about 22,000 deaths each year. Moreover, patients who recovered from one serotype are still susceptible to other serotypes with an increased likelihood of a more severe disease due to existing antibodies. Zika has caused three major outbreaks in Pacific Ocean islands, Brazil, and other American countries, in which more than 1 million infections were reported and a large number of patients sought medical treatment. [0015] More seriously, Zika infection has been correlated with a 20-fold increased incidence of serious neurological disorders, including Guillain-Barré syndrome and more than 4,000 cases of microcephaly in newborns. Zika has quickly spread to 48 Pan-American countries, and has recently been found to be transmitted through sex or body fluids. The World Health Organization has announced that Zika is a public health emergency of international concern. However, despite these serious outcomes, as well as possible future outbreaks, there have been no antiviral drugs to prevent or treat Zika and dengue infections. [0016] As such, a need exists for more effective compositions and methods for treating viral infections caused by flaviviruses. Various embodiments of the present disclosure address the aforementioned need. [0017] In some embodiments, the present disclosure pertains to compositions that include various compounds. In some embodiments illustrated in FIG. 1A, the compositions of the present disclosure are in the form of compound 10, which can include various functional groups 12 (indicated as R1, R2, and R3). In some embodiments, compound 10 can include elements 14 (indicated as X, Y, and Z). [0018] Additional embodiments of the present disclosure pertain to methods of treating a viral infection in a subject. In some embodiments illustrated in FIG. 1B, the methods of the present disclosure include a step of administering a composition to a subject (step 20) to result in the inhibition of the virus in the subject (step 22). In some embodiments, the methods of the present disclosure are used to treat a viral infection caused by a flavivirus, such as, but not limited to, dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof. [0019] As set forth in more detail herein, the compositions and methods of the present disclosure can have numerous embodiments. For instance, the compounds in the compositions of the present disclosure can include various chemical configurations, functional groups, elements, and moieties. Furthermore, various methods may be utilized to treat various viral infections in various subjects. [0020] Compositions [0021] As set forth in more detail herein, the compositions of the present disclosure can include various compounds. For instance, in some embodiments, the compositions of the present disclosure can include the following compound:

. [0022] In some embodiments, R1, R2, and R3 can each independently include, without limitation, hydrogen, aromatic groups, phenyl groups, benzyl, furan groups, phenyl furan groups, pyridine groups, phenyl pyridine groups, biphenyl groups, phenyl piperidine groups, pyrazole groups, amine groups, piperidine groups, amine groups, alkyl amine groups, aniline groups, methyl piperidine groups, benzene groups, cyclohexane groups, methyl benzoate groups, benzyl piperidine groups, imidazole groups, piperidine amine groups, -NHCH₃ groups, - CH(CH₃)CH₂CH₂NH₂ groups, -NH₂ groups, furan, 3-phenylfuran, 2-phenylfuran, 3- phenyloxolane, phenylmethanamine, propan-2-ylbenzene, tert-butylbenzene, phenylpyrrolidine, 1H-1,3-benzodiazole, benzimidazole, 3-phenylthiophene, 4-phenyl-1H-pyrazole, 4-phenyl- 1,2,3,6-tetrahydropyridine, 1-(pyridin-2-yl)piperazine, biphenyl, 3-phenylpyridine, 4- phenylpyridine, pyridine, 4-(1H-pyrazol-1-yl)piperidine, 1-tert-butyl-3-methoxybenzene, 1- phenylethan-1-one, 2-methoxybenzamide, 2-phenylpropan-2-amine, 2-fluorobenzamide, benzoylpiperidine, 4-phenyloxane, 2-(oxan-4-yl)pyridine, phenol, 2-methylpyridine, 3- propylpyridine, 4-methylpyridine, benzylpiperidine, piperidine, benzene, cyclohexane, methylcyclohexane, 3-methylbutan-1-amine, butan-1-amine, NHCH₃, CH(CH₃)CH₂CH₂NH₂, NH₂, aniline, p-toluidine, 4-ethylaniline, methylpiperidine, methyl benzoate, methyl 4- methylbenzoate, 4-benzylpiperidine, imidazole, piperidin-4-amine, 1,4-dimethylpiperidine, acetophenone, N-methylethanamine, 4-isopropylbenzne, 1-(4-methylpiperidin-1-yl)ethan-1-one, 1-(piperidin-1-yl)ethan-1-one, phenyl(piperidin-1-yl)methanone, 3-phenyltetrahydrofuran, 1- ethyl-1H-imidazole, 4-(1-phenyl)piperidine, 3-methylpiperidine, 4-ethylpiperidine, 4- bromobenzene (4-Br-Ph), 4-tertbutyl-benzene (4-t-Bu-Ph), 4-benzylamine (4-(NH₂CH₂)-Ph), 4- benzamide (4-(NH₂CO)-Ph), N-methyl-4-benzylamine (4-(MeNHCH₂)-Ph), 5-indole(Indol-5- yl), 4-(3-phenylfuran) (4-(furan-3-yl)-Ph), (4-(pyrazol-4-yl)-Ph), or combinations thereof. [0023] In some embodiments, X can include, without limitation, O, NH, C=O, CH₂, CONH, or combinations thereof. In some embodiments, Y can include, without limitation, N, CH, or combinations thereof. In some embodiments, Z can include, without limitation, N, CH, or combinations thereof. In some embodiments, m is an integer. In some embodiments, m is greater than or equal to 0. [0024] In some embodiments, the compounds in the compositions of the present disclosure can include, without limitation:

, _,

, or combinations thereof.

[0025] In some embodiments, the compounds in the compositions of the present disclosure can include, without limitation:

,

,

,

^,

, ,

or combinations thereof. [0026] In some embodiments, the compounds in the compositions of the present disclosure can include, without limitation:

. [0027] In some embodiments, n is an integer. In some embodiments, n can include, without limitation, 0, 1, 2, 3, or 4. In some embodiments, the compounds in the compositions of the present disclosure can include, without limitation:

or combinations thereof.

[0028] In some embodiments, the compositions of the present disclosure are suitable for treating various viral infections in various subjects. In some embodiments, the viral infection is caused by a flavivirus, such as, but not limited to, dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof. [0029] In some embodiments, the compositions of the present disclosure inhibit linked or binary ZIKV protease (ZVpro). In some embodiments, the compositions of the present disclosure inhibit DENV serotype-2 protease (DV2pro), DENV serotype-3 protease (DV3pro), West Nile protease (WVpro), or combinations thereof. In some embodiments, the compositions of the present disclosure inhibit ZIKV replication. In some embodiments, the compositions of the present disclosure bind to an allosteric pocket of a flavivirus protease. [0030] In some embodiments, the compositions of the present disclosure are associated with a delivery agent. In some embodiments, the delivery agent is a nanoparticle. In some embodiments, the nanoparticles have diameters ranging from about 50 nm to about 500 nm. In some embodiments, the nanoparticles have diameters of about 100 nm to about 150 nm. In some embodiments, the nanoparticles can be, for example, polymeric nanoparticles, lipid-based nanoparticles, single wall or multiwall carbon nanotubes, fullerenes, and combinations thereof. [0031] In some embodiments, the compositions of the present disclosure include a solubilizing agent. Solubilizing agents generally refer to one or more compounds that are capable of facilitating the solubilization of the compositions of the present disclosure in liquid formulations. Solubilizing agents may also be referred to as co-solvents or carriers. In some embodiments, the solubilizing agents include, but are not limited to, polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides, polyoxyethylated oleic glycerides, polysorbates, sorbitan monooleate, hydroxypropyl-beta-cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil, polyoxyl hydroxystearates, or combinations thereof. [0032] In some embodiments, the solubilizing agents of the present disclosure include water miscible organic solvents. In some embodiments, the solubilizing agents of the present disclosure include, without limitation, polyethylene glycol (e.g., PEG 400 and/or PEG 300), glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides (e.g., Labrafil M- 2125CS), polyoxyethylated oleic glycerides (e.g., Labrafil M-1944CS, Polyoxyl 35 castor oil, and/or Cremophor EL), polysorbates (e.g., polysorbate 20 and/or polysorbate 80), sorbitan monooleate, hydroxypropyl-beta-cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil (i.e., Cremophor RH 40), polyoxyl hydroxystearates (e.g., Solutol HS 15), and combinations thereof. [0033] In some embodiments, the compositions of the present disclosure can include at least one excipient agent. In some embodiments, the excipient agents can include, without limitation, anti- adherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, vehicles, or combinations thereof. [0034] Treatment of Viral Infections in Subjects [0035] The compositions of the present disclosure may be administered to various subjects in various manners to treat various viral infections in the subject. For instance, in some embodiments, the compositions of the present disclosure can be used to treat a viral infection caused by a flavivirus. In some embodiments, the flavivirus includes, but is not limited to, dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof. [0036] Without being bound by theory, the compositions of the present disclosure can treat a viral infection in a subject through various mechanisms. For instance, in some embodiments, the compositions of the present disclosure inhibit a viral protease. In some embodiments, the compositions of the present disclosure inhibit linked or binary ZIKV protease (ZVpro). In some embodiments, the compositions of the present disclosure inhibit DENV serotype-2 protease (DV2pro), DENV serotype-3 protease (DV3pro), West Nile protease (WVpro), or combinations thereof. [0037] In some embodiments, the compositions of the present disclosure bind to an allosteric pocket of a flavivirus protease. In some embodiments, the compositions of the present disclosure inhibit viral replication. [0038] Various methods may be utilized to administer the compositions of the present disclosure to a subject. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, oral administration, inhalation, subcutaneous administration, intravenous administration, intra-nasal administration, intra-dermal administration, trans-dermal administration, intraperitoneal administration, intramuscular administration, intrathecal injection, topical administration, central administration, peripheral administration, transdermal administration, intraarterial administration, intracranial administration, intraspinal administration, intranasal administration, intraocular administration, intratumor administration, intramuscular administration, intranasal administration, subcutaneous administration, intra- or trans-dermal administration, intravenous administration, topical administration, or combinations thereof. [0039] In some embodiments, the compositions of the present disclosure can be administered to the subject via delivery agents. In some embodiments, the delivery agents can include various types of particles and/or targeting agents associated with the compositions of the present disclosure. For instance, in some embodiments, the delivery agents can be particles associated with the compositions of the present disclosure. In some embodiments, the delivery agent is a nanoparticle. [0040] The compositions of the present disclosure can be administered to various subjects. For instance, in some embodiments, the subject is a human being suffering from a viral infection. In some embodiments, the viral infection is caused by a flavivirus. [0041] Applications and Advantages [0042] The present disclosure can have various advantages. For instance, in some embodiments, the compositions of the present disclosure can exhibit significant anti-viral activities against various flaviviruses. In some embodiments, the compositions of the present disclosure can have IC50 values between 25 µM to 200 nM for various flavivirus proteases. In some embodiments, the compositions of the present disclosure can have IC50 values between 25 µM to 5 µM for various flavivirus proteases. In some embodiments, the compositions of the present disclosure can have IC₅₀ values between 5 µM to 200 nM for various flavivirus proteases. In some embodiments, the compositions of the present disclosure can have IC₅₀ values between 1 µM to 200 nM for various flavivirus proteases. In some embodiments, the compositions of the present disclosure can be broadly active inhibitors of flavivirus proteases with a high selectivity. [0043] As such, the compositions of the present disclosure can be utilized in various manners and for various purposes. For instance, in some embodiments, the compositions of the present disclosure can be used for effectively treating various flavivirus infections in various subjects. [0044] Additional Embodiments [0045] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way. [0046] Example 1.1. Discovery, X-ray Crystallography and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease [0047] This Example describes discovery, X-ray crystallography, and antiviral activity of allosteric inhibitors of flavivirus NS2B-NS3 protease. [0048] Flaviviruses, including dengue, West Nile and recently emerged Zika virus, are human pathogens, but there are no drugs to prevent or treat these viral infections. The highly conserved flavivirus NS2B-NS3 protease is used for viral replication and therefore a drug target. Compound screening followed by medicinal chemistry yielded a series of drug-like, broadly active inhibitors of flavivirus proteases with IC50 as low as 120 nM. The inhibitor exhibited significant antiviral activities in cells (EC68: 300-600 nM) and in a mouse model of Zika virus infection. X-ray studies reveal that the inhibitors bind to an allosteric, mostly hydrophobic pocket of dengue NS3 and hold the protease in an open, catalytically inactive conformation. The inhibitors and their binding structures would be useful for rational drug development targeting Zika, dengue and other flaviviruses. [0049] Dengue (DENV), West Nile and recently emerged Zika (ZIKV) viruses belong to the genus Flavivirus in the Flaviviridae family of RNA viruses. These viruses are transmitted primarily by Aedes mosquitos. Four serotypes of DENV infect ~400 million people each year with 100 million developing dengue fever. ~500,000 cases develop serious dengue hemorrhagic fever, causing ~22,000 deaths each year. Moreover, patients recovered from one serotype are still susceptible to other serotypes with an increased likelihood of a more severe disease due to existing antibodies. ZIKV has caused three major outbreaks in Pacific Ocean islands (2007 and 2013), Brazil and other American countries (2015-2016), in which >1 million infections were reported and a large number of patients sought medical treatment. More seriously, ZIKV infection has been correlated with a 20-fold increased incidence of serious neurological disorders, including Guillain-Barré syndrome and >4,000 cases of microcephaly in newborns. Since 2015, ZIKV has quickly spread to 48 pan-American countries. Recently, ZIKV was found to be transmitted through sex or body fluids. Despite these serious outcomes as well as possible future outbreaks, there have been no antiviral drugs to prevent or treat ZIKV and DENV infections. A licensed dengue vaccine, Dengvaxia, has raised concerns about efficacy and increased risk of severe disease for seronegative people during clinical trials. [0050] ZIKV/DENV contain a single-stranded, positive-sense RNA with ~10,800 nucleotides, encoding a viral polyprotein. The polyprotein is site-specifically cleaved by the viral NS2B-NS3 protease and several host proteases to produce functional proteins. The NS2B-NS3 protease is used in viral replication and, therefore, a promising drug target. A number of peptide-based covalent inhibitors of flavivirus proteases have been reported, but they did not demonstrate significant antiviral activities in cells or animal models due to low cell permeability and metabolic stability. Non-peptidic inhibitors have also been reported, but their inhibitory activities are relatively weak and how these compounds bind to the protease is unknown. [0051] Homology analysis showed flavivirus proteases are evolutionally conserved and highly stable. NS3 contains an N-terminal serine protease domain, but complexation with NS2B is required to become an active enzyme. Previous X-ray and NMR studies show the protease can adopt a“closed” or“open” conformation. In the closed state that is catalytically active, NS2B is fully tied around NS3, and becomes part of the active site. In the open and inactive conformation, NS2B is partially bound to NS3 and far from the active site. [0052] Applicants produced a Gly4-Ser-Gly4 linked and binary form of recombinant ZIKV protease (ZVpro), containing NS2B (47-95) and NS3 (1-170). ~1,200 compounds in Applicants' laboratory that were synthesized targeting histone modifying enzymes including lysine specific demethylase 1 (LSD1) were screened against the linked-ZVpro. Compounds 1 and 2 were identified to be novel inhibitors with IC50 of 21.7 and 3.1 µM (Table 1). Table 1 shows structures and activity of compounds 1-9. Table 1

g y y

[0053] Scheme 1 shows the general synthesis for medicinal chemistry studies. 6-Chloro-2- aminopyrazine (10) was selectively iodized using N-iodosuccinimide, and the 2-amino group was converted to a hydroxyl, which was alkylated using a Mitsunobu reaction to give 12 with a protected piperidin-4-yl-methoxy group. Two selective Suzuki reactions were performed to introduce different aryl groups R⁵ and R⁶ to produce, after deprotection, compounds 3-5, 7 and 9. Mono-substitution of 1,6-dibromo-pyridine or -pyrazine (15) with (N-Boc-piperidin-4- yl)methylamine, followed by iodination produced the intermediate 16. Selective replacement of the 5-iodo and 6-bromo substituent using a Suzuki reaction gave compound 6 or 8. Scheme 1 shows general synthesis for compounds 2-9. Reagents and conditions: (i) N-Iodosuccinimide, DMSO; (ii) NaNO2, H₂SO4(Conc.); (iii) N-Boc-piperidin-4-ylmethanol, PPh3, diisopropyl azodicarboxylate, THF; (iv) R⁵-boronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane-H₂O, 80 ºC; (v) R⁶-boronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane-H₂O, 110 ºC; (vi) 4M HCl, CH₂Cl₂, 0 ºC; (vii) (N-Boc-piperidin-4-yl)methylamine, K2CO3, DMF, 100 °C. Scheme 1

[0054] Compound 9 was found to be a potent inhibitor of the linked- and binary-ZVpro with IC₅₀ of 200 and 220 nM, respectively. Table 1 and Table 2 summarize the inhibitory activities of selected analogs 3-8. Changing the -O- linkage at 2-position to an -NH- in 8 (IC₅₀: 400 nM) resulted in a 2-fold activity reduction. Changing the central pyrazine ring in 8 to a pyridine in 6 (IC50: 790 nM) further reduced the potency. As compared with 7 (IC50: 530 nM) with a N-methyl secondary amine or 4 (IC50: 1.1 mM) with an amide at the 5-position, the primary amine in 9 is more favored. Changing the furan-3-yl group in 9 to a pyrazol-4-yl in 5 (IC₅₀: 710 nM) or a fused pyrrole ring in 3 (IC₅₀: 1.1 mM) also decrease the inhibitory activity. Table 2 shows structures and activity of compounds 1-9. Table 2

[0055] Compounds 3-9 also inhibited DENV serotype-2, -3, and West Nile protease (DV2pro, DV3pro and WVpro) with IC50 values of 120-1340 nM (Table 3). However, 9 exhibited negligible activities against several human serine-, cysteine-, aspartic- and metallo-proteases (Table 4). These results show 9 is a broadly active inhibitor of flavivirus proteases with a high selectivity. Table 3 shows IC50 (mM) of 3-9 against flavivirus proteases. Table 3

[0056] Table 4 shows inhibitory activity of compound 9 against 5 human proteases, including serine proteases trypsin and dipeptidyl peptidase 4 (DPP4), aspartic protease pepsin, cysteine protease caspase-3, and metalloprotease matrix metalloprotease 8 (MMP-8). Table 4

[0057] Applicants determined X-ray structures of DV2pro in complex with compounds 5, 8 and 9 at 2.7-3.0 Å. Statistics for diffraction data and structure refinement are shown in Table 5. The three structures are very similar to each other, with each asymmetric unit containing two inhibitor-bound proteins (FIGS. 2A-C). Similar to the apo-protein, the DV2pro-inhibitor complexes adopt an open conformation, with NS2B binding partially to NS3. The inhibitor- bound NS3 does not deviate significantly from the apo- or substrate-bound protein, except that the residues 152-164 are disordered (with no observed electron density) upon inhibitor binding. In contrast, the U-shaped peptide segment is well organized in both the apo- and substrate-bound NS3. In the latter case, residues 152-164 constitute part of the S1 and S2 pockets of the active site and have interactions with the substrate. Inhibitor binding pushes the loops 71-75 and 117- 122 outwards by ~1.3 Å and 3 Å. All of these movements remodel the surface of NS3 and create a deep, L-shaped pocket (FIG. 2D) that accommodates the inhibitors. The compounds are allosteric inhibitors, which do not occupy the substrate binding site (FIG. 2E). Mechanistically, these inhibitors bind to and stabilize DV2pro in the open conformation, which prevents NS2B from folding into the active site as well as the binding of the substrate. Table 5 shows data collection and refinement statistics (molecular replacement). Table 5

DV2 5 DV2 8 DV2 9

[0058] The inhibitor-protein interactions are illustrated in FIGS.2D/F. The central pyrazine ring of 9 is located at the junction of the L-shaped pocket. The furanylphenyl group is deeply inserted into the pocket with favorable hydrophobic interactions. The 2- and 5-substituents occupy a deep surface groove, having mostly hydrophobic interactions. The positively charged -NH₂ of 9 has hydrogen-bond and electrostatic interactions with Asp75, one of the protease catalytic triad. [0059] High structural and sequence similarities between DV2pro and ZVpro, particularly for the inhibitor-interacting residues, suggest compound 9 binds to ZVpro similarly. Enzyme kinetics studies showed that 9 is a non-competitive inhibitor of DV2pro, consistent with its X-ray structure. Similar enzyme kinetics results support 9 is also an allosteric inhibitor of ZVpro. [0060] Anti-ZIKV activity of 9 was evaluated in U87 glioma cells. The passage-3 stock of ZIKV FLR strain was used for clinical relevance. Upon infection with ZIKV-FLR, U87 cells were incubated with 9 for 48 h. Newly generated ZIKV viruses in the media were determined quantitatively. Compound 9 significantly reduced ZIKV RNA in a dose-dependent manner (FIG. 3A). Because ~1/10⁴ of RNAs represent infectious viruses, an end-point dilution assay was used to determine viral titers more accurately. Half-log (0.32×) serial dilutions of the media were added to Vero cells in quadruplicate. Upon incubation for 7 days, ZIKV infection in each sample was determined with cytopathic effects. TCID₅₀ (tissue culture infective dose) was calculated based on the highest dilution in which ³50% of the quadruplicate samples were infected with ZIKV. As shown in FIG. 3B for a representative experiment, compound 9 reduced infectious ZIKV viruses by 68% at 300 nM, 90% at 600 nM, 97% at 1.2 µM, 99% at 2.5 µM, and 99.7% at 5 µM. Multiple experiments showed that EC68 of 9 was 300 or 600 nM. Compound 9 exhibited similar antiviral activities against ZIKV HN16 strain (FIG. 3C). 9 also showed significant activity against DENV-2 (strain K0049), inhibiting viral replication in Vero cells by 97% at 5 µM. These results demonstrate compound 9 has potent cellular antiviral activity against Zika and dengue viruses. Moreover, cellular antiviral activities of compounds 1-9 are generally correlated with their biochemical activities against ZVpro (Table 1). Compound treatment also dose- dependently inhibited the viral proteins capsid, NS3 and NS5 in infected Vero cells. These results support ZVpro is the cellular target of these compounds. [0061] In vivo anti-ZIKV activity of 9 was evaluated in C57BL/6 mice with both interferon-a/b and -g receptor genes knocked out. Applicants found that intraperitoneal (ip) injection of 100 TCID₅₀ of ZIKV-FLR caused rapid viral replication and death of the mice in ~10 days. Started 1h before inoculation of ZIKV, ip treatment with 9 (15 mg/kg/12h) for 24h (when ZIKV replication is in a rapidly growing phase) reduced ZIKV RNA copies in both plasma and brains of the mice by 96% and 98% (FIG. 3D). Treatment at 30 and 20 mg/kg/day for 3 days significantly prolonged the survival of ZIKV-infected mice, with the average values for the control, 20 and 30 mg/kg groups (N=12) being 11.7, 13.7 and 15.1 days (FIG.3E). These results show that 9 can effectively inhibit ZIKV replication in vivo. [0062] In summary, compound 9 is a broadly active inhibitor of flavivirus proteases and exhibits significant cellular and in vivo activities against Zika virus. In addition, X-ray studies reveal that it binds to an allosteric pocket of NS3 and provide, for the first time, a druggable pocket of the flavivirus protease, as contrasted to the shallow active site recognizing polar and positively charged Arg or Lys of the substrate. Rational inhibitor development based on the pharmacological leads and structural platform could lead to compounds with improved potency. [0063] Example 1.2. Synthesis of Inhibitors

All chemicals for synthesis were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI). The identity of the synthesized compounds was characterized by ¹H and ¹³C NMR on a Varian (Palo Alto, CA) 400-MR spectrometer and mass spectrometer (Shimadzu LCMS-2020). The identity the potent inhibitors was confirmed with high resolution mass spectra (HRMS) using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer with electrospray ionization (ESI). The purities of the final compounds were determined to be >95% with a Shimadzu Prominence HPLC using a Zorbax C18 (or C8) column (4.6 x 250 mm) monitored by UV at 254 nm. Synthesis of compound 1 was reported in Applicants' previous publication. Scheme 2 shows general method of synthesizing 2-5, 7, and 9. ^aReagents and conditions: (i) N-Iodosuccinimide, DMSO, rt, 72 h, 80%; (ii) NaNO₂, H₂SO₄(Conc.), 1 h; (iii) N- Boc-4-piperidinemethanol, PPh₃, DIAD, THF, 77% for 2 steps; (iv) Arylboronic acid or Aryl- 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, Na2CO3, p-dioxane-H₂O, 80 ºC, 73-90%; (v) Arylboronic acid or Aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, Na2CO3, p- dioxane-H₂O, 110 ºC, 60-80%; (vi) HCl (4 N in p-dioxane), CH₂Cl2, 0 ºC, 90%.

Scheme 2

[0064] To a solution of 2-amino-6-chloropyrazine (10, 8 g, 62 mmol) in DMSO (50 mL) was added N-iodosuccinimide (NIS, 15.3 g, 68 mmol) in portions. After stirring at room temperature for 72 h, the reaction was quenched with sodium thiosulfate aqueous solution (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 2:1) to afford 6-chloro-5-iodopyrazin-2-amine (11, 12.7 g, 80%) as a yellow solid.¹H NMR (400 MHz, CDCl₃) d 7.72 (s, 1H), and 4.73 (br, 2H). [0065] To a suspension of 11 (3.78 g, 14.9 mmol) in sulfuric acid (18 mL) at 0 °C was added sodium nitrite (1.09 g, 15.8 mmol) in 3 portions. The resulting reaction mixture was stirred at 0 °C for 1 h. The mixture was then poured into a beaker with ice while stirring. The resulting precipitate was collected by filtration, washed with water and dried under vacuum to afford 6- chloro-5-iodopyrazin-2-ol (3.6 g) as a yellowish solid, which is used directly for the next step.¹H NMR (400 MHz, DMSO-d₆) d 7.98 (s, 1H). Crude product 6-chloro-5-iodopyrazin-2-ol (3.6 g, 14 mmol), N-Boc-4-piperidinemethanol (3.1 g, 14.5 mmol), and triphenylphosphine (5.9 g, 22.5 mmol) were dissolved in THF (40 mL) and cooled to 0 °C. Diisopropyl azodicarboxylate (4.55 g, 22.5 mmol) was added dropwise under nitrogen atmosphere. The mixture was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 10:1 to 5:1) to afford compound 12 (5.2 g, 77% for 2 steps) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) d 7.96 (s, 1H), 4.16 (d, J = 6.4 Hz, 4H), 2.73 (t, J = 12.0 Hz, 2H), 1.96 (s, 1H), 1.77 (d, J = 12.8 Hz, 2H), 1.46 (s, 9H), and 1.33– 1.19 (m, 2H). [0066] Compound 12 (1.2 g, 2.66 mmol), arylboronic acid or aryl-4,4,5,5-tetramethyl-1,3,2- dioxaborolane, (2.66 mmol), tetrakis(triphenylphosphine)palladium (154 mg, 0.13 mmol), and sodium carbonate (564 mg, 5.32 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 80 °C for 24 h. The reaction was cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:1) to afford the product 13 as a yellow solid. [0067] For Compound 13a, ¹H NMR (400 MHz, CDCl₃) d 8.19 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 5.04 (s, 1H), 4.34 (d, J = 5.2 Hz, 2H), 4.21 (d, J = 6.4 Hz, 2H), 4.15 (s, 2H), 2.73 (t, J = 11.2 Hz, 2H), 1.99– 1.92 (m, 1H), 1.79 (d, J = 12.4 Hz, 2H), 1.44 (s, 18H), and 1.30– 1.20 (m, 2H).

[0068] For Compound 13b, ¹H NMR (400 MHz, CDCl₃) d 8.21 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.31 (s, 2H), 4.47 (s, 2H), 4.22 (d, J = 6.5 Hz, 2H), 4.18– 3.98 (m, 2H), 3.01– 2.58 (m, 5H), 2.01– 1.93 (m, 1H), 1.81 (d, J = 13.1 Hz, 2H), 1.46 (s, 18H), and 1.31– 1.24 (m, 2H). [0069] For Compound

8.24 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 6.12 (br, 1H), 5.64 (br, 1H), 4.25 (d, J = 6.5 Hz, 2H), 4.17 (br, 2H), 2.78 (t, J = 14.2 Hz, 2H), 2.01 (br, 1H), 1.82 (d, J = 13.0 Hz, 2H), 1.47 (s, 9H), and 1.36– 1.26 (m, 2H). [0070] Compound 13 (1.94 mmol), arylboronic acid or aryl-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (2.51 mmol), tetrakis(triphenylphosphine)palladium (110 mg, 0.095 mmol), and sodium carbonate (610 mg, 5.75 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 110 °C for 24 h. The reaction was then cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:2) to afford the product 14 as a pale yellow solid. [0071] For compound 14a,

NMR (400 MHz, CDCl₃) d 8.22 (s, 1H), 7.75 (s, 1H), 7.50– 7.34 (m, 7H), 7.20 (d, J = 7.8 Hz, 2H), 6.70 (s, 1H), 4.85 (s, 1H), 4.36– 4.24 (m, 4H), 4.20– 4.07 (m, 2H), 2.76 (t, J = 12.4 Hz, 2H), 2.03– 1.97 (m, 1H), 1.84 (d, J = 12.4 Hz, 3H), 1.54– 1.40 (m, 18H), and 1.37– 1.27 (m, 2H). [0072] For compound 14b,

NMR (400 MHz, CDCl₃) d 8.22 (s, 1H), 7.75 (s, 1H), 7.49– 7.39 (m, 5H), 7.38 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 7.3 Hz, 2H), 6.70 (s, 1H), 4.42 (d, J = 17.3 Hz, 2H), 4.30 (d, J = 6.4 Hz, 2H), 4.17 (s, 2H), 2.85 (s, 2H), 2.76 (s, 3H), 2.02 (s, 1H), 1.84 (d, J = 12.8 Hz, 2H), 1.51– 1.42 (m, 18H), and 1.31 (dd, J = 12.6, 4.0 Hz, 2H). [0073] For compound 14c,

NMR (400 MHz, CDCl₃) d 8.24 (s, 1H), 7.78-7.73 (m, 3H), 7.53 (s, 1H), 7.51 (s, 1H), 7.48 (s, 1H), 7.43 (s, 4H), 6.70 (s, 1H), 6.05 (br, 1H), 5.60 (br, 1H), 4.31 (d, J = 6.5 Hz, 2H), 4.18 (br, 2H), 2.77 (t, J = 14.2 Hz, 2H), 2.03 (br, 1H), 1.84 (d, J = 13.4 Hz, 2H), 1.47 (s, 9H), and 1.37– 1.27 (m, 2H). [0074] For compound 14d,

NMR (400 MHz, CDCl₃) d 8.84 (s, 1H), 8.19 (s, 1H), 7.82 (s, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.19 (s, 1H), 7.17– 7.13 (m, 2H), 7.11 (d, J = 7.9 Hz, 2H), 6.50 (s, 1H), 4.95 (s, 1H), 4.31 (d, J = 6.4 Hz, 2H), 4.26 (d, J = 5.0 Hz, 2H), 4.18– 4.11 (m, 2H), 2.76 (t, J = 11.3 Hz, 2H), 2.02 (s, 1H), 1.84 (d, J = 12.3 Hz, 2H), 1.48 (s, 9H), 1.44 (s, 9H), and 1.31 (dd, J = 12.2, 3.9 Hz, 2H). [0075] For compound 14e, ¹H NMR (400 MHz, CDCl₃) d 8.21 (s, 1H), 7.84 (s, 2H), 7.49– 7.40 (m, 4H), 7.38 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 4.92 (br, 1H), 4.29 (d, J = 6.5 Hz, 4H), 4.23– 4.11 (m, 2H), 2.76 (t, J = 12.6 Hz, 2H), 2.02 (br, 1H), 1.84 (d, J = 12.5 Hz, 2H), 1.47 (s, 9H), 1.44 (s, 9H), and 1.35– 1.27 (m, 2H). [0076] For compound 14f, 1H NMR (400 MHz, CDCl₃): d 8.17 (s, 1H), 7.43 (d, J = 7.6 Hz, 2H), 7.34-7.27 (m, 6H), 4.27 (d, J = 6.8 Hz, 2H), 4.14 (m, 2H), 2.75 (t, J = 11.6 Hz, 2H), 1.99 (br, 1H), 1.82 (d, J = 12 Hz, 2H), 1.45 (s, 9H), 1.29 (s, 18H), and 1.35-1.26 (m, 2H). [0077] To a solution of compound 14 (1.5 mmol) in DCM (5 mL) was added dropwise HCl (1.2 mL, 4 N in p-dioxane) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford an oil, which was triturated in diether ether and solidified to give the final product hydrochloric salt as a white or pale yellow powder. [0078] 2,3-bis(4-(tert-butyl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazine (2) ¹H NMR (400 MHz, DMSO-d₆) d 9.00– 8.27 (br, 4H), 8.30 (s, 1H), 7.38– 7.26 (m, 8H), 4.27 (d, J = 6.2 Hz, 2H), 3.33– 3.24 (m, 2H), 2.90 (t, J = 11.8 Hz, 2H), 2.13 (br, 1H), 1.93 (d, J = 13.0 Hz, 2H), 1.58 – 1.48 (m, 2H), and 1.26 (s, 18H); ¹³C NMR (100 MHz, DMSO-d6) 157.4, 151.2, 150.3, 147.4, 144.0, 135.7, 135.4, 131.9, 129.2, 129.0, 125.0, 124.9, 69.4, 42.7, 34.4, 34.3, 33.0, 31.1, 31.0, and 25.2; HRMS (ESI⁺) calcd for C30H40N3O [M+H]⁺ 458.3166, found 458.3179. [0079] (4-(3-(1H-indol-5-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride (3) ¹H NMR (400 MHz, D₂O) d 8.27 (s, 1H), 7.71 (s, 1H), 7.47– 7.35 (m, 6H), 7.32 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 4.36 (s, 2H), 4.17 (s, 2H), 3.51 (d, J = 11.5 Hz, 2H), 3.06 (t, J = 13.1 Hz, 2H), 2.22 (s, 1H), 2.10 (d, J = 13.5 Hz, 2H), and 1.65 (dd, J = 24.7, 12.0 Hz, 2H) ; ¹³C NMR (100 MHz, D₂O) d 158.6, 151.1, 143.4, 139.3, 136.1, 132.8, 130.6, 130.3, 129.1, 128.8, 127.6, 126.6, 123.6, 122.5, 111.6, 70.2, 43.7, 42.7, 33.2, and 25.3; MS (ESI) [M+H]⁺ 414.5. [0080] 4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)benzamide hydrochloride (4)

NMR (400 MHz, DMSO-d6) d 8.77 (br, 2H), 8.40 (s, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.99 (s, 1H), 7.81 (d, J = 7.7 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J = 7.7 Hz, 2H), 7.43– 7.40 (m, 4H), 6.98 (s, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.09– 3.05 (m, 2H), 2.92 (br, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), and 1.56– 1.47 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) 167.6, 158.0, 147.8, 144.6, 143.4, 141.4, 140.1, 136.3, 133.4, 132.5, 130.2, 129.4, 127.53, 127.46, 125.4, 125.3, 108.7, 69.7, 42.7, 33.0, and 25.3; MS (ESI) [M+H]⁺ 455.5. [0081] (4-(3-(4-(1H-Pyrazol-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methan-amine hydrochloride (5) ¹H NMR (400 MHz, D2O) d 8.18 (s, 2H), 8.15 (s, 1H), 7.41 (s, 2H), 7.34– 7.27 (m, 6H), 4.20 (s, 2H), 4.14 (s, 2H), 3.49 (d, J = 12.9 Hz, 2H), 3.02 (t, J = 13.2 Hz, 2H), 2.12 (br, 1H), 2.03 (d, J = 15.3 Hz, 2H), and 1.64– 1.54 (m, 2H); ¹³C NMR (100 MHz, D2O) 158.5, 148.8, 143.2, 138.1, 135.9, 132.7, 131.11, 131.04, 131.02, 131.00, 130.95, 130.4, 130.1, 128.8, 125.1, 70.1, 43.5, 42.6, 32.9, and 25.0; MS (ESI) [M+H]⁺ 441.5. [0082] 1-(4-(3-(4-(furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2-yl)phenyl)-N- methylmethanamine hydrochloride (7) ¹H NMR (400 MHz, D₂O) d 8.07 (s, 1H), 7.76 (s, 1H), 7.54 (s, 1H), 7.28 (d, J = 7.9 Hz, 2H), 7.24– 7.16 (m, 4H), 7.12 (d, J = 7.8 Hz, 2H), 6.67 (s, 1H), 4.1– 4.00 (m, 4H), 3.43 (d, J = 6.1 Hz, 2H), 2.97 (t, J = 12.6 Hz, 2H), 2.64 (s, 3H), 2.06 (s, 2H), 1.95 (d, J = 13.2 Hz, 2H), and 1.59– 1.48 (m, 2H) ; ¹³C NMR (100 MHz, D₂O) d 158.5, 148.8, 144.7, 143.2, 139.5, 138.9, 135.6, 132.6, 131.3, 130.8, 130.4, 130.2, 129.9, 125.1, 116.0, 108.3, 70.1, 52.3, 43.7, 33.07, 32.11, and 25.0; MS (ESI) [M+H]⁺ 455.6. [0083] (4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride (9) ¹H NMR (400 MHz, D2O) d 8.24 (s, 1H), 7.94 (s, 1H), 7.61 (s, 1H), 7.51 (dd, J = 8.4, 1.8 Hz, 2H), 7.41 (br, 6H), 6.85 (s, 1H), 4.35 (br, 2H), 4.17 (s, 2H), 3.50 (d, J = 13.3 Hz, 2H), 3.06 (t, J = 13.3 Hz, 2H), 2.124 (br, 1H), 2.11 (d, J = 14.6 Hz, 2H), and 1.68– 1.58 (m, 2H); ¹³C NMR (100 MHz, D2O) 158.4, 148.8, 144.5, 143.3, 139.4 (2), 138.3, 135.6, 132.6, 132.4, 131.1, 130.3, 130.0, 128.7, 125.0, 108.1, 70.0, 43.5, 42.6, 32.9, and 24.9; HRMS (ESI⁺) calcd for C₂₇H₂₉N₄O₂ [M+H]⁺ 441.2291, found 441.2285. [0084] Synthesis of 6 and 8 is shown in Scheme 3. Reagents and conditions: (i) tert-butyl 4- (aminomethyl)piperidine-1-carboxylate, K2CO3, DMF, 100 °C, 12 h; (ii) N-Iodosuccinimide, CH₃CN-DMSO, 24 h, 60% for 2 steps; (iii) 4-((N-Boc-amino)-methyl)phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, p-dioxane-H₂O, 80 ºC, 73%; (iv) 2-(4-(furan-3-yl)phenyl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane, Pd(PPh3)4, Na2CO3, p-dioxane-H₂O, 100 ºC, 78%; (v) HCl (4 N in p-dioxane), CH₂Cl2, 0 ºC, >90%.

Scheme 3

[0085] 2,6-Dibromopyrazine (15a, 7.14 g, 30 mmol) or 2,6-dibromopyridine (15b, 7.14 g, 30 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (7.07 g, 33 mmol) and potassium carbonate (8.28 g, 60 mmol) in DMF (30 mL) were placed in a sealed tube. The mixture was stirred at 100 °C for 12 h. The reaction was cooled and quenched with saturated brine (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford 18 as a white solid, which was used in the next step without further purification. For compound 18a,

NMR (400 MHz, CDCl₃): d 7.85 (s, 1H), 7.76 (s, 1H), 5.00 (s, 1H), 4.14– 4.11 (m, 2H), 3.25 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 11.8 Hz, 2H), 1.73– 1.70 (m, 3H), 1.44 (s, 9H), and 1.21– 1.09 (m, 2H). For compound 18b,

NMR (400 MHz, CDCl₃): d 7.23 (t, J = 7.8 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.26 (d, J = 7.8 Hz, 1H), 4.76 (t, J = 5.4 Hz, 1H), 4.10 (br, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.67 (t, J = 12.8 Hz, 2H), 1.77– 1.67 (m, 3H), 1.44 (s, 9H), and 1.19– 1.09 (m, 2H). [0086] To a solution of compound 18 (～30 mmol) in CH₃CN/DMSO (20/10 mL) was added N- Iodosuccinimide (8.1 g, 36 mmol). After stirring at room temperature for 24 h, the reaction was quenched with sodium thiosulfate aqueous solution (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 10:1 to 3:1) to give 16 as a white solid (yield > 60% over two steps). For compound 16a, ¹H NMR (400 MHz, CDCl₃): d 7.64 (s, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.14– 4.11 (m, 2H), 3.24 (t, J = 5.9 Hz, 2H), 2.69 (t, J = 12.2 Hz, 2H), 1.73– 1.69 (m, 3H), 1.45 (s, 9H), and 1.21– 1.13 (m, 2H). For compound 16b, ¹H

NMR (400 MHz, CDCl₃): d 7.64 (d, J = 8.5 Hz, 1H), 6.10 (d, J = 8.5 Hz, 1H), 4.74 (t, J = 5.4 Hz, 1H), 4.14– 4.09 (m, 2H), 3.14 (t, J = 5.9 Hz, 2H), 2.69 (t, J = 12.2 Hz, 2H), 1.73– 1.69 (m, 3H), 1.45 (s, 9H), and 1.20– 1.11 (m, 2H). [0087] Compound 16 (994 mg, 2 mmol), 4-((N-Boc-amino)methyl)phenylboronic acid (527 mg, 2.1 mmol), tetrakis(triphenylphosphine)palladium (69 mg, 3 mol%), and sodium carbonate (424 mg, 4 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 80 °C for 24 h. The reaction was then cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine, dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:1) to give product 19 as a white solid (yield > 75%). For Compound 19a, ¹H NMR (400 MHz, CDCl₃): d 7.88 (s, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 4.94 (t, J = 5.9 Hz, 1H), 4.88 (br, 1H), 4.37 (d, J = 5.9 Hz, 2H), 4.16– 4.11 (m, 2H), 3.31 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 12.2 Hz, 2H), 1.77– 1.74 (m, 3H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.25– 1.15 (m, 2H). For Compound 19b,

NMR (400 MHz, CDCl₃): d 7.38 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.36 (d, J = 8.4 Hz, 1H), 4.88 (br, 2H), 4.36 (br, 2H), 4.16– 4.11 (m, 2H), 3.18 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 12.2 Hz, 2H), 2.10 (br, 1H), 1.77– 1.74 (m, 2H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.22– 1.15 (m, 2H). [0088] Compound 19 (1 mmol), 2-(4-(furan-3-yl)phenyl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (1.1 mmol), tetrakis(triphenylphosphine)palladium (57.7 mg, 5 mol%), and sodium carbonate (216 mg, 2 mmol) in p-dioxane/H₂O (8/2 mL) were placed in a sealed tube. The mixture was degassed and heated to 100 °C for 24 h. The reaction was then cooled and quenched with brine (10 mL). The mixture was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:2) to afford 17 as a white solid (yield > 75%). For Compound 17a,

NMR (400 MHz, CDCl₃): d 7.90 (s, 1H), 7.73 (s, 1H), 7.46 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 6.69 (s, 1H), 4.89 (t, J = 5.9 Hz, 1H), 4.85 (br, 1H), 4.28 (d, J = 5.9 Hz, 2H), 4.16– 4.11 (m, 2H), 3.36 (t, J = 6.1 Hz, 2H), 2.71 (t, J = 11.8 Hz, 2H), 1.79– 1.76 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H), and 1.26– 1.16 (m, 2H). For Compound 17b,

NMR (400 MHz, CDCl₃): d 7.70 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.45 (br, 1H), 7.34 (br, 4H), 7.13 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.67 (s, 1H), 6.42 (d, J = 8.4 Hz, 1H), 4.83 (t, J = 5.5 Hz, 2H), 4.28 (d, J = 5.3 Hz, 2H), 4.14– 4.09 (m, 2H), 3.25 (t, J = 5.8 Hz, 2H), 2.71 (t, J = 13.4 Hz, 2H), 1.81– 1.78 (m, 3H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.27– 1.20 (m, 2H). [0089] Compound 17 (0.2 mmol) was dissolved in dichloromethane (2 mL). HCl (0.2 mL, 4 N in p-dioxane) was slowly added to the reaction mixture at 0 °C. After 0.5 h, the reaction was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford an oil, which was triturated in diether ether and solidified to give 6 or 8 hydrochloric salt as a white or pale yellow powder (yield > 90%). [0090] 5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperidin-4- ylmethyl)pyridin-2-amine hydrochloride (6) ¹H NMR (400 MHz, DMSO-d6) d 9.05 (br, 1H), 8.83 (br, 2H), 8.44 (br, 3H), 8.28 (s, 1H), 7.91 (br, 2H), 7.77 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 7.01 (s, 1H), 3.97 (d, J = 6.3 Hz, 2H), 3.38 (br, 2H), 3.28 (d, J = 13.5 Hz, 2H), 2.85 (q, J = 12.3 Hz, 2H), 1.95– 1.91 (m, 3H), and 1.48– 1.40 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) 153.9, 144.6, 140.4, 136.6, 133.2, 130.4, 129.5, 129.0, 125.3, 124.9, 123.1, 108.5, 46.3, 42.5, 41.6, 33.1, and 25.9; MS (ESI) [M+H]⁺ 439.6. [0091] 5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperidin-4- ylmethyl)pyrazin-2-amine hydrochloride (8).¹H NMR (400 MHz, DMSO-d6): d 9.00 (br, 2H), 8.67 (br, 2H), 8.38 (br, 4H), 8.21 (s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 7.54 (d, J = 7.3 Hz, 2H), 7.37– 7.30 (m, 6H), 6.96 (s, 1H), 3.98 (br, 2H), 3.28– 3.24 (m, 4H), 2.83 (q, J = 10.8 Hz, 2H), 1.89– 1.85 (m, 3H), and 1.46– 1.38 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6): 153.0, 148.1, 144.5, 143.8, 139.8, 139.6, 137.9, 137.6, 132.5, 131.7, 130.0, 129.3, 128.5, 125.3, 125.1, 108.6, 55.0, 42.9, 41.9, 33.5, and 26.4; MS (ESI) [M+H]⁺ 440.5. [0092] Example 1.3. Constructs for Recombinant Proteins [0093] Two expression plasmids for ZVpro were constructed according to reported methods. cDNA encoding NS2B (residues 47-95) and NS3 (residues 1-170) of Zika virus (GenBank: KU729217.2) connected with a Gly4-Ser-Gly4 linker was synthesized by GenScript. To avoid autoproteolytic cleavage, Arg95 of NS2B and Arg29 of NS3 were replaced by Ala and Gly, respectively. It was inserted into the NdeI/XhoI sites of pET-28a vector. For the binary construct, NS2B and NS3 were inserted into the NdeI/XhoI and NcoI/HindIII sites of pET-Duet-1 vector, respectively. The pET-28a expression plasmids for DV2pro (GenBank: AY037116) including NS2B (residues 50-95) and NS3 (residues 1-182), DV3pro (GenBank: AAW66479) including NS2B (residues 50-95) and NS3 (residues 1-182), and WVpro (GenBank: AAV54504) including NS2B (residues 49-96) and NS3 (residues 2-184) were constructed similarly. The correctness of all of the inserted DNA was verified by sequencing. [0094] Example 1.4. Expression and Purification of Flavivirus Proteases [0095] E. coli BL21 (Rosetta strain, Agilent) was transformed with the expression plasmids and cultured at 37 °C in LB medium in the presence of an appropriate antibiotics according to the vector. Upon reaching an optical density of ~1.3 at 600 nm, protein expression was induced by adding 0.5 mM isopropylthiogalactoside at 18 °C for 20 hours. Cells were harvested, lysed, and centrifuged at 20,000 rpm for 20 min. The supernatant was collected and applied to an affinity column chromatography using immobilized metal affinity chromatography (IMAC) beads (GE Healthcare). The target protein was eluted with 300 mM imidazole buffer. To remove the His- tag, thrombin was added to the eluted fractions and the mixture was dialyzed against buffer containing 20 mM HEPES, 150 mM NaCl, 2 mM DTT, pH 7.5 for 20 hours. Subsequently, the protein was further purified to be >95% purity (SDS-PAGE) with a size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column. [0096] Example 1.5. Activity and Inhibition Assays for Flavivirus Proteases [0097] Similar to a reported method, activity and inhibition assay for the linked- and binary- ZVpro was performed using the enzyme (1 nM) and benzoyl-norleucine-lysine-lysine-arginine 7- amino-4-methylcoumarine (Bz-Nle-Lys-Lys-Arg-AMC, 20 mM) as the substrate in a HEPES buffer (20mM, pH 7.3) containing 0.05% Triton X-100. To determine IC₅₀, triplicate samples of a compound with concentrations ranging from 1 nM to 10 mM were incubated with the enzyme for 10 min before adding the substrate to initiate the reaction in 96-well plate (100 µL final volume). The fluorescence signal (Ex: 360 nm, Em: 460 nm) of each well was monitored every 30s, using a Beckman DTX-880 microplate reader. The initial velocity data were imported into Prism (version 5.0), and IC50 values from 3 independent experiments with standard deviation were obtained by using a standard dose-response curve fitting. Enzyme inhibition assays for DV2pro, DV3pro and WVpro were performed similarly. [0098] To determine the K_m value of the substrate for ZVpro, the assay was performed with ZVpro (1 nM) and increasing concentrations of the substrate (2.5 mM to 200 mM). The initial velocities were fitted to the Michaelis-Menten model in Prism (version 5.0) to give a Km value of 14.6 mM, which is comparable to a reported value of 18.3 mM. The K_m value for DV2pro (3 nM) was similarly determined to be 140 mM with increasing concentrations of the substrate (10-1000 mM). [0099] Example 1.6. Inhibition Assays for Human Proteases [00100] Inhibition of the 5 human proteases were evaluated using commercially available kits according to manufacturers’ protocols. The fluorescent assay kits for Dipeptidyl peptidase-4 (DPP4) and Caspase-3 were from BPS Bioscience. Inhibition of Matrix metalloproteinase 8 (MMP-8) was determined using SensoLyte 490 MMP-8 Assay Kit (AnaSpec). Pepsin and trypsin were purchased from Sigma. Their inhibition assay was performed using a FRET protease assay kit from Thermo Scientific. [00101] Example 1.7. Crystallization, X-Ray Data Collection and Structural Determination [00102] Recombinant DV2pro was expressed and purified as described above. The protein was concentrated to 10 mg/mL in a buffer containing 20 mM Tris (pH 7.2) and 200 mM NaCl. Co- crystallization with an inhibitor (5 mM) was set up by hanging drops with 1:1 ratio mixtures of 1 mL of protein solution and 1 mL of well solution containing 35% PEG 200, 100 mM MES, pH 8.5. Crystals of the DV2pro-inhibitor complex were grown at 22 °C for 2 weeks, which were harvested in crystal freezing buffer containing 20% glycerol, 35% PEG 200, 100 mM MES, pH 8.5. The crystals were then flash-frozen in liquid N₂ for data collection. X-ray diffraction data were collected at the Advanced Photon Source beamline 19-ID. Data were processed using HKL3000. The initial molecular replacement structures were solved by program CCP4 PHASER using the coordinates of 2FOM as a template. The program COOT was used for model building. Models of the inhibitors were built based on the difference maps. The program CCP4 refmac was used for structure refinement. The similes files of inhibitors were loaded onto CCP4 module sketcher to generate cif files. The generated cif files were used in CCP4 refinement of the DV2pro-inhibitor structures. The final refinement statistics were summarized in Table 4 and the coordinates were deposited into Protein Data Bank as entries 6MO0, 6MO1 and 6MO2. All figures were generated using the programs Maestro or PyMOL in Schrödinger Suite. Ligand- protein interactions were determined and illustrated using Maestro. Analysis of the Ramachandran plots for the DV2pro-5, -8 and -9 showed that 99.4% of the residues are in the preferred or allowed regions. [00103] Example 1.8. Cellular Antiviral Activity Testing [00104] Anti-ZIKV activity of compound 9 was evaluated in human U87 glioma cells, in which ZIKV replicates rapidly, but does not cause cytopathic effects (CPE. ZIKV also replicates rapidly in monkey Vero cells lacking interferon-mediated defense and causes significant CPE and cell lysis. The passage-3 stock of the ZIKV FLR strain, which was isolated from the serum of a patient infected in Colombia in 2015, was used for clinical relevance. In addition, ZIKV HN16 strain (Honduras, 2016) and DENV K0049 strain (Thailand, 1995) were used to determine antiviral activities of compound 9. 2x10⁴ U87 cells/well were seeded in 96-well plates and cultured in DMEM media with 2% FBS to form a monolayer of cells.0.01 MOI (multiplicity of infection) of ZIKV was added. After incubation for 1h, the supernatant was removed and cells were washed with PBS. Fresh medium (150 µL/well) containing various concentrations of a compound in triplicate were added. Upon incubation at 37 °C for 48h, aliquots of the supernatant from each well were used to determine ZIKV RNA copies and titers using the methods discussed below. [00105] Example 1.9. Quantitative RT-PCR to Determine ZIKV RNA Copies. [00106] Viral RNA was extracted from the supernatant (50 µL) using TRIzol (ThermoFisher) according to the manufacturer’s instructions. qPCR is based on amplification of ZIKV envelope gene region 3, using ZIKV-specific primers and probes. PCR was performed using a TaqMan Fast Virus 1-step Master Mix kit on a StepOnePlus RT-PCR system (Applied Biosystems). Concentrations of ZIKV RNA (copies/mL) were calculated by using a standard curve. Although this is a quick method to evaluate activity, only ~1/10⁴ copies of RNA equals 1 TCID. Infectious ZIKV titer was determined with an end-point dilution assay. [00107] Example 1.10. End-Point Dilution Assay to Determine TCID50 (Tissue Culture Infectious Dose) [00108] Half-log serial dilution of the viral supernatant (50 µL) was added to a monolayer of Vero cells in quadruplicate in 96-well plates and cultured for 7 days. CPE/cell lysis was determined with microscope followed by MTT assay. TCID₅₀ was calculated based on the highest dilution in which ³50% (i.e., ³2 out of the 4 quadruplicate wells) of Vero cells were infected with ZIKV. Compared to controls, the ability for a compound to reduce TCID50 can be determined. The results were from at least 2 independent experiments. [00109] Example 1.11. Western Blot [00110] 8 x 10⁵ Vero cells in 6-well plate were infected with ZIKA (MOI = 0.1) for 2h. Upon removal of the media, the cells were washed 3x with PBS and cultured with fresh media contain 2% FBS and compound 9 for 48h. The cells were harvested and the total cellular proteins extracted using ice-cold radioimmunoprecipitation assay buffer (Invitrogen). Equal amounts of total proteins were separated on SDS-PAGE and transferred to PVDF membranes. The blots were probed with primary antibodies against Zika virus NS3 (GTX133309, GeneTex), Zika virus capsid (GTX133317, GeneTex), Zika virus NS5 (GTX133327, GeneTex), and human b-Actin (4967S, Cell Signaling), followed by antirabbit IgG (Invitrogen) secondary antibodies. [00111] Example 1.12. In Vivo Anti-ZIKV Activity Evaluation [00112] The animal studies were performed according to an approved animal protocol by IACUC of Applicants' institute. 100 TCID50 of ZIKV in 0.1 mL of medical grade saline was injected i.p. into C57BL/6 mice with IFNa/bR-/- and IFNgR-/- (both male and female, 8-12 weeks old, Jackson Laboratory, stock # 029098) to initiate infection. Mice were randomly separated into control (N=11) and treatment (N=10) groups. Compound 9 in 0.1 mL of medical grade saline was injected i.p.1 h before virus inoculation. Medical grade saline was administered for the control group of mice. After treatment, mice were euthanized and their blood and brain samples were obtained and processed to determine copies of ZIKV RNA. The brain sample (90 mg) was homogenized in TRIzol (1 mL), centrifuged for 5 min, and the supernatant transferred to a fresh tube. 200 µL of chloroform was added and RNA was then extracted, which was dissolved in DEPC water (400 µL) for RT-PCR. Plasma (50 µL) was separated from the blood samples and mixed with TRIzol (600 µL). RNA was extracted following the above procedure and subjected to qPCR determination. [00113] For survival experiments, mice were randomly separated into control and treatment groups (N=12). Upon compound treatment as described in the text, mice were weighed and monitored daily for symptoms of viral infection and mortality. Although 10-15% weight loss was observed, compound 9 at these doses did not cause overt toxicity to the mice. Mice showing severe weight loss (>20%), inactivity with ruffled fur, or developing severe neurological dysfunctions were humanely euthanized. [00114] Example 1.13. Statistics [00115] The significance of experimental differences in ZIKV RNA copies for in vivo studies was evaluated by use of the Student’s t test (Prism 5.0). The significance of experimental differences in mouse survival studies was evaluated by the Log-rank test (Prism 5.0). [00116] Example 1.14. Synthesis and Characterization of Compounds [00117] Scheme 4 illustrates a general method of synthesizing SYC-1110, 1598, 1622, 1617 and 1307. Reagents and conditions: (i) N-Iodosuccinimide, DMSO, rt, 72 h, 80%; (ii) NaNO2, H₂SO4(Conc.), 1 h; (iii) N-Boc-4-piperidinemethanol, PPh3, DIAD, THF, 77% for 2 steps; (iv) Arylboronic acid or Aryl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, p- dioxane-H₂O, 80 ºC, 73-90%; (v) Arylboronic acid or Aryl-4,4,5,5-tetramethyl-1,3,2- dioxaborolane, Pd(PPh3)4, Na2CO3, p-dioxane-H₂O, 110 ºC, 60-80%; (vi) HCl (4 N in p- dioxane), CH₂Cl₂, 0 ºC, 90%.

Scheme 4

[00118] To a solution of 2-amino-6-chloropyrazine (1, 8 g, 62 mmol) in DMSO (50 mL) was added N-iodosuccinimide (NIS, 15.3 g, 68 mmol) in portions. After stirring at room temperature for 72 h, the reaction was quenched with sodium thiosulfate aqueous solution (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 2:1) to afford 6-chloro-5-iodopyrazin-2-amine (2, 12.7 g, 80%) as a yellow solid.¹

H NMR (400 MHz, CDCl₃) d 7.72 (s, 1H), and 4.73 (br, 2H). [00119] To a suspension of 2 (3.78 g, 14.9 mmol) in sulfuric acid (18 mL) at 0 °C was added sodium nitrite (1.09 g, 15.8 mmol) in 3 portions. The resulting reaction mixture was stirred at 0 °C for 1 h. The mixture was then poured into a beaker with ice while stirring. The resulting precipitate was collected by filtration, washed with water and dried under vacuum to afford 6- chloro-5-iodopyrazin-2-ol (3.6 g) as a yellowish solid, which is used directly for the next step.¹H NMR (400 MHz, DMSO-d₆) d 7.98 (s, 1H). Crude product 6-chloro-5-iodopyrazin-2-ol (3.6 g, 14 mmol), N-Boc-4-piperidinemethanol (3.1 g, 14.5 mmol), and triphenylphosphine (5.9 g, 22.5 mmol) were dissolved in THF (40 mL) and cooled to 0 °C. Diisopropyl azodicarboxylate (4.55 g, 22.5 mmol) was added dropwise under nitrogen atmosphere. The mixture was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 10:1 to 5:1) to afford compound 3 (5.2 g, 77% for 2 steps) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) d 7.96 (s, 1H), 4.16 (d, J = 6.4 Hz, 4H), 2.73 (t, J = 12.0 Hz, 2H), 1.96 (s, 1H), 1.77 (d, J = 12.8 Hz, 2H), 1.46 (s, 9H), and 1.33– 1.19 (m, 2H). [00120] Compound 3 (1.2 g, 2.66 mmol), arylboronic acid or aryl-4,4,5,5-tetramethyl-1,3,2- dioxaborolane, (2.66 mmol), tetrakis(triphenylphosphine)palladium (154 mg, 0.13 mmol), and sodium carbonate (564 mg, 5.32 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 80 °C for 24 h. The reaction was cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:1) to afford the product 4 as a yellow solid. [00121] For Compound

8.19 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 5.04 (s, 1H), 4.34 (d, J = 5.2 Hz, 2H), 4.21 (d, J = 6.4 Hz, 2H), 4.15 (s, 2H), 2.73 (t, J = 11.2 Hz, 2H), 1.99– 1.92 (m, 1H), 1.79 (d, J = 12.4 Hz, 2H), 1.44 (s, 18H), and 1.30– 1.20 (m, 2H). [00122] For Compound 4b, ¹

8.21 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.31 (s, 2H), 4.47 (s, 2H), 4.22 (d, J = 6.5 Hz, 2H), 4.18– 3.98 (m, 2H), 3.01– 2.58 (m, 5H), 2.01– 1.93 (m, 1H), 1.81 (d, J = 13.1 Hz, 2H), 1.46 (s, 18H), and 1.31– 1.24 (m, 2H). [00123] For Compound

8.24 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 6.12 (br, 1H), 5.64 (br, 1H), 4.25 (d, J = 6.5 Hz, 2H), 4.17 (br, 2H), 2.78 (t, J = 14.2 Hz, 2H), 2.01 (br, 1H), 1.82 (d, J = 13.0 Hz, 2H), 1.47 (s, 9H), and 1.36– 1.26 (m, 2H). [00124] Compound 4 (1.94 mmol), arylboronic acid or aryl-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (2.51 mmol), tetrakis(triphenylphosphine)palladium (110 mg, 0.095 mmol), and sodium carbonate (610 mg, 5.75 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 110 °C for 24 h. The reaction was then cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:2) to afford the product 5 as a pale yellow solid. [00125] For compound 5a,

NMR (400 MHz, CDCl₃) d 8.22 (s, 1H), 7.75 (s, 1H), 7.50– 7.34 (m, 7H), 7.20 (d, J = 7.8 Hz, 2H), 6.70 (s, 1H), 4.85 (s, 1H), 4.36– 4.24 (m, 4H), 4.20– 4.07 (m, 2H), 2.76 (t, J = 12.4 Hz, 2H), 2.03– 1.97 (m, 1H), 1.84 (d, J = 12.4 Hz, 3H), 1.54– 1.40 (m, 18H), and 1.37– 1.27 (m, 2H). [00126] For compound 5b,

NMR (400 MHz, CDCl₃) d 8.22 (s, 1H), 7.75 (s, 1H), 7.49– 7.39 (m, 5H), 7.38 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 7.3 Hz, 2H), 6.70 (s, 1H), 4.42 (d, J = 17.3 Hz, 2H), 4.30 (d, J = 6.4 Hz, 2H), 4.17 (s, 2H), 2.85 (s, 2H), 2.76 (s, 3H), 2.02 (s, 1H), 1.84 (d, J = 12.8 Hz, 2H), 1.51– 1.42 (m, 18H), and 1.31 (dd, J = 12.6, 4.0 Hz, 2H). [00127] For compound 5c,

NMR (400 MHz, CDCl₃) d 8.24 (s, 1H), 7.78-7.73 (m, 3H), 7.53 (s, 1H), 7.51 (s, 1H), 7.48 (s, 1H), 7.43 (s, 4H), 6.70 (s, 1H), 6.05 (br, 1H), 5.60 (br, 1H), 4.31 (d, J = 6.5 Hz, 2H), 4.18 (br, 2H), 2.77 (t, J = 14.2 Hz, 2H), 2.03 (br, 1H), 1.84 (d, J = 13.4 Hz, 2H), 1.47 (s, 9H), and 1.37– 1.27 (m, 2H). [00128] For compound 5d,

NMR (400 MHz, CDCl₃) d 8.84 (s, 1H), 8.19 (s, 1H), 7.82 (s, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.19 (s, 1H), 7.17– 7.13 (m, 2H), 7.11 (d, J = 7.9 Hz, 2H), 6.50 (s, 1H), 4.95 (s, 1H), 4.31 (d, J = 6.4 Hz, 2H), 4.26 (d, J = 5.0 Hz, 2H), 4.18– 4.11 (m, 2H), 2.76 (t, J = 11.3 Hz, 2H), 2.02 (s, 1H), 1.84 (d, J = 12.3 Hz, 2H), 1.48 (s, 9H), 1.44 (s, 9H), and 1.31 (dd, J = 12.2, 3.9 Hz, 2H). [00129] For compound 5e,

NMR (400 MHz, CDCl₃) d 8.21 (s, 1H), 7.84 (s, 2H), 7.49– 7.40 (m, 4H), 7.38 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 4.92 (br, 1H), 4.29 (d, J = 6.5 Hz, 4H), 4.23– 4.11 (m, 2H), 2.76 (t, J = 12.6 Hz, 2H), 2.02 (br, 1H), 1.84 (d, J = 12.5 Hz, 2H), 1.47 (s, 9H), 1.44 (s, 9H), and 1.35– 1.27 (m, 2H). [00130] For compound 5f, 1H NMR (400 MHz, CDCl₃): d 8.17 (s, 1H), 7.43 (d, J = 7.6 Hz, 2H), 7.34-7.27 (m, 6H), 4.27 (d, J = 6.8 Hz, 2H), 4.14 (m, 2H), 2.75 (t, J = 11.6 Hz, 2H), 1.99 (br, 1H), 1.82 (d, J = 12 Hz, 2H), 1.45 (s, 9H), 1.29 (s, 18H), and 1.35-1.26 (m, 2H). [00131] To a solution of compound 5 (1.5 mmol) in DCM (5 mL) was added dropwise HCl (1.2 mL, 4 N in p-dioxane) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford an oil, which was triturated in diether ether and solidified to give the final product hydrochloric salt as a white or pale yellow powder. [00132] Example 1.15. Synthesis of SYC-1623 and 1600 [00133] Scheme 5 illustrates synthesis of SYC-1623 and 1600. Reagents and conditions: (i) tert-butyl 4-(aminomethyl)piperidine-1-carboxylate, K₂CO₃, DMF, 100 °C, 12 h; (ii) N- Iodosuccinimide, CH₃CN-DMSO, 24 h, 60% for 2 steps; (iii) 4-((N-Boc-amino)- methyl)phenylboronic acid, Pd(PPh3)4, Na2CO3, p-dioxane-H₂O, 80 ºC, 73%; (iv) 2-(4-(furan-3- yl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, Na₂CO₃, p-dioxane-H₂O, 100 ºC, 78%; (v) HCl (4 N in p-dioxane), CH₂Cl2, 0 ºC, >90%.

Scheme 5

[00134] 2,6-Dibromopyrazine (6, 7.14 g, 30 mmol) or 2,6-dibromopyridine (7, 7.14 g, 30 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (7.07 g, 33 mmol) and potassium carbonate (8.28 g, 60 mmol) in DMF (30 mL) were placed in a sealed tube. The mixture was stirred at 100 °C for 12 h. The reaction was cooled and quenched with saturated brine (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford 8 or 9 as a white solid, which was used in the next step without further purification. For compound 8, ¹H NMR (400 MHz, CDCl₃): d 7.85 (s, 1H), 7.76 (s, 1H), 5.00 (s, 1H), 4.14– 4.11 (m, 2H), 3.25 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 11.8 Hz, 2H), 1.73– 1.70 (m, 3H), 1.44 (s, 9H), and 1.21– 1.09

(m, 2H). For compound

7.23 (t, J = 7.8 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.26 (d, J = 7.8 Hz, 1H), 4.76 (t, J = 5.4 Hz, 1H), 4.10 (br, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.67 (t, J = 12.8 Hz, 2H), 1.77– 1.67 (m, 3H), 1.44 (s, 9H), and 1.19– 1.09 (m, 2H). [00135] To a solution of compound 8 or 9 (～30 mmol) in CH₃CN/DMSO (20/10 mL) was added N-Iodosuccinimide (8.1 g, 36 mmol). After stirring at room temperature for 24 h, the reaction was quenched with sodium thiosulfate aqueous solution (50 mL). The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 10:1 to 3:1) to give 10 or 11 as a white solid (8.95 g, 60% over two steps). For compound 10, ¹H NMR (400 MHz, CDCl₃): d 7.64 (s, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.14– 4.11 (m, 2H), 3.24 (t, J = 5.9 Hz, 2H), 2.69 (t, J = 12.2 Hz, 2H), 1.73– 1.69 (m, 3H), 1.45 (s, 9H), and 1.21– 1.13 (m, 2H). For compound 11,

NMR (400 MHz, CDCl₃): d 7.64 (d, J = 8.5 Hz, 1H), 6.10 (d, J = 8.5 Hz, 1H), 4.74 (t, J = 5.4 Hz, 1H), 4.14– 4.09 (m, 2H), 3.14 (t, J = 5.9 Hz, 2H), 2.69 (t, J = 12.2 Hz, 2H), 1.73– 1.69 (m, 3H), 1.45 (s, 9H), and 1.20– 1.11 (m, 2H). [00136] Compound 10 or 11 (994 mg, 2 mmol), 4-((N-Boc-amino)methyl)phenylboronic acid (527 mg, 2.1 mmol), tetrakis(triphenylphosphine)palladium (69 mg, 3 mol%), and sodium carbonate (424 mg, 4 mmol) in p-dioxane/H₂O (15/3 mL) were placed in a sealed tube. The mixture was degassed and heated to 80 °C for 24 h. The reaction was then cooled and quenched with brine (20 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine, dried over Na₂SO₄. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:1) to give product 12 or 13 as a white solid (842 mg, 73% yield). For Compound 12,

NMR (400 MHz, CDCl₃): d 7.88 (s, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.34 (d, J = 7.8 Hz, 2H), 4.94 (t, J = 5.9 Hz, 1H), 4.88 (br, 1H), 4.37 (d, J = 5.9 Hz, 2H), 4.16– 4.11 (m, 2H), 3.31 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 12.2 Hz, 2H), 1.77– 1.74 (m, 3H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.25– 1.15 (m, 2H). For Compound 13,

NMR (400 MHz, CDCl₃): d 7.38 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.36 (d, J = 8.4 Hz, 1H), 4.88 (br, 2H), 4.36 (br, 2H), 4.16– 4.11 (m, 2H), 3.18 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 12.2 Hz, 2H), 2.10 (br, 1H), 1.77– 1.74 (m, 2H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.22– 1.15 (m, 2H). [00137] Compound 12 or 13 (1 mmol), 2-(4-(furan-3-yl)phenyl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (1.1 mmol), tetrakis(triphenylphosphine)palladium (57.7 mg, 5 mol%), and sodium carbonate (216 mg, 2 mmol) in p-dioxane/H₂O (8/2 mL) were placed in a sealed tube. The mixture was degassed and heated to 100 °C for 24 h. The reaction was then cooled and quenched with brine (10 mL). The mixture was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 1:2) to afford 14 or 15 as a white solid (78% yield). For Compound 14,

NMR (400 MHz, CDCl₃): d 7.90 (s, 1H), 7.73 (s, 1H), 7.46 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 6.69 (s, 1H), 4.89 (t, J = 5.9 Hz, 1H), 4.85 (br, 1H), 4.28 (d, J = 5.9 Hz, 2H), 4.16– 4.11 (m, 2H), 3.36 (t, J = 6.1 Hz, 2H), 2.71 (t, J = 11.8 Hz, 2H), 1.79– 1.76 (m, 3H), 1.45 (s, 9H), 1.44 (s, 9H), and 1.26– 1.16 (m, 2H). For Compound 15,

NMR (400 MHz, CDCl₃): d 7.70 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.45 (br, 1H), 7.34 (br, 4H), 7.13 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.67 (s, 1H), 6.42 (d, J = 8.4 Hz, 1H), 4.83 (t, J = 5.5 Hz, 2H), 4.28 (d, J = 5.3 Hz, 2H), 4.14– 4.09 (m, 2H), 3.25 (t, J = 5.8 Hz, 2H), 2.71 (t, J = 13.4 Hz, 2H), 1.81– 1.78 (m, 3H), 1.46 (s, 9H), 1.45 (s, 9H), and 1.27– 1.20 (m, 2H). [00138] Compound 14 or 15 (0.2 mmol) was dissolved in dichloromethane (2 mL). HCl (0.2 mL, 4 N in p-dioxane) was slowly added to the reaction mixture at 0 °C. After 0.5 h, the reaction was warmed to room temperature and stirred for 12 h. The volatiles were removed in vacuo to afford an oil, which was triturated in diether ether and solidified to give SYC-1600 or SYC-1623 hydrochloric salt as a white or pale yellow powder (>90% yield). [00139] Example 1.16. Synthesis of Compound SYC-1012 [00140] Scheme 6 illustrates synthesis of compound SYC-1012. Reagents and conditions: (i) NaOH, MeOH, reflux; (ii) N-Boc-4-piperidinemethanol, PPh3, DIAD, THF; (iii) HCl (in p- dioxane), CH₂Cl₂, 0 ºC, >90%.

Scheme 6

[00141] Sodium hydroxide solution (12.5 M, 3.2 mL, 40 mmol) was added over 30 min to a refluxing mixture of glycine amide hydrogen chloride (2.21 g, 20 mmol), 4,4’-dibromobenzil (16, 7.36 g, 20 mmol) and 50 mL methanol. After refluxing for another 30 min, the mixture was treated with HCl (12 N, 2.5 mL), followed by KHCO3 (2 g). The following yellow solid formed was filtered off, washed well with water and recrystallized from ^tBuOH. Yellow needles of 5,6- bis(4-bromophenyl)pyrazin-2-ol (17, 5.94 g) was obtained after filtration. ¹H NMR (400 MHz, CDCl₃): d 8.12 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), and 7.15 (d, J = 8.4 Hz, 2H). [00142] To a solution of 2-hydroxypyrazine 17 (1.12 g, 2.76 mmol) and N-Boc-4- piperidinemethanol (594 mg, 2.76 mmol) in anhydrous THF (27 mL) was added triphenylphosphine (1.16 g, 4.42 mmol) and DIAD (894 mg, 4.42 mmol). The reaction mixture was stirred at room temp. for 17 h. The solution was then concentrated and purified to give the product tert-butyl 4-(((5,6-bis(4-bromophenyl)pyrazin-2-yl)oxy)methyl)piperidine-1-carboxylate

8.22 (s, 1H), 7.47-7.41 (m, 4H), 7.30-7.24 (m, 4H), 4.26 (d, J = 6.8 Hz, 2H), 4.15 (br, 2H), 2.75 (t, J = 12.8 Hz, 2H), 2.03 (br, 1H), 1.81 (d, J = 13.2 Hz, 2H), 1.46 (s, 9H), and 1.35– 1.25 (m, 2H). [00143] To a solution of 18 (1.5 g, 2.49 mmol) in CH₂Cl2 (25 mL) was added dropwise hydrogen chloride (4 N in p-dioxane, 12.5 mL) at 0 °C. The solution was stirred overnight and precipitated solid was filtered off to give 1.1 g compound SYC-1012 as pale yellow powder. [00144] Example 1.17. Compound Characterization of the Final Compounds [00145] SYC-1012. 2,3-bis(4-Bromophenyl)-5-(piperidin-4-ylmethoxy)pyrazine hydrochloride:

s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 4.26 (d, J = 5.6 Hz, 2H), 3.26 (d, J = 12.0 Hz, 2H), 2.88 (t, J = 12.4 Hz, 2H), 2.11 (br, 1H), 1.90 (d, J = 12.8 Hz, 2H), and 1.57-1.47 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): d 158.2, 147.1, 143.4, 137.8, 137.4, 133.3, 132.1, 131.9, 131.79, 131.70, 122.9, 122.0, 70.1, 43.1, 33.3, and 25.5. MS (ESI) [M+H]⁺ 504.2. [00147] SYC-1110. 2,3-bis(4-(tert-butyl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazine hydrochloride:

IC503.14 uM

[00148] ¹H NMR (400 MHz, DMSO-d₆) d 9.00– 8.27 (br, 4H), 8.30 (s, 1H), 7.38– 7.26 (m, 8H), 4.27 (d, J = 6.2 Hz, 2H), 3.33– 3.24 (m, 2H), 2.90 (t, J = 11.8 Hz, 2H), 2.13 (br, 1H), 1.93 (d, J = 13.0 Hz, 2H), 1.58– 1.48 (m, 2H), and 1.26 (s, 18H); ¹³C NMR (100 MHz, DMSO-d6) 157.4, 151.2, 150.3, 147.4, 144.0, 135.7, 135.4, 131.9, 129.2, 129.0, 125.0, 124.9, 69.4, 42.7, 34.4, 34.3, 33.0, 31.1, 31.0, and 25.2; HRMS (ESI⁺) calcd for C30H40N3O [M+H]⁺ 458.3166, found 458.3179. [00149] SYC-1598. (4-(3-(1H-indol-5-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

IC₅₀1.12 uM

[00150] ¹ NMR (400 MHz, D2O) d 8.27 (s, 1H), 7.71 (s, 1H), 7.47– 7.35 (m, 6H), 7.32 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 4.36 (s, 2H), 4.17 (s, 2H), 3.51 (d, J = 11.5 Hz, 2H), 3.06 (t, J = 13.1 Hz, 2H), 2.22 (s, 1H), 2.10 (d, J = 13.5 Hz, 2H), and 1.65 (dd, J = 24.7, 12.0 Hz, 2H) ; ¹³C NMR (100 MHz, D₂O) d 158.6, 151.1, 143.4, 139.3, 136.1, 132.8, 130.6, 130.3, 129.1, 128.8, 127.6, 126.6, 123.6, 122.5, 111.6, 70.2, 43.7, 42.7, 33.2, and 25.3; MS (ESI) [M+H]⁺ 414.2. [00151] SYC-1622.4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)benzamide hydrochloride:

[00152] ¹H NMR (400 MHz, DMSO-d6) d 8.77 (br, 2H), 8.40 (s, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.99 (s, 1H), 7.81 (d, J = 7.7 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J = 7.7 Hz, 2H), 7.43– 7.40 (m, 4H), 6.98 (s, 1H), 4.33 (d, J = 5.6 Hz, 2H), 3.09– 3.05 (m, 2H), 2.92 (br, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), and 1.56– 1.47 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) 167.6, 158.0, 147.8, 144.6, 143.4, 141.4, 140.1, 136.3, 133.4, 132.5, 130.2, 129.4, 127.53, 127.46, 125.4, 125.3, 108.7, 69.7, 42.7, 33.0, and 25.3; MS (ESI) [M+H]⁺ 455.2. [00153] SYC-1617. (4-(3-(4-(1H-Pyrazol-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methan-amine hydrochloride:

[00154] ¹H NMR (400 MHz, D2O) d 8.18 (s, 2H), 8.15 (s, 1H), 7.41 (s, 2H), 7.34– 7.27 (m, 6H), 4.20 (s, 2H), 4.14 (s, 2H), 3.49 (d, J = 12.9 Hz, 2H), 3.02 (t, J = 13.2 Hz, 2H), 2.12 (br, 1H), 2.03 (d, J = 15.3 Hz, 2H), and 1.64– 1.54 (m, 2H); ¹³C NMR (100 MHz, D₂O) 158.5, 148.8, 143.2, 138.1, 135.9, 132.7, 131.11, 131.04, 131.02, 131.00, 130.95, 130.4, 130.1, 128.8, 125.1, 70.1, 43.5, 42.6, 32.9, and 25.0; MS (ESI) [M+H]⁺ 441.2. [00155] SYC-1623. 5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperidin-4- ylmethyl)pyridin-2-amine hydrochloride:

[00156] ¹H NMR (400 MHz, DMSO-d6) d 9.05 (br, 1H), 8.83 (br, 2H), 8.44 (br, 3H), 8.28 (s, 1H), 7.91 (br, 2H), 7.77 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 7.01 (s, 1H), 3.97 (d, J = 6.3 Hz, 2H), 3.38 (br, 2H), 3.28 (d, J = 13.5 Hz, 2H), 2.85 (q, J = 12.3 Hz, 2H), 1.95– 1.91 (m, 3H), and 1.48– 1.40 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) 153.9, 144.6, 140.4, 136.6, 133.2, 130.4, 129.5, 129.0, 125.3, 124.9, 123.1, 108.5, 46.3, 42.5, 41.6, 33.1, and 25.9; MS (ESI) [M+H]⁺ 439.2. [00157] SYC-1293. 1-(4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)-N-methylmethanamine hydrochloride:

4 (s, 1H), 7.28 (d, J = 7.9 Hz, 2H), 7.24– 7.16 (m, 4H), 7.12 (d, J = 7.8 Hz, 2H), 6.67 (s, 1H), 4.1– 4.00 (m, 4H), 3.43 (d, J = 6.1 Hz, 2H), 2.97 (t, J = 12.6 Hz, 2H), 2.64 (s, 3H), 2.06 (s, 2H), 1.95 (d, J = 13.2 Hz, 2H), and 1.59– 1.48 (m, 2H) ; ¹³C NMR (100 MHz, D2O) d 158.5, 148.8, 144.7, 143.2, 139.5, 138.9, 135.6, 132.6, 131.3, 130.8, 130.4, 130.2, 129.9, 125.1, 116.0, 108.3, 70.1, 52.3, 43.7, 33.07, 32.11, and 25.0; MS (ESI) [M+H]⁺ 455.2. [00159] SYC-1600. 5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)-N-(piperidin-4- ylmethyl)pyrazin-2-amine hydrochloride:

IC500.40 uM [00160] ¹H NMR (400 MHz, DMSO-d6): d 9.00 (br, 2H), 8.67 (br, 2H), 8.38 (br, 4H), 8.21 (s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 7.54 (d, J = 7.3 Hz, 2H), 7.37– 7.30 (m, 6H), 6.96 (s, 1H), 3.98 (br, 2H), 3.28– 3.24 (m, 4H), 2.83 (q, J = 10.8 Hz, 2H), 1.89– 1.85 (m, 3H), and 1.46– 1.38 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6): 153.0, 148.1, 144.5, 143.8, 139.8, 139.6, 137.9, 137.6, 132.5, 131.7, 130.0, 129.3, 128.5, 125.3, 125.1, 108.6, 55.0, 42.9, 41.9, 33.5, and 26.4; MS (ESI) [M+H]⁺ 440.2. [00161] SYC-1307. (4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

[00162] ¹H NMR (400 MHz, D2O) d 8.24 (s, 1H), 7.94 (s, 1H), 7.61 (s, 1H), 7.51 (dd, J = 8.4, 1.8 Hz, 2H), 7.41 (br, 6H), 6.85 (s, 1H), 4.35 (br, 2H), 4.17 (s, 2H), 3.50 (d, J = 13.3 Hz, 2H), 3.06 (t, J = 13.3 Hz, 2H), 2.124 (br, 1H), 2.11 (d, J = 14.6 Hz, 2H), and 1.68– 1.58 (m, 2H); ¹³C NMR (100 MHz, D2O) 158.4, 148.8, 144.5, 143.3, 139.4 (2), 138.3, 135.6, 132.6, 132.4, 131.1, 130.3, 130.0, 128.7, 125.0, 108.1, 70.0, 43.5, 42.6, 32.9, and 24.9; HRMS (ESI⁺) calcd for C₂₇H₂₉N₄O₂ [M+H]⁺ 441.2291, found 441.2285. [00163] SYC-1686.2-Methoxy-4-(5-(piperidin-4-ylmethoxy)-3-(4-(thiophen-3- yl)phenyl)pyrazin-2-yl)benzamide hydrochloride:

1H), 8.39 (s, 1H), 7.96 (br, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 8.0 Hz, 1H), 7.65-7.59 (m, 2H), 7.55 (br, 1H), 7.47 (d, J = 7.9 Hz, 2H), 7.11 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 4.33 (d, J = 6.2 Hz, 2H), 3.68 (s, 3H), 3.31 (d, J = 12.5 Hz, 2H), 2.96-2.88 (m, 2H), 2.16 (br, 1H), 1.94 (d, J = 13.6 Hz, 2H), and 1.58- 1.49 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) 165.9, 157.9, 147.9, 143.2, 142.4, 140.6, 136.5, 135.4, 132.4, 132.1, 131.5, 130.7, 130.2, 128.8, 127.4, 126.2, 125.9, 121.9, 113.2, 69.6, 66.4, 42.8, 32.9, and 26.3; MS (ESI) [M+H]⁺ 501.2. [00165] SYC-1688. 2-Fluoro-4-(3-(4-(furan-3-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin- 2-yl)benzamide hydrochloride:

1H), 8.39 (s, 1H), 7.77-7.75 (m, 1H), 7.74 (br, 1H), 7.66 (br, 1H), 7.63 (d, J = 8.3 Hz, 2H), 7.59 (t, J = 7.8 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.26-7.19 (m, 2H), 7.00 (s, 1H), 4.33 (d, J = 6.3 Hz, 2H), 3.33-3.30 (m, 2H), 2.96- 2.87 (m, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), and 1.56-1.48 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) 164.8, 159.0 (¹J _F-C = 249.3 Hz), 158.0, 147.9, 144.6, 142.9 (³J _F-C = 8.5 Hz), 142.0, 140.1, 135.9, 132.6 (³J F-C = 8.5 Hz), 131.5 (²J F-C = 10.0 Hz), 130.2, 128.9, 128.7, 125.4, 125.3 (²J F-C = 19.3 Hz), 108.6, 70.0, 43.2, 35.3, and 25.6; MS (ESI) [M+H]⁺ 473.2. [00167] SYC-1689. 2-Fluoro-4-(5-(piperidin-4-ylmethoxy)-3-(4-(thiophen-3- yl)phenyl)pyrazin-2-yl)benzamide hydrochloride:

[00168] ¹H NMR (400 MHz, DMSO-d₆) d 8.91 (br, 1H), 8.61 (br, 1H), 8.46 (s, 1H), 8.39 (s, 1H), 8.00 (br, 1H), 7.67-7.64 (m, 2H), 7.61 (br, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.26-7.21 (m, 3H), 4.33 (d, J = 6.3 Hz, 2H), 3.31 (d, J = 13.6 Hz, 2H), 2.96-2.87 (m, 2H), 2.16 (br, 1H), 1.94 (d, J = 14.6 Hz, 2H), and 1.59-1.50 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) 165.2, 158.4, 158.1, 148.3, 143.2, 142.4, 140.9, 136.4, 135.9, 133.0, 132.6, 131.9, 130.6, 127.9, 126.6, 126.3, 125.8, 122.4, 117.3, 70.1, 43.1, 33.3, and 25.6; MS (ESI) [M+H]⁺ 489.2. [00169] SYC-1169. (4-(3-(4-(Tert-butyl)-2-methoxyphenyl)-6-(piperidin-4- ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride:

[00170] ¹H NMR (400 MHz, D2O) d 8.24 (s, 1H), 7.36 (dd, J = 17.2, 8.2 Hz, 4H), 7.26 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 4.34 (d, J = 5.2 Hz, 2H), 4.14 (s, 2H), 3.51 (d, J = 12.6 Hz, 2H), 3.28 (s, 3H), 3.06 (t, J = 12.0 Hz, 2H), 2.24 (s, 1H), 2.11 (d, J = 14.4 Hz, 2H), 1.63 (dd, J = 24.0, 11.8 Hz, 2H), 1.28 (s, 9H); ¹³C NMR (100 MHz, D2O) d 158.7, 155.4, 155.2, 149.6, 141.7, 138.6, 133.1, 131.8, 130.8, 129.1, 128.5, 123.7, 118.3, 109.4, 70.1, 55.0, 43.6, 42.6, 34.5, 32.9, 30.4, 25.0; MS (ESI) [M+H]⁺ 461.3. [00171] SYC-1211:

[00172] ¹H NMR (400 MHz, DMSO-d6) d 8.74 (s, 4H), 8.45 (d, J = 7.6 Hz, 1H), 7.73-7.63 (m, 4H), 4.51 (d, J = 4.4 Hz, 2H), 4.10 (d, J = 7.2 Hz, 4H), 3.28 (d, J = 11.2 Hz, 2H), 2.89 (d, J = 10.4 Hz, 2H), 2.15 (s, 1H), 1.90 (d, J = 13.2 Hz, 2H), 1.61 (d, J = 13.2 Hz, 2H); ¹³C NMR (400 MHz, DMSO-d6) d 165.6, 161.4, 156.7, 137.1, 136.5, 134.1, 132.7, 129.4, 129.3, 129.04, 128.0, 119.2, 69.8, 42.6, 41.9, 33.0, 28.3, 28.2, 25.2; MS (ESI) [M+H]⁺ 404.2. [00173] SYC-1212. 4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenol hydrochloride:

IC₅₀0.31 or 0.76 uM [00174] ¹H NMR (400 MHz, DMSO-d6) d 9.79 (s, 1H), 8.25-9.82 (m, 5H), 7.34-7.40 (m, 4H), 7.20 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.27 (d, J = 6.4 Hz, 2H), 4.00 (d, J = 5.2 Hz, 2H), 3.27 (br, 2H), 2.89 (d, J = 11.2 Hz, 2H), 2.11 (br, 1H), 1.91 (d, J = 13.2 Hz, 2H), 1.48-1.51 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.5, 158.0, 148.4, 143.5, 139.5, 133.8, 131.8, 131.4, 129.8, 129.0, 128.8, 115.5, 69.8, 43.2, 42.3, 33.4, 25.6; MS (ESI) [M+H]⁺ 391.5. [00175] SYC-1764. (4-(3-(1H-benzo[d]imidazol-6-yl)-5-(piperidin-3-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

[00176] ¹H NMR (400 MHz, DMSO-d6) d 9.61 (s, 1H), 9.18-9.28 (m, 2H), 8.49 (br, 2H), 8.45 (s, 1H), 7.95 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.28-4.38 (m, 2H), 3.97 (d, J = 4.8 Hz, 2H), 3.21 (d, J = 12.8 Hz, 1H), 2.77 (dd, J = 10.4 Hz, 21.2 Hz, 2H), 2.39 (br, 1H), 1.81 (dd, J = 13.2 Hz, 30 Hz, 2H), 1.35-1.38 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.0, 147.3, 144.4, 141.8, 138.6, 136.5, 134.3, 133.6, 131.0, 131.0, 130.0, 129.2, 128.1, 126.5, 116.1, 114.9, 114.6, 68.3, 65.4, 45.7, 42.2, 31.7, 25.2, 15.6; MS (ESI) [M+H]⁺ 415.3. [00177] SYC-1783. (4-(5-(Piperidin-4-ylmethoxy)-3-(4-(tetrahydrofuran-3- yl)phenyl)pyrazin-2-yl)phenyl)methanamine hydrochloride:

IC500.59 uM [00178] ¹H NMR (400 MHz, DMSO-d6) d 9.16 (br, 1H), 8.86 (br, 1H), 8.48 (br, 3H), 8.31 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.32-7.40 (m, 4H), 7.21 (d, J = 8.4 Hz, 2H), 4.26 (d, J = 5.6 Hz, 2H), 3.99 (d, J = 7.6 Hz, 3H), 3.88-3.93 (m, 1H), 3.76 (dd, J = 8 Hz, 15.6 Hz, 1H), 3.35 (t, J = 8.4 Hz, 1H), 3.25 (d, J = 13.2 Hz, 2H), 2.87 (d, J = 11.2 Hz, 2H), 2.26-2.29 (m, 1H), 2.12 (br, 1H), 1.88-1.92 (m, 3H), 1.52-1.56 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 158.1, 148.0, 143.9, 143.8, 139.0, 136.5, 134.0, 132.7, 130.1, 129.9, 129.1, 127.6, 74.1, 69.9, 68.1, 55.4, 44.3, 43.0, 34.2, 33.4, 25.6; MS (ESI) [M+H]⁺ 445.2. [00179] SYC-1782. (4-(5-(Piperidin-4-ylmethoxy)-3-(6-(tetrahydro-2H-pyran-4-yl)pyridin- 3-yl)pyrazin-2-yl)phenyl)methanamine hydrochloride:

5 (m, 1H), 8.60 (s, 1H), 8.49 (br, 3H), 8.43 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 8 Hz, 1H), 7.46 (d, J = 8 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 4.28 (d, J = 6.0 Hz, 2H), 4.00 (d, J = 5.2 Hz, 2H), 3.93 (d, J = 10.8, 2H), 3.32-3.62 (m, 4H), 3.26 (d, J = 11.6 Hz, 2H), 3.14 (br, 1H), 2.88 (d, J = 12 Hz, 2H), 2.14 (br, 1H), 1.90 (d, J = 12.8 Hz, 2H), 1.59-1.74 (m, 4H), 1.51-1.59 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 158.3, 146.2, 144.6, 144.0, 138.0, 134.6, 134.2, 133.6, 130.1, 129.4, 129.3, 129.2, 122.8, 70.3, 67.2, 65.4, 43.0, 42.2, 41.1, 34.6, 33. 3, 31.8, 31.7, 25.5, 15.6; MS (ESI) [M+H]⁺ 460.3. [00181] SYC-1749. (4-(5-(Piperidin-4-ylmethoxy)-3-(4-(tetrahydro-2H-pyran-4- yl)phenyl)pyrazin-2-yl)phenyl)methanamine hydrochloride:

2 (m, 1H), 8.40 (br, 3H), 8.32 (s, 1H), 7.32-7.41 (m, 6H), 7.20 (d, J = 8.0 Hz, 2H), 4.26 (d, J = 6.0 Hz, 2H), 3.99 (d, J = 5.2 Hz, 2H), 3.91 (d, J = 10.8 Hz, 2H), 2.88 (dd, J = 11.2 Hz, 22.8 Hz, 2H), 2.73-2.76 (m, 1H), 2.11 (br, 1H), 1.90 (d, J = 13.2 Hz, 2H), 1.64 (br, 4H), 1.51 (d, J = 12.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.1, 148.1, 147.0, 143.8, 139.1, 136.3, 134.0, 132.6, 130.1, 129.9, 129.0, 127.0, 69.9, 67.7, 43.0, 42.21, 40.54, 33.75, 33.34, 25.60; MS (ESI) [M+H]⁺ 459.3. [00183] SYC-1216. 2,2'-((5-(Piperidin-4-ylmethoxy)pyrazine-2,3-diyl)bis(4,1- phenylene))bis(propan-2-amine) hydrochloride:

IC500.47 uM [00184] ¹H NMR (400 MHz, DMSO-d₆) d 9.01 (br, 1H), 8.75 (br, 3H), 8.69 (br, 3H), 8.36 (s, 1H), 7.50-7.54 (m, 4H), 7.47 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8 Hz, 2H), 4.27 (d, J = 6.4 Hz, 2H), 2.83-2.97 (m, 2H), 2.17 (br, 1H), 1.89-1.94 (m, 2H), 1.61 (s, 12H), 1.46-1.58 (m, 2H); MS (ESI) [M+H]⁺ 460.3. [00185] SYC-1758. 2-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)-N-methylethan-1-amine hydrochloride:

[00186] ¹H NMR (400 MHz, DMSO-d₆) d 8.32 (br, 1H), 8.20 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 7.2 Hz, 2H), 7.38-7.40 (m, 3H), 7.26-7.28 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.96 (s, 1H), 4.53 (t, J = 5.2 Hz, 2H), 4.10 (d, J = 5.2 Hz, 2H), 2.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d6) d 157.8, 156.3, 144.9, 144.4, 140.4, 140.3, 137.4, 137.0, 132.8, 132.6, 130.4, 129.7, 127.0, 125.7, 125.6, 109.0, 78.9, 78.3, 63.8, 43.5; MS (ESI) [M+H]+ 401.2. [00187] SYC-1759. (4-(3-(1H-benzo[d]imidazol-4-yl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

IC501.56 uM [00188] ¹H NMR (400 MHz, DMSO-d6) d 9.43 (s, 1H), 9.06 (br, 1H), 8.78 (br, 1H), 8.50 (s, 1H), 8.39 (br, 3H), 7.83 (d, J = 7.6 Hz, 2H), 7.38-7.40 (m, 2H), 7.23 (d, J = 7.2 Hz, 2H), 4.25 (d, J = 6.4 Hz, 2H), 3.93 (d, J = 4.8 Hz, 2H), 2.87 (d, J = 11.6 Hz, 4H), 2.11 (br, 1H), 1.89 (d, J = 12.4 Hz, 2H), 1.51 (d, J = 11.2 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 170.8, 158.2, 144.9, 144.2, 142.0, 137.9, 134.6, 134.3, 129.8, 129.0, 127.0, 126.5, 125.8, 115.6, 114.9, 70.3, 65.4, 60.2, 34.6, 21.2; MS (ESI) [M+H]⁺ 415.2. [00189] SYC-1755. (4-(5-(Benzyloxy)-3-(4-(furan-3-yl)phenyl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

[00190] ¹H NMR (400 MHz, DMSO-d₆) d = 8.40-8.42 (m, 3H), 8.23 (s, 1H), 7.74 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.52-7.57 (m, 3H), 7.38-7.41 (m, 8H), 7.34-7.36 (m, 2H), 6.70 (s, 1H), 5.48 (s, 2H), 3.99 (d, J = 5.6 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 157.9, 147.9, 145.0, 144.1, 140.4, 139.1, 137.0, 136.8, 134.0, 133.0, 132.7, 130.5, 130.0, 129.1, 128.9, 128.8, 128.5, 125.7, 125.6, 109.0, 68.1, 42.3; MS (ESI) [M+H]⁺ 434.2. [00191] SYC-1201. 1-(4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)ethan-1-one hydrochloride:

1H), 8.41 (br, 4H), 7.89 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.29 (d, J = 6 Hz, 2H), 3.99 (d, J = 5.2 Hz, 2H), 3.27 (d, J = 12.0 Hz, 2H), 2.88 (d, J = 10.4 Hz, 2H), 2.57 (s, 3H), 2.15 (br, 1H), 1.91 (d, J = 11.6 Hz, 2H), 1.53 (d, J = 12 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 198.0, 158.2, 147.2, 144.4, 143.0, 138.5, 136.9, 134.3, 133.6, 130.4, 130.0, 129.2, 128.6, 70.1, 55.4, 43.0, 42.2, 33.4, 25.6; MS (ESI) [M+H]⁺ 417.3. [00193] SYC-1752. (4-(5-(Cyclohexylmethoxy)-3-(4-(furan-3-yl)phenyl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

[00194] ¹H NMR (400 MHz, DMSO-d₆) d 8.45 (br, 3H), 8.33 (s, 1H), 8.21 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.38-7.42 (m, 6H), 6.96 (s, 1H), 4.20 (d, J = 5.2 Hz, 2H), 3.99 (d, J = 4.8 Hz, 2H), 1.80 (d, J = 10.8 Hz, 2H), 1.70 (d, J = 11.2 Hz, 2H), 1.64 (d, J = 10.8 Hz, 1H), 1.21 (dd, J = 12.8 Hz, 25.6 Hz, 4H), 1.04-1.07 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 158.4, 147.9, 144.9, 143.7, 140.4, 139.1, 137.0, 134.0, 132.8, 132.6, 130.5, 130.0, 129.1, 125.7, 125.6, 109.0, 71.6, 42.2, 37.3, 29.7, 26.4, 25.7; MS (ESI) [M+H]⁺ 440.2. [00195] SYC-1756. 3-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)propan-1-amine hydrochloride:

s, 1H), 8.22 (s, 1H), 8.09 (br, 3H), 7.74 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.37-7.42 (m, 6H), 6.97 (s, 1H), 4.48 (t, J = 6.0 Hz, 2H), 3.99 (d, J = 5.6 Hz, 2H), 2.96 (d, J = 6.0 Hz, 2H), 2.09-2.13 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 158.0, 147.9, 145.0, 144.0, 140.4, 139.0, 136.8, 134.1, 132.9, 132.7, 130.5, 129.9, 129.1, 125.7, 125.6, 109.0, 64.0, 42.2, 36.6, 34.6; MS (ESI) [M+H]⁺ 401.2. [00197] SYC-1757. (4-(3-(4-(Furan-3-yl)phenyl)-5-(piperidin-4-yloxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

1H), 7.73 (s, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.38-7.40 (m, 4H), 7.29 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 6.96 (s, 1H), 5.27 (br, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.65 (br, 2H), 3.23 (br, 2H), 2.00 (br, 2H), 1.64-1.66 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.2, 156.3, 154.4, 147.6, 144.9, 144.3, 140.3, 137.4, 137.0, 133.1, 132.6, 130.4, 129.7, 127.0, 125.7, 109.0, 79.2, 78.3, 71.6, 43.5; MS (ESI) [M+H]⁺ 427.2. [00199] SYC-1894. 5-(4-(Piperidin-1-ylmethyl)phenyl)-N-(piperidin-4-yl)pyrazine-2- carboxamide hydrochloride:

IC₅₀0.3 uM [00200] ¹H NMR (400 MHz, DMSO-d6) d 10.89 (s, 1H), 9.33 (d, J = 1.2 Hz, 1H), 9.23 (d, J = 1.2 Hz, 1H), 9.09– 8.96 (m, 2H), 8.80– 8.68 (m, 1H), 8.30 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 4.33 (d, J = 5.2 Hz, 2H), 4.19– 4.05 (m, 1H), 3.35– 3.25 (m, 4H), 3.08– 2.95 (m, 2H), 2.89– 2.78 (m, 2H), 1.91– 1.69 (m, 8H), 1.39– 1.25 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 162.5, 152.8, 143.5, 143.2, 140.4, 136.0, 132.3, 132.2, 127.5, 58.3, 51.6, 44.4, 42.2, 28.0, 22.1, 21.4; MS (ESI) [M+H]⁺ 380.2. [00201] SYC-1198. (4-(3-(4-Isopropylphenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

H), 7.44 (d, J = 7.6 Hz, 2H), 7.41– 7.29 (m, 4H), 7.21 (d, J = 7.2 Hz, 2H), 4.29 (d, J = 6.0 Hz, 2H), 4.01 (s, 2H), 3.28 (d, J = 12.4 Hz, 2H), 2.90 (t, J = 10.8 Hz, 3H), 2.14 (s, 1H), 1.93 (d, J = 13.2 Hz, 2H), 1.55 (dd, J = 24.2, 11.8 Hz, 2H), 1.20 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d6) d 157.6, 149.0, 147.8, 143.4, 138.6, 135.6, 133.6, 132.1, 129.5, 129.4, 128.6, 126.2, 69.5, 42.6, 41.8, 33.1, 33.0, 25.2, 23.7; MS (ESI) [M+H]⁺ 417.3. [00203] SYC-1754. 4-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)butan-1-amine hydrochloride:

2H), 7.73 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.52– 7.34 (m, 5H), 4.45 (d, J = 7.6 Hz, 2H), 4.09 (s, 2H), 4.01 (s, 2H), 2.85 (s, 2H), 1.80 (d, J = 38.7 Hz, 4H); MS (ESI) [M+H]⁺ 415.2. [00205] SYC-1761. 4-(((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)methyl)aniline hydrochloride:

IC501.52 uM [00206] ¹H NMR (400 MHz, DMSO-d₆) d 8.28 (d, J = 11.6 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.76 (s, 1H), 7.61 (d, J = 7.2 Hz, 1H), 7.45– 7.19 (m, 6H), 7.00 (s, 1H), 6.79 (d, J = 8.4 Hz, 1H), 3.97 (s, 2H); MS (ESI) [M+H]⁺ 449.2. [00207] SYC-1767. 4-(2-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)ethyl)aniline hydrochloride:

, 7.88 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.52 - 7.27 (m, 10H), 6.98 (s, 1H), 4.63 (t, J = 6.0 Hz, 2H), 4.02 (d, J = 4.4 Hz, 2H), 3.14 (d, J = 6.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.6, 147.6, 144.6, 143.5, 140.0, 138.7, 138.4, 136.5, 133.6, 132.3, 130.3, 130.1, 129.6, 128.7, 126.4, 125.3, 125.2, 123.2, 115.3, 109.6, 108.6, 66.6, 41.9, 34.0; MS (ESI) [M+H]⁺ 463.2. [00209] SYC-1775. (4-(3-(4-(Furan-3-yl)phenyl)-5-(3-(pyridin-3-yl)propoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

5.2 Hz, 1H), 8.65– 8.45 (m, 4H), 8.31 (d, J = 16.0 Hz, 1H), 8.03– 7.95 (m, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.60– 7.25 (m, 7H), 4.51– 4.42 (m, 2H), 4.01 (d, J = 4.8 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.27– 2.13 (m, 2H); MS (ESI) [M+H]⁺ 463.2. [00211] SYC-1776. (4-(5-(2-(1H-imidazol-1-yl)ethoxy)-3-(4-(furan-3-yl)phenyl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

IC500.44 uM [00212] ¹H NMR (400 MHz, DMSO-d6) d 9.32 (s, 1H), 8.62 (s, 3H), 8.41– 8.33 (m, 1H), 7.90 (d, J = 10.0 Hz, 2H), 7.76– 7.64 (m, 2H), 7.56– 7.28 (m, 6H), 4.82 (s, 2H), 4.72 (s, 2H), 4.01 (s, 2H); MS (ESI) [M+H]⁺ 438.2. [00213] SYC-1787. (4-(5-(3-(Pyridin-3-yl)propoxy)-3-(4-(thiophen-3-yl)phenyl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

IC502.22 uM [00214] ¹H NMR (400 MHz, DMSO-d₆) d 8.87 (s, 1H), 8.74 (s, 1H), 8.58 (s, 1H), 8.48 (d, J = 7.6 Hz, 1H), 8.28 (t, J = 9.2 Hz, 1H), 7.92 (s, 2H), 7.62 (ddd, J = 28.0, 9.6, 4.8 Hz, 3H), 7.49– 7.17 (m, 6H), 4.44 (s, 2H), 3.97 (s, 2H), 3.03– 2.90 (m, 2H), 2.17 (d, J = 5.9 Hz, 2H); MS (ESI) [M+H]⁺ 479.2. [00215] SYC-1770. (4-(3-(4-(Furan-3-yl)phenyl)-5-(2-(piperidin-4-yl)ethoxy)pyrazin-2-

IC₅₀0.72 uM [00216] ¹H NMR (400 MHz, DMSO-d6) d 8.31 (d, J = 9.2 Hz, 1H), 8.19 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.39 (s, 4H), 7.25 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.43 (s, 2H), 4.00 (s, 2H), 3.22 (d, J = 11.2 Hz, 2H), 2.87– 2.75 (m, 2H), 1.87 (d, J = 13.2 Hz, 2H), 1.74 (s, 2H), 1.58 (s, 1H), 1.45– 1.30 (m, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.1, 149.8, 148.0, 144.9, 140.2, 136.8, 132.6, 131.0, 130.5, 130.0, 129.1, 126.8, 125.6, 124.2, 115.7, 108.9, 64.2, 43.4, 42.1, 34.7, 30.5, 28.5; MS (ESI) [M+H]⁺ 455.2. [00217] SYC-1772. Methyl 4-(((5-(4-(aminomethyl)phenyl)-6-(4-(furan-3- yl)phenyl)pyrazin-2-yl)oxy)methyl)benzoate hydrochloride:

IC₅₀3 uM [00218] ¹H NMR (400 MHz, DMSO-d₆) d 8.54– 8.36 (m, 3H), 8.31– 8.20 (m, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 1H), 7.52– 7.32 (m, 6H), 6.99 (s, 1H), 5.60 (s, 2H), 4.01 (s, 2H), 3.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) d 166.0, 157.3, 147.4, 144.5, 142.1, 140.0, 138.5, 136.2, 133.7, 132.3, 130.50, 130.1, 129.6, 129.3, 129.1, 128.7, 128.0, 126.3, 125.2, 115.3, 108.6, 67.0, 52.2, 41.8; MS (ESI) [M+H]⁺ 492.2. [00219] SYC-1773. (4-(3-(4-(Furan-3-yl)phenyl)-5-((1-methylpiperidin-4- yl)methoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride:

IC503.0 uM [00220] ¹H NMR (400 MHz, DMSO-d6) d 8.38 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.61 (s, 1H), 7.48 (dd, J = 18.4, 6.4 Hz, 4H), 7.42– 7.29 (m, 5H), 4.30 (d, J = 6.0 Hz, 2H), 4.00 (d, J = 5.2 Hz, 2H), 2.98 (d, J = 11.2 Hz, 2H), 2.75– 2.62 (m, 3H), 2.10 (s, 1H), 1.96 (d, J = 12.4 Hz, 2H), 1.68 (d, J = 12.4 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.1, 149.9, 147.6, 143.9, 138.7, 137.5, 134.2, 130.5, 130.2, 130.0, 129.2, 128.4, 127.4, 126.8, 126.8, 112.7, 69.9, 53.2, 43.0, 42.2, 33.0, 26.2; MS (ESI) [M+H]⁺ 455.2. [00221] SYC-1774. 4-((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)-3-methylbutan-1-amine hydrochloride:

), 7.85 (s, 2H), 7.50-7.30(m, 8H), 4.24 (s, 2H), 4.00 (s, 2H), 2.88 (s, 2H), 2.06 (s, 1H), 1.54 (s, 2H), 1.00 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.9, 149.5, 147.2, 143.6, 140.9, 138.5, 133.8, 132.4, 131.2, 129.8, 129.6, 128.8, 128.4, 128.1, 126.9, 126.4, 70.6, 41.8, 36.9, 30.8, 30.0, 16.6; MS (ESI) [M+H]⁺ 429.2. [00223] SYC-1775. (4-(3-(4-(Furan-3-yl)phenyl)-5-(3-(pyridin-3-yl)propoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

8.0 Hz, 2H), 8.54 (d, J = 8.0 Hz, 2H), 8.33 (s, 1H), 8.03– 7.94 (m, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.55– 7.30 (m, 8H), 4.46 (s, 2H), 4.01 (d, J = 4.8 Hz, 3H), 3.01 (t, J = 7.2 Hz, 2H), 2.20 (s, 2H);¹³C NMR (100 MHz, DMSO- d6) d 157.6, 149.4, 147.1, 145.8, 143.5, 141.5, 141.2, 140.9, 139.5, 139.2, 138.4, 133.8, 132.8, 129.8, 129.6, 128.8, 128.7, 128.0, 126.8, 126.4, 109.5, 59.5, 41.8, 28.9, 28.5; MS (ESI) [M+H]⁺ 463.2. [00225] SYC-1788. 1-(4-(((5-(4-(Aminomethyl)phenyl)-6-(4-(furan-3-yl)phenyl)pyrazin-2- yl)oxy)methyl)piperidin-1-yl)ethan-1-one hydrochloride:

8.4 Hz, 1H), 7.46– 7.25 (m, 6H), 7.66-7.59 (m, 2H), 6.99 (s, 1H), 4.39 (d, J = 12.0 Hz, 2H), 4.27 (s, 2H), 4.01 (s, 2H), 3.09– 2.98 (m, 2H), 2.07 (s, 1H), 1.99 (s, 5H), 1.83– 1.75 (m, 2H); MS (ESI) [M+H]⁺ 483.2. [00227] SYC-1789. (4-(3-(4-(Furan-3-yl)phenyl)-5-(pyridin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

), 8.60 (s, 3H), 8.10 (d, J = 4.1 Hz, 2H), 7.92– 7.79 (m, 2H), 7.45-7.33 (m, 7H), 5.81 (s, 2H), 4.01 (s, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 163.5, 156.7, 156.0, 149.6, 149.1, 147.4, 142.6, 141.0, 138.1, 134.0, 129.8, 129.6, 128.8, 128.0, 127.0, 126.4, 124.2, 123.7, 123.5, 61.4, 41.8; MS (ESI) [M+H]⁺ 435.2. [00229] SYC-1118. 2,3-Di([1,1'-biphenyl]-4-yl)-5-(piperidin-4-ylmethoxy)pyrazine hydrochloride:

4 (m, 8H), 7.57 (d, J = 8.4 Hz, 2H), 7.51– 7.42 (m, 6H), 7.37 (d, J = 5.2 Hz, 2H), 4.36– 4.28 (m, 2H), 3.33– 3.30 (m, 8H), 2.92 (s, 2H), 2.17 (s, 1H), 1.96 (d, J = 13.2 Hz, 2H), 1.54 (dd, J = 25.2, 12.4 Hz, 2H); MS (ESI) [M+H]⁺ 498.2. [00231] SYC-1122. 5-(Piperidin-4-ylmethoxy)-2,3-bis(4-(pyridin-3-yl)phenyl)pyrazine hydrochloride:

IC₅₀0.44 uM [00232] NMR (400 MHz, DMSO-d6) d 9.25 (s, 2H), 8.87– 8.75 (m, 4H), 8.46 (s, 1H), 8.07– 7.96 (m, 2H), 7.94– 7.83 (m, 4H), 7.66 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 4.34 (d, J = 6.0 Hz, 2H), 3.28 (s, 2H), 2.91 (dd, J = 21.2, 9.8 Hz, 2H), 2.17 (s, 1H), 1.95 (d, J = 12.4 Hz, 2H), 1.59 (dd, J = 23.2, 11.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.9, 147.2, 143.4, 142.1, 141.8, 141.6, 141.4, 141.2, 140.9, 139.5, 139.1, 137.4, 137.2, 134.4, 133.5, 133.0, 130.6, 130.4, 127.2, 127.1, 126.8, 126.7, 69.7, 42.6, 33.0, 25.1; MS (ESI) [M+H]⁺ 500.2. [00233] SYC-1586. (4-(5-(Piperidin-4-ylmethoxy)-3-(4-(pyrrolidin-1-yl)phenyl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

H), 8.45 (s, 3H), 8.19 (s, 1H), 7.43 (q, J = 8.4 Hz, 4H), 7.26 (d, J = 8.8 Hz, 2H), 6.45 (d, J = 8.8 Hz, 2H), 4.29 (d, J = 6.4 Hz, 2H), 4.02 (d, J = 6.0 Hz, 2H), 3.29 (d, J = 12.0 Hz, 2H), 3.23 (s, 4H), 2.90 (dd, J = 23.6, 12.4 Hz, 2H), 2.14 (s, 1H), 1.95 (s, 7H), 1.54 (dd, J = 24.2, 10.8 Hz, 2H); ¹³C NMR (100 MHz, DMSO- d6) d 157.5, 148.2, 147.7, 142.6, 139.6, 133.3, 130.7, 130.3, 129.3, 128.6, 123.90, 111.1, 69.2, 47.2, 42.7, 41.8, 33.0, 25.2, 25.0; MS (ESI) [M+H]⁺ 444.3. [00235] SYC-1939. (4-(3-(4-(Aminomethyl)phenyl)-6-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)(piperidin-1-yl)methanone hydrochloride:

, 1H, NH), 8.47 (s, 3H, NH), 8.37 (s, 1H), 7.46– 7.20 (m, 8H), 4.26 (s, 2H), 3.97 (s, 2H), 3.53 (s, 2H), 3.24 (s, 4H), 2.86 (s, 2H), 2.11 (s, 1H), 1.90 (d, J = 13.2 Hz, 2H), 1.60– 1.32 (m, 8H); ¹³C NMR (100 MHz, DMSO- d6) d 168.3, 157.7, 147.2, 143.7, 138.8, 138.3, 136.5, 133.8, 132.8, 129.6, 129.5, 128.7, 126.6, 69.6, 42.6, 41.8, 32.9, 25.1, 24.0; MS (ESI) [M+H]⁺ 486.3. [00237] SYC-2026. 2-(4-(Tert-butyl)phenyl)-3-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-5- (piperidin-4-ylmethoxy)pyrazine hydrochloride:

IC₅₀3.1 uM [00238] ¹H NMR (400 MHz, DMSO-d6) d 9.36 (s, 1H, NH), 9.19 (s, 2H, NH), 8.98 (s, 1H, NH), 8.14 (s, 1H), 7.71 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.07 (s, 1H), 4.46 (s, 1H), 4.30 (d, J = 6.4 Hz, 2H), 3.29 (s, 4H), 3.03– 2.84 (m, 4H), 2.09 (s, 5H), 1.92 (d, J = 13.2 Hz, 2H), 1.58 (dd, J = 24.0, 11.6 Hz, 2H), 1.33 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆) d 157.7, 151.1, 143.2, 141.0, 138.3, 136.1, 130.5, 129.0, 128.7, 125.3, 119.7, 69.3, 55.2, 42.6, 41.9, 34.5, 33.0, 31.2, 28.4, 25.2; MS (ESI) [M+H]⁺ 475.3. [00239] SYC-1123. 5-(Piperidin-4-ylmethoxy)-2,3-bis(4-(pyridin-4-yl)phenyl)pyrazine hydrochloride:

[00240] ¹H NMR (400 MHz, DMSO-d₆) d 9.06 (s, 1H), 8.72 (s, 1H), 8.36 (s, 1H), 7.53 (s, 1H), 7.31 (s, 15H), 4.29 (s, 2H), 3.27 (s, 2H), 2.89 (s, 2H), 2.13 (s, 1H), 1.91 (s, 2H), 1.53 (s, 2H); MS (ESI) [M+H]⁺ 500.2. [00241] SYC-1119. 2,3-Bis(4-(furan-2-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazine hydrochloride:

H), 8.35 (s, 1H), 7.61 (s, 1H), 7.29 (s, 13H), 4.28 (s, 2H), 3.28 (s, 2H), 2.89 (s, 2H), 2.14 (s, 1H), 1.92 (s, 2H), 1.53 (s, 3H); MS (ESI) [M+H]⁺ 478.2. [00243] SYC-1700. (4-(3-(4-(Tert-butyl)-2-methoxyphenyl)-5-(piperidin-4- ylmethoxy)pyrazin-2-yl)phenyl)methanamine hydrochloride:

1H), 8.39 (s, 2H), 8.33 (s, 1H), 7.31 (s, 5H), 7.06 (s, 1H), 6.88 (s, 1H), 4.21 (s, 2H), 3.97 (s, 2H), 3.56 (s, 3H), 3.30 (s, 2H), 2.88 (s, 2H), 2.09 (s, 1H), 1.89 (s, 2H), 1.50 (s, 2H), 1.30 (s, 9H); MS (ESI) [M+H]⁺ 460.3. [00245] SYC-1220. (4-(6-(Piperidin-4-ylmethoxy)-3-(pyridin-4-yl)pyrazin-2- yl)phenyl)methanamine hydrochloride:

0 (s, 1H), 7.81 (d, J = 6.0 Hz, 2H), 7.51 (dd, J = 21.2, 8.0 Hz, 4H), 4.42 (d, J = 6.0 Hz, 2H), 4.24 (s, 2H), 3.53 (d, J = 12.4 Hz, 2H), 3.09 (t, J = 12.4 Hz, 2H), 2.29 (s, 1H), 2.14 (d, J = 13.2 Hz, 2H), 1.66 (dd, J = 24.0, 11.6 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d6) d 158.3, 152.7, 149.3, 142.3, 138.7, 136.1, 135.1, 133.3, 131.7, 129.6, 128.8, 126.2, 69.6, 42.2, 41.4, 32.4, 24.6; MS (ESI) [M+H]⁺ 476.2. [00247] SYC-1166. (4-(3-(Furan-3-yl)-6-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

IC₅₀1.09 uM [00248] NMR (400 MHz, D2O) d 7.82 (s, 1H), 7.21– 7.04 (m, 5H), 7.01 (s, 1H), 6.93 (s, 1H), 3.87 (s, 2H), 3.82 (s, 2H), 3.06 (d, J = 11.6 Hz, 2H), 2.62 (t, J = 12.4 Hz, 2H), 1.79 (s, 1H), 1.66 (d, J = 13.2 Hz, 2H), 1.27– 1.12 (m, 2H); MS (ESI) [M+H]⁺ 465.2. [00249] SYC-1821. (4-(3-(4-(Piperidin-4-yl)phenyl)-5-(piperidin-4-ylmethoxy)pyrazin-2- yl)phenyl)methanamine hydrochloride:

25 (d, J = 7.6 Hz, 2H), 4.36 (d, J = 5.2 Hz, 2H), 4.18 (s, 2H), 3.51 (t, J = 12.0 Hz, 4H), 3.18– 3.01 (m, 4H), 2.97– 2.88 (m, 1H), 2.25 (s, 1H), 2.16– 2.02 (m, 4H), 1.87 (dd, J = 26.4, 12.8 Hz, 2H), 1.63 (dd, J = 25.6, 12.0 Hz, 2H); ¹³C NMR (100 MHz, D2O) d 158.7, 149.2, 145.21, 143.6, 138.4, 135.7, 132.5, 131.3, 130.1, 130.0, 128.7, 126.5, 70.1, 44.2, 43.5, 42.6, 38.8, 32.8, 29.1, 24.9; MS (ESI) [M+H]⁺ 458.3. [00251] SYC-1778. (4-(5-(Piperidin-4-ylmethoxy)-3-(4-(1,2,3,6-tetrahydropyridin-4- yl)phenyl)pyrazin-2-yl)phenyl)methanamine hydrochloride:

IC502.9 uM or 6 uM [00252] ¹H NMR (400 MHz, DMSO-d6) d 9.52 (s, 2H, NH), 9.15 (s, 1H, NH), 8.86 (s, 1H, NH), 8.49 (s, 4H), 8.34 (s, 1H), 7.44– 7.32 (m, 8H), 6.24 (s, 6H), 4.26 (d, J = 5.2 Hz, 2H), 3.97 (d, J = 4.0 Hz, 2H), 3.69.18 (s, 2H), 3.60– 3.24 (m, 4H), 2.90– 2.85 (m, 2H), 2.65 (s, 2H), 2.11 (s, 1H), 1.88 (d, J = 12.0 Hz, 2H), 1.54 (dd, J = 25.6, 12.0 Hz, 2H); MS (ESI) [M+H]⁺ 456.3. [00253] SYC-1926. (4-(3-(6-(Piperazin-1-yl)pyridin-3-yl)-5-(piperidin-4-ylmethoxy)pyrazin- 2-yl)phenyl)methanamine hydrochloride:

[00254] ¹H NMR (400 MHz, D2O) d 8.20 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 12.0 Hz, 1H), 7.35 (s, 4H), 7.04 (d, J = 12.0 Hz, 1H), 4.27 (s, 2H), 4.09 (s, 1H), 3.83 (s, 4H), 3.35 (s, 6H), 2.94 (t, J = 12.0 Hz, 4H), 2.15 (s, 2H), 1.99 (d, J = 12.0 Hz, 2H), 1.50 (d, J = 12.0 Hz, 2H); ¹³C NMR (100 MHz, D₂O) d 158.9, 152.1, 144.6, 143.7, 143.3, 138.5, 137.4, 133.4, 133.2, 130.2, 129.2, 124.5, 111.8, 70.2, 43.6, 42.9, 42.6, 42.2, 32.8, 25.0; MS (ESI) [M+H]⁺ 460.3. [00255] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

WHAT IS CLAIMED IS: 1. A composition comprising a compound with the following structure:

,

wherein R₁, R₂, and R₃ are each independently selected from the group consisting of hydrogen, aromatic groups, phenyl groups, benzyl, furan groups, phenyl furan groups, pyridine groups, phenyl pyridine groups, biphenyl groups, phenyl piperidine groups, pyrazole groups, amine groups, piperidine groups, amine groups, alkyl amine groups, aniline groups, methyl piperidine groups, benzene groups, cyclohexane groups, methyl benzoate groups, benzyl piperidine groups, imidazole groups, piperidine amine groups, -NHCH₃ groups, - CH(CH₃)CH₂CH₂NH₂ groups, -NH₂ groups, furan, 3-phenylfuran, 2-phenylfuran, 3- phenyloxolane, phenylmethanamine, propan-2-ylbenzene, tert-butylbenzene, phenylpyrrolidine, 1H-1,3-benzodiazole, benzimidazole, 3-phenylthiophene, 4-phenyl-1H-pyrazole, 4-phenyl- 1,2,3,6-tetrahydropyridine, 1-(pyridin-2-yl)piperazine, biphenyl, 3-phenylpyridine, 4- phenylpyridine, pyridine, 4-(1H-pyrazol-1-yl)piperidine, 1-tert-butyl-3-methoxybenzene, 1- phenylethan-1-one, 2-methoxybenzamide, 2-phenylpropan-2-amine, 2-fluorobenzamide, benzoylpiperidine, 4-phenyloxane, 2-(oxan-4-yl)pyridine, phenol, 2-methylpyridine, 3- propylpyridine, 4-methylpyridine, benzylpiperidine, piperidine, benzene, cyclohexane, methylcyclohexane, 3-methylbutan-1-amine, butan-1-amine, NHCH₃, CH(CH₃)CH₂CH₂NH₂, NH₂, aniline, p-toluidine, 4-ethylaniline, methylpiperidine, methyl benzoate, methyl 4- methylbenzoate, 4-benzylpiperidine, imidazole, piperidin-4-amine, 1,4-dimethylpiperidine, acetophenone, N-methylethanamine, 4-isopropylbenzne, 1-(4-methylpiperidin-1-yl)ethan-1-one, 1-(piperidin-1-yl)ethan-1-one, phenyl(piperidin-1-yl)methanone, 3-phenyltetrahydrofuran, 1- ethyl-1H-imidazole, 4-(1-phenyl)piperidine, 3-methylpiperidine, 4-ethylpiperidine, 4- bromobenzene (4-Br-Ph), 4-tertbutyl-benzene (4-t-Bu-Ph), 4-benzylamine (4-(NH₂CH₂)-Ph), 4- benzamide (4-(NH₂CO)-Ph), N-methyl-4-benzylamine (4-(MeNHCH₂)-Ph), 5-indole(Indol-5- yl), 4-(3-phenylfuran) (4-(furan-3-yl)-Ph), (4-(pyrazol-4-yl)-Ph), or combinations thereof,

wherein X is selected from the group consisting of O, NH, C=O, CH₂, CONH, or combinations thereof,

wherein Y is selected from the group consisting of N, CH, or combinations thereof, wherein Z is selected from the group consisting of N, CH, or combinations thereof, and wherein m is an integer greater than or equal to 0.

2. The composition of claim 1, wherein the compound is selected from the group consisting of:

,

, or combinations thereof.

3. The composition of claim 1, wherein the compound is selected from the group consisting of:

,

,

, ,

,

,

, ,

,

, or combinations thereof.

4. The composition of claim 1, wherein the compound comprises the following structure:

,

wherein n is an integer selected from the group consisting of 0, 1, 2, 3, or 4.

5. The composition of claim 4, wherein the compound is selected from the group consisting of:

,

, or combinations thereof.

6. The composition of claim 1, wherein the composition is suitable for treating or inhibiting a virus, wherein the virus is a flavivirus selected from the group consisting of dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof.

7. The composition of claim 1, wherein the composition inhibits linked or binary ZIKV protease (ZVpro).

8. The composition of claim 1, wherein the composition inhibits DENV serotype-2 protease (DV2pro), DENV serotype-3 protease (DV3pro), West Nile protease (WVpro), or combinations thereof.

9. The composition of claim 1, wherein the composition inhibits ZIKV replication.

10. The composition of claim 1, wherein the composition binds to an allosteric pocket of a flavivirus protease.

11. The composition of claim 1, wherein the composition is associated with a delivery agent.

12. The composition of claim 11, wherein the delivery agent is a nanoparticle.

13. The composition of claim 1, wherein the composition comprises at least one excipient agent selected from the group consisting of anti-adherents, binders, coatings, colors,

disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, vehicles, or combinations thereof.

14. The composition of claim 1, wherein the composition comprises at least one solubilizing agent selected from the group consisting of polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides, polyoxyethylated oleic glycerides, polysorbates, sorbitan monooleate, hydroxypropyl-beta-cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil, polyoxyl hydroxystearates, or combinations thereof.

15. A method of treating a viral infection in a subject, said method comprising:

administering a composition to the subject, wherein the compositions comprises a compound with the following formula:

,

wherein R₁, R₂, and R₃ are each independently selected from the group consisting of hydrogen, aromatic groups, phenyl groups, benzyl, furan groups, phenyl furan groups, pyridine groups, phenyl pyridine groups, biphenyl groups, phenyl piperidine groups, pyrazole groups, amine groups, piperidine groups, amine groups, alkyl amine groups, aniline groups, methyl piperidine groups, benzene groups, cyclohexane groups, methyl benzoate groups, benzyl piperidine groups, imidazole groups, piperidine amine groups, -NHCH₃ groups, - CH(CH₃)CH₂CH₂NH₂ groups, -NH₂ groups, furan, 3-phenylfuran, 2-phenylfuran, 3- phenyloxolane, phenylmethanamine, propan-2-ylbenzene, tert-butylbenzene, phenylpyrrolidine, 1H-1,3-benzodiazole, benzimidazole, 3-phenylthiophene, 4-phenyl-1H-pyrazole, 4-phenyl- 1,2,3,6-tetrahydropyridine, 1-(pyridin-2-yl)piperazine, biphenyl, 3-phenylpyridine, 4- phenylpyridine, pyridine, 4-(1H-pyrazol-1-yl)piperidine, 1-tert-butyl-3-methoxybenzene, 1- phenylethan-1-one, 2-methoxybenzamide, 2-phenylpropan-2-amine, 2-fluorobenzamide, benzoylpiperidine, 4-phenyloxane, 2-(oxan-4-yl)pyridine, phenol, 2-methylpyridine, 3- propylpyridine, 4-methylpyridine, benzylpiperidine, piperidine, benzene, cyclohexane, methylcyclohexane, 3-methylbutan-1-amine, butan-1-amine, NHCH₃, CH(CH₃)CH₂CH₂NH₂, NH₂, aniline, p-toluidine, 4-ethylaniline, methylpiperidine, methyl benzoate, methyl 4- methylbenzoate, 4-benzylpiperidine, imidazole, piperidin-4-amine, 1,4-dimethylpiperidine, acetophenone, N-methylethanamine, 4-isopropylbenzne, 1-(4-methylpiperidin-1-yl)ethan-1-one, 1-(piperidin-1-yl)ethan-1-one, phenyl(piperidin-1-yl)methanone, 3-phenyltetrahydrofuran, 1- ethyl-1H-imidazole, 4-(1-phenyl)piperidine, 3-methylpiperidine, 4-ethylpiperidine, 4- bromobenzene (4-Br-Ph), 4-tertbutyl-benzene (4-t-Bu-Ph), 4-benzylamine (4-(NH₂CH₂)-Ph), 4- benzamide (4-(NH₂CO)-Ph), N-methyl-4-benzylamine (4-(MeNHCH₂)-Ph), 5-indole(Indol-5- yl), 4-(3-phenylfuran) (4-(furan-3-yl)-Ph), (4-(pyrazol-4-yl)-Ph), or combinations thereof, wherein X is selected from the group consisting of O, NH, C=O, CH₂, CONH, or combinations thereof,

16. The method of claim 15, wherein the compound is selected from the group consisting of:

,

17. The method of claim 15, wherein the compound is selected from the group consisting of:

,

,

, ,

,

,

, ,

,

, or combinations thereof.

18

,

wherein n is an integer selected from the group consisting of 0, 1, 2, 3, or 4.

19. The method of claim 18, wherein the compound is selected from the group consisting of:

,

, or combinations thereof.

20. The method of claim 15, wherein the viral infection in the subject is caused by flavivirus selected from the group consisting of dengue virus, West Nile virus, Zika virus, tick-borne encephalitis virus, yellow fever virus, viruses causing encephalitis, insect-specific flaviviruses, cell fusing agent viruses, Palm Creek virus, Parramatta River virus, or combinations thereof.

21. The method of claim 15, wherein the composition treats the viral replication in the subject by inhibiting a viral protease.

22. The method of claim 21, wherein the viral protease is selected from the group consisting of linked or binary ZIKV protease (ZVpro), DENV serotype-2 protease (DV2pro), DENV serotype- 3 protease (DV3pro), West Nile protease (WVpro), or combinations thereof.

23. The method of claim 15, wherein the subject is a human being.

24. The method of claim 15, wherein the administering is performed by a method selected from the group consisting of oral administration, inhalation, subcutaneous administration, intravenous administration, intra-nasal administration, intra-dermal administration, trans-dermal administration, intraperitoneal administration, intramuscular administration, intrathecal injection, topical administration, central administration, peripheral administration, transdermal

administration, intraarterial administration, intracranial administration, intraspinal

administration, intranasal administration, intraocular administration, intratumor administration, intramuscular administration, intranasal administration, subcutaneous administration, intra- or trans-dermal administration, intravenous administration, topical administration, or combinations thereof.

25. The method of claim 15, wherein the composition is associated with a delivery agent.

26. The method of claim 25, wherein the delivery agent is a nanoparticle.

27. The method of claim 15, wherein the composition comprises at least one excipient agent selected from the group consisting of anti-adherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, vehicles, or combinations thereof.

28. The method of claim 15, wherein the composition comprises a solubilizing agent selected from the group consisting of water miscible organic solvents, polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides, polyoxyethylated oleic glycerides, polysorbates, sorbitan monooleate, hydroxypropyl-beta-cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil, polyoxyl hydroxystearates, and combinations thereof.