WO2025080695A1

WO2025080695A1 - 1-sulfonyl-3-amino-1h-1,2,4-triazoles as yellow fever virus inhibitors

Info

Publication number: WO2025080695A1
Application number: PCT/US2024/050549
Authority: WO
Inventors: Thane JONES; Thomas R. Lane; Vadim Makarov; Sean Ekins
Original assignee: Collaborations Pharmaceuticals Inc
Current assignee: Collaborations Pharmaceuticals Inc
Priority date: 2023-10-10
Filing date: 2024-10-09
Publication date: 2025-04-17
Anticipated expiration: 2026-04-10

Abstract

Methods of inhibiting yellow fever virus (YFV) and treating diseases caused by YFV infections are described. For example, the methods can include administering a compound that includes a 1-sulfonyl-3-amino-1H-1,2,4-triazole group to a subject in need of treatment for yellow fever. The sulfonyl moiety of the l-sulfonyl-3-amino-1H-1,2,4-triazole group can be attached to a substituted or unsubstituted aryl group. For example, the sulfonyl moiety can be attached to a substituted or unsubstituted naphthyl moiety.

Description

DESCRIPTION

1 -SULF ONYL-3 -AMINO- 1H-1, 2, 4-TRIAZOLES AS YELLOW FEVER VIRUS

INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application Serial No. 63/543,431, filed October 10, 2023, which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Numbers R44GM122196, 1R01NS102164, and R43ES031038 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter relates to compounds comprising 1- sulfonyl-3-amino-lH-l,2,4-triazole moieties and their use in treating viral infections, e.g., yellow fever virus infections.

ABBREVIATIONS °C = degrees Celsius

% = percent pg = micrograms pL = microliter pm = micrometer pM = micromolar

A = angstrom

ACN = acetonitrile

AUC = area under the curve

CCso = 50% cytotoxic concentration

DMSO = dimethyl sulfoxide

EC50 = 50% effective concentration

ECFP = extended-connectivity fingerprint EtOH = ethanol

FPR = false positive rate h = hour

HBV = Hepatitis B virus

HCV = Hepatitis C virus

HIV = human immunodeficiency virus

ICso = 50% inhibitory concentration kg = kilogram

Ireg = linear regression

M = molar

MeOH = methanol mg = milligram min = minute mL = milliliter mm = millimeter ng = nanogram

PK = pharmacokinetic

ROC = receiver operating characteristic

SAR = structure-activity relationship

SD = standard deviation

SI50 = selectivity index

SVC = support vector machine classification TPR = true positive rate t-SNE = t-distributed stochastic neighbor embedding VYR = virus yield reduction

YF = yellow fever

YFV = yellow fever virus

BACKGROUND Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Yellow fever virus (YFV), a mosquito-borne flavivirus [1,2]. The highest prevalence of YF is found in Africa and South America [3] with mortality rates up to 50% [4], Despite the availability of a safe and effective vaccine [5], there has been a rise in unvaccinated populations with outbreaks in Brazil and Angola in 2016 [6-8], This could be due to various reasons, such as vaccine hesitancy, inadequate healthcare infrastructure, and low awareness about the importance of vaccination. At the same time, this opens the possibility of developing small-molecule drugs. There are currently no approved small-molecule specific antivirals for the treatment of yellow fever virus infection. Nucleoside analogs, such as ribavirin [9], sofosbuvirflO], galidesivir [11], and favipiravir [12] have mostly been tested in animal models and have not yet reached clinical trials. Moreover, in recent years most of the flavivirus drug discovery campaigns have focused on broad-spectrum agents for dengue and Zika viruses, while the need for small-molecule YF inhibitors has not yet been met [13].

Accordingly, there is an ongoing need for small molecules that can inhibit YFV and treat diseases caused by YFV, i.e., yellow fever.

SUMMARY

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

In some embodiments, the presently disclosed subject matter provides a method of treating a disease caused by a yellow fever virus infection in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of a compound comprising a l-sulfonyl-3-amino- lH-l,2,4-triazole group or a pharmaceutically acceptable salt of the compound.

In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group has a structure of Formula (I): H₂N,

^{Ar2 N}-_NA_N-^Ar2 ⁰ O ^H (I), wherein An and An are each independently selected from the group comprising aryl, substituted aryl, heteroaryl, and substituted heteroaryl. In some embodiments, An is phenyl, naphthyl, substituted phenyl or substituted naphthyl, optionally naphthyl or substituted naphthyl, further optionally wherein said substituted naphthyl is naphthyl substituted with a substituent selected from the group comprising halo, nitro, acyl, and cyano-substuted alkyl. In some embodiments, An is substituted phenyl, optionally wherein An is phenyl substituted with one, two, or three substituents independently selected from the group comprising halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O- alkyl.

In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group has a structure of Formula (II):

wherein R3 and R4 are independently selected from the group comprising H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H. In some embodiments, R3 and R4 are each H or one of R3 and R4 is selected from Cl, F, cyano, CH=CHCN, and C(=O)CH3. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, CHCN, -C(=O)-OEt, and -C(=O)-OMe, and wherein one, two, three or four of Rs, Re, R7, Rs, and R9 are H.

In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group is selected from the group comprising:

or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the presently disclosed subject matter provides the a use of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group, or a pharmaceutically acceptable salt of said compound, in preparing a medicament for treating a disease caused by a yellow fever virus infection. In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group has a structure of Formula (I):

wherein An and An are each independently selected from the group comprising aryl, substituted aryl, heteroaryl, and substituted heteroaryl. In some embodiments, An is phenyl, naphthyl, substituted phenyl or substituted naphthyl, optionally naphthyl or substituted naphthyl, further optionally wherein said substituted naphthyl is naphthyl substituted with a substituent selected from the group comprising halo, nitro, acyl, and cyano-substuted alkyl. In some embodiments, An is substituted phenyl, optionally wherein An is phenyl substituted with one, two, or three substituents independently selected from the group comprising halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O- alkyl.

wherein R3 and R4 are independently selected from the group comprising H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H. In some embodiments, R3 and R4 are each H or wherein one of R3 and R4 is selected from Cl, F, cyano, CH=CHCN, and C(=0)CH3. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, CHCN, -C(=O)-OEt, and -C(=O)-OMe, and wherein one, two, three or four of Rs, Re, R7, Rs, and R9 are H.

or a pharmaceutically acceptable salt thereof.

In some embodiments, the presently disclosed subject matter provides a method of inhibiting yellow fever virus in a sample comprising yellow fever virus, wherein the method comprises contacting the sample with an effective amount of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group or a pharmaceutically acceptable salt of the compound, optionally wherein the compound comprises a structure of Formula (I):

H₂N

^{Ar2 N}-_NA_N-^Ar2 ⁰ O ^H (I), wherein An and An are each independently selected from the group consisitng of aryl, substituted aryl, heteroaryl, and substituted heteroaryl. In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group has a structure of Formula (II):

wherein R3 and R4 are independently selected from the group comprising H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H; further optionally wherein the compound is selected from the group comprising:

or a pharmaceutically acceptable salt thereof.

Accordingly, it is an obj ect of the presently disclosed subj ect matter to provide methods of treating diseases caused by a yellow fever virus infection; methods of inhibiting yellow fever virus; and compounds for use in preparing medicaments for treating a disease caused by a yellow fever virus infection.

An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic drawing showing the chemical structure of the presently disclosed compounds comprising a l-sulfonyl-3-amino-l-H-l,2,4-triazole scaffold and summarizing substituent group effects on compound anti-yellow fever potency.

Figures 2A and 2B are graphs showing the pharmacokinetic data for mice dosed with 250 mg/kg of an exemplary compound of the presently disclosed subject matter, RCB16007, via intragastric intubation administration in either plasma (Figure 2A) or brain (Figure 2B). Figure 2A shows the plasma concentration (nanograms per milliliter (ng/mL)) of RCB16007 as a function of time (in hours h)), while Figure 2B shows the brain concentration (ng/mL) of RCB16007 as a function of time (h). Pharmacokinetic parameters were calculated with a Noncompartmental Pharmacokinetics Analysis method. Error bars represent standard deviation (SD).

Figures 3A and 3B are a pair of graphs showing the receiver operating characteristic (ROC) plots (true positive rate (TPR) versus false positive rate (FPR)) of different classification machine learning model algorithms, linear regression (Ireg; Figure 3 A) and support vector machine (svc; Figure 3B) for yellow fever virus (YFV) inhibition at a 20 micromolar (pM) threshold for activity built with training data from the literature and a chemical database from the European Molecular Biology Laboratory (ChEMBL). In the plots, each distinct line represents a single fold from nested, 5-fold cross validation studies.

Figures 4 is a graph showing the t-distributed stochastic neighbor embedding (t-SNE) visualization plot (tsne-x versus tsne-y) of the training and test data for the yellow fever inhibition models using extended-connectivity fingerprint 6 (ECFP6) descriptors. Data is from compounds from the literature (ML Publication), a chemical database from the European Molecular Biology Laboratory (ChEMBL) or compounds tested as disclosed herein (N-phenyl-l-(arylsulfonyl)-lH-l,2,4-triazol-3- amines). The box highlights the cluster containing the compounds tested in this study. For simplicity, duplicate compounds (found in reference 29 and ChEMBL) are identified as ChEMBL only.

Figures 5A-5I are a series of graphs showing the comparison of simple chemical descriptors between active (bars or bar sections above 0.0 relative frequency) and inactive (bars or bar sections below 0.0 relative frequency) classes (20 micromolar (pM) threshold) of the A-phenyl-l-(arylsulfonyl)-U/-l,2,4-triazol-3- amines of the presently disclosed subject matter. Statistically significant differences, comparison tests assumed non-parametric data distributions (Mann Whitney tests). Unless noted, differences between groups are not significantly different. The chemical descriptors are as follows: molecular weight (Figure 5 A), logP (Figure 5B), molecular fraction polar surface area (PSA) (Figure 5C), number of aromatic rings (Figure 5D), number of hydrogen bond acceptors (Figure 5E), number of hydrogen bond donors (Figure 5F), number of rings (Figure 5G), number of rotatable bonds (Figure 5H), and logD at pH 7.4 (Figure 51).

Figure 6A and 6B are two sets of receiver operating characteristic (ROC) plots (true positive rate (TPR) versus false positive rate (FPR)) of different classification machine learning model algorithms for yellow fever inhibition using training data. The training data from these models originated from a dataset described in reference 29. The 50% inhibitory concentration (IC50) thresholds of (Figure 6A) 6.7 micromolar (pM) and (Figure 6B) 80 pM were based on those chosen in reference 29. Values are the nested 5-fold cross validation scores. In the plots, each distinct line represents a single fold from nested, 5-fold cross validation studies. The machine learning model algorithms include DL = deep learning, ada = AdaBoosted decision trees, bnb = Naive Bayesian, knn = k-nearest neighbors, Ireg = linear regression, rf = random forest, svc = support vector machine, xgb = xgboost).

Figures 7A and 7B are two sets of receiver operating characteristic (ROC) plots (true positive rate (TPR) versus false positive rate (FPR)) of different classification machine learning model algorithms for yellow fever inhibition using training data. The training data from these models originated from a dataset described in reference 29 combined with data from the chemical database from the European Molecular Biology Laboratory (ChEMBL). The 50% inhibitory concentration (IC50) thresholds of (Figure 7A) 10 micromolar (pM) and (Figure 7B) 20 pM were chosen based on the most potent activities that still achieved a reasonable active/inactive ratio. Values are the nested 5-fold cross validation scores. In the plots, each distinct line represents a single fold from nested, 5-fold cross validation studies. The machine learning model algorithms include DL = deep learning, ada = AdaBoosted decision trees, bnb = Naive Bayesian, knn = k-nearest neighbors, Ireg = linear regression, rf = random forest, svc = support vector machine, xgb = xgboost).

Figures 8A and 8B are two sets of receiver operating characteristic (ROC) plots (true positive rate (TPR) versus false positive rate (FPR)) of different classification machine learning model algorithms for yellow fever inhibition using training data from 7V-phenyl-l-(arylsulfonyl)-U/-l,2,4-triazol-3-amines tested herein only. The 50% inhibitory concentration (IC50) thresholds of (Figure 8 A) 10 micromolar (pM) and (Figure 8B) 20 pM were chosen based on the most potent activities that still achieved a reasonable active/inactive ratio. Values are the nested 5-fold cross validation scores. In the plots, each distinct line represents a single fold from nested, 5 -fold cross validation studies. The machine learning model algorithms include DL = deep learning, ada = AdaBoosted decision trees, bnb = Naive Bayesian, knn = k-nearest neighbors, Ireg = linear regression, rf = random forest, svc = support vector machine, xgb = xgboost). Figures 9A-9I are a series of graphs showing the comparison of simple chemical descriptors between active (bars or bar sections above 0.0 relative frequency) and inactive (bars or bar sections below 0.0 relative frequence) classes (20 micromolar (pM) threshold) of from a combined dataset obtained from reference 29 combined with data from the chemical databased from the European Molecular Biology Laboratory (ChEMBL). Statistically significant differences, comparison tests assumed non-parametric data distributions (Mann Whitney tests). Unless noted, differences between groups are not significantly different. The chemical descriptors are as follows: molecular weight (Figure 9A), logP (Figure 9B), molecular fraction polar surface area (PSA) (Figure 9C), number of aromatic rings (Figure 9D), number of hydrogen bond acceptors (Figure 9E), number of hydrogen bond donors (Figure 9F), number of rings (Figure 9G), number of rotatable bonds (Figure 9H), and logD at pH 7.4 (Figure 91).

DETAILED DESCRIPTION

Yellow fever virus (YFV) is transmitted by infected mosquitoes and causes an acute viral disease for which there are no approved small-molecule therapeutics. Recently developed machine learning models for YFV inhibitors led to the selection of a new pyrazolesulfonamide derivative RCB16003 with acceptable in vitro activity. According to an aspect of the presently disclosed subject matter, the 7V-phenyl-l- (phenylsulfonyl)-U/-l,2,4-triazol-3-amine class, which was recently identified as active non-nucleoside reverse transcriptase inhibitors against HIV-1, can also be repositioned as inhibitors of yellow fever virus replication.

As compared to other Flaviviridae or Togaviridae family viruses tested, both compounds RCB16003 (a pyrazolesulfonamide) and RCB16007 (a N-penyl-1- (phenylsulfonyl-lH-l,2,4-triazol-3-amine) demonstrate selectivity for YFV over related viruses, with RCB16007 also showing some inhibition of West Nile virus (ECso 7.9 pM, CC50 17 pM, SI 2.2). The absorption, distribution, metabolism and excretion (ADME) in vitro and pharmacokinetics (PK) for RCB16007 in mice is also described herein. This compound was previously shown to not inhibit hERG, and it is described herein that it has good metabolic stability in mouse and human liver microsomes, low levels of CYP inhibition, high protein binding and no indication of efflux in Caco-2 cells. A single-dose oral PK study in mice has a T1/2 of 3.4 h and Cmax 1, 190 ng/mL, suggesting good availability and stability. The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

I. DEFINITIONS

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.

Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims.

The term “and/or” when used in describing two or more items or conditions, refers to situations where all named items or conditions are present or applicable, or to situations wherein only one (or less than all) of the items or conditions is present or applicable.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” can mean at least a second or more.

The term “comprising”, which is synonymous with “including,” “containing,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim. As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

Unless otherwise indicated, all numbers expressing quantities of time, concentration, dosage and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

As used herein, the term “about”, when referring to a value is meant to encompass variations of in one example ±20% or ±10%, in another example ±5%, in another example ±1%, and in still another example ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.

The term “arene” as used herein refers to any aromatic compound or moiety, including, but not limited to arene compounds and moieties (i.e., “heteroarenes”) comprising heteroatoms, such as nitrogen, oxygen, selenium, and sulfur.

As used herein the term “alkyl” refers to C1-20 inclusive, linear (i.e., "straightchain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms; 1 to about 6 carbon atoms (i.e., a C1-6 alkyl), or 1 to 5 carbon atoms (i.e., a C1-5 alkyl). "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, "alkyl" refers, in particular, to Ci-8 straight-chain alkyls. In some embodiments, “alkyl” refers, in particular, to Ci-8 branched-chain alkyls. In some embodiments, “alkyl” refers, in particular to Ci- 6 or Ci-5 straight-chain and/or branched-chain alkyls (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl.)

Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxy carbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

“Cyclic” and "cycloalkyl" refer to a non-aromatic mono- or multicyclic ring system of about 4 to about 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an alkyl group substituent as defined herein, oxo and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl, or aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl. Representative multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl. Representative nitrogen-containing heterocyclic groups include, but are not limited to, aziridine, piperidine, pyrroline, pyrrolidine, thiomorpholine, morpholine, azocane, and azepane.

"Alkoxyl" and “alkoxy” refer to an alkyl-O— group wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, and pentoxy.

The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

“Heteroaryl” as used herein refers to an aryl group that contains one or more non-carbon atoms (e.g., O, N, S, Se, etc) in the backbone of a ring structure. Nitrogen- containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, pyrimidine, and the like.

“Aralkyl” refers to an -alkyl-aryl group, optionally wherein the alkyl and/or aryl moiety is substituted. Exemplary aralkyl groups include, but are not limited to, benzyl and ethyl-phenyl.

"Alkylene" refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents." There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexylene (-CeHio-); -CH=CH— CH=CH-; -CH=CH-CH₂-; -(CH₂)_q-N(R)- (CH2)I— , wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedi oxy 1 (-O-CH2-O-); and ethylenedioxyl (-O-(CH2)2-O- ). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

The term “arylene” refers to a bivalent aromatic group, e.g., a bivalent phenyl or napthyl group. The arylene group can optionally be substituted with one or more aryl group substituents and/or include one or more heteroatoms.

The term “aralkylene” refers to a bivalent group that includes both aromatic and non-aromatic groups.

The term “amino” refers to the group -N(R)2 wherein each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. The terms “aminoalkyl” and “alkylamino” can refer to the group -N(R)2 wherein each R is H, alkyl or substituted alkyl, and wherein at least one R is alkyl or substituted alkyl. “Aralkylamine” can refer to the group -N(R)2 wherein each R is H, aralkyl, or substituted alkyl and wherein at least one R is aralkyl or substituted aralkyl, e.g., aminobenyzl. “Dialkylamino” refers to an alkylamino group where both R are alkyl or substituted alkyl. The term “carboxylic acid” refers to a group -C(=O)-OH as well as to the deprotonated acid, i.e., the group where the H is replaced by a negative charge (also referred to as “carboxylate”).

The terms “hydroxy” and “hydroxyl” as used herein refer to a -OH group.

The term “halo” as used herein refers to fluoro (-F), chloro (-C1), bromo (-Br), and iodo (-1).

The term “acyl” as used herein refers to a group having the structure -C(=O)- R, where R is H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl.

The term “formyl” as used herein refers to a group having the structure - C(=O)-H.

The term “nitro” as used herein refers to the group -NO2.

The term “cyano” as used herein refers to the group -C=N.

The term “perhaloalkyl” as used herein refers to an alkyl group where each hydrogen atom is replaced by a halo group. An exemplary perhaloalkyl group is - CF₃.

As use herein, the terms “administration of’ and/or “administering” a compound can be understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment. As used herein “administering” includes administration of a compound or compounds by any number of routes and modes including, but not limited to, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.

As used herein, the term “pharmaceutically acceptable carrier” means a composition with which an appropriate compound can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.

As used herein, the term “pharmaceutically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered. As used herein, an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to produce a selected and/or detectable effect, such as but not limited to alleviating symptoms of a condition, disease, or disorder. In the context of administering compounds in the form of a combination, such as multiple compounds, the amount of each compound, when administered in combination with one or more other compounds, can be different from when that compound is administered alone. Thus, an effective amount of a combination of compounds refers collectively to the combination as a whole, although the actual amounts of each compound can vary. The term “more effective” means that the selected effect occurs to a greater extent by one treatment relative to the second treatment to which it is being compared.

The term “prevent” as used herein means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, “prevention” generally refers to action taken to decrease the chance of getting a disease or condition. It is noted that “prevention” need not be absolute, and thus can occur as a matter of degree.

The terms “treatment” and “treating” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, and/or lower the chances of the individual developing a condition, disease, or disorder, even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition (e.g., those diagnosed with the condition or already experiencing symptoms of the conditions) as well as those prone to have or predisposed to having a condition, disease, or disorder, or those in whom the condition is to be prevented.

Code numbers used herein to refer to particular compounds can be used to refer to the compound as well as any salts thereof. For example, RCB16007 can be used here to refer to the compound having the structure:

or to a salt thereof, e.g., to the HC1 salt thereof. II. GENERAL CONSIDERATIONS

Efforts are still needed to cost-effectively identify new molecules for YFV [14], such as high-throughput screening (HTS) [15] and machine learning methods [16], In its simplest form, machine learning approaches use an array of algorithms (e.g. naive-Bayesian classifier [17-19], random forests (rf) [20,21] or support vector machines (svm)) [22-24], and molecular descriptors (fingerprints or physicochemical properties like logP and others) to generate models that can be used to score compound libraries [25-27], Rajput and Kumar have previously developed svm and rf regression models with data for many different flaviviruses, although the amount of YFV data used in the training set was comparatively small [28], In another recent study, various machine learning approaches were applied to YFV [29], First, YFV cell-based assay data from the literature and public databases was curated and then numerous machine learning models were generated. These were validated with an external test set before use for prioritizing and ultimately selecting five compounds for in vitro testing. While the training set was limited, one molecule, a pyrazolesulfonamide derivative referred to as RCB16003 showed a low micromolar potency (ECso 3.2 pM) and acceptable cytotoxicity (CCso 24 pM) against YFV and represented a new scaffold suitable for hit-to-lead optimization [29], While sulfonamides are a well-known class of drugs with antiviral activity for HBV [30], HIV [31], HCV [30], they had not previously been described for YFV.

According to the presently disclosed subject matter, the l-sulfonyl-3-amino- U/-l,2,4-triazole scaffold and its structure-activity relationship analysis for YFV is described. An exemplary l-sulfonyl-3-amino-U/-l,2,4-triazole compound, RCB16007, was studied in detail, generating absorption, distribution, metabolism, and excretion (ADME) and in vivo pharmacokinetics (PK) in mice.

III. METHODS OF INHIBITNG YELLOW FEVER VIRUS

Accordingly, in some embodiments, the presently disclosed subject matter provides a method of inhibiting yellow fever virus (YFV) in a sample comprising YFV, wherein the method comprises contacting the sample with an effective amount of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group or a pharmaceutically acceptable salt said compound. In some embodiments, the compound is a compound as described in one of Tables 1-3 or 5 below. In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group is a compound having a structure of Formula (I):

wherein An and An are each independently selected from the group comprising aryl (e.g., phenyl or naphthyl), substituted aryl, heteroaryl (e.g., pyridine), and substituted heteroaryl. In some embodiments, the substituted aryl or substituted heteroaryl groups are substituted with electron-withdrawing substituents, such as, but not limited to, halo (e.g., Cl, F, Br, or I), nitro, cyano, carboxylic acid, acyl (i.e., -C(=O)-R, where R is alkyl (e.g., C1-C6 alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl), formyl (-C(=C)-H), ester (-C(=O)-OR, where R is alkyl (e.g., Cl- C6 alkyl), substituted alkyl, aralkyl, substituted aralkyl, aryl, or substituted aryl), perhaloalkyl, and quaternary amino.

In some embodiments, An is phenyl, naphthyl, substituted phenyl or substituted naphthyl. In some embodiments, An is naphthyl or substituted naphthyl. In some embodiments, substituted naphthyl is naphthyl substituted with a substituent selected from the group comprising halo (e.g., Cl or F), nitro, acyl (e.g., C(=O)-CH3), and cyano-substuted alkyl (e.g., -CH2CN or -CH=CH-CN).

In some embodiments, An is substituted phenyl. In some embodiments, Ar² is mono-, di- or tri -substituted phenyl. In some embodiments, An is phenyl substituted with one, two, or three substituents selected from the group including, but not limited to, halo (e.g., Cl or F), cyano, acyl (e.g., -C(=O)-CH3), formyl, alkyl (e.g., C1-C6 alkyl), cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl (e g., -C(=O)-OCH₃ or -C(=O)-OCH₂CH₃).

In some embodiments, the compound has a structure of Formula (II):

wherein R3 and R4 are independently selected from the group comprising H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl. In some embodiments, at least one of Rs, Re, R7, Rs, and R9 is not H. In some embodiments, 1, 2, 3, or 4 of Rs, Re, R7, Rs, and R9 is H. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, -C(=O)-OEt, and -C(=O)-OMe. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, and -C(=O)-OEt. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, -C(=O)-OEt, and -C(=O)-OMe, and one, two, three or four of Rs, Re, R7, Rs, and R9 are H. In some embodiments, one or two of Rs, Re, R7, Rs, and R9 is halo. In some embodiments, one of Rs, Re, R7, Rs, and R9 is cyano. In some embodiments, R7 is other than H. In some embodiments, Re is other than H. In some embodiments, both Re and R7 are other than H.

In some embodiments, at least one of R3 and R4 is H. In some embodiments, R3 and R4 are each H. In some embodiments, one of R3 and R4 is selected from halo, cyano, cyano-substituted alkyl (e.g., cyano-substituted C1-C6 alkyl), and acyl. In some embodiments, one of R3 and R4 is Cl, F, cyano, CH=CHCN, or C(=O)CH3.

4-((5- Amino- 1 -(naphthal en-2-yl sulfonyl)- 1H- 1 ,2,4-triazol-3 -yl)amino)benzoic acid ethyl ester:

(also referred to herein as RCB17024);

5-((5- Amino-3 -((4-chlorophenyl)amino)- 1H- 1 ,2,4-triazol- 1 -yl)sulfonyl)-2-naphtho- nitrile:

(also referred to herein as RCB17099);

5-((5- Amino-3 -((4-fluorophenyl)amino)- 1H- 1 ,2,4-triazol- 1 -yl)sulfonyl)- 1 -naphthonitrile:

(also referred to herein as RCB17150); A³-(4-Chl oro-3 -methylphenyl)-! -(naphthal en-2 -yl sulfonyl)- 1/7-1, 2, 4-triazole-3, 5- diamine:

(also referred to herein as RCB17154);

4-((5- Amino- 1 -(naphthal en-2-yl sulfonyl)- 1H- 1 ,2,4-triazol-3 -yl)amino)-3 - methy lb enzo-nitril e

(also referred to herein as RCB17162);

4-(( l-((6-Acetylnaphthalen- l -yl)sulfonyl)-5-amino- l//- l ,2,4-triazol-3-yl)amino)-2- chi orob enzonitril e :

(also referred to as RCB22055); 4-((5-Amino-l-((6-(2-cyanovinyl)naphthalen-l-yl)sulfonyl)-lH-l,2,4-triazol-3- yl)amino)phthalonitrile:

(also referred to herein as RCB22057);

N³-(4-chlorophenyl)-l -naphthal en- 1-yl sulfonyl)- 1H-1, 2, 4-triazole-3,5-diamine:

(also referred to herein as RCB16007);

4-((5- Amino- 1 -(naphthal en- 1 -yl sulfonyl)- 1H- 1 ,2,4-triazol-3 -yl)amino)-2-chloro-

(also referred to herein as RCB17158);

N³-(3,4-di chlorophenyl)-! -naphthal en- 1-yl sulfonyl)- 1H-1, 2, 4-triazole-3,5-diamine:

(also referred to herein as RCB17159);

1 -((6-chloronaphthalen- 1 -yl)sulfonyl)-N³-(4-chlorophenyl)- 1H- 1 ,2,4-triazole-3 ,5-

(also referred to herein as RCB18320); and

4-((5- Amino- 1 -((6-cyanonaphthalen- 1 -yl)sulfonyl)- 1H- 1 ,2,4-triazol-3 -yl)amino)- phthalobenzonitrile:

(also referred to herein as RCB21055); or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is RCB16007 or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has an ECso against a YFV infection

(e.g., a YFV 17D infection) of Huh7 cells of about 10 pM or less. In some embodiments, the compound has a CCso of about 10 pM or more, 25 pM or more of about 30 pM or more.

In some embodiments, the sample comprises a biological extract or a biological fluid (e.g., blood, plasma, saliva, a cell extract, a tissue extract, or an organ extract). In some embodiments, the sample comprises a cell (e.g., a cell culture) or tissue. In some embodiments, the sample comprises a living organism, e.g., mosquito, a domesticated animal, a non-domesticated mammal, or a human.

IV. METHODS OF TREATING DISEASES CAUSED BY YELLOW FEVER VIRUS AND RELATED USES

According to some embodiments, the presently disclosed subject matter provides a method of treating a yellow fever virus infection in a subject in need of treatment thereof. In some embodiments, the presently disclosed subject matter provides a method of treating a disease caused by a yellow fever virus infection in a subject in need of treatment thereof. Thus, in some embodiments, the presently disclosed subject matter provides a method of treating yellow fever. In some embodiments, the compound is a compound as described in one of Tables 1-3 or 5 below.

In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a compound comprising a l-sulfonyl-3-amino- lH-l,2,4-triazole group or a pharmaceutically acceptable salt of the compound. In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH-l,2,4- triazole group is a compound having a structure of Formula (I):

In some embodiments, the compound has a structure of Formula (II):

(also referred to herein as RCB17024);

(also referred to herein as RCB17099);

(also referred to herein as RCB17154);

(also referred to herein as RCB17162);

(also referred to as RCB22055);

4-((5-Amino-l-((6-(2-cyanovinyl)naphthalen-l-yl)sulfonyl)-lH-l,2,4-triazol-3- yl)amino)phthalonitrile:

(also referred to herein as RCB22057);

(also referred to herein as RCB16007);

(also referred to herein as RCB17158);

(also referred to herein as RCB17159);

(also referred to herein as RCB18320); and

In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject in need of treatment is a subject (e.g., a human subject) that has yellow fever and/or who has a yellow fever virus infection (e.g., as diagnosed via a blood test for yellow fever). In some embodiments, the subject is a subject (e.g., a human subject) who has been in contact with another individual with a yellow fever virus infection or who has been to a geographical location where there is an outbreak of yellow fever or where there is a risk of yellow fever outbreak (e.g., tropical areas in Africa or South America).

In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal, particularly those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans), and/or of social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos or as pets (e.g., parrots), as well as fowl, and more particularly domesticated fowl, for example, poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock including, but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

In some embodiments, the presently disclosed subject matter provides the use of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group, or a pharmaceutically acceptable salt of said compound, in preparing a medicament for treating a yellow fever virus infection or a disease caused by a yellow fever virus infection. Thus, in some embodiments, the presently disclosed subject matter provides the use of compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group, or a pharmaceutically acceptable salt of said compound, in preparing a medicament to treat yellow fever. In some embodiments, the compound is a compound as described in one of Tables 1-3 or 5 below.

In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group is a compound having a structure of Formula (I):

In some embodiments, An is substituted phenyl. In some embodiments, An is mono-, di- or tri -substituted phenyl. In some embodiments, An is phenyl substituted with one, two, or three substituents selected from the group including, but not limited to, halo (e.g., Cl or F), cyano, acyl (e.g., -C(=O)-CH3), formyl, alkyl (e.g., C1-C6 alkyl), cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl (e g., -C(=O)-OCH₃ or -C(=O)-OCH₂CH₃).

In some embodiments, the compound has a structure of Formula (II):

wherein R3 and R4 are independently selected from the group comprising H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl. In some embodiments, at least one of Rs, Re, R7, Rs, and R9 is not H. In some embodiments, 1, 2, 3, or 4 of Rs, Re, R7, Rs, and R9 are H. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, -C(=O)-OEt, and -C(=O)-OMe. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, and -C(=O)-OEt. In some embodiments, Rs, Re, R7, Rs and R9 are independently selected from the group comprising H, F, Cl, methyl, CN, -CHCN, -C(=O)-OEt, and -C(=O)-OMe, and one, two, three or four of Rs, Re, R7, Rs, and R9 are H. In some embodiments, one or two of Rs, Re, R7, Rs, and R9 is halo. In some embodiments, one of Rs, Re, R7, Rs, and R9 is cyano. In some embodiments, R7 is other than H. In some embodiments, Re is other than H. In some embodiments, both Re and R7 are other than H.

In some embodiments, the compound comprising a l-sulfonyl-3-amino-lH- 1,2,4-triazole group is selected from the group comprising: RCB17024, RCB17099, RCB17150, RCB17154, RCB17162, RCB22055, RCB22057, RCB16007, RCB17158, RCB17159, RCB18320, and RCB21055, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from RCB16007 and RCB17159, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is RCB16007 or a pharmaceutically acceptable salt thereof. Thus, in one aspect, the presently disclosed subject matter is directed to methods of administering the compounds of the presently disclosed subject matter or pharmaceutical compositions thereof to a subject. Pharmaceutical compositions comprising the present compounds are administered to a subject in need thereof by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal approaches.

In accordance with one embodiment, a method for treating a subject in need of treatment is provided. The method comprises administering a pharmaceutical composition comprising at least one compound of the presently disclosed subject matter (e.g., a compound of Formula (I) or (II)) to a subject in need thereof. Compositions provided by the methods of the presently disclosed subject matter can be administered with known compounds or other medications as well.

The pharmaceutical compositions useful for practicing the presently disclosed subject matter may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.

The presently disclosed subject matter encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of the diseases and disorders disclosed herein as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

For in vivo applications, the compositions of the presently disclosed subject matter may comprise a pharmaceutically acceptable salt. Suitable acids which are capable of forming such salts with the compounds of the presently disclosed subject matter include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are in some embodiments sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical compositions according to the presently disclosed subject matter can be prepared in a manner fully within the skill of the art.

The compositions of the presently disclosed subject matter or pharmaceutical compositions comprising these compositions can be administered so that the compositions can have a physiological effect. Administration can occur enterally or parenterally; for example, orally, rectally, intraci stemally, intravaginally, intraperitoneally, locally (e.g., with powders, ointments or drops), or as a buccal or nasal spray or aerosol. Parenteral administration is an approach. Particular parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection, subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, for example by a catheter or other placement device.

Where the administration of the composition is by injection or direct application, the injection or direct application can be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion can be a single sustained dose over a prolonged period of time or multiple infusions.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition of the presently disclosed subject matter can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the presently disclosed subject matter may further comprise one or more additional pharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceutical composition of the presently disclosed subject matter may be made using conventional technology. As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.

Other components such as preservatives, antioxidants, surfactants, absorption enhancers, viscosity enhancers or film forming polymers, bulking agents, diluents, coloring agents, flavoring agents, pH modifiers, sweeteners or taste-masking agents may also be incorporated into the composition. Suitable coloring agents include red, black, and yellow iron oxides and FD&C dyes such as FD&C Blue No. 2, FD&C Red No. 40, and the like. Suitable flavoring agents include mint, raspberry, licorice, orange, lemon, grapefruit, caramel, vanilla, cherry grape flavors, combinations thereof, and the like. Suitable pH modifiers include citric acid, tartaric acid, phosphoric acid, hydrochloric acid, maleic acid, sodium hydroxide, and the like. Suitable sweeteners include aspartame, acesulfame K, thaumatic, and the like. Suitable taste-masking agents include sodium bicarbonate, ion-exchange resins, cyclodextrin inclusion compounds, adsorbates, and the like.

Typically, dosages of the compound of the presently disclosed subject matter which may be administered to an animal, in some embodiments a human, range in amount from 1 pg to about 100 g per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal, the size of the animal, the gender of the animal, the condition of the animal, and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. In another aspect, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The compositions may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.

Suitable preparations include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation can also be emulsified, or the compositions encapsulated in liposomes. The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants.

Various aspects and embodiments of the presently disclosed subject matter are described in further detail below.

The compounds of the presently disclosed subject matter may be administered to, for example, a cell, a tissue, or a subject by any of several methods described herein and by others which are known to those of skill in the art.

V. COMPOUNDS

In some embodiments, the presently disclosed subject matter provides a novel compound of Formula (I) or (II). In some embodiments, the compound is selected from the group comprising RCB14103, RCB16008, RCB16025, RCB16036, RCB16086, RCB16178, RCB16185, RCB17017, RCB17019, RCB17024,

RCB17099, RCB17150, RCB17153, RCB17154, RCB17160, RCB17162,

RCB17166, RCB22055, RCB22056, and RCB22057, or a pharmaceutically acceptable salt thereof. See Example 2, below. In some embodiments, the compound is selected from the group comprising RCB17024, RCB17099, RCB17150, RCB17154, RCB17162, RCB22055, and RCB22057, or a pharmaceutically acceptable salt thereof.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

EXAMPLE 1

Synthesis and General Methods

Data Curation

The curation of yellow fever inhibition data was previously described [29], Additional inhibition data was downloaded from the chemical database from the European Molecular Biology Laboratory (ChEMBL), (CHEMBL375): ChEMBL ID CHEMBL613731 (yellow fever virus), ECso only. This data was combined with the previously curated data and was then sanitized using proprietary software (designated “E-Clean”) which uses open-source RD-Kit tools to remove duplicate compounds, salts, and neutralize charges. There was significant overlap between these datasets. Prior to combining, each compound was binarized at the model threshold and only data that had an 80% classification agreement was retained. Based on this, there was some variation in the final dataset sizes based on threshold. Datasets were further standardized within the latest version of software sold under the tradename ASSAY CENTRAL® (Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina, United States of America) which uses the Indigo Toolkit [32], Machine Learning

Briefly, regression and classification models were built from the existing literature using several machine learning algorithms and were validated using 5-fold cross validation. More particularly, software sold under the tradename ASSAY CENTRAL® (Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina, United States of America) was used to generate multiple machine learning algorithms to build classification and regression models with ECFP6 descriptors as described previously [33], The algorithms used included Bernoulli naive Bayes, Linear Logistic Regression, AdaBoost Decision Tree, Random Forest, Support Vector Machine, Deep Neural Networks and XGBoost. Machine learning model validation was performed using a nested 5-fold cross validation. Nested 5-fold cross validation initially selects a random, stratified 20% hold out set that is removed from the training set prior to model building. The model is then built with the other 80% of the training data and the hyperparameters (if applicable) are optimized using a grid search using 5-fold dataset splits (20% validation sets). This model is then used to predict the initial 20% hold out set and this process is then repeated until all compounds have been in a hold-out set (total 20 models trained). The final nested 5-fold cross validation scores are an average of each of the hold-out set metrics. Due to its high computational requirement deep learning (DL) uses a 20% leave out set instead. t-SNE Visualization

T-distributed stochastic neighbor embedding (t-SNE) [34] plots embed data into a lower-dimensional space. For each compound, 1024-bit extended-connectivity fingerprint 6 (ECFP6) fingerprints were generated and embedded into a 2- dimensional vector using t-SNE. All t-SNE values were generated using the scikit- leam library in python with default hyperparameters (n components = 2, perplexity = 30, early exaggeration = 12.0, learning rate = 200, n_iter = 1000).

Synthetic Procedures

All reagents and solvents were purchased from commercial suppliers (AlfaAesar, Acros, Chimmed) and used without further purification. The ^JH and ¹³C spectra were recorded on a Bruker AC-300 (300 MHz, ¹ H) or a Bruker AC-200 (50 MHz, ¹³C) NMR spectrometer (Bruker Nano GmbH, Berlin, Germany). Chemical shifts were measured in deuterated dimethyl sulfoxide (DMSO-de) or deuterated chloroform (CDCh) using tetramethylsilane as an internal standard and reported as parts-per-million (ppm) values. The following abbreviations are used to indicate multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; brs, broad singlet; brm, broad multiplet. Mass spectra were recorded on a quadrupole mass spectrometer sold under the tradename Finnigan MAT INCOS™ 50 (Thermo Finnigan, San Jose, California, United States of America) (El, 70 eV) with direct injection. The purity of the final compounds was analyzed by analytical high- performance liquid chromatography (HPLC) on an HPLC system sold under the tradename ELUTE™ (Bruker Daltonik, Heidelberg, Germany) equipped with an Azura UVD 2. IS UV detector (Knauer, Berlin, Germany) with a wavelength at 254 nm and acquisition rate at 1 Hertz (Hz). The chromatographic separation was carried out on a column sold under the tradename ACQUITY® HSS T3 (2.1 x 100 mm, 1.3 pm, 100 A; Waters Corporation, Milford, Massachusetts, United States of America) at 30 °C, sample injection volume - 2.0 pL. A mobile phase consisting of 0.1 % formic acid in water (A), 0.1 % formic acid in acetonitrile (B) was programmed with gradient elution of 30-95% at a flow rate of 250 pL/min. Mass spectrometric detection was operated in the positive ion mode. Data were processed using Compass DataAnalysis 5.1 software (Bruker Daltonik, Heidelberg, Germany). All final compounds were > 95 % pure. Elemental analysis (% C, H, N) was performed on a EURO EA elemental analyzer (HEKAtech, Wegberg, Germany).

Melting points were determined on an Electrothermal 9001 melting point apparatus (Electrothermal, Basildon, United Kingdom) (10 °C per min) and were uncorrected. Merck KGaA silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany) were used for analytical thin-layer chromatography. Compound spots were visualized by a UV lamp. Column chromatography was performed using silica gel Merck 60 (70-230 mesh; Merck KGaA, Darmstadt, Germany). Yields refer to purified products and were not optimized.

Synthesis of compounds RCB16007, RCB17152, RCB17158, RCB17159, RCB18320, RCB20108, RCB20116, RCB21055, RCB21065, RCB21066 and their physicochemical properties were described previously [35],

Additional compounds were prepared as shown in Scheme 1 or Scheme 2 in Example 2, below.

Yellow Fever Virus Inhibition Assay in Huh7 Cells

YFV inhibition assays were performed through the National Institute of Allergy and Infectious Diseases (NIAID) In Vitro Assessment for Antimicrobial Activity program and are detailed in the supporting information. Briefly, compounds were tested for cytopathic effect against YFV 19D-infected Huh7 cells. Four- concentration assays were performed using Infergen as a positive control and visualized using neutral red dye. EC50 and CC50 values were obtained by linear regression analysis and the selectivity index (SI) was calculated by dividing the EC50 by the CC50.

More particularly, for primary testing, four-concentration cytopathic effect (CPE) inhibition assays were performed. Confluent or near-confluent cell culture monolayers in 96-well disposable microplates were prepared. Huh7 cells were maintained in MEM or DMEM supplemented with FBS as required for each cell line. The test compound was prepared at four logio final concentrations, 0.1, 1.0, 10, and 100 pM. Controls for the experiment consisted of six microwells that were infected (virus controls) and six that were untreated (cell controls). In parallel, Infergen was tested as a positive control drug using the same method applied for test compounds. The assay was initiated by first removing growth media from the 96-well plates of cells. Afterwards, the test compound was applied in 0.1 mL volume to wells at 2X concentration. The YFV 17D strain, at <100 50% cell culture infectious doses (CCIDso) in 0.1 mL volume, was placed in those wells designated for virus infection. Medium devoid of virus was placed in toxicity control wells and cell control wells. Virus control wells were treated similarly with virus. Plates were incubated at 37 °C with 5% CO2 until maximum CPE was observed microscopically in virus control wells. The plates were then stained with 0.011% neutral red for approximately two hours at 37 °C in a 5% CO2 incubator. The neutral red medium was removed by complete aspiration, and the cells rinsed IX with phosphate buffered solution (PBS) to remove residual dye. The PBS was completely removed, and the incorporated neutral red was eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well was quantified using a 96-well spectrophotometer at 540 nm wavelength. The dye content in each set of wells was converted to a percentage of dye present in untreated control wells. Both EC50 and CC50 concentrations were calculated by linear regression analysis of the data obtained. The quotient of CC50 divided by EC50 gives the selectivity index (SI) value.

For secondary testing, the methodology to calculate IC50 was identical except eight half-logio concentrations of inhibitor were used to test antiviral activity and cytotoxicity. The secondary assay was run independently of the primary test by using fresh cells, fresh culture medium, freshly prepared virus (from frozen stock), and newly prepared compound dilutions. After sufficient virus replication occurred, a sample of supernatant was taken from each infected well (three replicate wells are pooled) and frozen for the virus yield reduction (VYR) portion of this test. The VYR test is a direct determination of the concentration of the test compound that inhibits virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. Titration of pooled viral sample is performed by endpoint dilution. This is accomplished by titrating logio dilutions of virus using 3 microwells per dilution on fresh monolayers of cells in 96- well plates. Wells are scored for presence (+) or absence (-) of virus after distinct CPE is observed. Plotting the inhibitor concentration versus logio of virus produced at each concentration allows calculation of the 90% (one logio reduction) effective concentration by linear regression. Dividing EC90 by the CC50 gives the SI90 value for this test.

In Vitro ADME/T ox Assays

In vitro ADME studies were performed by BioDuro (San Diego, California, United States of America). Studies were performed using standard methods as described below. Briefly, kinetic solubility was calculated from the concentration of each compound in Universal Aqueous buffer with reference to a standard curve. Caco-2 permeability was calculated by treating pre-incubated Caco-2 cells with each compound and measuring the change in concentration between the apical and basolateral sides of the cells. Human CYP inhibition was measured by LCMS after treating each of the compounds with human liver microsome solution. Mouse/human liver microsome stability and clearance were determined by LCMS analysis of each compound after treatment with liver microsome solution. Plasma protein binding was determined by equilibrium dialysis using LCMS to measure concentration. Cytotoxicity was determined by treating Huh-7D12 and THP-1 cells with each compound and cell viability after 72 hours was assessed by resazurin fluorescence. Mutagenicity was evaluated using Escherichia coli PQ37 [36] using 4-nitroquinoline A-oxide as a positive control. Pharmacokinetic studies were performed using male Balb/C mice blood, brain, and liver samples collected up to 24 hours after intragastric dosing of each compound.

Bioanalytical Method for In Vitro ADME Studies:

Test compounds were analyzed by reverse phase HPLC with a Kinetex Cl 8 100A column (3.0 mm x 50 mm, 2.6 pm; Phenomenex (Torrance, California, United States of America)) using a Shimadzu LC-20AD system (Shimadzu Corporation, Kyoto, Japan). The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The MS detection was performed using a mass spectrometer system sold under the tradename API 4000 Q TRAP® (AB SCIEX PTE. LTD, Singapore). The amount of parent compound was determined on the basis of the peak area ratio (compound area to internal standard area).

Kinetic Solubility

Universal Aqueous buffer (396 pL, pH 7.4) was added to a 50 mM DMSO stock solution of each compound (4 pL). Wells were agitated at 20 °C for 4 h and then filtered. The compound was then diluted to serial concentrations with DMSO, followed by serial dilutions with acetonitrile:H2O (1 : 1) prior to LC-MS/MS analysis. The calculated concentration of soluble compound was determined in reference to a standard curve.

Caco-2 Permeability

Caco-2 cells (American Type Culture Collection (ATCC), Manassas, Virginia, United States of America) were grown on 24-well (pore size: 0.4 pm) polycarbonate filters. The monolayers were pre-incubated with pre-warmed HBSS (Hank’s balanced salt solution) containing 2.5% HEPES buffer (pH 7.4) at 37 °C for 0.5 h. After pre-incubation, the buffer was removed, and the experimental compounds were added to reach a final concentration of 10 pM. 2% bovine serum albumin (BSA) was added to the receiver buffer for the study. The total volume was 400 pL for the apical (A) side and 1200 pL for the basolateral (B) side. For apical to basolateral transport study (A-B), 100 pL each was collected from both sides for sample analysis at the start of the assay and then 200 pL was collected from the apical side at 90 minutes (end of the study). The same timepoints and amounts were used for the basolateral to apical transport study (B-A).

The apparent permeability coefficient (Papp) was calculated from the following equation.

P / / \ v dC/dt

Papp (cm / sec) = - - —

A » C

Where: v = Volume of the receiver cell

A = Exposed surface area (0.64 cm²)

C = Initial donor concentration

5C/5Z ₌

j_n receiver concentration over time

Human CYP Inhibition

Human liver microsome solution (0.2 mg/mL final concentration) (Sekisui Xenotech, Kansas City, Kansas, United States of America), along with substrate, was aliquoted into a 0.05 M phosphate buffer (pH 7.4) in 1.1 mL tubes. Study samples (containing either control inhibitor or test compound) were into added into the tubes, vortexed gently and pre-incubated for 5 min at 37 °C. 20 pL of NADPH solution was aliquoted into all tubes, then vortexed to start the reaction and to assure adequate mixture of the NADPH. After mixing, the tubes were incubated for 20 min at 37 °C in a shaking water bath and then quenched in 300 mL formic acid/acetonitrile solution. After quenching the samples were vortexed vigorously for 1 min and centrifuged at 4 °C at 4,000 rpm for 15 min. Supernatant (100 pL) was transferred to 0.65 mL tubes, for LCMS analysis by the bioanalytical method described earlier. The CYP450 substrates and control inhibitors for each enzyme were as follows: CYP1 A2 (phenacetin, naphthoflavone), CYP2C9 (diclofenac, sulfaphenazole), CYP2C19 (omeprazole, tranylcypromine), CYP2D6 (dextromethorphan, quinidine), CYP3A4 (midazolam, ketoconazole).

Mouse/Human Liver Microsome Stability

The liver microsome solution (197.5 pL, 1.27 mg/mL protein concentration) (Sekisui Xenotech, Kansas City, Kansas, United States of America) was aliquoted into 1.1 mL tubes, to which 2.5 pL of positive control and compound stock solutions (100 pM in DMSO) were added. The tubes were vortexed gently, pre-incubated at 37 °C for 5 min, then 50 pL of 5 mM NADPH or LM buffer (no NADPH buffer) was added into the tubes. For analysis, an aliquot of 15 pL was removed from each tube at 0, 5, 15, 30 and 60 min (without-NADPH reaction: 0, 30 and 60 min) and quenched with 300 pL of 25 ng/mL propranolol in acetonitrile. Samples were vigorously vortexed for 1 min and then centrifuged at 4,000 rpm for 15 min at 4°C. 100 pL of supernatant from each sample was transferred to 0.65 mL tubes for LCMS analysis. The amount of parent compound was determined on the basis of the peak area ratio (compound area to IS area) for each time point. Clearance rates were calculated by the equation:

CLint (pL/min/mg protein) = Ln (2)* 1000 /T1/2/ Protein Cone.

Plasma Protein Binding

The donor side of dialysis inserts were filled with 200 pL plasma (human and mouse; source BioDuro, San Diego, California, United States of America) containing 5 pM drug and 0.5% of DMSO and the receiver side of the dialysis inserts was filled with 350 pL of PBS buffer (100 mM, pH 7.4). The prepared dialysis apparatus was placed in a shaker at 37 °C at 100 rpm for 5 h. Two tubes with plasma containing 5 pM experimental compound were also prepared for stability test, one tube was placed in the freezer at 4 °C for 5 h, and the other tube was placed in shaker at 3 °C at 100 rpm for 5 h. Samples were collected from the donor and receiver sides of each dialysis insert. The same volume of blank plasma was added to buffer samples and blank buffer to plasma samples to make sure all sample mixtures contain 50% plasma and 50% buffer. Each sample (50 pL) was mixed with acetonitrile (300 pL) containing 25 ng/mL internal standard (propranolol). All samples were vortexed for 1 min and then centrifuged at 4 °C at 4000 rpm for 15 min. The supernatant (100 pL) was transferred to 0.65 mL tube for LC-MS analysis. The amount of compound was determined on the basis of the peak area ratio (compound area to internal standard area) for the two sides, and protein binding is determined using the following equations: %Bound = 100 x([Area Ratio of Donor] 5h *5 - [Area Ratio of Receiver] 5h) / ([Area Ratio of Donor] 5h *5). The percentage remaining at 37 °C after 5 h was calculated on the basis of the amount measured at 0 °C after 5 h.

Cytotoxicity Determination

The human hepatoma cell line Huh-7D12 and human monocytic leukemia cell line THP-1 were obtained from the EC ACC (European Collection of Authenticated Cell Cultures, Porton Down, United Kingdom) and were maintained in DMEM medium (Huh-7D12) and RPMI (THP-1) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in 5.5% CO2. Continuous cultures were maintained by sub-culturing flasks every 7 days at 2.2 x 10⁶ cells/75 cm² flask by trypsination. For the experiments, cells were cultured in FBS-free medium and Huh-7D12 cells and THP-1 were seeded into 96-well plates in 200 pL of complete culture medium at a final concentration of 5 x 10⁴ cells/well and treated by the experimental compound for 72 h. Redox status was estimated using resazurin reduction (0.025% [wt/vol] to 1/10 of well volume). The resazurin assay was performed with a fluorimetric method according to a previously described procedure [32], Three hours before the end of incubation, 10 pL resazurin/well were added, yielding a final concentration of 10% resazurin. Plates were returned to the incubator and the fluorescence was read after 6 h. The plates were exposed to an excitation - 46 -avelength of 560 nm and emission at 590 nm was recorded on a plate reader sold under the tradename INFINITE® 200 PRO (TECAN Life Sciences, Tecan, Mannedorf, Switzerland). The percent viability was expressed as fluorescence emitted by treated cells compared to control (medium or vehicle only).

Mutagenicity Evaluation

Escherichia coli PQ37 [36] was developed from E. coli K-12 by means of a sfiA .lacZ operon fusion in which the [3-galactosidase gene lacZ is placed under the control of sfiA, one of the SOS genes. In addition to this operon fusion, there is a deletion in the lac region so that the activity of ^-galactosidase is entirely dependent upon the expression of sfiA. The construction of this bacterial strain was previously described [36], Escherichia coli PQ37 strain was grown at 37 °C overnight, with shaking, in Luria Broth Base supplemented with 50 pg/mL Ampicillin. Bacterial were grown to mid-logarithmic phase and adjusted to an optical density of 0.4. The second day, Luria Broth Base supplemented with 1.5% Agar was autoclaved at 121 °C for 15 minutes and then put into a water bath at 55 °C for 1 h. Warm agar (80 mL) was transferred into a plastic bottle (Milian SA, Geneva, Switzerland; PETG 2019-0125) supplemented with 50 mg/mL ampicillin and 0.005% X-Gal (dissolved into DMF). E. coli PQ37 (4 mL) was added to the mixture and 70 mL of it was transferred into a square petri dish (Grenier Bio-One, Frickenhausen, Germany; 120 x 120 mm). Petri dish containing the mixture was dried 15 minutes at RT and once the mixture became solid, 6 mm absorbent disks were stuck on the plate. Each compound (5 pL) was added on the disk with a pipette sold under the tradename PIPETMAN® P20 (Gilson S.A.S., Villiers-Le-Bel, France) and the petri dish was incubated at 37 °C overnight. 4-Nitroquinoline A-oxide (4NQO, Sigma-Aldrich, St. Louis, Missouri, United States of America) was used as a positive control. The third day, zones of inhibition were recorded with a ruler and a picture of the plate was taken with a Canon 8800F scanner (Canon U.S.A., Melville, New York, United States of America). Zone of inhibition was calculated by eye using a ruler and presence/absence of blue halo was reported. In Vivo Acute Toxicity Study

Fifteen five-to-six-weeks-old male Balb/C mice weighing 20.0±0.5 g were raised in the laboratory animal nursery of the Central Institute for Tuberculosis (CIT). Prior to the experiment, the animals were fed standard dry pellets and water ad libitum. The animals were deprived of food and water for 1.5-2 h before compound administration. Animals were randomly divided into three groups (n = 5). Compound RCB16007 was administered intragastrically at doses of 40, 80, 120 mg per mouse as a 10% Cremophor EL solution (Sigma-Aldrich, St. Louis, Missouri, United States of America). All animals were observed for any signs of toxicity for 14 days. Gross pathological changes were not recorded.

Pharmacokinetics Study

Study design'. An additional twenty-four five-to-six-weeks-old male Balb/C mice weighing 20.0±0.5 g from the CIT were randomly divided into three groups (n = 5). Prior to the experiment, the animals were fed standard dry pellets and water ad libitum. The animals were deprived of food with free access to water for 1 h before and after compound administration. Compound RCB 16007 was administered intragastrically at a dose of 250 mg/kg of body weight at a volume of 0.5 mL of compound suspension in 1% CMC and 0.05% Tween 80. The animals were euthanized by decapitation for blood and brain sampling. Blood, brain, and liver samples (3 animals per time point) were taken at 0.5, 1, 2, 3, 5, 8 and 24 h after administration for pharmacokinetic evaluation. Blood was collected in heparinized tubes and centrifuged at 3500 RPM. Plasma was separated from formed elements and immediately frozen at -20 °C in freezer. This storage continued before transferring of plasma for analysis. Mice brains were immediately frozen at -120 °C. Details for tissue-specific separation are given in the below.

Sample preparation'. Acetonitrile (300 pL) was added to the plasma (100 pL), vortexed for 30 sec, and then centrifuged at 13,400 rpm for 5 min. The supernatant (300 pL) was removed, dried using a vacuum concentrator and stored at -80 °C. For analysis, the supernatant was reconstituted in 150 pL of 5% acetonitrile. For brain separation, samples of brain were dispersed with water (1 : 1 w/w) for 3 minutes, 200 mg of resulting mixture (100 mg of brain) was balanced into a fresh container, treated by zirconium balls in vortex mixer for 1 min, 400 pL of I was added, mixed for 30 sec and centrifuged at 13400 rpm for 5 min, 400 pL of supernatant removed, dried using vacuum concentrator and stored at -80 °C. Reconstituted in 300 pL of 50% ACN and filtered through 0.2 pm PVDF membrane filter for LC-MS analysis. 20 pL injection.

HPLC-MS analysis conditions'. Compound concentration in plasma was determined using an HPLC system sold under the tradename ELUTE™ UPLC (Bruker Daltonik, Germany) equipped with an Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany) with Apollo II ESI ion source (Bruker Daltonik, Germany). The chromatographic separation was performed at 40 °C on Intensity solo Cl 8-2 (2.1 * 100 mm, 1.8 pm) reverse phase column (Bruker Daltonik, Germany) with the following conditions: gradient elution at 0.3 mL/min from 5% to 95% B in 8 min (A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile), ion source in positive mode, HV capillary at 4.5 kV, spray gas - nitrogen at 2.0 bar, dry gas - nitrogen at 6 L/min 220 °C, full spectra scan range m/z 50-1500 at 3 Hz scan rate, automatic internal calibration with sodium trifluoroacetate solution. The sample at a volume of 10 pL was injected into the HPLC-MS/MS system. The target compound peak area was measured by automatic integration of EIC chromatogram (m/z 476.0691±0.01). The data were processed with Compass DataAnalysis 5.1 (Bruker Daltonik, Germany). Plasma concentration-time data were analyzed by a non-compartment model using an open-source software [34],

EXAMPLE 2

Synthesis of Target Compounds

The target compounds were synthesized according to Schemes 1 and 2, below. The key intermediate A-cyano-A-substituted phenylcarbamimidothioic acid methyl esters 1 can be synthesized by two different methods, as shown in Scheme 1. Method A is a two-step process involving the preparation of aryl thiocyanates followed by treatment with sodium ethoxide and cyanamide. Method B consists of a direct reaction of the corresponding aniline with dimethyl cyanocarbonimidodithioate. The A-cyano-A-substituted phenylcarbamimidothioic acid methyl esters 1 were then reacted with hydrazine hydrate to cyclize into a 1,2,4-triazole ring. Treatment of the corresponding substituted phenyl- 1A-1, 2, 4-triazole-3,5-diamines 2 with various arylsulfonyl chlorides afforded the final triazoles 3. In some cases, another isomer was also isolated by column chromatography (compound RCB17160). According to Scheme 2, the intermediate 4-cyano-5-amino-3-(phenylamino)-lA-pyrazoles 5 were prepared in situ after the treatment of the aniline and malononitrile reaction mixture with hydrazine hydrate. Subsequent treatment of 5 with naphthalenesulfonyl chloride resulted in the target pyrazoles 6.

Scheme 1. Synthesis of l-sulfonyl-3-amino-lA-l,2,4-triazoles 3 and 4^a ^a(a) dimethiocarbamoyl chloride, toluene or benzene, reflux; (b) NaOEt, NH2CN, Mel, EtOH, reflux; (c) EtOH, reflux; (d) hydrazine hydrate, EtOH, reflul(e) PI1SO2CI or NapSChCl, pyridine, rt

Scheme 2. Synthesis of 2-sulfonyl-4-cyano- l//-pyrazoles 6^a ^a(a) EtOH, reflux; (b) hydrazine hydrate, EtOH, reflux; (c) NapSO2Cl, pyridine, rt Synthesis of A’-cyano-A-substituted phenylcarbamimidothioic acid methyl esters 1: Method A : a) Dimethylthiocarbamoyl chloride (1.0-1.1 equiv) was added to a solution of the corresponding aniline (1.0 equiv) in dry toluene or benzene, and the reaction mixture was stirred at reflux for 2-3 h. The mixture was then cooled to room temperature, treated with hexane, and the resulting precipitate was filtered off. The organic solution was evaporated in vacuo, and the corresponding isothiocyanatobenzene was obtained and used without further purification. b) A mixture of sodium ethoxide (1.0-1.2 equiv) and cyanamide (1.0 equiv) in ethanol (20 mL) was stirred at room temperature for 30-40 min. The corresponding isothiocyanatobenzene from the previous step (1.0-1.2 equiv) was then added to the mixture. After stirring at room temperature for additional 1.5 h, iodomethane (2.0-2.5 equiv) was added to the mixture, and the reaction mixture was stirred at reflux for 1- 2 h and at room temperature overnight. The resulting precipitate was filtered and dried to give the corresponding phenylcarbamimidothioic acid methyl esters 1.

Method B: c) A mixture of the corresponding aniline (1.0-1.2 equiv) and dimethyl cyanodithioiminocarbonate (1.0 equiv) in EtOH (10 mL) was stirred at reflux for 3-4 h. The reaction mixture was then cooled, and the precipitate formed was filtered, washed with hexane and recrystallized from ethanol to give the corresponding phenylcarbamimidothioic acid methyl esters 1.

Synthesis of /'/’-substituted phenyl- l//- l ,2,4-triazole-3,5-di amines 2

A solution of hydrazine hydrate (3.0-5.0 evq) in water (50 mL) was added to a solution of phenylcarbamimidothioic acid methyl esters 1 (1.0-1.5 evq) in ethanol (30 mL), and the reaction mixture was stirred at 70 °C for 3-4 h. The mixture was then cooled to room temperature and poured in ice water. The precipitate formed was filtered, dried and recrystallized from ethanol to give the corresponding 1 H- 1,2,4- triazole-3,5-diamine 2.

Synthesis of l-sulfonyl-3-amino-U/-l,2,4-triazoles 3 and 4

Substituted benzenesulfonyl chloride or naphthalenesulfonyl chloride (1.0-1.2 equiv) was added to the suspension of the corresponding phenyl- H- 1,2, 4-triazole- 3,5-diamine 2 (1.0 equiv) in pyridine (5 mL), and the reaction mixture was stirred at room temperature overnight (12 h). The mixture was then diluted with water and stored in the fridge at 4°C for 6-24 h. The resulting precipitate was filtered and purified as indicated below to give the corresponding final product.

1 -((4-F1 uorophenyl )sulfonyl )-A³-(naphthal en- 1 -yl)- 1H- 1 ,2,4-triazole-3 ,5-diamine

RCB14103

Yield 49 %, mp 188-90 °C (EtOH). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.44 (s, 2H, NH₂), 7.37-7.65 (m, 4H, HC(3",4",6",7")), 7.79 (d, 1H, HC(5 "), J = 6.8), 7.87 (dd, 1H, HC(8 "), J= 7.0, 1.8), 8.04 (dd, 2H, HC(2',6 ), J= 8.9, 5.1), 8.15 (dd, 1H, HC(2 "), J = 7.4, 1.8), 9.07 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 116.07 (C(2")), 117.08 (d, C(3',5'), J= 22.2), 122.16 (C(8")), 122.45 (C(4")),

125.16 (C(8a")), 125.79 (C(3",6",7")), 127.96 (C(5")), 130.70 (d, C((2',6'), J = 10.0), 132.28 (d, C(l'), J = 3.0), 133.71 (C(4a")), 135.71 (C(l")), 157.71 (C(5)),

161.16 (C(3)), 165.50 (d, C(4'), J = 253.0). MS (El): m/z 383. Anal. Calcd for C18H14FN5O2S: C, 56.39; H, 3.68; N, 18.27. Found: C, 56.47; H, 3.74; N, 18.24. Y-(4-Fluorophenyl )- l -(naphthal en-2-yl sulfonyl)-! 77- 1 , 2, 4-triazole-3,5-di amine

RCB16008

Yield 42 %, mp 235-7 °C (EtOH/DMF). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.08 (t, 2H, HC(3",5"), J= 8.9), 7.45 (m, 4H, NH₂, HC(2",6")), 7.60-7.80 (m, 2H, HC(6',7')), 7.90 (dd, 1H, HC(8 ), J = 8.7, 1.7), 8.05 (d, 1H, HC(3 ), J = 7.2), 8.19 (d, 1H, HC(5'), J = 9.2), 8.24 (d, 1H, HC(4 ), J= 7.1), 8.74 (s, 1H, HC(1')), 9.21 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 115.10 (d, J= 22.0), 117.93 (d, J = 7.5), 121.79, 127.95, 128.10, 129.47, 129.52, 129.77, 129.89, 131.37, 132.92, 134.99, 137.03 (d, J= 2.0), 156.31 (d, J= 235.0), 157.48, 159.75. MS (El): m/z 383. Anal. Calcd for C18H14FN5O2S: C, 56.39; H, 3.68; N, 18.27. Found: C, 56.45; H, 3.73; N, 18.23. l-((4-Fluorophenyl)sulfonyl)-A³-( >-tolyl)-lZ7-l,2,4-triazole-3,5-diamine RCB16025

Yield 26 %, mp 184-86 °C (column, hexane:ethyl acetate 1 : 1). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 2.21 (s, 3H, CH₃), 7.04 (d, 2H, HC(3",5"), J= 8.4), 7.34 (d, , 2H, HC(2",6"), J = 8.5), 7.38 (s, 2H, NH2), 7.53 (t, 2H, HC(3',5 ), J = 8.8), 7.90-8.12 (dd, 2H, HC(2',6 ), J = 5.0, 8.9), 9.09 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 20.22, 116.67, 116.99 (d, J= 25.4), 128.86, 129.01, 130.62 (d, J = 10.5), 132.22 (d, J = 4.2), 138.07, 157.42, 160.00, 162.91, 165.44 (d, J = 252.0). MS (El): m/z 347. Anal. Calcd for C15H14FN5O2S: C, 51.87; H, 4.06; N, 20.16. Found: C, 51.95; H, 4.11; N, 20.20.

Y³-(/?-Tolyl)-l -tosyl- 1/7-1, 2, 4-triazole-3,5-diamine RCB16036

Yield

-de; 5, ppm; J, Hz): 2.21 (s, 3H, H₃C(C4 ")), 2.37 (s, 3H, H₃C(C4')), 7.03 (d, 2H, HC(3",5"), J = 8.4), 7.32 (s, 2H, NH₂), 7.34 (d, 2H, HC(2",6"), J= 8.2), 7.45 (d, 2H, HC(3',5 ), J= 8.3), 7.83 (d, 2H, HC(2',6 ), J= 8.3), 9.05 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 20.22, 21.07, 116.62, 127.34, 128.77, 128.98, 130.01, 133.07, 138.15, 145.63, 157.42, 159.82. MS (El): m/z 343. Anal. Calcd for C16H17N5O2S: C, 55.96; H, 4.99; N, 20.39. Found: C, 56.02; H, 5.04; N, 20.34.

A³-(4-Methoxyphenyl)- 1 -(naphthalen- 1 -ylsulfonyl)- 1H- 1 ,2,4-triazole-3 ,5-diamine

RCB16086

Yield 26 %, mp 201-2 °C (hexane:ethyl acetate). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 3.69 (s, 3H, CH₃), 6.80 (d, 2H, HC(3",5"), J = 9.0), 7.29 (d, 2H, HC(2",6"), J= 9.0), 7.40 (s, 2H, NH₂), 7.61-7.85 (m, 3H, HC(3',6',7')),8.11 (d, 1H, HC(5 ), J= 7.6), 8.38 (d, 1H, HC(4 ), J= 8.2), 8.43 (d, 1H, HC(8 ), J= 8.3), 8.88 (s, 1H, NH), 8.96 (d, 1H, HC(2 ), J = 8.5). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 56.77, 113.80, 117.82, 121.75, 122.16, 124.61, 124.83, 127.77, 128.97, 129.29, 132.00, 133.68, 133.92, 136.23, 136.49, 156.33, 159.15. MS (El): m/z 395. Anal. Calcd for C19H17N5O3S: C, 57.71; H, 4.33; N, 17.71. Found: C, 57.75; H, 4.26; N, 17.67.

A³-(3,4-Difluorophenyl)-l-(naphthalen-2 -ylsulfonyl)- 177-1, 2, 4-triazole-3,5-diamine

RCB16178

Yield 23 %, mp 228-30 °C (column, hexane:ethyl acetate 6:4). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.10-7.20 (m, 1H, HC(5 ")), 7.29 (q, 1H, HC(2 "), J= 9.3), 7.50 (s, 2H, NH2), 7.56 (dd, J = 7.3, 2.5 Hz, 1H, HC(6 ")), 7.65-7.82 (m, 2H, HC(6',7')), 7.89 (dd, 1H, HC(4 ), J= 9.0, 1.7), 8.06 (d, 1H, HC(5 ), J= 7.4), 8.19 (d, 1H, HC(3 ), J= 8.7), 8.23 (d, 1H, HC(8 ), J= 7.4), 8.74 (s, 1H, HC(l')), 9.44 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 105.17 (d, J= 22.1), 112.66 (dd, J= 2.6, 6.0), 117.31 (d, J = 18.0), 121.70, 127.97, 128.13, 129.52, 129.86, 129.94, 131.37, 132.92, 135.02, 137.77 (dd, J = 2.6, 9.1), 143.35 (dd, J= 13.0, 238.0), 148.88 (dd, J= 14.0, 252.0), 159.40. MS (El): m/z 401. Anal. Calcd for C18H13F2N5O2S: C, 53.86; H, 3.26; N, 17.45. Found: C, 53.91; H, 3.316; N, 17.39. l-((4-Butylphenyl)sulfonyl)-A³-(4-chlorophenyl)-U7-l,2,4-triazole-3,5-diamine

RCB16185

Yield 25 %, mp 156-8 °C (column hexane:ethyl acetate 6:4). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 0.85 (t, 3H, CH3, J = 7.2), 1.25 (h, 2H, H₂C(C3 "'), J = 7.2), 1.53 (p, 2H, H₂C(C2 "'), J= 8.1, 7.4), 2.64 (t, 2H, H₂Car, J= 7.6), 7.28 (d, 2H, HC(2 ",6 "), J= 8.9), 7.37 (s, 2H, NH2), 7.47 (d, 4H, HC(3',5',3",5"), J= 9.2), 7.86 (d, 2H, HC(2', 6'), J = 8.3), 9.36 (brs, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 13.58, 21.65, 32.34, 34.60, 118.06, 123.64, 127.42, 128.43, 129.42, 133.28, 139.59, 150.24, 157.31, 159.42. MS (El): m/z 405. Anal. Calcd for C18H20CIN5O2S: C, 53.26; H, 4.97; N, 17.25. Found: C, 53.32; H, 5.03; N, 17.29.

4-((5-Amino-l-(naphthalen-2-ylsulfonyl)-U7-l,2,4-triazol-3-yl)amino)benzonitrile

RCB17017

Yield 31 %, mp 228-9 °C (CHCh). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.55 (s, 2H, NH2), 7.58 (d, 2H, HC(2",6"), J= 8.8), 7.68 (d, 2H, HC(3",5"), J = 8.8), 7.72-7.81 (m, 2H, HC(6',7')), 7.90 (dd, 1H, HC(8 ), J = 8.7, 2.1), 8.05 (d, 1H, HC(3'), J = 7.3), 8.19 (d, 1H, HC(5 ), J= 9.0), 8.24 (d, 1H, HC(4 ), J= 7.9), 8.76 (s, 1H, HC(l')), 9.86 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 101.42, 116.62, 119.55, 121.69, 127.97, 128.16, 129.55, 129.92, 130.00, 131.35, 132.82, 133.17, 135.04, 144.65, 157.35, 159.00. MS (El): m/z 390. Anal. Calcd for C19H14N6O2S: C, 58.45; H, 3.61; N, 21.53. Found: C, 58.50; H, 3.67; N, 21.58.

3 -((5-Amino-l -(naphthal en-2-yl sulfonyl)- 1/7-1, 2, 4-triazol-3-yl)amino)benzonitrile

RCB17019

Yield 19 %, mp 237-9 °C (EtOH). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.27 (d, 1H, HC(6 "), J= 7.6), 7.44 (t, 1H, HC(5 "), J= 7.9), 7.54 (brs, 2H, NH2), 7.64-7.81 (m, 3H, HC(6',7',4")),7.83 (s, 1H, HC(2")), 7.90 (dd, 1H, HC(4 ), J= 8.8, 1.8), 8.06 (d, 1H, HC(5'), J= 7.3), 8.19 (d, 1H, HC(3 ), J= 8.3), 8.23 (d, 1H, HC(8 ), J = 8.6), 8.74 (d, 1H, HC(l'), J = 1.7), 9.65 (brs, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 111.40, 118.84, 119.03, 121.16, 121.72, 123.55, 127.98, 128.16, 129.52, 129.87, 129.98, 130.07, 131.37, 132.92, 135.04, 141.39, 157.41, 159.27. MS (El): m/z 390. Anal. Calcd for C19H14N6O2S: C, 58.45; H, 3.61; N, 21.53. Found: C, 58.52; H, 364; N, 21.48.

4-((5-Amino-l-(naphthalen-2-ylsulfonyl)-lH-l,2,4-triazol-3-yl)amino)benzoic acid ethyl ester RCB17024

Yield 36 %, mp 232-4 °C (CH3CN). ¹ H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 1.29 (t, 3H, CH₃, J= 7.1), 4.25 (q, 2H, CH2, J= 7.1), 7.53 (m, 4H, HC(3",5"), NH2), 7.65-7.74 (m, 2H, HC(6',7')), 7.84 (d, 2H, HC(2",6"), J= 8.8), 7.93 (dd, 1H, HC(8 ), J= 8.7, 1.8), 8.05 (d, 1H, HC(3 ), J= 7.2), 8.19 (d, 1H, HC(5 ), J= 9.0), 8.25 (d, 1H, HC(4 ), J= 7.4), 8.77 (s, 1H, HC(l')), 9.71 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO- de; 5, ppm): 14.21, 60.09, 115.91, 121.10, 121.79, 127.95, 128.13, 129.56, 129.86, 129.98, 130.29, 131.38, 132.86, 135.04, 144.89, 157.36, 159.21, 165.46. MS (El): m/z 437. Anal. Calcd for C21H19N5O4S: C, 57.66; H, 4.38; N, 16.01. Found: C, 57.71; H, 4.45; N, 16.05.

5-((5- Amino-3 -((4-chlorophenyl)amino)- 1H- 1 ,2,4-triazol- 1 -yl)sulfonyl)-2- naphthonitrile RCB17099

Yield 17 %, mp 215-7 °C (column, CHC13:MeOH 9: 1). *H NMR (200 MHz; DMSO- de; 5, ppm; J, Hz): 7.28 (d, 2H, HC(3",5"), J = 9.1), 7.38 (d, 2H, HC(2",6"), J = 9.1), 7.53 (s, 2H, NH2), 7.93 (t, 1H, HC(7 ), J= 7.9), 8.16 (d, 1H, HC(3 ), J = 8.9), 8.49 (d, 1H, HC(8 ), J= 8.0), 8.62 (d, 1H, HC(6 ), J= 7.5), 8.78 (s, 1H, HC(l')), 9.13 (d, 1H, HC(4 ), J= 9.1), 9.34 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 110.02, 117.95, 118.18, 123.76, 126.43, 126.61, 128.40, 128.54, 128.85, 132.18, 132.76, 133.46, 135.31, 136.88, 139.28, 156.44, 159.03. MS (El): m/z 424. Anal. Calcd for C₂iHi3ClN₆O₂S: C, 53.71; H, 3.08; N, 19.78. Found: C, 53.79; H, 3.13; N, 19.74.

5 -((5- Amino-3 -((4-fluorophenyl)amino)- 1H- 1 ,2,4-triazol- 1 -yl)sulfonyl)- 1 - naphthonitrile RCB17150

Yield 21 %, mp 228-30 °C (EtOH). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.08 (t, 2H, HC(3 ", 5"), J = 8.9), 7.38 (dd, 2H, HC(2",6"), J = 9.1, 4.8), 7.51 (s, 2H, NH₂), 7.91-8.10 (m, 2H, HC(3',7')), 8.35 (d, 1H, HC(2 ), J = 7.2), 8.53 (d, 1H, HC(6'), J= 8.5), 8.62 (d, 1H, HC(8 ), J= 7.5), 9.21 (s, 1H, NH), 9.38 (d, 1H, HC(4 ), J = 8.8). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 116.02, 121.15, 122.69, 123.60, 123.75, 130.75, 132.48, 133.52, 133.73, 133.78, 136.45, 137.79, 138.06, 138.27, 138.88, 140.48, 142.60, 159.84, 162.32, 163.29, 165.11. MS (El): m/z 408. Anal. Calcd for C19H13FN6O2S: C, 55.88; H, 3.21; N, 20.58. Found: C, 55.95; H, 3.26; N, 20.63.

5-((5-Amino-3-((4-fluorophenyl)amino)-lJ/-l,2,4-triazol-l-yl)sulfonyl)-2- naphthonitrile RCB17153

Yield 20 %, mp 213-5 °C (column, CHC1₃:CH₃OH 9: 1). 'HNMR (200 MHz; DMSO- de; 5, ppm; J, Hz): 7.08 (t, 2H, HC(3",5"), J= 8.9), 7.36 (dd, 2H, HC(2 ",6 "), J = 9.1, 4.7), 7.50 (brs, 2H, NH2), 7.93 (t, 1H, HC(7 ), J = 7.8), 8.14 (dd, 1H, HC(3 ), J = 9.1, 1.9), 8.50 (d, 1H, HC(8 ), J= 8.2), 8.61 (d, 1H, HC(6 ), J= 7.4), 8.78 (d, 1H, HC(l'), J= 1.8), 9.14 (d, 1H, HC(4 ), J= 9.0), 9.19 (s, 1H). ¹³C NMR (126 MHz; DMSO-de; 5, ppm): 110.55, 115.54 (d, J= 9.0), 118.40 (d, J= 2.5), 118.70, 121.15, 126.98, 127.12, 129.28, 129.86, 132.82, 133.29, 133.93, 135.80, 137.34, 156.86 (d, J = 224.0), 157.75, 159.80. MS (El): m/z 408. Anal. Calcd for Ci9Hi₃FNeO₂S: C, 55.88; H, 3.21; N, 20.58. Found: C, 55.94; H, 3.24; N, 20.51.

TV³ -(4-Chl oro-3 -methylphenyl)- 1 -(naphthalen-2-yl sulfonyl)- 1H-1 ,2,4-triazole-3 , 5 - diamine RCB17154

Yield 20 %, mp 218-20 °C (iPrOH:DMF). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 2.23 (s, 3H, CH₃), 7.24 (d, 1H, HC(5 "), J= 8.7), 7.32 (s, 1H, HC(2 ")), 7.34 (dd, 1H, HC(6 "), J = 2.5, 8.7), 7.46 (brs, 2H, NH₂), 7.70-7.84 (m, 2H, HC(6',7')),7.90 (dd, 1H, HC(4'), J= 8.7, 1.8), 8.05 (d, 1H, HC(5 ), J= 7.2), 8.18 (d, 1H, HC(3 ), J= 8.3), 8.23 (d, 1H, HC(8 ), J= 8.6), 8.74 (d, 1H, HC(l'), J= 1.7), 9.26 (brs, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 19.91, 115.79, 118.94, 121.81, 124.04, 127.95, 128.10, 128.84, 129.52, 129.77, 129.90, 131.36, 132.93, 135.01, 135.16, 139.50, 157.39, 159.54. MS (El): m/z 413. Anal. Calcd for C19H16CIN5O2S: C, 55.14; H, 3.90; N, 16.92. Found: C, 55.21; H, 3.98; N, 16.87. V⁵-(3,4-di chi orophenyl)-l -(naphthal en-2-yl sulfonyl)- 1/7-1, 2, 4-triazole-3,5-diamine

RCB17160

Yield 13 %, mp 223-5 °C (column, CHC1₃:CH₃OH 9: 1). 'HNMR (200 MHz; DMSO- de; 5, ppm; J, Hz): 6.11 (brs, 2H, NH₂), 7.60 (d, 1H, HC(5 "), J= 8.8), 7.66 (dd, 1H, HC(6 "), J= 2.0, 8.8), 7.73-7.85 (m, 2H, HC(6',7')), 8.05 (d, 1H, HC(2 "), J = 2.0), 8.07 (d, 1H, HC(5 ), J= 7.5), 8.16 (d, 1H, HC(3 ), J= 8.3), 8.20 (d, 1H, HC(8 ), J= 8.6), 8.64 (s, 1H, HC(l’)), 9.51 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 119.61, 120.52, 121.78, 124.52, 127.95, 128.10, 129.55, 129.82, 129.99, 130.50, 130.98, 131.35, 132.59, 135.03, 138.67, 154.39, 163.40. MS (El): m/z 434. Anal. Calcd for CisHuChNsChS: C, 49.78; H, 3.02; N, 16.13. Found: C, 49.85; H, 3.08; N, 16.07.

4-((5-Amino-l -(naphthal en-2-yl sulfonyl)- 1H- 1,2, 4-tri azol-3-yl)amino)-3- methylbenzonitrile RCB17162

Yield 34 %, mp 125-7 °C (EtOH). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 2.18 (s, 3H, CH₃), 7.46-7.62 (m, 4H, NH₂, HC(2",6")), 7.66-7.84 (m, 2H, HC(6',7')), 7.90 (dd, 1H, HC(4 ), J= 8.7, 1.8), 7.94 (d, 1H, HC(5 "), J= 8.3), 8.07 (d, 1H, HC(5 ), J= 7.2), 8.21 (d, 1H, HC(3 ), J= 8.3), 8.25 (d, 1H, HC(8 ), J= 8.2), 8.53 (s, 1H, NH), 8.75 (d, 1H, HC(l'), J= 1.7). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 17.48, 102.45, 118.09, 119.42, 121.70, 127.19, 127.98, 128.16, 129.58, 129.96 (2C), 130.67, 131.40, 132.94, 133.56, 135.07, 143.02, 157.42, 159.48. MS (El): m/z 404. Anal. Calcd for C₂oHi₆Ne0₂S: C, 59.39; H, 3.99; N, 20.78. Found: C, 59.45; H, 4.04; N, 20.71.

Y³-(4-Chlorophenyl)-l-((4-phenoxyphenyl)sulfonyl)- 1/7-1, 2, 4-tri azole-3,5-diamine RCB17166

Yield 21 %, mp 210-2 °C (CHC1₃, then CH₃CN). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.10-7.21 (m, 4H, HC(3',5',2"',6"')), 7.23-7.29 (m, 3H, HC(3"',4"',5"')),7.30 (s, 2H, NH₂), 7.40 (d, 2H, HC(3",5"), J= 9.0), 7.44-7.51 (m, 4H, HC(2",6")), 7.95 (d, 2H, HC(2',6 ), J = 8.8), 9.39 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 117.38, 118.08, 120.54, 123.64, 125.42, 128.45, 129.35, 130.19, 130.44, 139.59, 154.02, 157.22, 159.42, 162.51. MS (El): m/z 441. Anal. Calcd for C₂oHieClN₅0₃S: C, 54.36; H, 3.65; N, 15.85. Found: C, 54.41; H, 3.70; N, 15.88.

4-(( 1 -((6- Acetylnaphthal en- 1 -yl)sulfonyl)-5-amino- 1H- 1 ,2,4-triazol-3 -yl)amino)-2- chlorobenzonitrile RCB22055

Yield 36 %, mp 270-3 °C (column, CHC1₃:CH₃OH 9: 1). 'HNMR (200 MHz; DMSO- de; 5, ppm; J, Hz): 2.70 (s, 3H, CH₃), 7.36 (dd, 1H, HC(6 "), J= 8.7, 2.2), 7.67 (s, 2H, NH₂), 7.73 (d, 1H, HC(2 "), J = 2.2), 7.75 (d, 1H, HC(5 "), J = 8.7), 7.88 (t, 1H, HC(3'), J = 7.9), 8.22 (dd, 1H, HC(7 ), J= 9.1, 1.8), 8.60 (d, 2H, HC(2',4'), J= 7.7), 8.83 (d, 1H, HC(5 ), J= 1.7), 8.99 (d, 1H, HC(8 ), J= 9.0), 10.07 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 26.79, 101.84, 115.44, 116.22, 116.73, 124.80, 125.82, 126.31, 129.78, 131.14, 131.80, 133.14, 133.40, 134.92 (2C), 136.08, 138.25, 145.69, 156.45, 158.06, 197.45. MS (El): m/z 466. Anal. Calcd for C2iHi₅ClN₆O₃S: C, 54.02; H, 3.24; N, 18.00. Found: C, 54.11; H, 3.31; N, 18.06.

4-(( 1 -((6- Acetylnaphthal en- 1 -yl)sulfonyl)-5-amino- 1H- 1 ,2,4-triazol-3 - yl)amino)benzonitrile RCB22056

Yield 24 %, mp 210-5 °C (column, CHC1₃:CH₃OH 9: 1). 'HNMR (200 MHz; DMSO- de; 5, ppm; J, Hz): 2.70 (s, 3H, CH₃), 3.89 (s, 2H, CH₂), 7.16 (d, 2H, HC(2",6"), J = 8.4), 7.36 (d, 2H, HC(3",5"), J= 8.4), 7.52 (brs, 2H, NH₂), 7.87 (t, 1H, HC(3 ), J= 7.9), 8.19 (dd, 1H, HC(7 ), J= 9.1, 1.8), 8.57 (d, 2H, HC(2',4'), J= 7.8), 8.82 (d, 1H, HC(5 ), J= 1.8), 9.03 (d, 1H, HC(8 ), J= 9.1), 9.23 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 21.68, 26.80, 116.88, 119.52, 122.52, 125.26, 125.77, 126.03, 128.37, 129.89, 130.99, 132.08, 132.95, 133.35, 134.83, 137.93, 139.93, 156.51, 159.09, 197.51. MS (El): m/z 446. Anal. Calcd for C₂₂HI₈N₆O₃S: C, 59.18; H, 4.06; N, 18.82. Found: C, 59.25; H, 4.13; N, 18.89.

4-((5-Amino-l-((6-(2-cyanovinyl)naphthalen-l-yl)sulfonyl)-lH-l,2,4-triazol-3- yl)amino)-2-ethynylbenzonitrile RCB22057

Yield 19 %, mp 298-300 °C (column, CHC1₃:CH₃OH 9: 1). 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 6.67 (d, 1H, HC=(2 "'), J = 16.7), 7.66-7.89 (m, 5H, HC( 1' " ,3 " ,6" , NH₂)), 7.96 (d, 1 H, HC(5 " ), J = 8.8), 8.10 (dd, 1 H, HC(7' ), J = 9.1 , 1.8),v8.33 (d, 1H, HC(5'), J= 1.8), 8.41 (d, 1H, HC(2 ), J= 8.2), 8.53 (d, 1H, HC(4 ), J= 7.4), 8.87 (d, 1H, HC(8 ), J= 9.1), 10.30 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO- de; 5, ppm): 99.08, 103.94, 115.28, 116.16, 116.57, 118.46, 120.15 (2C), 124.83, 125.91, 125.97, 128.52, 130.22, 131.80, 132.28, 132.65, 133.65, 134.90, 137.32, 144.87, 149.24, 156.42, 157.88. MS (El): m/z 466. Anal. Calcd for C₂₃HI₃N₈O₂S: C, 59.22; H, 3.03; N, 24.02. Found: C, 59.31; H, 3.09; N, 24.05. Synthesis of 5 -amino-3 -(di substituted phenylamino)- l/Z-pyrazole-4-carbonitriles 5

A mixture of the corresponding aniline (1.0-1.2 equiv) and 2- (bis(methylthio)methylene)malononitrile (1.0 equiv) in EtOH (20 mL) was stirred at reflux for 3 h. After completion of the reaction (monitored by TLC hexane: acetone 2:1), the mixture was cooled to room temperature. When a precipitate appeared, hydrazine hydrate (2.0-2.5 equiv) was added, and the reaction mixture was stirred at reflux for additional 2 h. After completion of the reaction, the mixture was cooled to room temperature and poured into cold water. The precipitate of the corresponding product was filtered, dried and used without further purification.

Synthesis of A-(4-cyano-3 -(di substituted phenylamino)- lJT-pyrazol-5- yl)naphthalene-2-sulfonamides 6

A mixture of naphthalenesulfonyl chloride (1.0- 1.3 equiv) and the corresponding aminopyrazole (1.0 equiv) in pyridine (5 mL) was stirred at room temperature for 2-3 days. After completion of the reaction (monitored by TLC hexane: acetone 2:1), the mixture was poured into ice water, and the precipitate was filtered, dried and recrystallized from ethanol to give the corresponding final product.

A-(3-((4-Chlorophenyl)amino)-4-cyano-U/-pyrazol-5-yl)naphthalene-2- sulfonamide RCB16002

Yield 18 %, mp 226-9 °C. 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.27 (d, 2H, HC(3 ",5 "), J= 8.9), 7.50 (d, 2H, HC(2 ", 6"), J = 8.9), 7.63 (brs, 2H, 2NH), 7.67-7.83 (m, 2H, HC(6", 7')), 7.89 (dd, 1H, HC(4"), J = 8.7, 1.9), 8.06 (d, 1H, HC(5 ), J= 7.2), 8.20 (d, 1H, HC(3"), J= 8.9), 8.25 (d, 1H, HC(8"), J= 7.6), 8.74 (d, 1H, HC(l'), J= 1.6), 8.89 (brs, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 112.48, 119.04, 121.70, 124.40, 128.00, 128.19, 128.37, 129.61, 129.93, 130.08, 131.37, 132.40, 135.11, 139.50, 152.78, 156.02. MS (El): m/z 423. Anal. Calcd for C20H14CIN5O2S: C, 56.67; H, 3.33; N, 16.52. Found: C, 56.73; H, 3.39; N, 16.48. Y-(4-Cyano-3-((4-fluorophenyl)amino)- IT/-pyrazol-5-yl)naphthalene-2- sulfonamide RCB16003

Yield 59 %, mp 175-7 °C. 'H NMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.09 (t, 2H, HC(3",5"), J= 8.9), 7.52 (dd, 2H, HC(2",6"), J= 9.0, 4.8), 7.66-7.83 (m, 4H, 2NH, HC(6',7')), 7.88 (dd, 1H, HC(8 ), J= 8.7, 1.8), 8.07 (d, 1H, HC(3 ), J= 7.2), 8.21 (d, 1H, HC(5'), J = 9.2), 8.26 (d, 1H, HC(4 ), J= 7.2), 8.76 (s, 1H, HC(1')), 8.88 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 65.18, 112.57, 115.04 (d, J = 22.0), 119.11 (d, J = 7.5), 121.70, 128.00, 128.19, 129.59, 129.83, 129.91, 130.06, 131.37, 132.43, 135.10, 136.96, 153.08, 156.09, 156.70 (d, J= 237.0). MS (El): m/z 407. Anal. Calcd for C20H14FN5O2S: C, 58.96; H, 3.46; N, 17.19. Found: C, 59.02; H, 3.52; N, 17.17.

A-(4-Cyano-3-((3,4-difluorophenyl)amino)-U/-pyrazol-5-yl)naphthalene-2- sulfonamide RCB16004

Yield 21 %, mp 239-42 °C. 'HNMR (200 MHz; DMSO-de; 5, ppm; J, Hz): 7.20-7.40 (m, 2H, HC(2 ",5 ")), 7.50-7.65 (m, 1H, HC(6 ")), 7.76 (s, 2H, 2NH), 7.67-7.82 (m, 1H, HC(5',6')), 7.87 (dd, 1H, HC(7 ), J = 9.0, 1.9), 8.07 (d, 1H, HC(4 ), J = 7.4), 8.20 (d, 1H, HC(3'), J= 8.1), 8.24 (d, 1H, HC(8 ), J= 9.0), 8.76 (d, 1H, HC(l'), J= 1.5), 9.12 (s, 1H, NH). ¹³C NMR (50 MHz; DMSO-de; 5, ppm): 106.27 (d, J= 22.1), 112.42, 113.66 (dd,J= 2.7, 6.0), 117.23 (d,J= 17.5), 121.61, 128.01, 128.22, 129.58, 129.79, 130.01, 130.12, 131.34, 132.42, 135.13, 137.65 (dd, J= 2.6, 9.1), 143.88 (dd, J= 14.0, 254.0), 148.70 (dd, J= 14.0, 255.0), 152.69, 155.90. MS (El): m/z 425. Anal. Calcd for C20H13F2N5O2S: C, 56.47; H, 3.08; N, 16.46. Found: C, 56.53; H, 3.12; N, 16.48. EXAMPLE 3

Antiviral Activity

The synthesized triazoles and pyrazoles were then tested for activity against YFV-infected Huh7 cells. The in vitro antiviral activity was assayed by neutral red dye uptake, using Infergen as positive control (ECso < 0.01 ng/mL, CCso > 10 ng/mL). The activities of the compounds against YFV are shown in Tables 1-4, below, separated based on structural modifications. Table 1. In vitro effects of the phenyl sulfonyl aminotriazoles on YFV 17D infection of Huh7 cells.

"P Table 2. In vitro effects of the 2-naphthalenesulfonyl aminotriazoles on YFV 17D infection of Huh7 cells.

Table 3. In vitro effects of the 1 -naphthalenesulfonyl aminotriazoles on YFV 17D infection of Huh7 cells.

"Previously described [35]

Table 4. In vitro effects of the aminopyrazoles on YFV 17D infection of Huh7 cells.

After identification of the initial hit compound RCB16007, the SAR of this structure was explored by testing a panel of previously synthesized compounds [35], The compounds are composed of either a 1,2,4-triazole ring or a pyrazole ring between a functionalized aniline moiety and an arylsulfonyl moiety. A summary of the SAR is shown in Figure 1. Of the tested compounds, those bearing more electronwithdrawing groups on the aniline ring exhibited greater potency, with the exception of the difluoro-containing compound RCB16178. Aniline fragments bearing two cyano substituents, such as RCB22057, were also more active, supporting the idea that electron-deficient, yet sterically bulky aniline rings are important for potency. For example, compound RCB22055, which bears electron-withdrawing chloro and cyano groups, displays low micromolar activity while RCB22056, which bears an acetonitrile group on the aniline, is inactive. Similarly, electron-withdrawing groups on the sulfonyl arene moiety exhibit more potency, with electron-rich arenes, such as those present in RCB16086, resulting in a loss of potency. The presence of a naphthalene ring in the sulfonyl moieties were generally more active than those containing a single aromatic ring, and more so with more strongly electronwithdrawing substituents such as cyano and acrylonitrile groups. Compound RCB17160, in which the aniline and sulfonyl moieties are on adjacent atoms in the triazole and pyrazole rings, did not display significant activity. While this supports the requirement for 1,3 substituted triazoles, more analogs are needed to gauge the role of the 1,3-pyrazole core. Pyrazoles RCB16002 and RCB16004 showed similar activity to the analogous triazoles RCB16178 and RCB18320, supporting both heterocyclic cores are potential anti-YFV scaffolds.

Overall, these analogs suggest that both the aniline and arylsulfonyl moieties are sensitive to substitutions. However, compounds from the initial screen have been improved with compound RCB17159 displaying a 3-fold increase in activity and a similar 3-fold improvement in SI relative to RCB16007 in a primary screen. Compound RCB22055 displays similar antiviral activity with a slightly lower SI.

EXAMPLE 4

Secondary In Vitro Testing

RCB16003 was previously identified after prioritizing compounds using a machine learning model for YFV. Based on the initial hit, secondary testing was performed on RCB16003, RCB16007 (an initial lead compound against HIV) and RCB17159, as it had the best SI in the primary screen. These results suggested that these compounds all had similar in vitro against YFV. See Table 5, below. Table 5. Secondary in vitro antiviral screening results against YFV (17D) for compounds RCB16003, RCB16007 and RCB17159 in Huh7 cells.

yield reduction; Infergen was used as positive control with ECso < 0.01 ng/mL and CC50 > 10 ng/mL ^a Previously described [35],

EXAMPLE 5

In Vitro ADME/Tox for RCB 16007 The compound RCB16007 was chosen as a promising representative of the presently disclosed compounds and was used in in vitro ADME/Tox studies. The results are summarized below in Table 6. While relatively poor solubility and slight CYP2C19 inhibition was observed, RCB16007 displayed good human and mouse liver microsomal stability, and no signs of efflux in Caco-2 cells. Also, this compound at 10 mg/mL did not appear to be mutagenic in E. coli PQ37 in the SOS- chromotest assay, compared with the control 4NQO. The TD99 in TEIP-1 and Huh- 7D12 was > 11 pg /mL. This molecule additionally had no inhibition of hERG (> 10 pM) [35], Table 6. Summary ADME data for compound RCB16007.

EXAMPLE 6 Preliminary In Vivo Toxicity and Pharmacokinetic Studies for RCB16007

Toxicity studies on mice revealed that the LDso value for RCB16007 was 3,930 mg/kg, allowing it to be classified as "non-toxic". A single-dose PK study in mice with intragastrically administered RCB16007 at a dose 250 mg/kg showed a TI/2 value of 3.4 h and a C_max value of 1,190 ng/mL (see Table 7, below), suggesting it has good bioavailability. See also, Figures 2A and 2B, which show the plasma and brain PK concentration versus time curves. Table 7. Plasma Pharmacokinetic Parameters for RCB 16007 (values are Mean+SD

(n=3).

EXAMPLE 7

Machine Learning and Predictions

YFV machine learning models were rebuilt using the most recent version of proprietary software available under the tradename ASSAY CENTRAL® (Collaborations Pharmaceuticals, Inc., Raleigh, North Carolina, United States of America) from the dataset and thresholds from a previously published study [29] . The new models overall showed good cross validation statistics using these additional algorithms (See Tables 8A and 8B, below and Figures 6A and 6B) and performed similarly to the original models. Table 8A. Yellow Fever Virus Inhibition: Literature Compounds (Threshold < 8.7 pM).

Table 8B. Yellow Fever Virus Inhibition: Literature Compounds (Threshold < 80 pM).

This dataset was expanded to include more recent publicly available data from

ChEMBL with classification models built using the activity thresholds of 10 and 20 pM. While the training datasets were quite unbalanced (60/701 or 106/691 active/inactives), several algorithms had reasonable CV scores, with knn, Ireg and svc performing well at both thresholds. See Tables 9A and 9B, below, and Figures 7A and 7B.

Table 9A. Yellow Fever Virus Inhibition: Literature and ChEMBL Compounds (Threshold < 10 pM).

Table 9B. Yellow Fever Virus Inhibition: Literature and ChEMBL Compounds (Threshold < 20 pM).

To additionally attempt to validate these models we used our novel class of compounds as an external validation set. Cross validation and external test stats for models built using two of the best performing algorithms, Ireg and svc, are shown in Figures 3A and 3B and in Tables 10-12, below. While CV statistics suggested these were predictive models, the external test set validation scores strongly suggest that these models are not able to predict our new class of yellow fever virus inhibitors.

Based on this poor performance, models were also built with the currently generated data only. While the training data is small (40 compounds), some of these models showed surprisingly good CV statistics at a 20 pM threshold. This is in contrast to the random predictions at 10 pM. See Tables 13A and 13B, below. See also Figures 8A and 8B.

Table 10. Yellow Fever Virus Inhibition (Threshold < 20 pM, 106 Active/691

Inactive).

Table 12. Method: SVC

Table 13A. Yellow Fever Virus Inhibition: N-phenyl-l-(phenylsulfonyl)-lH-l,2,4,- triazol-3 -amines (Threshold < 10 pM).

Table 13B. Yellow Fever Virus Inhibition: N-phenyl-l-(phenylsulfonyl)-lH-l,2,4,- triazol-3 -amines (Threshold < 20 pM).

A t-SNE visualization using ECFP6 descriptors show that the compounds tested in this study are distinct from those that have been tested against YFV previously (ChEMBL and literature [29]), which suggests a reasonable explanation of why this external test set is predicted so poorly. See Figure 4. The majority of the most potent compounds were grouped into distinct clusters, which upon inspection were found to be analogs of an initial hit molecule.

To determine if some chemical descriptors can be useful to help distinguish between active and inactive compounds for our compound class, we compared these two groups using several simple chemical descriptors (molecular weight, log P, molecular fraction polar surface area, log D (pH 7.4), as well as the number of aromatic rings, hydrogen bond acceptor and donor, rings, and rotatable bonds) using a 20 pM activity threshold cutoff. See Figures 5A-5I. The majority of the descriptors did not have a statistically significant difference, although the number of rings was the exception. This exception was also noted in the SAR analysis, where the presence of more aromatic rings were generally more active, particularly in the sulfonyl moiety.

This analysis was extended to the larger, concatenated dataset using the same activity threshold cutoff. See Figures 9A-9I. These comparisons showed statistically significant differences for all the descriptors assessed, with the largest rank and distribution differences found in MW, PSA, number of rings (aromatic and total), and rotatable bonds. To test for statistically significant differences, comparison tests assumed non-parametric data distributions (Mann Whitney tests).

EXAMPLE 8

Discussion of Examples 1-7

As new viral pandemics emerge [37], there is a continued need for the identification of broad-spectrum antivirals [38], Many examples of antivirals for YFV have been described that are nucleosides [39, 40, 41] and, in contrast, there appear fewer examples of heterocyclic compounds demonstrating activity [42, 43], The pyrazolesulfonamide derivative RCB16003 was previously described as having low micromolar potency against YFV [29], This represented a starting point for hit-to- lead optimization that was identified by machine learning. The presently disclosed studies describe how this molecule is selective for YF. In an effort to expand SAR studies and possibly develop a broader spectrum molecule, a second class of molecules was selected that was under investigation against HIV [35], Compound RCB16007 had similar in vitro activity as the initial hit RCB16003 but was also found to be active against West Nile virus as well as YFV. See Table 14, below. It possessed good in vitro ADME properties and excellent mouse PK. RCB16007 also readily passed the BBB in vivo, which can be of interest since there is evidence of YFV BBB penetration [44-46], The most substantial evidence of this originates from an outbreak in 2018 in Brazil, where autopsies from 5 of 13 patients that had succumbed to yellow fever showed significant YFV RNA in their CSF [46], We explored a further 27 analogs of these two initial hits were studied and were found to improve the in vitro efficacy by 3-fold and the SI 3-fold. RCB16003, RCB16007 and RCB17159, in particular, appear to be of interest of interest against YFV. Table 14. Screening of RCB16003 and RCB16007 against a range of viruses in different cell types. Effective concentration and cytotoxicity were calculated using the uptake of neutral red (cytopathic effect/toxicity).

*Sho value

Earlier machine learning models for YFV were expanded to include additional algorithms as well as create further models based on updated datasets. These all showed robust CV scores but failed to be predictive for this new class of compounds. A t-SNE visualization using ECFP6 descriptors showed that the 7V-phenyl-l- (phenylsulfonyl)- IT/- l ,2,4-triazol-3-amines are substantially different than previously tested compounds, giving a probable reason for the inability of these models to accurately predict the activity of these compounds. While these may not have been useful to predict the activity of these particular compounds, based on the CV score these could be helpful for the prediction of compounds with more model overlap. Models built with the 7V-phenyl-l-(phenylsulfonyl)-U/-l,2,4-triazol-3- amines alone showed mixed CV results, making their use for further optimization ambiguous. Analysis of simple chemical descriptors for the compounds tested against YFV showed the potential importance of the number of rings linked to activity for this class. Expansion of this analysis included the public dataset, where multiple descriptors are suggested to be important for activity. While these represent multiple classes of compounds, it is interesting that the distribution of actives tended to require an increase in the number of rings over inactives, similarly to what was observed in the compounds tested in the present study. Other simple chemical descriptors also showed significant differences between the active and inactive classes, which can be helpful to help predict the activity of novel compounds with activity against YFV in future.

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All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to UniProt, EMBL, and GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, and/or teach methodology, techniques, and/or compositions employed herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicant reserves the right to challenge the accuracy and pertinence of any cited reference.

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It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. A method of treating a disease caused by a yellow fever virus infection in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of a compound comprising a l-sulfonyl-3- amino-lH-l,2,4-triazole group or a pharmaceutically acceptable salt of the compound.

2. The method of claim 1, wherein the compound comprising a l-sulfonyl-3- amino-lH-l,2,4-triazole group has a structure of Formula (I):

wherein An and An are each independently selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

3. The method of claim 2, wherein An is phenyl, naphthyl, substituted phenyl or substituted naphthyl, optionally naphthyl or substituted naphthyl, further optionally wherein said substituted naphthyl is naphthyl substituted with a substituent selected from the group consisting of halo, nitro, acyl, and cyano-substuted alkyl.

4. The method of claim 2 or 3, wherein An is substituted phenyl, optionally wherein An is phenyl substituted with one, two, or three substituents independently selected from the group consisting of halo, cyano, acyl, formyl, alkyl, cyanosubstituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl.

5. The method of any one of claims 1-4, wherein the compound comprising a 1- sulfonyl-3-amino-lH-l,2,4-triazole group has a structure of Formula (II):

wherein

R3 and R4 are independently selected from the group consisting of H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and

Rs, Re, R7, Rs and R9 are independently selected from the group consisting of H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H.

6. The method of claim 5, wherein R3 and R4 are each H or wherein one of R3 and R4 is selected from Cl, F, cyano, CH=CHCN, and C(=O)CH3.

7. The method of claim 5, wherein Rs, Re, R7, Rs and R9 are independently selected from the group consisting of H, F, Cl, methyl, CN, CHCN, -C(=O)-OEt, and -C(=O)-OMe, and wherein one, two, three or four of Rs, Re, R7, Rs, and R9 are H.

8. The method of any one of claims 1-7, wherein the compound comprising a 1- sulfonyl-3-amino-lH-l,2,4-triazole group is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the compound is selected from:

or a pharmaceutically acceptable salt thereof.

10. Use of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group, or a pharmaceutically acceptable salt of said compound, in preparing a medicament for treating a disease caused by a yellow fever virus infection.

11. The use of claim 10, wherein the compound comprising a l-sulfonyl-3-amino- lH-l,2,4-triazole group has a structure of Formula (I):

12. The use of claim 11, wherein An is phenyl, naphthyl, substituted phenyl or substituted naphthyl, optionally naphthyl or substituted naphthyl, further optionally wherein said substituted naphthyl is naphthyl substituted with a substituent selected from the group consisting of halo, nitro, acyl, and cyano-substuted alkyl.

13. The use of claim 11 or 12, wherein An is substituted phenyl, optionally wherein An is phenyl substituted with one, two, or three substituents independently selected from the group consisting of halo, cyano, acyl, formyl, alkyl, cyanosubstituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl.

14. The use of any one of claims 10-13, wherein the compound comprising a 1- sulfonyl-3-amino-lH-l,2,4-triazole group has a structure of Formula (II):

wherein

R.3 and R.4 are independently selected from the group consisting of H, halo, cyano, nitro, acyl, formyl, -C(=O)OH, -C(=O)-O-alkyl, perhaloalkyl, and cyanosubstituted alkyl; and Rs, Re, R7, Rs and R9 are independently selected from the group consisting of H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H.

15. The use of claim 14, wherein R3 and R4 are each H or wherein one of R3 and R4 is selected from Cl, F, cyano, CH=CHCN, and C(=O)CH3.

16. The use of claim 14, wherein Rs, Re, R7, Rs and R9 are independently selected from the group consisting of H, F, Cl, methyl, CN, CHCN, -C(=O)-OEt, and -C(=O)- OMe, and wherein one, two, three or four of Rs, Re, R7, Rs, and R9 are H.

17. The use of any one of claims 10-16, wherein the compound comprising a 1- sulfonyl-3-amino-lH-l,2,4-triazole group is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

18. The use of claim 17, wherein the compound is selected from:

or a pharmaceutically acceptable salt thereof.

19. A method of inhibiting yellow fever virus in a sample comprising yellow fever virus, wherein the method comprises contacting the sample with an effective amount of a compound comprising a l-sulfonyl-3-amino-lH-l,2,4-triazole group or a pharmaceutically acceptable salt of the compound, optionally wherein the compound comprises a structure of Formula (I):

20. The method of claim 19, wherein the compound comprising a l-sulfonyl-3- amino-lH-l,2,4-triazole group has a structure of Formula (II):

wherein

Rs, Re, R7, Rs and R9 are independently selected from the group consisting of H, halo, cyano, acyl, formyl, alkyl, cyano-substituted alkyl, perhaloalkyl, alkoxy, aryl, nitro, -C(=O)OH, and -C(=O)-O-alkyl, optionally wherein at least one of Rs, Re, R7, Rs, and R9 is not H; further optionally wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.