RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application serial numbers 62/566,663, filed October 2, 2017, 62/566,667, filed October 2, 2017, and 62/593,787, filed December 1, 2017, the specifications of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Flavivirus is a genus of viruses that include West Nile virus, dengue virus, yellow fever virus, Zika virus, and several others. Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. Animal model studies also suggest that Zika- infection may lead to male infertility (Cell, 2016; 167: 1511-1524; Nature, 2016; 540:438-442) and severe neurological and other systemic complications in adults.

ZIKV is an RNA virus of the Flaviviridae family and is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. However, several studies identified various modalities that can inhibit Zika's replication including interferons: IFN-a, IFN-β and IFN-γ (J. Vis. Exp. 2016;

114:e54767), antibodies (Nature. 2016; 540:443-447), peptides (Nat Commun. 2017; 8: 15672; Antiviral Res. 2017;141 : 140-149), small molecules (Antiviral Res. 2017; 141 :29-37; Antiviral Res. 2017;143:218-229), and antibiotics (azithromycin, PNAS 2016; 113: 14408-14413).

Generally, bioactivity (ICso) reported to date for small molecules is in the micromolar range and for most active peptides in low nanomolar range with ICso values for antibodies ~1 ng/mL, depending on antibody and viral strain tested. Therefore further development of inexpensive and orally available anti-Zika drugs is of critical importance, with small organic compounds being obvious candidates for versatile use as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in general and high-risk populations, including infected pregnant women. SUMMARY OF INVENTION

Provided herein are compounds, compositions and methods useful in the treatment and/or prevention of a flavivirus infection. The flavivirus may be Zika virus, Dengue virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus or St. Louis encephalitis virus, preferably, Zika virus. In some embodiments, the methods comprise administering a compound disclosed herein or a pharmaceutically acceptable salt, ester, or prodrug thereof. In certain embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier.

In some embodiments, the compounds disclosed herein have a structure of Formula I or Formula II:

Formula I Formula II

wherein A, R ⁴ , R ⁵ , R ¹¹ , R ¹² X, Y, Th and p are as defined herein.

In some embodiments, provided herein, is a method of treating or preventing a virus infection from a virus from the Flaviviridae family in a subject, comprising administering to the subject a compound having the structure of Formula III or Formula IV

Formula III, Formula IV wherein R ¹ , R ² and R ³ are as defined herein.

In certain embodiments, the methods disclosed herein treat or prevent a flavivirus infection in a subject, e.g., by administering to the subject a compound of Formula I, Formula II, Formula III or Formula IV. In some embodiments, the methods disclosed herein treat or prevent a Zika virus infection, Dengue virus infection, West Nile virus infection, Yellow fever virus infection, Japanese encephalitis virus infection or St. Louis encephalitis virus infection. In certain preferred embodiments, the flavivirus is Zika virus.

In certain preferred embodiments, the subject is a human, e.g., a female human. For example, a subject can be a human female of child-bearing age or a human female who is pregnant.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a bar graph showing anti-Zika activity of compounds 57X, 57Y, 57Z, 78X, 78 Y, 78Z, and 9 (NE9) at 5μΜ compared to a DMSO control.

FIG. 2 shows the corresponding dose response curves.

FIG. 3 shows an overlay of minimized structures of NE9 (top), ASN 07115854 (middle) and (S)-(+)-rolipram (bottom). (A) front view, (B) top view.

FIG. 4 shows a screen of selected PDE4 inhibitors and adenosine receptors' antagonists.

FIG. 5 shows the corresponding dose response curves of selected PDE4 inhibitors and adenosine receptors' antagonists.

FIG. 6 shows the screening results for ASN compounds at 5 μΜ concentration.

FIG. 7 shows representative examples of dose response curves.

DETAILED DESCRD7TION

In certain aspects, provided herein are compounds, compositions and methods related to the treatment and/or prevention of a flavivirus infection, such as a Zika virus infection, Dengue virus infection, West Nile virus infection, Yellow fever virus infection, Japanese encephalitis virus infection or St. Louis encephalitis virus infection. Preferably, the flavivirus is Zika virus.

I. COMPOUNDS

In one aspect, provided herein are com ounds having a structure of Formula I

Formula I

or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein

each R ¹¹ is individually selected from halo, -NO2, optionally substituted alkyl, cyano, optionally substituted alkoxy, alkylthio, carboxy, amide, hydroxy, -SO2R ¹⁴ , acyl, -C(=0)OR ¹³ , - SO3R ¹³ , and -NHR ¹⁵ , preferably selected from halo, haloalkyl, -NO2, cyano, -C(=0)R ¹⁴ , and - C(=0)OR ¹³ ;

R ¹² is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aralkyl, or optionally substituted aryloxyalkyl;

R ¹³ is H or optionally substituted alkyl;

R ¹⁴ is optionally substituted alkyl;

R ¹⁵ is H or acyl;

X is selected from O or S;

ene, e.g., methylene;

p is an integer selected from 1, 2, 3, 4 or 5.

In certain embodiments, each R is independently selected from halo, haloalkyl, and - C(=0)R ¹⁴ . In some embodiments, each R ¹¹ is independently selected from -F, -CI, -Br, -I, -CF3, and acyl. In certain preferred embodiments, each R ¹¹ is independently selected from -F, -CI, -Br

In certain embodiments, the compound comprises one occurrence of R in the para position. In some embodiments, the compound comprises an occurrence of R ¹¹ is in a meta position. In other embodiments, at least one occurrence of R ¹¹ , e.g., an occurrence of R ¹¹ in the para position, is halo. In preferred embodiments, the compound comprises an occurrence of R ¹ in the para position, where R ¹¹ is halo, e.g., -F, -CI, -Br or -I, preferably CI.

In certain preferred embodiments, at least one occurrence of R ¹¹ , e.g., an occurrence of R ¹¹ in the meta position, is substituted alkyl, preferably haloalkyl, most preferably -CF3.

In some embodiments, at least one occurrence of R ¹¹ is -C(=0)R ¹⁴ , e.g., wherein R ¹⁴ is alkyl, such as methyl.

In some embodiments, p is 1 or 2. In certain embodiments, R is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aralkyl, or optionally substituted aryloxyalkyl.

In certain preferred embodiments, R is optionally substituted alkenyl, e.g.

In other preferred embodiments, R is optionally substituted alkynyl, e.g.

In some embodiments, R ¹² is optionally substituted aralkyl. In some embodiments, R optionally substituted aryloxyalkyl, e.g.,

In certain preferred embodiments, X is O. In some embodiments, X is S.

In certain preferred embodiments, Y is S or -NH-.

In certain embodime In certain embodiments, Th i In

certain embodiments, Th is . In certain preferred embodiments, Th is ₀ r

In certain embodiments, each R ¹¹ is independently halo or haloalkyl, preferably halo or - CF3, e.g., a halo at the para position and, optionally, -CF3 at a meta position;

R ¹² is optionally substituted alkenyl or optionally substituted alkynyl, preferably allyl or propargyl;

X is selected from O or S;

Y is selected from S or -NH- p is 1 or 2.

In certain embodiments, the compound is selected from a compound of Formula I identified in Table 1 or a pharmaceutically acceptable salt, ester, or prodrug thereof.

TABLE 1

In another aspect, provided herein are com ounds having the structure of Formula II:

Formula II

or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein

A is O or S;

R ⁴ is selected from optionally substituted aryl or heteroaryl;

R ⁵ is selected from CR ⁶ R ⁷ , OR ⁶ , NR ⁶ and optionally substituted alkyl;

R ⁶ is selected from H or optionally substituted alkyl; and

R ⁷ is selected from H or optionally substituted alkyl.

In certain embodiments, A is O.

In certain embodiments, R ⁴ is optionally substituted aryl, e.g., optionally substituted phenyl. For example, in some embodiments, the aryl or phenyl is substituted with one or more substituents selected from halo, nitro, cyano, hydroxy, carboxy, alkyl, alkoxy, ester, sulfonate, sulfone, sulfoxide, and -C(0)R ⁸ and R ⁸ is alkyl. In certain such embodiments, R ⁸ is lower alkyl,

e.g., methyl. In certain embodiments, R ⁴ is , e.g.

In certain embodiments, R ⁵ is selected from CHR ⁶ and OR ⁶ .

In certain embodiments, R ⁶ is substituted alkyl. In some embodim R ⁶ is substituted with an alkynyl or alkyl group. For example, in some embodiments R ⁶ is or an integer selected from 1, 2, 3, or 4, e.g., 1, 2, or 3; and n is an integer selected from 1, 2, 3, or 4, e.g., 1, 2, or 3.

In certain embodiments of Formula II, the compound is selected from

pharmaceutically acceptable salts thereof.

In another aspect, provided herein are compounds having the structure of Formula III or Formula IV:

Formula III, Formula IV or a pharmaceutically acceptable salt, ester, or prodrug thereof, wherein

R ¹ is selected from optionally substituted aryl and heteroaryl;

R ² is selected from H and optionally substituted alkyl; and

R ³ is selected from optionally substituted aryl, heteroaryl or alkyl.

In some embodiments, R ¹ is optionally substituted aryl, e.g., phenyl. In some embodiments, the phenyl is substituted with at least one of halo, e.g., CI, alkoxy or nitro,

preferably nitro. For example, in certain embodiments, R ¹

In some embodiments, R ² is optionally substituted alkyl. In some embodiments, the alkyl is substituted with a sulfonate, e.g., wherein R ² is -(CH2)3-SC H.

In some embodiments, R ³ is optionally substituted aryl, e.g., phenyl. In some embodiments, the phenyl is substituted with at least one of halo e.g., -F, nitro and heteroaryl.

For example, in certain embodiments, R is

In certain embodiments, R ³ is optionally substituted heteroaryl. In some embodiments, the heteroaryl substituted with alkyl, e.g., methyl.

In certain embodiments, R ³ is optionally substituted aryl. In some embodiments, R ³ is optionally substituted phenyl, e.g., para-substituted phenyl.

In certain embodiments, R ³ is phenyl substituted with at least one of halo, nitro, heteroaryl, acyl, acylamino, alkyl, -SC Me, cyano, carboxy, -CF3 and -OCF3. In some embodiments, R ³ is phenyl substituted with at least one -F, -CI, or alkyl selected from methyl, iso-propyl, tert-butyl or n-hexyl.

or methyl.

In another aspect, provided herein is a compound selected from

or a pharmaceutically acceptable salt, ester, or prodrug thereof.

In certain embodiments, the compound is selected from the compounds identified in Table 2 or a pharmaceutically acceptable salt, ester, or prodrug thereof.

TABLE 2

21

In certain embodiments, provided herein are compounds identified in Table 3 and pharmaceutically acceptable salts, esters, and prodrugs thereof.

TABLE 3

II. PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the invention relates to a pharmaceutical composition comprising any one of the aforementioned compounds and a pharmaceutically acceptable carrier.

Patients, including but not limited to humans, can be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated, and possible drug-drug interactions with antiretroviral medications. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient can be administered at once, or can be divided into a number of smaller doses to be administered at varying intervals of time.

In certain embodiments, the mode of administration of the active compound is oral. Oral compositions will generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, unit dosage forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup can contain, in addition to the active compound(s), sucrose or sweetener as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrug or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti-inflammatories or other antivirals, including but not limited to nucleoside compounds. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;

chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, carriers include physiological saline and phosphate buffered saline (PBS).

In certain embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including but not limited to implants and microencapsulated delivery systems, such as those disclosed in International Publication No. WO 2010/093944, hereby incorporated by reference in its entirety, and specifically with respect to the formulations disclosed therein. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. For example, enterically coated compounds can be used to protect cleavage by stomach acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Suitable materials can also be obtained commercially.

Liposomal suspensions (including but not limited to liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated by reference). For example, liposome formulations can be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. The compositions and methods of the present invention may be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non- human mammal. When administered to an animal, such as a human, the composition is preferably administered as a pharmaceutical composition comprising, for example, a composition of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, e.g., for parenteral administration, the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, powder, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and -S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.

Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize or to increase the absorption of a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue);

absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally;

intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compositions may also be formulated for inhalation. In certain embodiments, a composition may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

In some embodiments of the present invention, the composition that is suitable for use in the invention may be administered orally, topically or parenterally, and in particular topically.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The composition may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an antibiotic, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of the invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The composition of the invention may be formulated with an excipient and component that is common for such oral compositions or food supplements, e.g., especially fatty and/or aqueous components, humectants, thickeners, preserving agents, texturizers, flavor enhancers and/or coating agents, antioxidants and preserving agents. Formulating agents and excipients for oral compositions, and especially for food supplements, are known in this field and will not be the subject of a detailed description herein.

In the case of a composition in accordance with the invention for oral administration, the use of an ingestible support is preferred. The ingestible support may be of diverse nature according to the type of composition under consideration. Tablets, gel capsules or lozenges, suspensions, oral supplements in dry form and oral supplements in liquid form are especially suitable for use as food supports.

Formulation of the oral compositions according to the invention may be performed via any usual process known to those skilled in the art for producing drinkable solutions, sugar- coated tablets, gel capsules, gels, emulsions, tablets to be swallowed or chewed, wafer capsules, especially soft or hard wafer capsules, granules to be dissolved, syrups, solid or liquid foods, and hydrogels allowing controlled release. Formulation of the oral compositions according to the invention may be incorporated into any form of food supplement or enriched food, for example food bars, or compacted or loose powders. The powders may be diluted with water, with soda, with dairy products or soybean derivatives, or may be incorporated into food bars.

In some embodiments, the composition according to the invention administered orally may be formulated in the form of sugar-coated tablets, gel capsules, gels, emulsions, tablets, wafer capsules, hydrogels, food bars, compacted or loose powders, liquid suspensions or solutions, confectioneries, fermented milks, fermented cheeses, chewing gum, toothpaste or spray solutions. An effective amount of the composition may be administered in a single dose per day or in fractional doses over the day, for example two to three times a day. By way of example, the administration of a composition according to the invention may be performed at a rate, for example, of 3 times a day or more, generally over a prolonged period of at least a week, 2 weeks, 3 weeks, 4 weeks, or even 4 to 15 weeks, optionally comprising one or more periods of stoppage or being repeated after a period of stoppage.

In certain embodiments, the compound may be administered at a dose between 1 mg and 1,500 mg per day, such as between 5 mg and 1,300 mg per day, such as between 10 mg and 900 mg per day, such as between 20 mg and 600 mg per day, such as between 40 mg and 300 mg per day, such as between 150 mg and 350 mg per day , such as between 40 and 150 mg per day, such as between 25 mg and 150 mg per day , such as between 2.5 mg and 150 mg per day, such as between 20 mg and 80 mg per day, or such as between 1 mg and 30 mg per day. In certain embodiments, the compound may be administered at a dose of 1,300 mg/day, 900 mg/day, 600 mg/day, 350 mg/day, 300 mg/day, 250mg/day, 200 mg/day, 150 mg/day, 80 mg/day, 75 mg/day, 60 mg/day, 40 mg/day, 30 mg/day, 20 mg/day, 15 mg/day, 10 mg/day, 5 mg/day, or 2.5 mg/day.

This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. The term "pharmaceutically acceptable salt" as used herein includes salts derived from inorganic or organic acids including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic,

benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic, and other acids. Pharmaceutically acceptable salt forms can include forms wherein the ratio of molecules comprising the salt is not 1 : 1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of a compound. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of base, such as two molecules of a compound per molecule of tartaric acid.

In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N- methylglucamine, hydrabamine, lH-imidazole, L-lysine, magnesium, 4-(2- hydroxy ethyl)morpholine, piperazine, potassium, 1 -(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In further embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.

The pharmaceutically acceptable salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

As one of skill in the art will appreciate, compositions of the present invention, not having adverse effects upon administration to a subject, may be administered daily to the subject.

Preferred embodiments of this invention are described herein. Of course, variations, changes, modifications and substitution of equivalents of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations, changes, modifications and substitution of equivalents as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed, altered or modified to yield essentially similar results. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly

contradicted by context.

While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention.

III. METHODS

In some embodiments, provided herein is a method for protecting a subject from flavivirus, comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein. In some embodiments, provided herein is a method of treating a subject for a flavivirus infection comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein. In some embodiments, provided herein is a method of preventing a subject from a flavivirus infection comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein. In some embodiments, the flavivirus is Zika virus, Dengue virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus or St. Louis encephalitis virus. In some embodiments, the flavivirus is Zika virus.

In some embodiments, provided herein is a method for protecting a subject from Zika virus, comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein. In some embodiments, provided herein is a method of treating a subject for a Zika virus infection comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein. In some embodiments, provided herein is a method of preventing a subject for a Zika virus infection comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein.

In some embodiments, provided herein are compounds (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein for use in secondary clinical application as antivirals against flavivirus infections. In some embodiments, provided herein are compounds (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein for use in secondary clinical application as antivirals against Zika virus, Dengue virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus or St. Louis encephalitis virus.

A "subject," as used herein, can be any mammal. For example, a subject can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. In some embodiments, the subject is an infant (e.g., a human infant). In some embodiments, the subject is female (e.g., a human female). For example, a subject can be a human female of child-bearing age or a human female who is pregnant.

In certain embodiments, the subject is exposed to Zika virus due to the subject's exposure to a mosquito comprising the Zika virus. The subject may be exposed to a Aedes mosquitoes, particularly aegypti. Such a subject may be at risk of developing a Zika virus infection and disease states related to or caused by such an infection.

In certain embodiments, provided herein, is a method of treating or preventing a virus infection from a virus from the Flaviviridae family in a subject, comprising administering to the subject a compound (e.g., compound of Formula I, compound of Formula II, compound of Formula III, compound of Formula IV) or a composition disclosed herein.. In some

embodiments, the virus is Zika, Dengue, West Nile, Yellow fever, Japanese encephalitis or St. Louis encephalitis viruses, preferably Zika.

IV. DEFINITIONS

For purposes of the present invention, the following definitions will be used (unless expressly stated otherwise):

The terms "a," "an," "the" and similar referents used in the context of describing the present invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any unclaimed element is essential to the practice of the invention.

The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(0)NH-. The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(0)0-, preferably alkylC(0)0-.

The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert- butoxy and the like.

The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

The term "alkenyl", as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

An "alkyl" group or "alkane" is a straight chained or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 unless otherwise defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A Ci-C ₆ straight chained or branched alkyl group is also referred to as a "lower alkyl" group. An alkyl group with two open valences is sometimes referred to as an alkylene group, such as methylene, ethylene, propylene and the like.

Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, if not otherwise specified, can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

The term "C _x - _y " when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "Cx- _y alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2- tirfluoroethyl, etc. Co alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. The terms "C2- _y alkenyl" and "C2- _y alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. As applied to heteroalkyls, "C _x - _y " indicates that the group contains from x to y carbons and heteroatoms in the chain. As applied to carbocyclic structures, such as aryl and cycloalkyl groups, "C _x - _y " indicates that the ring comprises x to y carbon atoms. As applied to heterocyclic structures, such as heteroaryl and heterocyclyl groups, "C _x - _y " indicates that the ring contains from x to y carbons and heteroatoms. As applied to groups, such as aralkyl and heterocyclylalkyl groups, that have both ring and chain components, "C _x - _y " indicates that the ring and the chain together contain from x to y carbon atoms and, as appropriate heteroatoms.

The term "alkylamino", as used herein, refers to an amino group substituted with at least one alkyl group.

The term "alkylthio", as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

The term "alkynyl", as used herein, refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

The term "amide", as used herein, refers to a group

wherein each R ¹⁰ independently represent a hydrogen or hydrocarbyl group, or two R ¹⁰ are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by

wherein each R ¹⁰ independently represents a hydrogen or a hydrocarbyl group, or two R ¹⁰ are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term "aminoalkyl", as used herein, refers to an alkyl group substituted with an amino group.

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group.

The term "aryl" as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The term "carbamate" is art-recognized and refers to a group

wherein R ⁹ and R ¹⁰ independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, or R ⁹ and R ¹⁰ taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms "carbocycle", and "carbocyclic", as used herein, refers to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic carbocycles and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings, in which all carbon atoms are saturated, and cycloalkene rings, which contain at least one double bond. "Carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary "carbocycles" include cyclopentane, cyclohexane,

bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3- ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-lH-indene and bicyclo[4.1.0]hept-3-ene. "Carbocycles" may be substituted at any one or more positions capable of bearing a hydrogen atom.

A "cycloalkyl" group is a cyclic hydrocarbon which is completely saturated.

"Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms unless otherwise defined. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

The term "carbocyclylalkyl", as used herein, refers to an alkyl group substituted with a carbocycle group.

The term "carbonate" is art-recognized and refers to a group -OCO2-R ¹⁰ , wherein R ¹⁰ represents a hydrocarbyl group.

The term "carboxy", as used herein, refers to a group represented by the formula -CO2H.

The term "ester", as used herein, refers to a group -C(0)OR ¹⁰ wherein R ¹⁰ represents a hydrocarbyl group.

The term "ether", as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

The terms "halo" and "halogen" as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

The terms "hetaralkyl" and "heteroaralkyl", as used herein, refers to an alkyl group substituted with a hetaryl group.

The term "heteroalkyl", as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent. In analogy with alkyl groups, heteroalkyl groups with two open valences are sometimes referred to as heteroalkylene groups. Preferably, the heteroatoms in heteroalkyl groups are selected from O and N.

The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include poly cyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term "heterocyclylalkyl", as used herein, refers to an alkyl group substituted with a heterocycle group.

The term "hydrocarbyl", as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

The term "hydroxyalkyl", as used herein, refers to an alkyl group substituted with a hydroxy group.

The term "lower" when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer. A "lower alkyl", for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

As used herein, "mitigating" means reducing the negative effects caused by exposure to ionizing radiation, relative to a cell, organ, tissue, or organism exposed to the same level of radiation for the same amount of time, but untreated.

As used herein, a "therapeutically effective amount" is an amount sufficient to mitigate the effects of the ionizing radiation.

The terms "polycyclyl", "polycycle", and "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7. When a polycyclic substituent is attached through an aryl or heteroaryl ring, that substituent may be referred to herein as an aryl or heteroaryl group, while if the polycyclic substituent is attached through a cycloalkyl or heterocyclyl group, that substituent may be referred to herein as a cycloalkyl or heterocyclyl group. By way of example, a 1,2,3,4-tetrahydronaphthalen-l-yl group would be a cycloalkyl group, while a l,2,3,4-tetrahydronaphthalen-5-yl group would be an aryl group.

The term "silyl" refers to a silicon moiety with three hydrocarbyl moieties attached thereto.

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons or heteroatoms of the moiety. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds.

In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

The term "sulfate" is art-recognized and refers to the group -OSO3H, or a

pharmaceutically acceptable salt thereof.

The term "sulfonamide" is art-recognized and refers to the group represented by the general formulae

wherein R ⁹ and R ¹⁰ independently represents hydrogen or hydrocarbyl, such as alkyl, or R ⁹ and R ¹⁰ taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term "sulfoxide" is art-recognized and refers to the group -S(0)-R ¹⁰ , wherein R ¹⁰ represents a hydrocarbyl.

The term "sulfonate" is art-recognized and refers to the group SO3H, or a

pharmaceutically acceptable salt thereof.

The term "sulfone" is art-recognized and refers to the group -S(0)2-R ¹⁰ , wherein R ¹⁰ represents a hydrocarbyl.

The term "thioalkyl", as used herein, refers to an alkyl group substituted with a thiol group. The term "thioester", as used herein, refers to a group -C(0)SR or -SC(0)R wherein R ¹⁰ represents a hydrocarbyl.

The term "thioether", as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

The term "urea" is art-recognized and may be represented by the general formula

R ⁹ R ⁹

wherein R ⁹ and R ¹⁰ independently represent hydrogen or a hydrocarbyl, such as alkyl, or either occurrence of R ⁹ taken together with R ¹⁰ and the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.

As used herein, the term "administering" means the actual physical introduction of a composition into or onto (as appropriate) a subject. Any and all methods of introducing the composition into subject are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well known to those skilled in the art, and also are exemplified herein.

As used herein, the terms "effective amount", "effective dose", "sufficient amount", "amount effective to", "therapeutically effective amount" or grammatical equivalents thereof mean a dosage sufficient to produce a desired result, to ameliorate, or in some manner, reduce a symptom or stop or reverse progression of a condition and provide either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified observer. Amelioration of a symptom of a particular condition by administration of a pharmaceutical composition described herein refers to any lessening, whether permanent or temporary, lasting, or transitory, that can be associated with the administration of the

pharmaceutical composition.

As used herein, the term "prodrug" is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention. A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxyl in the parent compound is presented as an ester or a carbonate or carboxylic acid present in the parent compound is presented as an ester.

As used herein, the term "pharmaceutically acceptable" refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

The term "treating" is art-recognized and includes administration to the host of one or more of the subject compositions, e.g., to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 - Synthesis of compound 9

Scheme 1

9

Briefly, 6.49 g (40 mmoles) of 1 -phenylpiperazine (A) was placed in 250 mL Erlenmeyer flask equipped with magnetic stirrer. Subsequently, 50 mL of anhydrous N-methyl-2-pyrrolidone (NMP) and 8.8 mL of 4-methylmorpholine (NMM, 80 mmoles) were added and solution cooled in ice bath for 10 min with vigorous mixing. 10.66 g (40 mmoles) of pentafluorobenzenesulfonyl chloride (B) was dissolved in 50 mL of anhydrous NMP and added in 5 mL portions to the reaction mixture over the period of 10 min with mixing. Then flask was "capped" with the parafilm and mixing continued overnight (~18h). Subsequently, reaction mixture was diluted with 900 mL of cold water. Precipitated 9 was collected by filtration and dried under the vacuum (overnight) giving 14.45 g of crude compound (92.1% yield). Crude compound may be re-crystallized from hot ethyl acetate (86.9% yield).

Example 2 - Synthesis of compound 19

Scheme 2

A C

19

Briefly, 6.49 g (40 mmoles) of 1 -phenylpiperazine (A) was placed in 250 mL Erlenmeyer flask equipped with magnetic stirrer. Subsequently, 45 mL of anhydrous tetrahydrofuran (THF), 8.8 mL of 4-methylmorpholine (NMM, 80 mmoles) and 50 mL of anhydrous N-methyl-2- pyrrolidone (NMP) were added and solution cooled in ice bath for 10 min with vigorous mixing. 9.75 g (44 mmoles) of 4-nitrobenzenesulfonyl chloride (C) was dissolved in 25 mL of anhydrous THF and added in 2.5 mL portions to the reaction mixture over the period of 10 min with mixing. Then flask was "capped" with the parafilm and mixing continued overnight (~18h). Subsequently, reaction mixture was transferred to 500 mL round-bottom flask and THF evaporated using rotary evaporator (bath temp 35°C). Remaining residue was diluted with 350 mL of absolute ethanol. Precipitated intermediate D was collected by filtration, washed twice with absolute ethanol (2x25 mL) and dried under the vacuum (overnight) giving 12.9 g (37.14 mmoles) of solid material (92.8% yield). Crude D was re-crystallized from hot ethyl acetate (88.6% yield). Subsequently intermediate D (1 equivalent) was reacted with 3 equivalents of 1,3- propanesultone in anhydrous 1,4-dioxane (75°C/72h). Next, reaction mixture was evaporated using rotary evaporator and remaining residue extracted with hot water (95°C, 3 x). Combined extracts were lyophilized and obtained solid residue purified using preparative reversed-phase liquid chromatography giving pure 19 (36.7% yield for betainization).

Example 3 - Synthesis of compound 39

Scheme 4

39

Briefly, 10.09 g (40 mmoles) of 1 -(2,4-dinitrophenyl)piperazine (E) was placed in 250 mL Erlenmeyer flask equipped with magnetic stirrer. Subsequently, 50 mL of anhydrous N- methyl-2-pyrrolidone (NMP) and 8.8 mL of 4-methylmorpholine (NMM, 80 mmoles) were added and solution cooled in ice bath for 10 min with vigorous mixing. 9.75 g (44 mmoles) of 4- nitrobenzenesulfonyl chloride (C) was dissolved in 50 mL of anhydrous NMP and added in 5 mL portions to the reaction mixture over the period of 10 min with mixing. Then flask was "capped" with the parafilm and mixing continued overnight (~18h). Subsequently, reaction mixture was diluted with 900 mL of cold water. Precipitated 39 was collected by filtration, dried under the vacuum (overnight) and then re-crystallized from hot ethyl acetate giving 15.69 g of compound (89.7% yield). Example 4 - Characterization of compounds

Table 4 summarizes the characterization data for compounds disclosed herein.

LC-MS analytical data were acquired using Agilent 6460 Triple Quadrupole LC/MS System (Agilent Technologies, Santa Clara, CA) with mobile phases consisted of solvent A, 0.1% formic acid (FA) in water, and solvent B, 0.1% FA in acetonitrile (ACN). Analysis was performed with an analytical reversed-phase CORTECS ^® UPLC ^® Phenyl 1.6 μηι 2.1 x 100 mm Column (Waters, Milford, MA) applying linear gradient of solvent B from 0% to 100% over 50 min (flow rate: 200 μΙ7ηήη).

Mass spectra were acquired using Applied Biosystems Voyager-DE STR MALDI-TOF instrument.

TABLE 4

Example 5 - In vitro screening against Zika virus infection by plaque assay

Naive A549 cells were seeded at concentration of 2* 10 ⁵ cells per well using a 48-well plate, and allowed to grow for 48 hours to form a confluent monolayer. Subsequently, the tested compounds were added at indicated concentrations (using Opti-MEM medium) in biological triplicates. The cells were infected immediately after compound treatment, (MOI of 10, 1, 0.1, 0.01, 0.001, and 0.0001) in 100 μΕ per well and plates were incubated at 37°C with 5% CO2 for 4 hours. At 4 hr post-infection, the viral inoculum was replaced with serum supplemented media (250 μΕ per well, including the same concentration of the compounds) and plates incubated at 37°C for additional 44 hours. At 48 hr post-infection, the plaques in each well were counted using a phase contrast microscope.

The dose response anti-Zika activity of compounds 9, 19 and 39 using the plaque assay are shown in Table 5 and the dose response anti-Zika activity of compounds 61, 63, 89, 91, 92, 93, 95, 96, 98, 99 and 102 using the plaque assay are shown in Table 6.

Table 5 summarizes the plaque assay data for certain compounds disclosed herein. TABLE 5

Table 6 summarizes the chemical and assay data for certain compounds disclosed herein.

TABLE 6

(4S)-4-[3-(Cyclopentyloxy)-

96 (S)-(+)- 4-methoxyphenyl]pyrrolidin- C16H21NO3 275.35 25.8±4.7

Rolipram

2-one

5-[3-[(lS,2S,4R)- Bicyclo[2.2.1 ]hept-2-yloxy]- 182.0±3

98 CP 80633 C18H24N2O3 316.39

4-methoxyphenyl]tetrahydro- 0.5 2( 1 H)-pyrimidinone

(R)-5-(4-Methoxy-3-

769.1±1

99 Mesopram propoxyphenyl)-5-methyl-2- C14H19NO4 265.31

93.1 oxazolidinone

(6R,12aR)-6-(l,3- B enzodioxol- 5 -y 1) -

200.9±4

102 Tadalafil 2,3 ,6,7, 12, 12a-hexahy dro-2- C22¥il9N304 389.40

7.4 methylpyrazino[l',2': l,6]pyri

do[3 ,4-blindole- 1 ,4-dione

Example 6 - Synthesis and screening of cross-group derivatives

The compounds of Formula III take advantage of diversity and commercial availability of relatively rigid 1-phenylpiperazine scaffold which is present in compound 9. With 100+ analogs of substituted 1-phenylpiperazine available, a small library of compounds of Formula III can be easily created using either 3-butenyl chloroformate, 4-isothiocyanato-l-butene or 5-hexenoic acid, giving as a result urethanes, thiourethanes or piperazides respectively, with slightly different geometry/orientation of alkene-containing moiety. Preliminary experiments toward development of a library was performed to determine the optimal size and saturation (alkene versus alkyne) to ascertain bioactive hybrids. A group of compounds of Formula III were synthesized and tested. Compound 57Z showed significant anti-Zika inhibitory effects

(ICso=l 172.2 nM, plaque assay) against ZIKV (See FIG. 1).

Example 7 - Development of orally available anti-Zika drugs

The development of inexpensive and orally available anti-Zika drugs is of critical importance, small-molecule organic compounds are candidates for versatile use as a

prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in general and high-risk populations, including pregnant women. A library consisting of both commercially available and newly synthesized small-molecule analogs which was arbitrarily considered as "relevant" for the search was utilized for NE9 (compound 9), ASN 07115854 (compound 61), rolipram (compound 96R), (D) preladenant (compound 91). The tested library was not targeted toward any particular structural properties/features of its members. All compounds were screened using a viral plaque-forming assay and the A549 human lung carcinoma cell line, which was previously described as highly permissive to Zika infection reaching a virus titer almost 7 log PFU mL ^"1 within the 48h of infection. As a result, 4 "hits" were found showing varying anti-Zika inhibitory activity (See FIG. 2), all within low nanomolar or sub-nanomolar range as shown in Table 7.

TABLE 7

Newly found compounds were both proprietary (NE9) as well as commercially available entities (ASN07115854, rolipram, preladenant) with potential for further pharmaceutically - relevant modifications. The "hits" show certain common structural features, namely they contain an assembly of 3 aromatic/aliphatic rings (or 2 rings with rigid linker, see compound 2) in para- or meta-configuration and with or without additional peripheral modifications (acetyl, S-allyl, O- Me, etc.) which are usually small. An additional common denominator is the presence of N- phenyl-substituted piperazine or phenyl-substituted-piperazine-like moiety (4-phenyl-2- pyrrolidone in case of rolipram (compound 3)). Molecular modeling studies have shown that at least 3 of the leading compounds which possess similar size (namely compounds 1, 2 and 3) can adopt certain conformations allowing them to occupy dimmensionaly similar binding cavities (See FIG. 3).

All 4 compounds are very "drug-like" and conform to at least some of Lipinski's rule of five criteria. In addition, for compound 3 and 4, numerous bioactivity and toxicity data exist to aid further their development. Importantly, both compounds are also orally available. Therefore, the identified analogs are excellent leads for the development of anti-Zika drugs, including inexpensive and orally available candidates. Two identified inhibitors of ZIKV possess known molecular targets. Rolipram (compound 3) is a selective inhibitor of phospho-diesterase 4A and 4B and preladenant (compound 4) is a specific antagonist of adenosine receptor A2A, raising their involvement in the ZIKV life cycle. Further analysis of the content of the proprietary library shows that the pentafluorobenzene-sulfonyl (Pfbs) moiety is a necessary structural component to achieve high anti-Zika inhibitory activity for sulfonamide-based compounds.

To this end, 18 differently substituted sulfonamides of 1 -phenyl-piperazine were tested and found that only Pfbs derivative exhibits antiviral activity in sub-nanomolar range, with irtually all other compounds listed (compound 9 and the compounds of Formula I, wherein R ³ is

inactive. An enantiomeric mixture of rolipram which possesses the strong anti-Zika properties showing IC50 value of 44.6±11.9 nM was utilized.

Numerous structurally related inhibitors of PDE4 are available, including

enantiomerically pure isomers of rolipram: (R)-(-)-rolipram and (S)-(+)-rolipram (Tocris, Minneapolis, MN), and a selected group of additional PDE4 inhibitors tested in plaque assays: (R)-(-)-rolipram (compound 95), (S)-(+)-rolipram (compound 96), CDP-840 (compound 200), CP 80633 (compound 98), mesopram (compound 99), piclamilast (compound 201), roflumilast (compound 202) and tadalafil (compound 102, PDE5 inhibitor) were tested looking specifically at SAR properties and the common activity-related structural features which may provide insights into further modification of rolipram-derived analogs. Specifically (R)-(-)-rolipram, (S)- (+)-rolipram, CDP-840, CP 80633, mesopram, piclamilast, roflumilast and as an additional control, tadalafil (PDE5/PDE11 inhibitor) was tested. A similar approach was also applied to the second "hit", preladenant, which is a highly selective and orally available antagonist of adenosine A2A receptor (AR) with Ki value of 1.1 nM. The compound was being researched as a potential treatment for Parkinson's disease and is currently tested in combination therapy with pembrolizumab against advanced solid tumors. Interestingly, preladenant also exhibited potent anti-Zika activity (IC50 = 136.1=1=33.4 nM) in the initial screening campaign, prompting the interest in its homologs and related adenosine receptor antagonists.

A small group of adenosine receptors' antagonists (preladenant (compound 91), SLV 320 (compound 89), DPCPX (compound 203), SCH 442416 (compound 92), PSB 603 (compound 93), ZM 241385 (compound 63) and MRS 1220 (compound 204) were tested in the plaque assays to see whether specific receptor subtypes are involved in anti-Zika inhibitory effects. The major criteria for selection was specific receptor subtype selectivity. The group comprised the following molecules: SLV 320 (Al antagonist), DPCPX (Al antagonist), SCH 442416 (A2A antagonist), PSB 603 (A2B antagonist), ZM 241385 (A2A and A2B antagonist) and MRS 1220 (A3 antagonist).

The results of our screen for both PDE4 inhibitors and AR antagonists are presented in FIG. 4. Several compounds exhibited potent anti-Zika inhibitory effects and they belonged to both groups. Among tested PDE4 inhibitors, the most active compound was (S)-(+)-rolipram (IC50=25.8±4.7 nM) which was approximately 3 times more active than its second

enantiomerically pure form, (R)-(-)- rolipram (IC50=77.6±19.6 nM) (See FIG. 5). These results show no correlation with reported PDE4 inhibitory effects for both enantiomers, where (R)-(-)- rolipram was showing 2-10 times more potency than its (S)-(+)-rolipram counterpart. As some other highly active PDE4 inhibitors show no anti-Zika properties (e.g., roflumilast, piclamilast and CDP-840), and PDE5/PDE11 inhibitor tadalafil is highly active (IC50=200.9±47.4 nM), it shows that PDE4 inhibitory effects are not important for anti-Zika properties of identified compounds, but rather their common structural features. Table 8 shows the activity of selected PDE4 inhibitors and adenosine receptors' antagonists at 5 μΜ concentration.

TABLE 8

(S)-(+)-

96 C16H21 NO3 275.34 25.8±4.7

Rolipram

98 CP 80633 C18H24N2O3 316.39 182.0±30.5

99 Mesopram Ci ₄ Hi ₉ N0 ₄ 265.30 769.1 ±193.1

102 Tadalafil C22H 389.40 200.9±47.4

89 SLV 320 Ci ₈ H ₂₀ N ₄ O 308.38 58.6±10.2

92 SCH 442416 C20H19N7O2 389.41 3062.0±279.1

93 PSB 603 C ₂ 4H ₂ 5CIN ₆ 0 ₄ S 529.01 43.8±8.8

63 ZM 241385 C16H15N7O2 337.34 1066.6±79.1

A similar conclusion can be drawn from the analysis of the results for various adenosine receptors' antagonists. The most active compounds were PSB 60359 (A2B antagonists,

IC50=43.8±8.8 nM) and SLV 32060 (Al antagonists, IC50=58.6±10.2 nM). Since DPCPX (primarily Al antagonists) shows no activity against Zika virus it is not likely that anti-Zika activity attributed to SLV 320 is due to adenosine receptor Al inhibitory effects. Moreover, preladenant (selective A2A antagonist, IC50=136.1±33.4 nM) with very low activity against A2B subtype is highly active against Zika virus, and PSB 603 which is selective A2B receptor antagonist with very low activity against A2A subtype is also highly active, precluding neither of AR subtypes, A2A and A2B as the important players in the Zika virus life cycle. The same is true for A3 adenosine receptor subtype as its selective antagonist MRS 1220 shows no anti-Zika potency even though it is also partially active against A2B subtype like PSB 603. The latter is also particularly similar to our hit compound 1 (NE9) with additional bulky xanthine substituent in benzenesulfonyl moiety, emphasizing again the role of structural similarities between the identified compounds.

Although seemingly structurally unrelated to the other "hits", ASN 07115854 (compound 2) exhibited potent anti-Zika inhibitory effects with IC50=160.3±12.1 nM. Chemically, compound 2 is 1,3-disubstituted urea, containing on the one side the acetophenone and on the other 3-allylthio-l,2,4-thiadiazole moieties. Due to the presence of urea linker which is positioned between two aromatic rings, the entire structure is relatively rigid, providing convenient scaffold for synthesis of compound 2-related derivatives. A number of such derivatives is commercially available (Asinex Corp., Winston- Salem, NC), and a small group of compounds was selected to further delineate anti-Zika SAR properties within this class of compounds in plaque assays (ASN 07115854 (compound 61), ASN 07115861 (compound 351), ASN 07115862 (compound 352), ASN 07115865 (compound 353), ASN 07115866 (compound 354), ASN 07115873 (compound 355), ASN 07115881 (compound 356), ASN 07115764 (compound 357), ASN 07115629 (compound 358), ASN 07114909 (compound 359), ASN 07115449 (compound 360), ASN 07114414 (compound 361), ASN 08966910 (compound 362), ASN 07114826 (compound 363), ASN 08966916 (compound 364), ASN 08966912 (compound 365), ASN 07115927 (compound 366) and ASN 07115916 (compound 367). This new group was assembled to test the role of specific structural features in anti-Zika inhibitory effects, namely: (a) presence of acetyl group, and (b) presence of allyl/unsaturated alkyl group in position 3 of 1,2,4-thiadiazole. To this end, 17 new analogs were tested containing various substituents in both aromatic rings using the plaque assay (FIGs. 6 and 7). Obtained results suggest that presence of 3-acetyl-substituent is not crucial for antiviral activity as numerous compounds containing it were inactive (compounds 26-30). The better substituent seems to be CI in position 4 which, when combined with 3 -allythio- 1,2,4-thiadiazole moiety, produced extremely potent analog compound 23 (IC50=189.2±39.2 pM). An introduction of additional CF3 -substituent in position 2 of the same analog has detrimental effects (compound 24,

IC50=317.7±65.4 pM). The presence in position 4 of substituents other than CI, as seen in analogs containing -CH3 (compound 21), -CC Et (compound 20) and -F (compound 19), showed lower activity. The presence of allylthio-substituent in position 3 of 1 ,2,4-thiadiazole seems to be preferred over the propargylthio-substituent (compound 23 versus compound 34). However such effects might be limited as propargylthio-moiety-containing analog compound 35 is still active, even without the presence of the "preferred" 4-Cl substituent. Nonetheless, presence of the unsaturated alkane of proper length (allyl- or propargyl-) seems to support high anti-Zika inhibitory effects (compounds 23, 24 and 34) as shortening the distance between unsaturated bonds and 1,2,4-thiadiazole ring produced analogs with very low activity (compounds 30, 32 and 33). Table 9 shows the screening results for ASN compounds at 5 μΜ concentration.

TABLE 9 (g/mole)

ASN

61 C14H14N4O2S2 334.42 160.3±12.1 nM 07115854

ASN

351 C12H10F2N4OS2 328.36 4477.1 ±293.3 nM

07115861

ASN

355 C12H11CIN4OS2 326.82 189.2±39.2 M

07115873

ASN

356 C13H10CIF3N4OS2 394.82 317.7±65.4 M

07115881

ASN

363 C17H14F2N4O2S2 408.44 4943.1±508.5 nM

07114826

ASN

366 C13H8CIF3N4OS2 392.81 26.9±1.7 nM

07115927

ASN

367 C12H9CIN4OS2 324.81 1678.8±274.7 nM

07115916

Considering all the results and structural similarities between the identified "hits", a general structure/model of com ounds exhibiting anti-Zika inhibitory effects can be constructed:

Moreover, due to spatial similarities between the lead compounds, the compounds can occupy the same binding cavity and, by extension, possess the same mechanism of action. The model is based on the data obtained for the compounds, the study provides a general direction for the design of Zika inhibitors. Effective Zika antivirals (compounds 9, 61, 96, 205, 206, 207 and 208) should contain an assembly of 3 aromatic/aliphatic rings (or 2 rings with rigid linker) in para- or meta-configuration and with or without additional peripheral modifications (acetyl, S-allyl, O- Me, etc.). Ring A should be a six-membered aromatic or aliphatic entity. If A is an aromatic hydrocarbon, ring B should be 5- or 6-membered cycloaliphatic ring with or without heteroatoms (pyrrolidin-2-one, 2-oxazolidinone, tetrahydro-2-pyrimidinone) and linker X should be positioned in meta-configuration (X bottom). If A is a non-aromatic ring (e.g. piperazine), B ring is likely to be 5- or 6-membered aromatic moiety which may contain small substituents (-Ac, - CH3, -CF3, -CI) with X-linker in para-configuration (X top). The linker X-ring A ensemble may be substituted by urea moiety, provided both rings B and C are aromatic. Linker X may be: - CH2-, -0-, -S-, -SO2- but not an amide, especially when ring A is an aromatic entity. The ring C may be both 5- or 6-membered aromatic, heteroaromatic or cycloaliphatic ring which may contain alkylating groups (allyl- or propargyl-).

To ascertain the utility of most active analogs, a standard MTT cell viability assays using A549 cells was performed. As shown in Table 10, the identified compounds have limited or no toxicity at concentrations <1 μΜ. At higher concentrations, toxicity varies. Importantly, significant toxicity (>50%) is observed only in concentrations significantly higher than corresponding IC50 values for each compound.

TABLE 10

ZM 241385 107.4±0.6 109.7±1.7 77.8±0.7 32.6±6.1

ASN 07115854 103.6±2.0 104.6±1.6 96.7±2.8 2\.6±\.0

ASN 07115861 100.5±0.3 100.0±2.7 90.2±1.5 31.3±4.1

ASN 07115873 100.2±1.3 98.1±1.8 51.2±1.2 15.1±0.6

ASN 07115881 102.2±3.2 103.9±0.8 95.8±3.3 43.1±4.3

ASN 07114826 96.2±3.4 102.2±2.2 95.5±3.4 27.3±0.4

ASN 07115927 99.8±2.4 97.8±2.0 74.1±3.3 18.0±0.2

ASN 07115916 98.1±2.6 96.2±2.6 89.9±0.8 2\.4±\.6

Thus, a group of small-molecule inhibitors of Zika virus infection was identified using plaque assays and the A549 human lung carcinoma cell line. The identified compounds belong to 3 different classes of chemicals, namely simple sulfonamides, 1,3-disubstituted urea derivatives, and generally multi-cyclic entities containing an assembly of 3 or more aromatic/aliphatic rings, often containing N-phenyl-substituted piperazine or phenyl-substituted-piperazine-like moieties. Among the identified "hits", several compounds exhibited high inhibitory activity against Zika virus showing IC50 values in low nanomolar and sub-nanomolar range. The most active analog, ASN 07115873 possesses an exceptional IC50=189.2±39.2 pM and is a potent anti-ZIKV compound. The relatively low toxicity of identified leads, the simple structure and potential for further modifications, these compounds are excellent leads for the development of clinically viable Zika virus inhibitors.

Example 8 - Synthesis of analogs

Scheme 5

Representative substrates for the synthesis of analogs are (A) 4-chlorophenyl isocyanate, (B) 4-chlorophenyl isothiocyanate, (C) 4-bromophenyl isocyanate, (D) 4-bromophenyl isothiocyanate, (E) 4-iodophenyl isocyanate, (F) 4-iodophenyl isothiocyanate, (G) 4-chloro-3- (trifluoromethyl)phenyl isocyanate, (H) 4-chloro-3-(trifluoromethyl)phenyl isothiocyanate, (I) 4- bromo-2-(trifluoromethyl)phenyl isocyanate, (J) 4-bromo-2-(trifluoromethyl)phenyl

isothiocyanate, (1) 3-(prop-2-en-l-ylsulfanyl)-l,2,4-thiadiazol-5-amine, (2) 3-(prop-2-yn-l- ylthio)-l,2,4-thiadiazol-5-amine, (3) 3-amino-5-allylthio-l,2,4-thiadiazole, (4) 5-(prop-2-en-l- ylsulfanyl)- 1 ,3 ,4-thiadiazol-2-amine, (5) 3 -amino-5-allylamino- 1 ,2,4-thiadiazole.

All compounds were synthesized using commercially available substrates and the modified protocol described by Liu and co-workers (Bioorg Med Chem Lett (2016): 24 1866- 1871) in the condensation reaction of specific iso(thio)cyanates (A- J) with selected amine- containing thiadiazoles (1-5) which is schematically depicted in Table 6. Briefly, 15 mmoles of thiadiazole (1-5) was placed in 500 mL single-neck round-bottom flask equipped with magnetic stirrer. Subsequently, 250 mL of anhydrous tetrahydrofuran (THF) and 4.95 mL of 4- methylmorpholine (45 mmoles) were added and solution cooled in ice bath for 10 min with vigorous mixing. 16.5 mmoles (1.1 eq.) of specific iso(thio)cyanate (A-J) was dissolved in 30 mL of anhydrous THF and added in 3 mL portions to the reaction mixture over the period of 10 min with mixing. The mixing was continued overnight (~18h) at room temp, followed by 2h at 50oC. Subsequently, THF was evaporated using rotary evaporator. Remaining residue was diluted with 75 mL of absolute ethanol, and desired compound precipitated by addition of 180 mL of water. The crude analog was collected by filtration, washed twice with 25 mL of ice-cold 30% ethanol in water and dried under the vacuum (overnight). Crude compound was re- crystalized from EtOH/water system (yields=68.3%-84.1%).

Representative substrates for the synthesis of analogs and the activity are shown in Table

11. TABLE 11

Representative analogs and the activity are shown in Table 12.

TABLE 12

Example 9 - In vitro screening against Zika virus infection by plaque assay

Naive A549 cells were seeded at concentration of 2* 10 ⁵ cells per well using a 48-well plate, and allowed to grow for 48 hours to form a confluent monolayer. Subsequently, the tested compounds were added at indicated concentrations (using Opti-MEM medium) in biological triplicates. The cells were infected immediately after compound treatment, (MOI of 10, 1, 0.1, 0.01, 0.001, and 0.0001) in 100 μL· per well and plates were incubated at 37°C with 5% CC for 4 hours. At 4 hr post-infection, the viral inoculum was replaced with serum supplemented media (250 μL· per well, including the same concentration of the compounds) and plates incubated at 37°C for additional 44 hours. At 48 hr post-infection, the plaques in each well were counted using a phase contrast microscope.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.