ES2829254T3 - Pyrrolopyrimidine nucleosides and analogs thereof useful as antiviral agents - Google Patents

Abstract

A compound of formula IB: ** (See formula) ** or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, wherein: A is: ** (See formula) ** X1 is -CR11R12- or -OCH2CH2-, wherein the oxygen atom is distal to the RIB moiety in A; R11 and R12 are independently hydrogen or C1-C4 alkyl, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH, or -NH2; X2 is absent, it is -O-, -C (O) O-, or -OCH2-, wherein the oxygen atom is distal to the RIB moiety in A; each RIB is independently hydrogen, -C1-C6 alkyl, ** (See formula) ** or RIB is an amino acid residue attached via the carbonyl group, wherein the alkyl is optionally substituted with one or more halogen, -OH, -SH or -NH2; v is 0 or 1; n is 0, 1, 2 or 3 and when X2 is -C (O) O-, n is 0; p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; Rz is hydrogen, halogen, -C1-C4 alkylthio, -C1-C4 alkoxy, -C1-C4 alkyl, -C2-C4 alkenyl, -C2-C4 alkynyl, aryl, heteroaryl, -C3-C8 cycloalkyl, -C4 cycloalkenyl- C8 or 3- to 5-membered non-aromatic heterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionally substituted with one or more of halogen, -OH, -SH, or -NH2; Ra, Rb, Rx, and Ry are each independently selected from the group consisting of hydrogen, halogen, -OH, -SH, -C1-C6 alkoxy, aryloxy, -C1-C6 alkylthio, arylthio, -OC (O) C1 alkyl -C6, -OC (O) aryl, -C1-C6 alkyl, -C2-C6 alkenyl, -25 C2-C6 alkynyl, aryl, heteroaryl, -C3-C8 cycloalkyl, and -C4-C8 cycloalkenyl, wherein each alkoxy , aryloxy, alkylthio, arylthio, alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl or cycloalkenyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2; or any two Ra or Rb, together with the atom to which they are both attached, may combine to form a 3- to 8-membered spirocycloalkyl or spiroheterocycle; or any two Ra or Rb, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond; or any two Ra or Rb, when on adjacent atoms, may combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring-membered heterocycle; or any CRaRb can be replaced by -O-, -S-, -S (O) - or -SO2-; or any two Rx or Ry, together with the atom to which they are both attached, may combine to form a 3- to 8-membered - spirocycloalkyl or spiroheterocycle; or any two Rx or Ry, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond; or any two Rx or Ry, when on adjacent atoms, may combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring heterocycle; or any CRxRy can be replaced by -O-, -S-, -S (O) - or -SO2-; R1 and R45 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -C2 alkynyl -C6, -C8-C12 cycloalkynyl, -C 1-6 alkoxy or -C 1-6 alkylthio in which each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy or alkylthio is independently substituted with one or more halogen, - N3, -OH, -NH2 or -SH; R2, R3, R4, and R44 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6 alkyl, -C1-C6 alkoxy, or -C1-C6 alkylthio, wherein each alkyl , alkoxy or alkylthio is optionally substituted with one or more of halogen, -N3, -OH, -NH2 or -SH; or R3 and one of R4 and R44, together with the atoms to which they are attached may form a carbon-carbon double bond; R5 is independently hydrogen, -RIB, M +, aryl, aralkyl, -C1-C6 alkyl, -C1-C6 heteroalkyl, cycloalkyl, non-aromatic heterocyclic ring, or heteroaryl, wherein M + is a cation and wherein each aryl, aralkyl , alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH, and wherein R5 is not an amino acid; and Rc is -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -C2-C6 alkynyl, -C8-C12 cycloalkynyl or aryl, wherein each alkyl, cycloalkyl, alkenyl , cycloalkenyl or aryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2 or -SH.

Description

DESCRIPTION

Pyrrolopyrimidine nucleosides and analogs thereof useful as antiviral agents

Related requests

This application claims priority to and benefit from US Provisional Application No. 62/202010, filed August 6, 2015, and UK Application No. 1606645.8, filed April 15, 2016.

Technical field

This application relates to pyrrolopyrimidine nucleoside analogs and phospholipid conjugates thereof and methods of synthesis thereof. Pyrrolopyrimidine nucleoside analogs and their phospholipid conjugates can be used as antiviral agents to treat viral infections. This application also relates to pharmaceutical compositions comprising pyrrolopyrimidine nucleoside analogs and phospholipid conjugates thereof.

Background

Viral infections can have serious adverse effects on individuals and society as a whole. In addition to deadly viral infections such as the Ebola virus, even infections that are not fatal can have serious social and economic consequences. For example, human norovirus (NV) is the most common cause of epidemic acute gastroenteritis worldwide with an estimated 19-21 million cases each year in the United States, including 56,000-71,000 hospitalizations and 570-800 deaths. (Hall et al., Emerg.Infect.Dis. 2013 Aug; 19 (8): 1198-205).

Therefore, the development of an effective antiviral treatment that is effective against viruses is important to improve the health of infected individuals and as a public health measure to prevent outbreaks of other pathogenic viruses.

Summary

The present disclosure provides pyrrolopyrimidine nucleoside analogs and phospholipid conjugates thereof. Also included are pharmaceutical compositions comprising the same and methods of synthesis thereof.

The present disclosure also provides compounds and compositions of the invention for use in methods of treating and / or preventing viral infections or diseases or disorders associated with viral infections. The disclosure addresses the need for new treatments that can be used to treat and / or prevent virus-induced diseases using novel antivirals and delivery vehicles.

In one aspect, the present disclosure relates to compounds of formula IB:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, wherein:

A is:

X X1 is -CR11R12- or -OCH2CH2-, wherein the oxygen atom is distal to the RIB moiety in A;

R11 and R12 are independently hydrogen or C 1 -C4 alkyl, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH, or -NH2;

X2 is absent, it is -O-, -C (O) O- or -OCH2-, where the oxygen atom is distal to the RIB moiety in A;

each RIB is independently hydrogen, -C1-C6 alkyl,

or RIB is an amino acid residue linked via the carbonyl group, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2;

v is 0 or 1;

n is 0, 1,2 or 3 and when X2 is -C (O) O-, n is 0;

p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;

Rz is hydrogen, halogen, -C1-C4 alkylthio, -C1-C4 alkoxy, -C1-C4 alkyl, -C2-C4 alkenyl, -C2-C4 alkynyl, aryl, heteroaryl, -C3-C8 cycloalkyl, -C4 cycloalkenyl- C8 or non-aromatic 3- to 5-membered heterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionally substituted with one or more of halogen, -OH, -SH, or -NH2;

Ra, Rb, Rx, and Ry are each independently selected from the group consisting of hydrogen, halogen, -OH, -SH, -C1-C6 alkoxy, aryloxy, -C1-C6 alkylthio, arylthio, -OC (O) C1 alkyl -C6, -OC (O) aryl, -C1-C6 alkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, aryl, heteroaryl, -C3-C8 cycloalkyl and -C4-C8 cycloalkenyl, wherein each alkoxy, aryloxy, alkylthio, arylthio, alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl or cycloalkenyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2;

or any two Ra or Rb, together with the atom to which they are both attached, can combine to form a 3 to 8 membered spirocycloalkyl or spiroheterocycle;

or any two Ra or Rb, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond;

or any two Ra or Rb, when on adjacent atoms, can combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring-membered heterocycle;

or any CRaRb can be replaced by -O-, -S -, -S (O) - or -SO2-;

or any two Rx or Ry, together with the atom to which they are both attached, may combine to form a 3- to 8-membered -spyrocycloalkyl or spiroheterocycle;

or any two Rx or Ry, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond;

or any two Rx or Ry, when on adjacent atoms, can combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring-membered heterocycle;

or any CRxRy can be replaced by -O-, -S -, -S (O) - or -SO2-;

R1 and R45 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -alkynyl C2-C6, -C8-C12 cycloalkynyl, -C1-C6 alkoxy or -C1-C6 alkylthio, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, or alkylthio is independently substituted with one or more halogen , -N3, -OH, -NH2 or -SH;

R2, R3, R4, and R44 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6alkyl, -C1-C6alkoxy, or -C1-C6alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH;

or R3 and one of R4 and R44, together with the atoms to which they are attached may form a carbon-carbon double bond; R5 is independently hydrogen, -R IB, M +, aryl, aralkyl, -C1-C6 alkyl, -C1-C6 heteroalkyl, cycloalkyl, non-aromatic heterocyclic ring or heteroaryl, wherein M + is a cation and wherein each aryl, aralkyl, alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH, and wherein R5 is not an amino acid; Y

Rc is -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -C2-C6 alkynyl, -C8-C12 cycloalkynyl or aryl, wherein each alkyl, cycloalkyl, alkenyl, Cycloalkenyl or aryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH.

In another aspect, the present disclosure relates to compounds of formula II:

o h 2n

RbÓ ÓRa (II)

and pharmaceutically acceptable salts, solvates, enantiomers, diastereoisomers, racemates, or mixtures thereof, wherein:

Y is -C (O) -, or

wherein X 1 is independently O, NH, or S, X 2 is independently a bond, -O-, -S-, or -NH-, and X3 is independently -OR, -N HRM, or -S R M;

each RM is independently -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -C3-C8 cycloalkyl, -C4-C8 cycloalkenyl, aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl , alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl is optionally substituted with one or more of halogen, oxo, R1, -O R1, -N R1R2, -SR 1, -OC (O) R1, -C (O) OR1, -NHC (O) OR1 or -NHC (O) R1;

Ra and Rb are each independently, each occurrence, -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -C3-C8 cycloalkyl, -C4-C8 cycloalkenyl, aryl, heteroaryl or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, -OR 1, -N R1R2, -SR 1, -OC (O) R1, -C (O) OR1, -NHC (O) OR1 or -NHC (O) R1;

R1 and R2 are each independently, each occurrence, -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -C3-C8 cycloalkyl, -C4-C8 cycloalkenyl, aryl, heteroaryl or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, -R 3, -R 4, -O R3, -N R3R4, - SR 3, -OC (O) R3, -C (O) OR3, -NHC (O) OR3 or -NHC (O) R3;

R3 and R4 are each independently, each occurrence, -H, -C1-C20 alkyl, -C2-C20 alkenyl, -C2-C20 alkynyl, -C3-C8 cycloalkyl, -C4-C8 cycloalkenyl, aryl, heteroaryl or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, aryl, heteroaryl, -OH, -NH2, -SH, -OC (O ) H, -C (O) OH, -NHC (O) OH or -NHC (O) H;

Rd is independently -H or -D; Y

n is independently 0, 1,2 or 3.

In some embodiments, Ra is -H. In some embodiments, Rb is -H. In some embodiments, Rd is -H. In some embodiments, n is 0. In some embodiments, Ra, Rb, and Rd are -H. In some embodiments, Ra, Rb, and Rd are -H and n is 0.

In some embodiments, the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising a compound of formula IB, or formula II, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure refers to a pharmaceutical composition comprising a compound 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to treat a viral infection (eg, norovirus).

In another aspect, the present disclosure also refers to a pharmaceutical formulation of the compounds disclosed herein, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof, for use in a method of treating or preventing a viral infection or disease or disorder associated with viral infection, for example, a double-stranded DNA (dsDNA) or single-stranded RNA (ssRNA) virus infection. In some embodiments, the compound is compound 1. In some embodiments, the virus is norovirus.

The present disclosure also refers to a compound described herein, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, for use in treating or preventing a viral infection or associated disease or disorder. with viral infection. The compound can be a compound of formula IB or formula II. In some embodiments, the compound is compound 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In some embodiments, the virus is norovirus.

Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, shall prevail. In the specification, singular forms also include the plural, unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. References cited in this document are not admitted as prior art to the claimed disclosure. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other characteristic features and advantages of the present disclosure will be apparent from the following detailed description and claims.

Brief description of the drawings

Figure 1A shows the murine norovirus concentration (plaque-forming units per ml) in tissue and stool collected 3 days after infection as part of Study # 1.

Figure 1B shows the murine norovirus concentration (plaque-forming units per mg of tissue) in tissue and stool collected 3 days after infection as part of Study # 1.

Figure 2A shows the murine norovirus concentration (plaque-forming units per ml) in tissue and stool collected 3 days after infection as part of Study # 2.

Figure 2B shows the murine norovirus concentration (plaque-forming units per mg of tissue) in tissue and stool collected 3 days after infection as part of Study # 2.

Figure 3A shows the number of plaque-forming units per gram of cecum from Study 1 on a linear scale.

Figure 3B shows the number of plaque-forming units per gram of stool from Study 1 on a linear scale.

Figure 4 shows the first duplicate of the results demonstrating the in vitro efficacy of Compound 1 to inhibit human norovirus compared to 2'-C-methylcytidine triphosphate and Compound 2.

Figure 5 shows the second duplicate of results demonstrating the in vitro efficacy of Compound 1 to inhibit human norovirus compared to 2'-C-methylcytidine triphosphate and Compound 2.

Figure 6 shows an overlay of the first and second duplicates of results demonstrating the in vitro efficacy of Compound 1 in inhibiting human norovirus compared to 2'-C-methylcytidine triphosphate and Compound 2.

Figure 7a shows an HPLC diagram of compound 1.

Figure 7b shows an HPLC diagram of compound 1 after suspension for 3 hours at room temperature. Figure 7c shows an HPLC diagram of compound 1 after suspension for 3 hours at 50 ° C.

Figure 7d shows an HPLC diagram of compound 1 after suspension for 24 hours at about room temperature.

Figure 8a shows a 1H NMR spectrum of compound 1 from about -2 to about 14 ppm. Figure 8b shows a 1H NMR spectrum of compound 1 from about 2 to about 9 ppm. Figure 8c shows a 1H NMR spectrum of compound 1 from about 0 to about 9 ppm.

Detailed description

Nucleoside phosphonates (eg ribonucleoside derivatives) represent a target class of antivirals for inhibiting viruses, which are based on virus-encoded enzymes that use ribonucleotides or deoxyribonucleotides as substrates, such as certain viral polymerases for many RNA viruses and / or or viral helicases for RNA (eg ssRNA) or DNA viruses. However, without wishing to be bound by theory, an impediment to the efficacy of this class of antivirals is the need for biochemical modification of the administered agent within the target cells to form the active antiviral nucleoside triphosphate. In some In embodiments, if a nucleoside is administered, three phosphorylation steps are required to form the triphosphate. Administration of nucleoside phosphonates effectively prevents the first phosphorylation, but can exacerbate problems of delivering clinically useful amounts of the loaded drug through the lipid bilayers surrounding cells.

Without wishing to be bound by theory, lipid conjugation can be used to mask oral drugs, including nucleoside phosphonates, as naturally occurring compounds that are readily absorbed by the body. Specifically, in some embodiments, the nucleoside phosphonates can be modified to resemble partially metabolized phospholipids (monoacylphospholipids). In some embodiments, in contrast to normal diacylphospholipids, monacyl lipid-modified nucleotides can easily penetrate enterocytes lining the lumen of the intestine, enter the circulating blood and / or lymphatic system, and, unlike conventional drugs, remain intact. Consequently, the lipid moiety can do more than deliver the nucleoside to the plasma; can facilitate efficient uptake into target cells. The lipid can be cleaved in the cytoplasmic compartment of target cells and, in the case of nucleoside analog conjugates, can produce the corresponding monophosphate. Overall, this strategy can result in greatly increased levels of the active antiviral at the virus replication site.

The present disclosure provides compounds, pharmaceutical compositions, and methods of synthesis and use of the compounds to treat or prevent a viral infection or disease or disorder associated with viral infection, for example, a ssRNA virus infection.

In some embodiments, the compounds of the present disclosure have an improved efficacy / toxicity ratio compared to similarly used compounds in the art.

Definitions

Certain compounds of the present disclosure and definitions of specific functional groups are also described in more detail below.

It will be appreciated that compounds, as described herein, can be substituted with very many functional moieties or substituents. In general, the term "substituted", whether preceded by the term "optionally" or not, and the substituents contained in formulas disclosed herein, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. . When more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be the same or different at each position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For the purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. The nitrogen and sulfur heteroatoms can optionally be oxidized, and the nitrogen heteroatom can optionally be quaternized. Examples of substituents on the moieties disclosed herein (eg, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl, cycloalkenyl, non-aromatic heterocycle groups) include, but are not limited to, alkenyl, alkynyl, halogen, haloalkyl, alkoxy , alkylthio, alkylsulfinyl, alkylsulfonyl, heteroaryl, aryl, cycloalkyl, cycloalkenyl, non-aromatic heterocycle, hydroxyl, carbamoyl, oxo, amino, nitro, azido, -SH and -CN.

The present disclosure is intended to include all isotopes of atoms that appear in the present compounds. Isotopes include those atoms that have the same atomic number, but different mass numbers. In particular one, some or all of the hydrogens can be deuterium. Radioactive isotopes can be used, for example, for structural analysis or to facilitate tracking of the fate of compounds or their metabolic products after administration. By way of general example and without limitation, hydrogen isotopes include deuterium and tritium and carbon isotopes include C -13 and C -14. For example, compounds of formula I include those in which Ri is H or D; R2 and R3 are independently H, D, OH, OD, CH3 or CD3; and / or R4 is H, D, CH3 or CD3.

The term "independently" is used herein to indicate that the variable, such as the atom or functional group, which is applied independently, varies independently from one application to another. For example, when there is more than one substituent or atom (carbon or heteroatom, such as oxygen (O), sulfur (S), or nitrogen (N)), each substituent or atom is independent of another substituent or atom and said substituents or atoms they can also alternate.

The term "alkyl", as used herein, refers to straight chain or branched saturated hydrocarbon radicals containing, in certain embodiments, between one and twenty, including between one and ten, or between ten and six, atoms. carbon. Branched means that one or more C1-C6 lower alkyl groups, such as methyl, ethyl, or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl. Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, neopentyl, n-hexyl radicals; and examples of radicals Ci-Ca alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tere-butyl, neopentyl, n-hexyl, heptyl, octyl radicals. Examples of C1-C20 alkyl radicals include, but are not limited to, hexadecamethyl, hexadecaethyl, hexadecapropyl, octadecamethyl, octadecaethyl, octadecapropyl, and the like.

The term "alkenyl", as used herein, refers to a monovalent linear or branched group derived from a hydrocarbon moiety containing, in certain embodiments, two to six, or two to eight, or two to twenty carbon atoms, having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Examples of C2-C8 alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, and the like. As defined herein, "alkenyl" groups include both eis and trans isomers.

The term "alkynyl", as used herein, refers to a monovalent linear or branched group derived from a hydrocarbon moiety containing, in certain embodiments, two to six, or two to eight, or two to twenty carbon atoms, having at least one carbon-carbon triple bond. The triple bond may or may not be the point of adhesion to another group. Examples of C2-C8 alkynyl groups include, but are not limited to, for example, ethynyl, propynyl, butynyl, and the like.

The term "alkoxy" refers to an -O-alkyl radical.

The term "thioalkyl" or "alkylthio" refers to an -S-alkyl radical. In some embodiments, the thio group can be replaced by a sulfinyl (SO) or sulfonyl (SO2).

The terms "hal", "halo" or "halogen", as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.

The terms "haloalkyl", "haloalkenyl" or "haloalkynyl", as used herein, refer to an alkyl, alkenyl, or alkynyl that is substituted with one or more halogens or halo groups. Examples of haloalkyl include, but are not limited to, CF3, CH2CF3, CCl3.

The term "aryl", as used herein, refers to a mono- or polycyclic carbocyclic ring system having one or more aromatic rings, fused or uncondensed including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl , idenilo and the like. The term aryl includes indoline.

The term "cycloalkyl", as used herein, refers to a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic ring compound. Examples of C3-C8 cycloalkyl (3- to 8-membered cycloalkyl) include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, and cyclooctyl; and examples of C3-C12 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl and bicyclo [2.2.2] octyl and the like.

The term "cycloalkenyl", as used herein, refers to a monovalent group derived from a monocyclic or polycyclic partially unsaturated carbocyclic ring compound (ie, non-aromatic). In other words, it refers to a monovalent group derived from a monocyclic or polycyclic carbocyclic ring compound having at least one carbon-carbon double bond. Examples of such groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term "cycloalkynyl", as used herein, indicates a monovalent group derived from a partially unsaturated monocyclic or polycyclic (ie, non-aromatic) carbocyclic compound having at least one carbon-carbon triple bond. Examples include cyclooctin.

The term "heteroaryl", as used herein, refers to a mono- or polycyclic (eg, bi- or tricyclic or more) fused or unfused radical or ring system, having at least one aromatic ring, having five to ten ring atoms, of which at least one ring atom is selected from S, O, P and N. In other words, heteroaryl is aryl containing at least one hetero atom. Examples of heteroaryl include, but are not limited to, pyridinyl, furanyl, thiazolyl, imidazolyl, indolyl, benzofuranyl, and the like.

The term "5- or 6-membered heteroaryl" is accepted to mean a ring having from five to twelve ring atoms, of which at least one ring atom is selected from S, O, P, and N. Heteroaryl includes but not limited to pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalyl, and the like.

The term "non-aromatic heterocyclic" or "non-aromatic heterocycle" ring, as used herein, refers to a saturated or unsaturated, non-aromatic monocyclic or polycyclic, condensed or non-condensed system, where, for example, at least one ring contains between one and four heteroatoms independently selected from oxygen, sulfur, phosphorus, and nitrogen. The nitrogen and sulfur heteroatoms can optionally be oxidized, and the nitrogen heteroatom can optionally be quaternized. Representative non-aromatic heterocyclic groups include, but are not limited to, [1,3] dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

As used herein, the term "oxo" is understood to describe a carbonyl group (ie, C (O)).

As described herein, the compounds of the disclosure may optionally be substituted with one or more substituents, such as those generally illustrated above, or as exemplified by particular classes, subclasses, and species of the disclosure. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted". In general, the term "substituted", whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have one substituent at each substitutable position in the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be the same or different in each position.

The term "protected", as described herein, refers to functional groups or compounds of the present disclosure that have a protecting group used in synthesis to temporarily mask the characteristic chemistry of a functional group (such as hydroxyl, amino, carboxyl, etc.) because it interferes with another reaction. After completion of the reaction, these protecting groups are removed by common methods, or the protected compounds are used as prodrugs or as the compounds of the disclosure.

The term "prodrug" or "pharmaceutically acceptable prodrugs", as used herein, refers to compounds that are rapidly transformed in vivo to produce the parent compound, for example, by hydrolysis in blood (T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987).

The term "pharmaceutical" or "pharmaceutically acceptable", when used herein as an adjective, means substantially non-toxic and substantially non-injurious to the recipient. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers, and / or dosage forms that are, within reasonable medical judgment, suitable for use in contact with the tissues of human beings. humans and animals without causing toxicity, irritation, allergic response or other excessive problem or complication, and consistent with a reasonable benefit / risk ratio.

By "pharmaceutical formulation" it is further meant that the carrier, solvent, one or more excipients and salt must be compatible with the active ingredient of the formulation (eg, a compound of the disclosure). Those skilled in the art understand that the terms "pharmaceutical formulation" and "pharmaceutical composition" are, in general, interchangeable, and therefore are used for the purposes of this application and include preparations suitable for administration to mammals, for example, human beings. humans.

A "pharmaceutical composition", as used herein, refers to a formulation containing a compound of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms including, for example, a capsule, an IV bag, a tablet, a single pump in an aerosol inhaler, or a vial. The amount of active ingredient (eg, a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of the composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations in dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. As used herein, "pharmaceutically acceptable carrier" can include all solvents, diluents or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various vehicles used in the formulation of pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds, such as by producing some undesirable biological effect or otherwise interacting in a deleterious manner with any other components or components of the pharmaceutical composition, its use is contemplated within the scope of this disclosure. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, carbohydrates such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; Ethyl alcohol and phosphate buffer solutions, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and sodium stearate. Magnesium, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the criteria of the formulator. "Pharmaceutically acceptable excipient or carriers" also refers to an excipient or carrier that is useful in the preparation of a pharmaceutical composition that is generally safe, non-toxic, and neither biologically undesirable nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use, as well as pharmaceutical use in humans. A "pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one of said excipient.

The compounds disclosed herein include the compounds themselves, as well as their salts, solvates, and prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged group (eg, protonated amino) in a compound of this disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalene sulfonate. acetate (eg trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, a salt can also be formed between a cation and a negatively charged group (eg, carboxylate) in a compound of this disclosure. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. The compounds of this disclosure also include those salts that contain quaternary nitrogen atoms. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives which, upon administration to a subject, can provide active compounds of this disclosure.

Additionally, the physiologically acceptable, ie, pharmaceutically compatible salts can be salts of the compounds disclosed herein with inorganic or organic acids. Preference is given to salts with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or to salts with organic carboxylic or sulfonic acids such as, for example, acetic acid, trifluoroacetic acid, propionic acid, acid maleic, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid or naphthalenedisulfonic acid.

Other pharmaceutically compatible salts that may be mentioned are salts with customary bases such as, for example, alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example, calcium or magnesium salts) or ammonium salts. derived from ammonia or organic amines such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine or methylpiperidine.

As used herein, "pharmaceutically acceptable salts" can refer to derivatives of the compounds of the present disclosure in which the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acid residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1, 2-Ethanesulfonic, Fumaric, Glucoheptonic, Gluconic, Glutamic, Glycolic, Glycoliarsanilic, Hexylresorcinic, Hydrabamic, Hydrobromic, Hydrochloric, Hydroiodic, Hydroxymaleic, Hydroxynaphthoic, Isethionic, Lactic, Lactobionic, Mandelic, Nauryl, Methane sulfonic, Lactobionic, Lauryl, Naurylsulfonic, Male oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic and the naturally occurring amino acids, for example, glycine, alanine, phenylalanine , etc.

Other examples of pharmaceutically acceptable salts may include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid. , 4-methylbicyclo- [2.2.2] -oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound is replaced by a metal ion, for example an alkali metal ion or an alkaline earth metal ion, for example an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, lysine, arginine, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylglucamine, pyroxylethylenediamine, trimethylamine, pyrimethylamine, pyrimethylamine, trimethylamine tetramethylammonium and the like.

All references to pharmaceutically acceptable salts are to be understood to include solvent addition forms (solvates) or crystalline forms (polymorphs) as defined herein, of the same salt.

The compounds of the present disclosure can also be prepared as prodrugs. In certain embodiments, one or more compounds of the present disclosure are formulated as a prodrug. In certain Embodiments, upon in vivo administration, a prodrug is chemically converted to the most biologically, pharmaceutically, or therapeutically active form. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain cases, a prodrug may be more bioavailable (eg, by oral administration) than the corresponding active form is. In certain cases, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrugs are less soluble in water than the corresponding active form. In certain cases, these prodrugs have superior transmission through cell membranes, where solubility in water is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid after administration. In certain cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyamino acid) attached to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound so that the active compound will regenerate after administration in vivo . The prodrug can be designed to alter the metabolic stability or transport characteristics of a drug, to mask side effects or toxicity, to enhance the aroma of a drug, or to alter other characteristics or properties of a drug. As a consequence of the knowledge of the pharmacological processes and the metabolism of drugs in vivo , those skilled in the art, once they know a pharmaceutically active compound, will be able to design prodrugs of the compound (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).

Furthermore, the compounds of the present disclosure, eg, the salts of the compounds, can exist in their hydrated or non-hydrated (the anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

Some of the compounds of the present disclosure may exist in unsolvated as well as solvated forms such as, for example, hydrates.

"Solvate" means a solvent addition form that contains stoichiometric or non-stoichiometric amounts of solvent. Some compounds may have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water, the solvate formed is a hydrate, when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by combining one or more water molecules with one of the substances in which water retains its molecular state as H2O, and this combination can form one or more hydrates. In hydrates, water molecules are linked through secondary valences by intermolecular forces, in particular hydrogen bonds. Solid hydrates contain water such as so-called crystalline water in stoichiometric ratios, where the water molecules do not have to be equivalent with respect to their binding state. Examples of hydrates are sesquihydrates, monohydrates, dihydrates, or trihydrates. Equally suitable are the hydrates of salts of the compounds of the disclosure.

The disclosure also includes metabolites of the compounds described herein. The metabolites of chemical compounds, whether inherent or pharmaceutical, are formed as part of the natural biochemical process of degradation and elimination of the compounds. The rate of degradation of a compound is an important determinant of the duration and intensity of its action. Profiling the metabolites of pharmaceutical compounds, drug metabolism, is an important part of drug discovery, leading to an understanding of any undesirable side effects.

As used herein, the term "treating", "treating" or "treatment" means decreasing the symptoms, markers, and / or any negative effects of a condition to any appreciable degree in a patient who currently has the condition. In some embodiments, the treatment can be administered to a subject showing only early signs of the condition in order to decrease the risk of developing the disease, disorder, and / or condition.

As used herein, the term "prevent", "prevention" or "preventing" refers to any method of partially or completely preventing or delaying the appearance of one or more symptoms or characteristic features of a disease, disorder and / or condition. The preventive treatment can be administered to a subject showing no signs of a disease, disorder, and / or condition.

The term "therapeutically effective amount", as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to display a detectable therapeutic or inhibitory effect. The effect can be detected by any test method known in the art. As used herein, "therapeutically effective amount" can also mean that amount necessary to generate a clinically observed improvement in the patient. In some embodiments, the composition it is formulated such that it comprises an amount that would not cause one or more unwanted side effects.

An effective amount of a pharmaceutical agent can also mean that which provides an objectively identifiable improvement appreciated by the physician or other qualified observer. The precise effective amount for a subject will depend on the subject's body weight, size, and health; the nature and degree of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation, according to the skill and judgment of the physician.

As used herein, "subject" means a human or animal (in the case of an animal, more typically a mammal). In one aspect, the subject is a human being. In one aspect, the subject is male. In one respect, the subject is female.

The compounds of the present disclosure can also be prepared as esters, for example pharmaceutically acceptable esters. For example, a carboxylic acid functional group in a compound can be converted to its corresponding ester, eg, a methyl, ethyl, or other ester. Furthermore, an alcohol or hydroxyl group in a compound can be converted to its corresponding ester, for example, acetate, propionate, or other esters.

The present disclosure includes novel compounds generally represented by formula IB or formula II, or pharmaceutically acceptable salts thereof, and methods for the preparation and uses thereof.

Throughout the description, when describing compositions having, including, or comprising specific components, it is contemplated that the compositions also consist essentially of, or consist of, the listed components.

Compounds

The present disclosure provides a compound of formula IB:

and pharmaceutically acceptable salts, solvates, enantiomers, diastereoisomers, racemates, or mixtures thereof, wherein:

A is:

istal al resto RIB en A;

istal to the RIB residue in A;

R11 and R12 are independently hydrogen or C 1 -C4 alkyl, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH, or -NH2;

X2 is absent, it is -O-, -C (O) O- or -OCH2-, where the oxygen atom is distal to the RIB moiety in A;

each RIB is independently hydrogen, -C ^{1 -C 6} alkyl,

or RIB is an amino acid residue linked via the carbonyl group, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2;

v is 0 or 1;

n is 0, 1,2 or 3 and when X2 is -C (O) O-, n is 0;

p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11;

q is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18;

Rz is hydrogen, halogen, -C1-C4 alkylthio, -C1-C4 alkoxy, -C1-C4 alkyl, -C2-C4 alkenyl, -C2-C alkynyl heteroaryl, -C3-C8 cycloalkyl, -C4-C8 cycloalkenyl or heterocycle 3 to 5 membered non-aromatic, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionally substituted with one or more of halogen, -OH, -SH, or -NH2;

Ra, Rb, Rx, and Ry are each independently selected from the group consisting of hydrogen, halogen, -OH, -SH, -C1-C6 alkoxy, aryloxy, -C1-C6 alkylthio, arylthio, -OC (O) C1 alkyl -C6, -OC (O) aryl, -C1-C6 alkyl, -C2-C6 alkynyl, aryl, heteroaryl, -C3-C8 cycloalkyl and -C4-C8 cycloalkenyl, wherein each alkoxy, aryloxy, alkylthio,

arylthio, alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl, or cycloalkenyl is optionally substituted with one or more of halogen, -OH, -SH, or -NH2;

or any two Ra or Rb, together with the atom to which they are both attached, can combine to form a 3 to 8 membered spirocycloalkyl or spiroheterocycle;

or any two Ra or Rb, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond;

or any two Ra or Rb, when on adjacent atoms, may combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring-membered heterocycle;

or any CRaRb can be replaced by -O-, -S-, -S (O) - or -SO2-;

or any two Rx or Ry, together with the atom to which they are both attached, may combine to form a 3- to 8-membered -spyrocycloalkyl or spiroheterocycle;

or any two Rx or Ry, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond;

or any two Rx or Ry, when on adjacent atoms, may combine to form an aryl, heteroaryl, -C3-C10 -cycloalkyl, -C4-C10 -cycloalkenyl, or 5- to 10-membered ring heterocycle;

or any CRxRy can be replaced by -O-, -S-, -S (O) - or -SO2-;

R1 and R45 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -C2 alkynyl -C6, -C8-C12 cycloalkynyl, -C1-C6 alkoxy, or -C1-C6 alkylthio, wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, or alkylthio is independently substituted with one or more halogen, -N3, -OH, -NH2 or -SH;

R2, R3, R4, and R44 are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C1-C6 alkyl, -C1-C6 alkoxy, or -C1-C6 alkylthio, wherein each alkyl , alkoxy or alkylthio is optionally substituted with one or more of halogen, -N3, -OH, -NH2 or -SH;

or R3 and one of R4 and R44, together with the atoms to which they are attached may form a carbon-carbon double bond; R5 is independently hydrogen, -RIB, M +, aryl, aralkyl, -C1-C6 alkyl, -C1-C6 heteroalkyl, cycloalkyl, non-aromatic heterocyclic ring, or heteroaryl, wherein M + is a cation and wherein each aryl, aralkyl , alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH, and wherein R5 is not an amino acid; Y

Rc is -C1-C6 alkyl, -C3-C6 cycloalkyl, -C2-C6 alkenyl, -C4-C8 cycloalkenyl, -C2-C6 alkynyl, -C8-C12 cycloalkynyl or aryl, wherein each alkyl, cycloalkyl, alkenyl, Cycloalkenyl or aryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH.

In some embodiments, A is selected from A1 to A14:

In some embodiments, A is

In some embodiments, A is

In some embodiments, A is

In some embodiments, Ri is -H. In some embodiments, R2 is -OH. In some embodiments, R4 is -OH. In some embodiments, R3 is -H. In some embodiments, R44 is -H. In some embodiments, RIB is -H. In some embodiments, Rc is -CH3. In some embodiments, v is 1, X2 is -O-, n is 0, and RIB is -H. In some embodiments, R5 is -H or M +, where M + is Na +, Li +, K +, C a2 +, Mg2 +, or NRgRdReRf +, and where Rg, Rd, Re, and Rf are each independently hydrogen or -C1- alkyl C5.

In one or more embodiments, RIB is selected from: -H,

In one or more embodiments, the compound is of formula l-a:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In one or more embodiments, the compound is of formula l-b:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In one or more embodiments, the compound is of formula l-c:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof.

In one or more embodiments, the compound is of formula l-d:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof.

In one or more embodiments, the compound of the disclosure is compound 1:

(Compound 1; 4-amino-7 - ((2R, 3R, 4S, 5R) -3,4-d¡hydrox¡-5- (hydrox¡methyl) tetrahydrofuran-2-¡ l) -2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carboxamide) or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof.

In one or more embodiments, the compound of the disclosure is 1-triphosphate compound (1-TP compound or 1-PPP compound) or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof:

In one or more embodiments, the compound of the disclosure is selected from:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In one or more embodiments, the compound of the disclosure is selected from:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In one or more embodiments, the compound of the disclosure is selected from:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In one or more embodiments, the compound of the disclosure is selected from:

Ċ

In one or more embodiments, the compound of the disclosure is selected from:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof.

In some embodiments, the present disclosure provides a compound as described herein for use in treating a disease.

In some embodiments, the present disclosure provides the compound

for use in treating a disease. In some embodiments, the disease is a viral disease. In some embodiments, the viral infection is norovirus infection.

In some embodiments, the compound of the disclosure is selected from compound 1,2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143, or 145, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, or any combination thereof.

Synthesis methods

The compounds of the present disclosure can be prepared by a variety of methods, including conventional chemistry. Suitable synthetic routes are represented in the schemes given below.

The compounds described herein (eg, compounds of formula IB or formula II) can be prepared by methods known in the art of organic synthesis as set forth, in part, by the following synthetic schemes and examples. In the schemes described below, it is well understood that protecting groups are employed for sensitive or reactive groups where necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to conventional methods of organic synthesis (T. W. Greene and P. G. M. Wuts, "Protective Groups in Organic Synthesis", third edition, Wiley, New York 1999). These groups are removed at a convenient stage in the synthesis of the compound using methods that are well apparent to those of skill in the art. The selection processes, as well as the reaction conditions and the order of their execution, must be consistent with the preparation of compounds of formula IB or formula II.

Those skilled in the art will recognize whether a stereocenter exists in compounds of formula IB or formula II. Accordingly, the present disclosure includes both possible stereoisomers (unless specified in the synthesis) and includes not only racemic compounds, but also individual enantiomers and / or diastereoisomers. When a compound is desired as a single enantiomer or diastereoisomer, it can be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, an intermediate or a starting material can be achieved by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel, S. H. Wilen, and L. N. Mander (Wileylnterscience, 1994).

The compounds described herein can be prepared from commercially available starting materials or synthesized using known organic, inorganic and / or enzymatic processes.

Scheme 1: General synthesis of compounds of the invention

As shown above in Scheme 1, 4-chloro-2-methyl-7H-pyrrolo [2,3-d] pyrimidine ( a ; numbering from Scheme 1) can be iodinated in the presence of N-iodosuccinimide (NIS). The resulting 4-chloro-5-iodo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine ( b ) can be treated with a protected furan ( c ) as shown in step 2 to give compound ( d ). Radical substitution of ( d ) gives the corresponding cyano derivative ( e ) which can undergo deprotection and nucleophilic aromatic substitution at the chlorine-bound carbon to give the amine derivative ( f ). Finally, hydration of the nitrile of ( f ) gives 4-amino-7 - ((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2-methyl- 7H-pyrrolo [2,3-d] pyrimidine-5-carboxamide ( 1 ).

Scheme 2: General synthesis of compounds of the invention

Step 1: 4-Amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile, (3R, 4R, 5R) -2-acetoxy-5 - ((benzoyloxy) dibenzoate ) methyl) tetrahydrofuran-3,4-diyl and DCE were charged into a reactor. Stirring was started and DBU was added. TMSOTf (8.01 kg) was added slowly. The reaction mixture was diluted with DCM and slowly quenched with water while cooling. The reaction was extracted with DCM (19.90 kg) and washed with sat. NaHCO3. The aqueous phase was further extracted with DCM (19.71 kg) and washed with brine.

Step 2: (2R, 3R, 4R, 5R) -2- (4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidine dibenzoate was charged into a reactor -7-yl) -5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl, 10% Pd on C and THF. Hydrogen was sent to the reactor and the mixture was stirred for about 4 hours at room temperature at about 212.35 kPa (31 psi).

The reaction mixture was filtered over Celite (7.2 kg) and a filter polish and the filter residue was washed with THF. The combined filtrate and wash were transferred to a 100 L jacketed reactor with the aid of a THF wash. The reactor contents were vacuum distilled with a maximum batch temperature of 30.0 ° C for a period of approximately 6 hours to a final volume of 27 L. IPA was loaded into the reactor. The contents of the reactor were vacuum distilled. IPA was loaded into the reactor. The reactor contents were heated to about 60 ° C, stirred and slowly cooled to about 5 ° C. Cold stirring was continued for a period of approximately 9 h with a minimum temperature of approximately 1 ° C. The suspension was filtered and washed with IPA. The residue was dried under vacuum with a nitrogen purge to provide a LOD of 0.36%. Yield: (73.9%). 1H NMR confirms the structure. Purity: 97.78% (HPLC, ABC).

Step 3: A dibenzoate solution of (2R, 3R, 4R, 5R) -2- (4-amino-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidin-7-yl) - 5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl and THF was heated and the addition of NaOH was started. The initial addition gave a biphasic mixing and endothermic response, but as the addition continued a clear single phase solution formed which was accompanied by a rapid exotherm; the reaction temperature was maintained for the remainder of the addition and for an additional ~ 2 h. IPC showed that no (2R, 3R, 4R, 5R) -2- (4-amino-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidin-7-yl) - dibenzoate remained 5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl.

The reaction mixture was cooled to 21 ° C and neutralized with 3N HCl with external cooling to neutral pH. The mixture continued to cool and the resulting neutralized mixture was vacuum distilled until solids were observed in the vessel. The suspension was cooled and stirred for about 2 h at about 2 ° C. The beige suspension was filtered to produce a dark filtrate; the off-white residue was washed once with cold water.

In certain embodiments, compounds of the disclosure, eg, formula IB or formula II, can be prepared as enantiomers, diastereoisomers, and racemates. In some embodiments, the compounds of the disclosure, eg, formula IB or formula II, include all isomeric (eg, enantiomeric, diastereoisomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, double bond isomers (Z) and (E), and conformational isomers (Z) and (E). Therefore, individual stereochemical isomers, as well as enantiomeric, diastereoisomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure. Unless otherwise indicated, all tautomeric forms of the compounds disclosed herein are within the scope of the disclosure.

Compounds of the disclosure, eg, formula IB or formula II, can be synthesized with substantially no impurities. The compounds of the disclosure are greater than or equal to about 99% w / w pure. In certain embodiments, the phosphonate esters can be prepared on a large scale, eg, on an industrial production scale rather than on an experimental / laboratory scale. For example, a batch type process according to the methods of the disclosure allows the preparation of batches of at least 1 g, or at least 5 g, or at least 10 g, or at least 100 g, or at least 1 kg. , or at least 100 kg of phosphonate ester product. Furthermore, the methods allow the preparation of a phosphonate ester product having a purity of at least 98%, or at least 98.5%, as measured by HPLC. In preferred embodiments, these products are obtained in a reaction sequence that does not involve purification by any form of chromatography (eg, gas chromatography, HPLC, preparative LC, size exclusion chromatography, and the like).

Pharmaceutical compositions and treatment methods

As set forth above, provided herein are pharmaceutical compositions comprising compounds of the disclosure (eg, formula IB or formula II) or pharmaceutically acceptable salts thereof. In some embodiments, the present disclosure provides pharmaceutical compositions comprising compounds of formula IB or formula II or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, the present disclosure provides compounds of formula IB or formula II formulated as a pharmaceutical composition. In one embodiment, the compounds of formula IB or formula II are formulated as a tablet. In another embodiment, the compounds of formula IB or formula II are formulated as a suspension.

Techniques for the formulation and administration of the disclosed compounds can be found in Remington: the Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012).

In one embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described above.

The compounds of the disclosure, for example, compounds of formula IB or formula II or pharmaceutically acceptable salts thereof described herein can be combined with a pharmaceutically acceptable carrier in accordance with conventional pharmaceutical compounding techniques. Furthermore, the vehicle can take a wide variety of forms depending on the form of the preparation desired for administration, for example, oral, nasal, rectal, vaginal, parenteral (including intravenous injections or infusions). Any of the usual pharmaceutical media can be used in the preparation of the compositions for oral dosage form. Usual pharmaceutical media include, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, solutions, emulsions and elixirs); aerosol sprays; or vehicles such as starches, carbohydrates, cellulose microcrystalline, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, in the case of solid oral preparations (such as, for example, powders, capsules and tablets).

In another embodiment, the disclosure provides a compound of formula IB or formula II or pharmaceutically acceptable salts thereof, for use in a method for the therapeutic and / or prophylactic treatment of viral infections in a subject, eg, an immunocompromised subject. In some embodiments, the salt has a purity of equal to or greater than 91% w / w, for example, it has less than or equal to 9% w / w of impurities, for the subject.

Pharmaceutical compositions comprising the compounds of the present disclosure (eg, compounds of formula IB or formula II) can be formulated to have any desired concentration. In some embodiments, the composition is formulated to comprise at least a therapeutically effective amount.

Pharmaceutical compositions include those suitable for oral, sublingual, nasal, rectal, vaginal, topical, buccal, and parenteral administration (including subcutaneous, intramuscular, and intravenous), although the most suitable route will depend on the nature and severity of the condition being treated. . The compositions may be conveniently presented in unit dosage form, and prepared by any of the methods well known in the art of pharmacy. In certain embodiments, the pharmaceutical composition is formulated for oral administration in the form of a lozenge, capsule, lozenge, or tablet. In other embodiments, the pharmaceutical composition is in the form of a suspension.

When the compounds of the present disclosure are administered as pharmaceuticals to mammals, for example, humans, they can be administered per se or as a pharmaceutical composition containing, for example, from about 0.1% to 99.9%, from about 0.2 to 98%, from about 0.3% to 97%, from about 0.4% to 96%, or from about 0.5 to 95% active ingredient in combination with a pharmaceutically acceptable carrier. In one embodiment, a pharmaceutical composition containing from about 0.5% to 90% active ingredient in combination with a pharmaceutically acceptable carrier is suitable for administration to mammals, eg, humans. Some embodiments of the present disclosure provide a preparation of a pharmaceutical composition comprising from about 0.1% to 99.9%, from about 0.2 to 98%, from about 0.3% to 97%. %, from about 0.4% to 96% or from about 0.5 to 95% of the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof, for example, any one of the compounds of Table 7 or pharmaceutically acceptable salt thereof, for use in the treatment, prevention or prophylaxis of viral infections or disorders associated with viral infections. The present disclosure provides the use of about 0.1% to 99.9%, about 0.2 to 98%, about 0.3% to 97%, about 0.4% to 96% or from about 0.5 to 95% of the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof for the manufacture of a medicament containing effective amounts of the compound for use in treatment, prevention or prophylaxis of viral infections and diseases associated with viral infections.

The present disclosure provides a compound of formula IB or formula II for use in treating a viral infection or a disease or disorder associated with viral infection. The compounds may be in a pharmaceutical formulation comprising from about 0.1% to 99.9%, from about 0.2 to 98%, from about 0.3% to 97%, from about a 0.4% to 96% or from about 0.5 to 95% of the compounds of formula IB or formula II.

For any compound, the therapeutically effective amount of a compound or composition can be estimated initially in cell culture assays, for example, of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic / prophylactic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, the ED ⁵⁰ (the therapeutically effective dose in 50% of the population) and the LD ⁵⁰ (the lethal dose for the 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio of LD ⁵⁰ / ED ⁵⁰ . Pharmaceutical compositions showing large therapeutic indices are preferred. The dosage can vary within this range depending on the dosage form used, the sensitivity of the patient and the route of administration.

Pharmaceutical compositions containing compounds of formula IB or formula II of the present disclosure can be manufactured in a manner that is generally known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, processes. encapsulation, inclusion or lyophilization. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and / or auxiliaries that facilitate processing of the active compounds into pharmaceutically usable preparations. The appropriate formulation depends on the chosen route of administration.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the improvised preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be liquid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, carbohydrates, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filter sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and lyophilization which produces a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.

Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They are confined in gelatin capsules or compressed into tablets. For oral therapeutic administration purposes, excipients may be incorporated into the active compound and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a liquid vehicle for use as a mouthwash, wherein the compound in the liquid vehicle is applied orally and removed by rinsing and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjunct materials can be included as part of the composition. Tablets, lozenges, capsules, troches, and the like may 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 cornstarch; a lubricant such as magnesium stearate or Sterotes; an emollient 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.

Active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly (glycolic acid), collagen, polyorthoesters, and poly (lactic acid). Methods for the preparation of such formulations will be apparent to those skilled in the art. In some embodiments, the materials may also be commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells, with monoclonal antibodies against viral antigens) may also be used as pharmaceutically carriers. acceptable.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unit doses for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.

Compounds of formula IB or formula II or pharmaceutically acceptable salts thereof can be formulated as a pharmaceutical composition. Furthermore, the present disclosure provides a compound of formula IB or formula II, or a composition comprising a compound of formula IB or formula II for use in treating a viral infection or a disease or disorder associated with viral infection. The composition can be formulated as a tablet or suspension. Tablets of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are formulated comprising buffers, excipients, carriers, including emulsifiers, enhancers (eg, absorption enhancers), disintegrants (eg, polyvinylpolypyrrolidone (polyvinylpolypyrrolidone, PVPP, crospovidone, crospolividone or E1202), which is a highly cross-linked modification of polyvinylpyrrolidone (PVP)) and / or pharmacologically acceptable polymers disclosed in the present disclosure and well known in the art.

In one embodiment, the present disclosure provides the tablet formulation of the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof for use in treatment, prophylactic treatment or prevention of viral infections and / or diseases or disorders associated with infections. viral. The present disclosure provides the tablet formulation of the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof for use in treating subjects in need of such treatment including, but not limited to, immunocompromised subjects, or before or after subjects. organ and / or tissue transplantation.

In one embodiment, the present disclosure provides suspension formulations of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof for use in prophylactic treatment or prevention of viral infections and / or diseases and / or disorders associated with viral infections. . The present disclosure provides suspension formulation of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof for use in treating subjects in need of such treatment including, but not limited to, immunocompromised subjects, or before or after subjects. organ and / or tissue transplantation.

In another embodiment, additional excipients include, but are not limited to, sodium phosphate, dibasic citric acid (monohydrate) (approximately 0.01-5% by weight), sodium citrate (approximately 0.01-5% by weight). , xanthan gum (approximately 0.01-5% by weight), methyl paraben (sodium salt) (approximately 0.01-5% by weight), propyl paraben (sodium salt) (approximately 0.01-5% by weight), sucralose (approximately 0.01-5% by weight), microcrystalline cellulose and sodium carboxymethyl cellulose (VivaPur MCG 591) (approximately 0.5-10% by weight), high fructose corn syrup (approximately 10-70% by weight), lemon-lime flavor (WONF220J15) (approximately 0.01-5% by weight), sodium hydroxide granules, sodium hydroxide / hydrochloric acid and purified water (approximately one 68.93% by weight).

In another embodiment, the present disclosure provides compositions (eg, pharmaceutical compositions) with desirable pharmacokinetic characteristics. For example, the compositions of the disclosure can provide a blood level of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof which, after metabolism in the therapeutically active form (eg, diphosphate equivalent), causes blood levels of the metabolite that do not induce toxicity.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, or any combination thereof).

Diseases

Disclosed herein is a method of treating a viral infection or a disease or disorder associated with viral infection, which comprises administering to a subject in need thereof a compound of the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, or any combination thereof).

In some embodiments, the present disclosure provides a compound of the disclosure (e.g., compound 1, 2, 3, 4, 5, 6, 71, 77, 76, 107, 111, 126, 133, 137, 139, 141, 143 or 145, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof, or any combination thereof) for use in treating a disease (e.g., a viral infection or a disease or disorder associated with viral infection).

Disclosed herein is the treatment and / or prevention of a viral infection with the compounds disclosed herein and pharmaceutically acceptable salts thereof. The compounds represented by formula IB or formula II are used in the treatment, prevention and / or manufacture of a medicament for treating and / or preventing at least one virus selected from, but not limited to, ssRNA viruses. In some embodiments, the virus can be a norovirus, human cytomegalovirus (HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, JC virus (JCV), SV40, MC virus (MCV), KI virus. (KIV), WU virus (WUV), vaccinia virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpes virus 6 (HHV-6), human herpes 8 (HHV-8), hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV), severe smallpox, mild smallpox, smallpox, cowpox, camelid pox, ape pox, poliovirus, picornaviridae (eg rhinovirus), paramyxoviridae (eg respiratory syncytial virus, RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, enterovirus (eg EV68 and EV71, papilloma virus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Great Fault Valley fever virus and other flaviviruses, arenaviruses, bunyaviruses, a lfavirus and human immunodeficiency virus (HIV) infections, and any combination thereof.

In some embodiments, the present disclosure also provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of viral infections or diseases or disorders associated with viral infections (eg, norovirus, human cytomegalovirus ( HCMV), BK virus (BKV), Epstein-Barr virus (EBV), adenovirus, j C virus (JCV), SV40, MC virus (MCV), KI virus (KIV), WU virus (w Uv), vaccinia, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human herpes virus 6 (HHV-6), human herpes virus 8 (HHV-8), hepatitis B, hepatitis C virus, varicella-zoster virus (VZV), severe smallpox, mild smallpox, smallpox, cowpox, camelid pox, ape pox, poliovirus, picornaviridae (e.g. rhinovirus), paramyxoviridae ( for example, respiratory syncytial virus (RSV), Ebola virus, Marburg virus, Epstein-Barr virus (EBV), influenza virus, and nteroviruses (for example, EV68 and EV71, papillomavirus, West Nile virus, yellow fever virus, foot-and-mouth disease virus, Great Fault Valley fever virus, and other flaviviruses, arenaviruses, bunyaviruses, alphaviruses and human immunodeficiency virus (HIV) infections, and any combination thereof) by orally administering to a subject a pharmaceutical composition comprising a therapeutically effective dose of a compound of formula IB or Formula II or a pharmaceutically acceptable salt thereof, in combination with one or more of a compound or composition selected from an immunosuppressive agent and / or an antiviral agent.

In some embodiments, the present disclosure provides a compound of Formula IB or Formula II for use in treating, preventing, or delaying the onset of Marburg virus infection or Marburg virus infection-associated disease or disorder, by oral administration. to a subject who needs it.

In some embodiments, the present disclosure provides a compound of Formula IB or Formula II for use in treating, preventing, or delaying the onset of Ebola virus infection or Ebola virus infection-associated disease or disorder, by oral administration. to a subject who needs it.

In some embodiments, the present disclosure provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of influenza virus infection or disease or disorder associated with influenza virus infection, such as oral administration to a subject in need.

In some embodiments, the present disclosure provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of norovirus infection or disease or disorder associated with norovirus infection, by oral administration to a subject who you need it.

In some embodiments, the present disclosure provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of picornaviridae virus infection or disease or disorder associated with picornaviridae virus infection, by oral administration to a subject who needs it.

In some embodiments, the present disclosure provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of paramyxoviridae virus infection or disease or disorder associated with paramyxoviridae virus infection, by oral administration to a subject who needs it.

In some embodiments, the present disclosure provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of enterovirus infection or enterovirus infection-associated disease or disorder, by oral administration to a subject who you need it.

In any of the above embodiments, the compound can be compound 1.

The present disclosure also provides a compound of formula IB or formula II (eg, compound 1) for use in the prophylactic treatment, prevention or delay of the onset of norovirus infection or a disease or disorder associated with norovirus infection, administering orally to a subject a pharmaceutical composition comprising a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

The present disclosure also provides a compound of formula IB or formula II for use in the prophylactic treatment, prevention, or delay of the onset of enterovirus infection or a disease or disorder associated with enterovirus infection, by orally administering to a subject a pharmaceutical composition comprising a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof, in combination with one or more antiviral agents.

In one embodiment, compounds of formula IB or formula II are used to treat norovirus. In other embodiments, compounds of formula IB or formula II are used to treat norovirus associated with specific genotypes such as those of genogroups I, II and IV, VI, and VII that are known to infect humans (Phan et al., J. Med. Virol. 2007 Sep; 79 (9): 1388-1400).

Dosage guidelines

The administration schedule can achieve what constitutes a pharmaceutically effective amount. Compounds of formula IB or formula II or pharmaceutically acceptable salts thereof can be administered to the subject before or after the onset of a disease. In addition, several divided dosages can be administered, as well as staggered dosages daily or sequentially, or the dose can be infused continuously, or it can be a bolus injection. Furthermore, dosages may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In addition, the dosages can be co-administered in combination with another antiviral.

The dosage regimen using the compounds can also be selected according to a variety of factors including the type, species, age, weight, sex, and clinical status of the patient; the severity of the condition to be treated; the route of administration; the kidney and liver function of the patient; and the particular compound or salt of the same employee. A physician or veterinarian skilled in the art can easily determine and prescribe the effective amount of the drug required to prevent, counteract, or halt the progress of the condition.

In some embodiments, the subject treated for a viral infection (eg, a norovirus infection or a disease or disorder associated with norovirus infection) is administered once or twice a week about 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg or 250 mg of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof. The present disclosure provides treatment of a subject for norovirus infection or disease or disorder associated with norovirus infection by administering to the subject once a week (QW) about 200 mg or twice a week (BIW) about 100 mg of a compound. of formula IB or formula II, or a pharmaceutically acceptable salt thereof. In one embodiment, the subject is treated twice weekly (BIW) with approximately 100 mg of the compound. In another embodiment, the subject is treated once a week (QW) with about 200 mg, or twice a week (BIW) with about 100 mg of the compound.

In one embodiment, a compound of formula IB or formula II or pharmaceutically acceptable salt thereof having a purity equal to or greater than about 91% is administered orally to a subject, for example, at a dosage of about 0.01. mg / kg to about 10 mg / kg or more, for example, up to 100 mg / kg, or up to 400 mg / kg, or up to 1000 mg / kg.

In another embodiment, a compound of formula IB or formula II or pharmaceutically acceptable salt thereof having a purity equal to or greater than about 91% w / w is administered to a subject at a dosage of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7, 5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, or 10 mg / kg or more or any range therein.

In a preferred aspect, the disease or condition to be treated is viral infection.

Dosage and administration are adjusted to provide sufficient levels of the one or more active agents or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, the diet, the time and frequency of administration, the drug combination (s), the reaction sensitivities and tolerance / response to treatment. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, once every two weeks, or monthly depending on the half-life and clearance rate of the particular formulation.

In some embodiments, administration continues for a total of ten doses. For example, compounds of formula IB or formula II can be administered at dosages of about 100 mg twice weekly for five weeks (ie, a total of ten doses). Alternatively, the compounds of formula IB or formula II can be administered with a loading dose of about 200 mg followed by doses of about 100 mg continuing twice weekly. In some embodiments, administration continues for a total of ten doses. For example, compounds of formula IB or formula II can be administered at a loading dose of about 200 mg followed by nine additional doses of about 100 mg twice weekly for a total of ten doses. In one embodiment, the compounds of formula IB or formula II can be dosed daily in the range of about 20-200mg / day or weekly in the range of about 200mg-3000mg.

In one or more embodiments, the compounds of the disclosure are useful in treating a viral infection such as a norovirus infection or a disease or disorder associated with norovirus infection. In some embodiments, treating the infection, eg, norovirus infection, may comprise daily dosing, or dosing multiple times daily. In some embodiments, the total treatment regimen lasts only as long as the norovirus infection is active (eg, between 1-3 days). In some embodiments, the compounds of the disclosure can be dosed multiple times per day for 1-3 days to treat a norovirus infection.

In another embodiment, tablets or suspensions of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are administered at a dose of about 40-3000 mg per day, BID, TID, once a week (QW) or twice a week (BIW). In another embodiment, tablets or suspensions of compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are administered at a dose of approximately 40-400 mg per day, BID, TID, once a week (QW) or twice a week (BIW).

In therapeutic applications, the dosages of the pharmaceutical compositions disclosed in this document vary depending on the agent, the age, weight and clinical condition of the recipient patient, and the experience and judgment of the physician or practitioner administering the treatment, among other factors that affect the selected dosage. Dosages can range from about 0.01 mg / kg to about 100 mg / kg. In preferred aspects, dosages can range from about 0.1 mg / kg to about 10 mg / kg. In one aspect, the dose will range from about 1 mg to about 1 g; from about 10 mg to about 500 mg; from about 20 mg to about 400 mg; from about 40 mg to about 400 mg; or from about 50 mg to about 400 mg, in individual, divided or continuous doses (the dose of which can be adjusted for the patient's weight in kg, their body surface area in m2, and their age in years). In certain embodiments, the amount per dosage form may be from about 0.1 mg to about 3000 mg, for example, about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, of about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, of about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, of about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1250 mg, 1500 mg, about 1750 mg, about 2000 mg, about 2500 mg or about 3000 mg. In one embodiment, the amount can be about 20 mg. In one embodiment, the amount can be about 50 mg. In another embodiment, the dosage can be about 100 mg. In another embodiment, the dose can be 500 mg.

In another embodiment, the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are administered to a subject as a single dose. In another embodiment, the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are administered to a subject in multiple doses. Multiple doses can be given on a regular basis, for example, once every 12 hours, once a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days or every 15 days. For example, doses can be given twice a week. In addition, each individual dose can be administered with the same or a different dosage.

For example, a subject may be administered any one of the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof with a first dose of about 1-20 mg / kg (eg, about 1-1.1 mg / kg, about 1.1-1.2 mg / kg, about 1.2-1.3 mg / kg, about 1.3-1.4 mg / kg, about 1.4-1.5 mg / kg, about 1.5-1.6 mg / kg, about 1.6-1.7 mg / kg, about 1.7 - 1.8 mg / kg, about 1.8-1.9 mg / kg, about 1 , 9-2.0 mg / kg, approximately 2.0-2.1 mg / kg, approximately 2.2-2.2 mg / kg, approximately 2.2-2.3 mg / kg, approximately 2.3-2 , 4 mg / kg, about 2.4-2.5 mg / kg, about 2.5-2.6 mg / kg, about 2.6-2.7 mg / kg, about 2.7-2.8 mg / kg, about 2.8-2.9 mg / kg, about 2.9-3.0 mg / kg, about 3.0-3.1 mg / kg, about 3.1-3.2 mg / kg, about 3.2-3.3 mg / kg, about 3.3-3.4 mg / kg, about 3.4 -3.5 mg / kg, about 3.5-3.6 mg / kg, about 3.6-3.7 mg / kg, about 3.7-3.8 mg / kg, about 3.8-3.9 mg / kg, about 3.9-4.0 mg / kg, about 4.0-5.0 mg / kg, about 5.0- 6.0 mg / kg, about 6.0-7.0 mg / kg, about 7.0-8.0 mg / kg, about 8.0-9.0 mg / kg, about 9.0-10.0 mg / kg, or about 10-20 mg / kg) of any one of the compounds of formula IB or formula II (or a pharmaceutically acceptable salt thereof) followed by one or more additional doses at 1-4 mg / kg (e.g., about 1-1.1 mg / kg, about 1.1-1 , 2mg / kg, about 1.2-1.3mg / kg, about 1.3-1.4mg / kg, about 1.4-1.5mg / kg, about 1.5-1.6mg / kg, about 1.6-1.7mg / kg, about 1.7-1.8mg / kg, about 1.8-1.9mg / kg, about 1.9-2.0mg / kg, about 2.0-2.1 mg / kg, about 2.1-2.2 mg / kg, about 2.2-2.3 mg / kg, about 2.3-2 , 4 mg / kg, about 2.4-2.5 mg / kg, about 2.5-2.6 mg / kg, about 2.6-2.7 mg / kg, about 2.7-2.8 mg / kg, about 2.8-2.9 mg / kg, about 2.9-3.0 mg / kg, about 3.0-3.1 mg / kg, about 3.1-3.2 mg / kg, about 3.2-3.3 mg / kg, about 3.3-3.4 mg / kg, about 3.4-3.5 mg / kg, about 3.5-3.6 mg / kg, about 3.6-3 m, 7g / kg, about 3.7-3.8 mg / kg, about 3.8-3.9 mg / kg, or about 3.9-4.0 mg / kg) of any one of the compounds of formula IB or formula II (or a pharmaceutically acceptable salt thereof) in the same week or in the following week. For example, a subject may be administered a first dose of about 3 mg / kg followed by one or more additional doses at about 1 mg / kg. For example, a subject may be administered a first dose of approximately 2 mg / kg followed by one or more additional doses at approximately 3 mg / kg. For example, a subject may be administered a first dose of 4 mg / kg followed by one or more additional doses at about 4 mg / kg.

Multiple doses can also be administered at varying time intervals. For example, the first 2, 3, 4, 5, 6, 7, or 8 or more doses may be administered at a 6-day interval followed by additional doses administered at a 7-day interval. For example, the first 2, 3, 4, 5, 6, 7, or 8 or more doses can be administered at a 7-day interval followed by additional doses administered at a 3-day interval.

In one embodiment, the compounds of formula IB or formula II or pharmaceutically acceptable salts thereof are administered to a subject once a week at a dose of about 40-3000 mg, or twice a week at a dose of about 40-3000 mg.

In some embodiments, the pharmaceutical composition of the present disclosure is administered daily, BID, TID, once a week (QW) or twice a week (BIW) with about 40-3000 mg of compounds of formula IB or formula II. or pharmaceutically acceptable salts thereof. The pharmaceutical compositions of the present disclosure are administered daily, BID, TID, once a week (QW) or twice a week (BIW) with approximately 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 500-600 mg, 600-700 mg, 700-800 mg, 800-900 mg or 900-1000 mg, or twice weekly (BIW) with approximately 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 175 mg, 200 mg, 250 mg, 275 mg, 300 mg, 325 mg , 350 mg, 375 mg or 400 mg, 450 mg, 500 mg, 500-600 mg, 600-700 mg, 700-800 mg, 800-900 mg or 900-1000 mg of compounds of formula I, formula IA, formula IB or formula II or pharmaceutically acceptable salts thereof.

The present disclosure provides compounds of formula IB or formula II administered at a dose of about 1-100 mg / kg (eg, 10-20 mg / kg, 20-50 mg / kg, 50-75 mg / kg, 75- 100 mg / kg).

Administration routes

The compounds of the present disclosure, or pharmaceutically acceptable salts, esters, or derivatives thereof, can be administered orally, nasal, intranasal, transdermal, pulmonary, inhaled, jugal, sublingual, intraperitoneal, subcutaneous, intramuscular, intravenous, rectal, intrapleural, intrathecal and parenteral. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of certain routes of administration.

Pharmaceutical forms for topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

For administration by inhalation, the compounds are delivered as an aerosol spray from a pressurized container or dispenser, containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and derivatives of fusidic acid. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, balms, gels, or creams as is generally known in the art.

A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, 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 tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be confined in ampoules, disposable syringes, or multidose vials made of glass or plastic.

Polytherapy

The present disclosure provides methods for preventing or treating a viral infection in a subject (eg, a norovirus infection). The methods comprise administering to a subject a therapeutically effective amount of a compound described herein. The compounds can be used in a monotherapy or combination therapy regimen.

As used herein, "monotherapy" means or refers to the administration of a single active or therapeutic compound (eg, a compound of formula IB or formula II) to a subject in need thereof. Preferably, monotherapy will involve the administration of a therapeutically effective amount of an active compound. For example, monotherapy against norovirus with one of the compounds of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, to a subject in need of treatment against norovirus. Monotherapy can be as opposed to combination therapy, in which a combination of multiple active compounds is administered, preferably with each component of the combination present in a therapeutically effective amount. In one aspect, monotherapy with a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, is more effective than combination therapy in inducing a desired biological effect.

As used herein, "combination therapy" or "conjoint treatment" includes administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, and at least one second agent as part. of a specific treatment regimen intended to provide the beneficial effect of the joint action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic joint action of the combination of therapeutic agents. Administration of these therapeutic agents in combination is typically over a defined period of time (usually minutes, hours, days, or weeks depending on the combination selected). "Combination therapy" may be intended, but generally does not, encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that accidentally and arbitrarily elicit the combinations of the present disclosure.

"Combination therapy" is intended to encompass the administration of these therapeutic agents in a sequential manner, in which each therapeutic agent is administered at a different time, as well as the administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneously. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple individual capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. Therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the selected combination can be administered by intravenous injection while the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally or all therapeutic agents can be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not strictly crucial.

"Combination therapy" also encompasses the administration of the therapeutic agents as described above in additional combination with other biologically active ingredients and non-drug treatments. When the combination therapy further comprises drug-free treatment, the drug-free treatment can be performed at any suitable time provided that a beneficial effect is achieved from the joint action of the combination of the therapeutic agents and the drug-free treatment. For example, in appropriate cases, the beneficial effect is still achieved when the drug-free treatment is temporarily removed from the administration of the therapeutic agents, perhaps for days or even weeks.

The present disclosure also provides a compound of formula IB or formula II for use in treating, preventing, or delaying the onset of a viral infection (eg, norovirus viral infection or disease or disorder associated with norovirus viral infection; infection; influenza virus or disease or condition associated with influenza virus infection), by oral administration to a subject in need thereof of a pharmaceutical composition of a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically salt acceptable thereof in combination with one or more antiviral agents.

The present disclosure also provides a compound of formula IB or formula II for use in treating, preventing or delaying the onset of a picornaviridae viral infection or disease or disorder associated with picornaviridae viral infection, by oral administration to a subject who it requires a pharmaceutical composition of a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof in combination with one or more antiviral agents.

The present disclosure also provides a compound of formula IB or formula II for use in treating, preventing or delaying the onset of a paramyxoviridae viral infection or disease or disorder associated with paramyxoviridae viral infection, by oral administration to a subject who it requires a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof in combination with one or more antiviral agents.

In one embodiment, the method of treating a viral infection, eg, influenza virus or norovirus infection, further comprises administering at least one additional antiviral agent. In one embodiment, the compound Formula II may be for use in combination with an additional antiviral agent. In one embodiment, the additional antiviral agent is adamantane. In a further embodiment, the additional antiviral agent is amantadine or rimantadine. In another embodiment, the additional antiviral agent is a neuraminidase inhibitor (eg, oseltamivir, zanamivir, laninamivir, and peramivir). In a further embodiment, the additional antiviral agent is oseltamivir or zanamivir.

In some embodiments, the pharmaceutical composition of the present disclosure (eg, a compound of formula IB or formula II) is administered in combination with one or more compounds or compositions selected from midazolam, cyclosporin A, tacrolimus, ganciclovir, valganciclovir, foscavir, cidofovir, second-line anti-CMV drugs, second-line anti-HCV drugs, foscarnet, filgrastim, pegfilgrastim, corticosteroids such as budesonide, beclomethasone, and broad spectrum CYP inhibitor aminobenzotriazole or combinations thereof.

In additional embodiments, the compound is for administration in combination with at least one other immunosuppressive agent. In one embodiment, the immunosuppressive agent is administered simultaneously or sequentially. Immunosuppressive agents include, but are not limited to, daclizumab, basiliximab, tacrolimus, sirolimus, mycophenolate, cyclosporin A, glucocorticosteroids, anti-CD3 monoclonal antibodies, antithymocyte globulin, anti-CD52 monoclonal antibodies, azathioprine, everolimus, platinosfynomycin, , methotrexate, mercaptopurine, muromonab, IFN gamma, infliximab, etanercept, adalimumab, natalizumab, fingolimod, and combinations thereof.

The compounds or compositions provided herein can also be used in combination with an enhancing agent, with other active ingredients, or with an immunosuppressive agent. In certain embodiments, the compounds can be administered in combination, or sequentially, with another therapeutic agent or an enhancer. Such other therapeutic agents include those known for the treatment, prevention or amelioration of one or more symptoms associated with viral infections. It should be understood that any suitable combination of the compounds provided herein with one or more of the above-mentioned compounds and optionally one or more additional pharmacologically active substances is considered within the scope of the present disclosure. In another embodiment, the compound provided herein is administered before or after the one or more additional active ingredients. In one embodiment, two or more of the antiviral agents disclosed herein are administered in series or in combination. The amount of some enhancers can be selected using methods known in the art to enhance the bioavailability of the antiviral agent. Any amount that provides a response desired by some enhancers can be used. Dosages may range, in a non-limiting example, from 0.001 mg to about 3000 mg of compound per kilogram of body weight per day, for example 0.01 to 500 mg / kg, or for example 0.1-20 mg / kg.

The pharmacokinetic behavior of a composition will vary somewhat from subject to subject within a population. The numbers described above for the compositions disclosed herein are based on average behavior in a population. The present disclosure is intended to encompass compositions that are on average within the disclosed ranges, although it is understood that certain subjects may be outside the ranges.

The pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for their administration. The present disclosure provides a kit that includes, in addition to a pharmaceutical composition of any one of the disclosed compounds, a container, pack or dispenser with instructions for its administration.

A compound of the present disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, can be administered in combination with a second antiviral compound. For example, as indicated above, the compositions of the present disclosure may include the compounds as described above in combination with one or more (eg, 1, 2, 3) additional active agents as described in this section, of analogous to as is known in the art. Additional antiviral active agents that can be used with the compounds of the present disclosure in performing the present methods include, but are not limited to, those that target the M2 ion channels in influenza A viruses (e.g., adamantanes, such as amantadine and rimantadine); those that inhibit the loss of viral coat after entry into the cell, agents that block the release of newly formed virions from the surface of infected cells (eg, neuraminidase inhibitors, such as oseltamivir and zanamivir). Methods to prevent diseases or disorders due to reactivation of the virus.

Also disclosed herein is a method of preventing a disease or disorder in a subject at risk of reactivation of viral infection, orally administering to the subject a pharmaceutical composition of a therapeutically effective dose of a compound of formula IB or formula II or a pharmaceutically acceptable salt thereof. In some embodiments, the virus at risk of reactivation can be influenza virus, norovirus, EBV, Ebola virus, picornaviridae, paramyxoviridae, and Marburg virus. In some preferred embodiments, the virus at risk of reactivation can be the influenza virus.

Effect of food

In some embodiments, the pharmaceutical composition of the present embodiments, eg, tablet or suspension, can be provided to a subject when the subject is fasting or in a nourished state. In one embodiment, the composition comprising a compound of formula IB or formula II (or a pharmaceutically acceptable salt thereof) can be provided to a subject who has an empty stomach, for example, after helping for less than 24 hours, but longer. 12 hours, more than 11 hours, more than 10 hours, more than 8 hours or more than 5 hours.

In other embodiments, the composition comprising a compound of formula IB or formula II (or a pharmaceutically acceptable salt thereof) can be provided to a subject with food or after food has been taken. In one embodiment, a compound of Formula I, Formula IA, Formula IB, or Formula II (or a pharmaceutically acceptable salt thereof) can be taken by a subject on an empty stomach.

Patient population

In certain embodiments, compounds of formula IB or formula II (or a pharmaceutically acceptable salt thereof), a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a composition of formula IB or formula II are administered to a mammal in need of it (e.g. human) that is approximately 1 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10-15 years old, 15-20 years old, 20-25 years old, 25-30 years old, 30-35 years old, 35-40 years old, 40 to 45 years of age, 45 to 50 years of age, 50 to 55 years of age, 55 to 60 years of age, 60 to 65 years of age, 65 to 70 years of age, 70 to 75 years old, 75-80 years old, 80-85 years old, 85-90 years old, 90-95 years old, or 95-100 years old. In some embodiments, the mammal is suffering from a viral infection (eg, a ssRNA virus infection such as a norovirus infection).

In certain embodiments, a compound of formula IB or formula II, a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to a human at risk of developing a viral infection. In certain embodiments, a compound of formula IB or formula II, a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to a human with a viral infection. In certain embodiments, the patient is a human approximately 1 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old. , 10-15 years old, 15-20 years old, 13-19 years old, 20-25 years old, 25-30 years old, 20-65 years old, from 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, 60- 65 years old, 65-70 years old, 70-75 years old, 75-80 years old, 80-85 years old, 85-90 years old, 90-95 years old old or 95 to 100 years old.

In some embodiments, a compound of formula IB or formula II, a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to a human infant. In other embodiments, a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to a human infant. In other embodiments, a compound of formula IB or formula II, a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to a human adult. In still other embodiments, a compound of formula IB or formula II, a composition comprising a compound of formula IB or formula II, or a combination therapy comprising a compound of formula IB or formula II is administered to an elderly human.

All percentages and ratios used in this document, unless otherwise indicated, are by weight. Other characteristic features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in the practice of the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure, those skilled in the art can identify and employ other components and methodology useful in the practice of the present disclosure.

In some embodiments, the compound of formula II is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. In some embodiments, the compounds are:

or pharmaceutically acceptable salts, solvates, enantiomers, diastereoisomers, racemates or mixtures thereof (where * indicates compounds not in accordance with the present invention.

HPLC charts were recorded on a Zorbax Eclipse Plus C18 column (4.6 x 50 mm x 1.8 pm). The first mobile phase was 97.5% water: 2.5% acetonitrile: 0.05% trifluoroacetic acid. The second mobile phase was 97.5% acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

1H NMRs were recorded at 300 MHz unless otherwise indicated.

Antiviral and Cytotoxicity Assays

Cell culture and virus strains: Human foreskin fibroblasts (HFF) were prepared from human foreskin tissue. The tissue was incubated in cell culture medium consisting of minimal essential medium (MEM) with Earle's salts supplemented with 10% fetal bovine serum (FBS), and standard concentrations of L-glutamine, fungizone and vancomycin. The tissue was then placed in phosphate buffered saline (PBS), ground, rinsed to remove red blood cells, and resuspended in trypsin / EDTA solution. The tissue suspension was incubated at 37 ° C and gently shaken to disperse the cells, which were then collected by centrifugation. Cells were resuspended in medium and placed in a histoculture flask and incubated at 37 ° C in a humidified ^{CO 2 incubator.} The medium was then replaced with fresh medium and cell growth was monitored daily until a confluent cell monolayer was formed. The HFF cells were then expanded through serial passages in conventional MEM growth medium with Earle's salts supplemented with 10% FBS, L-glutamine, penicillin, and gentamicin. Cells were routinely passaged and used for assays at or below passage 10.

Influenza Viruses: Influenza viruses were passed into canine kidney cells to create working stock solutions, which were used for antiviral assays.

Antiviral Assays: Each experiment that evaluated the antiviral activity of compounds included both positive control and negative control compounds to ensure the performance of each assay. Simultaneous evaluation of cytotoxicity was performed when possible at equivalent levels of exposure to the compound. The methods are presented in effective concentrations that give 50% reductions in viral replication in vitro (EC ⁵⁰ ), concentrations that produce a 50% reduction in cell viability (CC ⁵⁰ ) and the selectivity index (SI, calculated as CC ⁵⁰ divided by CE ⁵⁰ ). When sufficient material was available, multiple tests were performed for each compound evaluation to obtain statistical data.

Cytotoxicity Assays: Each antiviral assay included a parallel cytotoxicity assay with the same cells used for each virus, the same number of cells, the same drug concentrations, and the same incubation times to provide the same drug exposure. To ensure that the cytotoxicity of all compounds could be directly compared, a standard neutral red uptake cytotoxicity assay was performed for all compounds on confluent HFF cells with an incubation period of 7 days.

Neutral Red Uptake Cytotoxicity Assays: Each compound was evaluated in a standard cytotoxicity assay by standard methods. Briefly, HFF cells were seeded in histoculture plates in conventional histoculture medium. After 24 h of incubation, the medium was replaced with maintenance cell culture medium and compounds were added and then serial dilutions of factor 5 were then used to generate a series of compound concentrations. Assay plates were incubated for 7 days, and neutral red solution in PBS was added to each well and the plates were incubated for 1 hr. The dye was then removed, the plates were rinsed with PBS, and the dye internalized by viable cells was solubilized in PBS supplemented with 50% ethanol and 1% glacial acetic acid. The optical density was then determined and the CC ⁵⁰ values were interpolated from the experimental data.

For all HFF cell plaque reduction assays, neutral red cytotoxicity assays were performed on a parallel set of 6-well plates containing uninfected HFF cells that received the same compound concentrations as used for antiviral assays. The cytotoxicity plates were removed from the incubator on the same day as each antiviral assay and the cell monolayer was stained with a solution of neutral red in PBS. The dye was then removed, residual dye was rinsed from the cells with PBS, and the cell monolayers were visually inspected for any signs of toxicity. Cell viability was determined using a cell proliferation assay according to the manufacturer's instructions.

Cell proliferation assays: Inhibition of HFF cell proliferation was used to refine estimates of cytotoxicity for some compounds and was performed according to a standard procedure used in the laboratory. Cells were plated at low density using standard culture medium. After 24 h, the medium was aspirated and a series of compound solutions were prepared in the growth medium. The plates were incubated for 72 hrs at 37 ° C, and the cells were then detached with trypsin and counted. Compound concentrations that reduced cell proliferation by 50% were interpolated from the experimental data.

Cytotoxicity in lymphocyte assays: Cell viability in all lymphocyte assays was evaluated with a luminescent cell viability assay. Briefly, assay plates were incubated at room temperature for 30 min, then luminescent reagent was added to each well and the plates were mixed to lyse cells. The plates were then incubated at room temperature and luminescence was quantified on a luminometer. Conventional methods were used to calculate drug concentrations that inhibited cell proliferation by 50% (CC ⁵⁰ ).

Mouse norovirus (MNV) assay: RAW cells were incubated at 37 ° C with 5% CO ² in cell culture medium. The mouse norovirus isolate used in this assay was isolated from a wild-type mouse.

Serial dilutions of test compounds in DMEM were added to histocultured plates. Cell suspension was added to the compound dilutions on the plate, and incubated at 37 ° C with 5% CO ² for 2 days. Each assay plate included uninfected cell controls and untreated virus control wells. After 2 days of incubation, aqueous MTS reagent as directed by the manufacturer was added to each well and incubated at 37 ° C until untreated cell controls revealed an absorbance at 490 nm between 1.1 and 1.8. Final absorbance readings were read and a computer program was used to calculate the concentration that protected RAW cells infected with MNV by 50% compared to uninfected cell controls (EC50).

Human Norovirus Assays: Antiviral activity against a human norovirus (NoV) was evaluated in a 3-day assay using a stable expression human norovirus replicon cell line, maintained as subconfluent cultures. In some embodiments, 4 doses (factor 10 or factor 3 steps) can be used, in triplicate. Antiviral activity was determined by intracellular NoV RNA blot hybridization analysis (normalized to the level of cellular p-actin RNA in each culture sample). Cytotoxicity was evaluated by neutral red dye uptake in cultures maintained in parallel plates (Korba and Gerin, 1992, Antivir. Res. 19:55).

EC ⁵⁰ and CC ⁵⁰ values were calculated by linear regression analysis using pooled data from all treated cultures (Korba and Gerin, 1992, Antivir. Res. 19:55; Okuse, et al., 2005, Antivir. Res. 65:23). The standard deviations for the EC ⁵⁰ and EC ⁹⁰ values were calculated from the standard errors generated by the regression analyzes. EC ⁵⁰ and EC ⁹⁰ are drug concentrations at which there is a factor 2 or factor 10 decrease in intracellular NoV RNA (relative to average levels in untreated cultures), respectively. The CC ⁵⁰ is the drug concentration at which a 2-fold lower level of neutral red dye uptake is observed (relative to average levels in untreated cultures). The selectivity index (IS) is calculated as CC ⁵⁰ / EC ⁵⁰ . Recombinant human interferon 2b (PBL laboratories, Inc.) is used as an assay control.

EC ⁵⁰ of BKV in VERO cells: Histoculture plates were seeded with Vero cells in DMEM containing 2% Hyclone standard fetal bovine serum and 1% Hyclone penicillin and streptomycin. Cells were inoculated with BKV. Serial dilutions of test compounds were added to the cells and the plates were incubated for 10 days at 37 ° C in 5% CO ² . After the 10 day incubation, the supernatant was mixed with lysis buffer. Each plate was incubated C. The BKV DNA from the supernatant was measured by quantitative polymerase chain reaction (qPCR) using the BKV PCR forward and reverse primers, and a FAM-labeled probe. Absolute quantification of virus copy number was performed using a standard curve with dilutions of BKV DNA amplicon containing sequences homologous to the amplified fragment.

Plaque Reduction Assays for HSV-1, HSV-2, VZV, and HCMV: Cell monolayers were prepared in six-well plates and incubated to allow cells to reach confluence. The medium was then aspirated from the wells and virus was added to each of the three wells to produce 20-30 plaques in each well. The virus was allowed to adsorb on the cells for 1 h and the plates were gently rocked every 15 min to redistribute the medium. Compounds were diluted in maintenance cell culture medium consisting of MEM with Earle's salts supplemented with 2% FBS, L-glutamine, penicillin, and gentamicin. The solutions were added to duplicate wells and the plates were incubated for various times, depending on the virus used. The plaque reduction assay with HSV-1 strain F was performed in a similar manner, but with infected Vero cells one day after plating. For HSV-1 and 2, monolayers were stained with 1% gentian violet in 20% methanol and unbound dye was washed away with dH ² O. For HCMV and VZV assays, the cell monolayer was stained with 1% neutral red solution for 4 h, then the dye was aspirated and the cells were washed with PBS. For all tests, the plaques were enumerated using a stereomicroscope and the concentration of compound that reduced plaque formation by 50% (EC50) was interpolated from the experimental data.

Plaque Reduction Assays for MCMV: Mouse embryo fibroblasts were prepared from mouse embryos using a procedure similar to that outlined above for HFF cells and suspended in histoculture medium as described above and plated and incubated. . The medium was aspirated and the cell monolayers were infected with MCMV. The infected cells were then incubated at 37 ° C for 1 h and the plates were occasionally rocked to ensure that the medium covered the entire monolayer. Compounds were serially diluted in tissue maintenance cell culture medium described above and the solutions were added to the infected monolayers. The infected monolayers were incubated for 7 days and then stained with neutral red solution as described above. Plates were numbered and EC50 values interpolated from experimental data by standard methods.

DNA Quantitation Assays for EBV and HHV-6B: The EBV assays were performed on Akata cells that were induced to undergo lytic infection with goat anti-human IgG antibody by standard methods. The experimental compounds were diluted on plates. The Akata cells were added to the plates and incubated. For HHV-6, compounds were serially diluted, then uninfected HSB-2 or Molt-3 cells were added to each well. The infection was initiated by adding HHV-6A infected H SB-2 cells or HHV-6B infected Molt-3 cells, and incubated for 7 days at 37 ° C. For all assays, denaturation buffer was added to each well to denature DNA and an aliquot was aspirated through a nylon membrane. The membranes were then allowed to dry before equilibration in DIG. Virus-specific digoxigenin-labeled (DIG) probes were prepared according to the manufacturer's protocol. Detection of specifically bound DIG probe was done with anti-DIG antibody using manufacturer's protocol. An image of the photographic film was captured and quantified and compound concentrations sufficient to reduce viral DNA accumulation by 50% (EC50) were interpolated from the experimental data.

DNA Quantification Assays for HHV-8: Test compounds were diluted in duplicate wells. BCBL-1 cells were induced to undergo lytic infection by the addition of phorbol 12-myristate 13 -acetate, the cells were added to each well in the plate. Cells were incubated for 7 days at 37 ° C in a ^{humidified CO 2} incubator, then total DNA was prepared. Viral DNA was quantified by flash PCR. Compound concentrations sufficient to reduce genome copy number by 50% were calculated from the experimental data.

Influenza Cell Assays: For dose response curves, single drugs were added to MDCK cells in 96-well microplates. Untreated wells of infected cells (virus controls) and uninfected cells (cell controls) were included in each assay plate. At three days after infection, the virus control wells showed 100% cytopathology. The degree of viral cytopathology in each well was determined microscopically by inspection and by neutral red (NR) staining. Briefly, cells were stained with NR diluted in MEM to determine cell viability. Two hours later, the plates were processed for the quantification of NR uptake in viable cells. The amount of NR collected by the cells was determined spectrophotometrically.

QPCR Assays for BKV and JCV: Primary assays for BK viruses were performed in 96-well plates containing HFF cell monolayers. Compound dilutions were prepared on plates containing cells that were subsequently infected with the Gardner strain of BK virus. After a 7 day incubation, total DNA was prepared with a purification kit and the genome copy number was quantified by flash PCR. Compounds that were positive in this assay were confirmed in a similar assay in 96-well plates with compounds added 1 hr after infection to identify compounds that inhibit early stages of replication, including adsorption and penetration. The copy number of the genome was determined by methods described above.

The main evaluation of the compounds against JC virus was also carried out by methods similar to those of the main tests for BK virus. Secondary assays against JC V were performed in CO S7 cells by methods similar to those of the bK virus to identify compounds that inhibited virus uptake or penetration.

Hepatitis C virus assays: Luciferase (replicon) / CytoTox-1 (toxicity) indicator. Compounds were screened for anti-HCV activity using a luciferase reporter gene (Luc) endpoint in the primary HCV assay. The Luc indicator was used as an indirect measure of HCV replication since its activity is directly proportional to the levels of HCV RNA. Cytotoxicity evaluation is performed in parallel. Drug stocks were prepared in DMSO unless otherwise noted and diluted with histoculture medium to the desired high assay concentration. For each assay, the compounds were then further diluted in histoculture medium as necessary. After incubation, cells were processed to derive, where applicable, the EC50 and EC90 (concentration of compound that reduces replicon replication by 50% and 90% respectively). The values of CC50 (concentration that decreases cell viability by 50%) and IS50 (CC50 / EC50) were determined and presented. The anti-HCV activity was evaluated with the Luc activity derived from the replicon (genotype 1b or 2a) or the HCVcc virus as reading; while the cytotoxic drug concentrations that reduce cell numbers were evaluated by the CytoTox-1 cell proliferation assay according to the manufacturer's protocol. Recombinant alpha interferon was used as a positive control drug to validate the performance of the assay.

Assays for influenza virus, RSV and SA RS CoV were cytopathic effect / toxicity assay using CellTiter-Glo. Antiviral cytoprotection assays examine the effects of compounds at indicated dose-response concentrations in specific cell types to test the efficacy of compounds in preventing virus-induced cytopathic effect. Ribavirin was included as a positive control drug for influenza and RSV viruses, while the IV calpain inhibitor was used for SARS antiviral assays. Subconfluent cell cultures were seeded in 96-well plates for analysis of cell viability (cytotoxicity) and antiviral activity (ECP). For the standard assay, the drugs were added to the cells 24 hours later. ECP wells also received 100 doses of histoculture infections (100 TCID50) of virus titrated. At 72 hours later, cell viability was determined.

Measurement of virus-induced CPE was based on quantification of ATP, an indicator of metabolically active cells. The ECP assay used a commercially available luminescent cell viability kit, and was a reliable method to determine cytotoxicity and cell proliferation in culture. The procedure involved adding the individual reagent directly to subconfluent cells previously cultured in the medium. This induced cell lysis and the production of a bioluminescent signal (half-life greater than 5 hours, depending on the cell type) that was proportional to the amount of ATP present (which is a biomarker of viability).

Assays for Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus (YFV), Great Falla Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV):

Primary Cytopathic Effect Reduction (CPE) Trial. Four concentration ECP inhibition assays were performed. Confluent or near confluent cell culture monolayers were prepared on microplates. Cells were kept in MEM or DMEM supplemented with FBS as needed for each cell line. For antiviral assays, the same medium is used, but with FBS reduced to 2% or less and supplemented with gentamicin. The test compound was prepared at different concentrations, u. Virus control and cell control wells were on each microplate. In parallel, an active drug known as a positive control drug was tested using the same method as that applied for the test compounds. The positive control was tested with each assay run. The assay was established by first removing the growth medium from the 96-well plates of cells. The test compound was then applied to the wells. The virus was placed in those wells indicated for virus infection. Medium devoid of virus was placed in the toxicity control wells and cell control wells. Virus control wells were similarly treated with virus. The plates were incubated at 37 ° C with 5% CO ² until a maximum CPE was observed in the virus control wells. The plates were then stained neutral red for approximately two hours at 37 ° C in a 5% CO ² incubator. The neutral red medium was removed by complete aspiration, and cells can be rinsed with phosphate buffered saline (PBS) to remove residual dye. The PBS was completely removed and the incorporated neutral red was eluted with buffer. The content of dye in each well is quantified using a spectrophotometer. The content of dye in each set of wells was converted to a percentage of dye present in untreated control wells using a Microsoft Excel-based computer spreadsheet. The 50% effective concentrations (EC50) and the 50% cytotoxic concentrations (CC50) were then calculated by linear regression analysis. The quotient of CC50 divided by EC50 gives the Selectivity Index (IS) value.

Assays for adenovirus (AdV), measles virus (MEV), poliovirus (POV), and enterovirus (ENTV): The main screening was a cytopathic effect reduction (CPE) assay. Briefly, cell cultures were infected with virus in the presence of test compounds and incubated for 4-7 days (depending on the specific virus / cells). Each virus was pre-scored so that control wells showed approximately 95% loss of cell viability due to virus replication. Therefore, an antiviral effect, or cytoprotection, was observed when the compounds prevented virus replication. Each assay plate contained cell control wells (cells only), virus control wells (cells plus virus), compound toxicity control wells (cells plus compound only), compound colorimetric control wells (compound only, without cells or virus), as well as experimental wells (composed plus cells plus virus). The cytoprotection and cytotoxicity of the compound was evaluated by dye reduction m Ts. The percentage reduction in the CPE of the virus (antiviral activity) and the percentage of cell viability (cytotoxicity) were determined and presented.

Vaccine variovirus (VACV) assays: The main trial was a cytopathic effect reduction (CPE) assay. Low-passage HFF cells were trypsinized, counted, and plated on histoculture plates. The cells were then incubated for 24 h at 37 ° C. The medium was then removed and MEM containing 2% FBS was added to all but the first row. In the first row, medium containing the test drug (eg, compound 1) was added in triplicate wells. Medium alone was added to both cell and virus control wells. The drug in the first row of wells was then serially diluted 1: 5 in all remaining wells. The plates were then incubated for 60 minutes and a virus suspension was added to each well, excluding the cell control wells that received MEM. The plates were then incubated at 37 ° C in a CO ² incubator. After the incubation period, the medium was aspirated and the cells were stained with gentian violet in formalin for 4 h. The dye was then removed and the plates were rinsed until all excess dye was removed. The plates were allowed to dry for 24 h and the amount of ECP in each row was determined. EC ⁵⁰ and CC ⁵⁰ values were determined by comparing drug-treated and untreated cells using a computer program.

As set forth in Example 1 below, Compound 1 has activity against mouse norovirus in vitro. In some embodiments, Compound 1 has an EC ⁵⁰ value of about 2.1 and a CC ⁵⁰ value of about 114 against mouse norovirus. Furthermore, Compound 1 has activity against a wide range of DNA and RNA viruses.

As set forth in Example 2 below, Compound 1 has activity against mouse norovirus in vivo. As set forth in Example 2, Compound 1 was able to reduce viral load in mice infected with norovirus. murine in a dose-dependent manner. The results are given in Figure 1A and Figure 1B, which show the reduction in viral concentration in the stool and tissue, respectively, of mice in Study 1. In addition, Figures 2A and 2B show the reduction in viral concentration. in the stool and tissue, respectively, of mice in Study 2. Figures 3A and 3B show the reduction in viral concentration in the tissue and stool, respectively, of the mice in Study 1. The results are shown on a scale linear. Mice were treated with 30 mg / kg / day, or 100 mg / kg / day, or 300 mg / kg / day of Compound 1 in Study 1. Mice were treated with 150 mg / kg / day, or 300 mg / kg / day of compound 1 in study 2.

Example 3 demonstrates that Compound 1 can inhibit norovirus polymerase in vitro. Without wishing to be bound by theory, it is proposed that Compound 1 can protect against and treat norovirus by inhibiting norovirus polymerase.

As set forth in Example 4, Compound 1 is converted to a triphosphate in vitro. As shown, when cells were incubated with Compound 1, the corresponding triphosphate (ie, Compound 1-TP) was produced. The level of Compound 1-TP was 12 to 23 times higher than Compound 1 after the incubation period.

Example 5, below, demonstrates that Compound 1 is more effective in inhibiting mouse norovirus than Compound 2, or 2'-C-methylcytidine triphosphate. A comparison with DMSO as a control is given by reference. The experiment was performed in duplicate and the results are shown in Figure 4 (first duplicate) and Figure 5 (second duplicate). Figure 6 shows an overlay of the results of the first and second duplicates of the experiment. As shown in Figures 4, 5 and 6, "A" is DMSO, "B" is compound 2, "C" is 2'-C-methylcytidine triphosphate (2'CmeC TP) and "D" is compound 1 Figures 4-6 demonstrate that the viral concentration increased by almost two orders of magnitude when treated with only DMSO or 2'CmeC TP. However, the viral concentration increased by less than an order of magnitude in the presence of compound 1.

Example 6 shows a comparison with compound 1 and other compounds of formula II (Table 7) and analogs thereof. Without wishing to be bound by theory, compounds that are even slightly different from the structure of formula I may have substantially reduced activity in vitro. For example, compound 7 has a hydroxy group in place of arylamine and has an EC ⁵⁰ value> 38 pM; compound 35 shows an analog of compound 1 lacking the arylamine group, and has an EC ⁵⁰ value> 100 pM. Furthermore, compound 11 has a cyano group in place of the arylamide and has an EC ⁵⁰ value> 121 pM. Also, compounds 36 and 37 show methyl substituted amines and have EC ⁵⁰ values> 100 pM. Those skilled in the art will understand additional comparisons.

Figure 7a shows an HPLC day of compound 1.

Without wishing to be bound by theory, peak # 2 was identified as benzoic acid.

Figure 7b shows an HPLC day of compound 1 after suspension for 3 hours at room temperature.

Figure 7c shows an HPLC day of compound 1 after suspension for 3 hours at 50 ° C.

Figure 7d shows an HPLC diagram of compound 1 after suspension for 24 hours at about room temperature.

Figure 8a shows a 1H NMR spectrum of compound 1 from about -2 to about 14 ppm.

Figure 8b shows a 1H NMR spectrum of compound 1 from about 2 to about 9 ppm.

Figure 8c shows a 1H NMR spectrum of compound 1 from about 0 to about 9 ppm.

EXAMPLES

General procedures

HPLC charts were recorded on a Zorbax Eclipse Plus C18 column (4.6 x 50 mm x 1.8 pm). The first mobile phase was 97.5% water: 2.5% acetonitrile: 0.05% trifluoroacetic acid. The second mobile phase was 97.5% acetonitrile: 2.5% water: 0.05% trifluoroacetic acid.

1H NMRs were recorded at 500 MHz unless otherwise indicated.

Example 1 - Antiviral and cytotoxicity tests

Cell Culture and Virus Strains: Human foreskin fibroblasts (HFF) were prepared from human foreskin tissue obtained from the University of Alabama at Birmingham Tissue Procurement Facility with the approval of its CIR. The tissue was incubated at 4 ° C for 4 h in cell culture medium consisting of minimal essential medium (MEM) with Earle's salts supplemented with 10% fetal bovine serum (FBS) (Hyclone, Inc. Logan UT), and conventional concentrations of L-glutamine, fungizone, and vancomycin. The tissue was then placed in phosphate buffered saline (PBS), ground, rinsed to remove red blood cells, and resuspended in trypsin / EDTA solution. The tissue suspension was incubated at 37 ° C and gently shaken to disperse the cells, which were then collected by centrifugation. The cells were resuspended in 4 ml of medium and placed in a 25 cm2 histoculture flask and incubated at 37 ° C in a ^{humidified CO 2} incubator for 24 h. The medium was then replaced with fresh medium and cell growth was monitored daily until a confluent cell monolayer was formed. The HFF cells were then expanded through serial passages in conventional MEM growth medium with Earle's salts supplemented with 10% FBS, L-glutamine, penicillin, and gentamicin. Cells were routinely passaged and used for assays at or below passage 10.

Akata cells latently infected with EBV were obtained from John Sixbey (Louisiana State University, Baton Rouge, LA). HHV-6A strain GS was obtained through the NIH AIDS Research and Reference Reagent Program. HSB-2 cells and BCBL-1 cells were obtained through the AIDS Research and Reference Reagent Program of the NIH, Division of AIDS, NIAID, NIH. Molt-3 cells were obtained from Scott Schmid at the Centers for Disease Control and Prevention, Atlanta, GA. All lymphocyte cultures were routinely maintained in RPMI 1640 (Mediatech, Inc., Herndon, VA) with 10% FBS, L-glutamine, and antibiotics and passaged twice weekly. Vero cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and were maintained in standard MEM growth medium with Earle's salts supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin.

Influenza viruses: Influenza viruses A / New Caledonia / 20/99 (H1N1) and A / Sydney / 05/97 (H3N2) were provided by the Centers for Disease Control and Prevention (Atlanta, GA). Viruses were passaged into Madin-Darby canine kidney cells (MDCK) (American Type Culture Collection, Manassas, VA) to create working stock solutions, which were used for antiviral assays.

HSV-1 strains E-377 and DM2.1 were used, as well as HSV-2 strains MS and 13078. HSV-1 strain F was obtained from ATCC. HCMV strains AD169 and Merlin were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and C8805 / 37-1-1 and 759RD100 were a gift from Karen Biron. The Ellen strain of VZV was obtained from ATCC. The HHV-6B Z29 strain was a gift from Scott Schmid at the Centers for Disease Control and Prevention, Atlanta GA. HHV-8 was obtained as latently infected BCBL-1 cells through the NIH AIDS Research and Reference Reagent Program.

Antiviral Assays: Each experiment that evaluated the antiviral activity of compounds included both positive control and negative control compounds to ensure the performance of each assay. Simultaneous evaluation of cytotoxicity was performed when possible at equivalent levels of exposure to the compound. The methods are presented following the tabulated data in effective concentrations that give 50% reductions in viral replication in vitro (EC50), concentrations that produce a 50% reduction in cell viability (CC50) and the selectivity index (IS , calculated as CC50 divided by EC50). When sufficient material was available, multiple tests were performed for each compound evaluation to obtain statistical data.

Cytotoxicity Assays: Each antiviral assay included a parallel cytotoxicity assay with the same cells used for each virus, the same number of cells, the same drug concentrations, and the same incubation times to provide the same drug exposure. To ensure that the cytotoxicity of all compounds could be directly compared, a standard neutral red uptake cytotoxicity assay was performed for all compounds on confluent HFF cells with an incubation period of 7 days.

Neutral Red Uptake Cytotoxicity Assays: Each compound was evaluated in a standard cytotoxicity assay by standard methods. Briefly, HFF cells were seeded in 96-well histoculture plates at 2.5 x 10 4 cells / well in standard histoculture medium. After 24 h of incubation, the medium was replaced with maintenance cell culture medium and the compounds were added to the first row and then serial dilutions of factor 5 were used to generate a series of compound concentrations with a maximum of 300 pM. Assay plates were incubated for 7 days, and 100 µl of a 0.66 mg / ml solution of neutral red in PBS was added to each well and the plates were incubated for 1 hr. The dye was then removed, the plates were rinsed with PBS, and the dye internalized by viable cells was solubilized in PBS supplemented with 50% ethanol and 1% glacial acetic acid. The optical density at 550 nm was then determined and the CC50 values from the experimental data were interpolated.

For all HFF cell plaque reduction assays, neutral red cytotoxicity assays were performed on a parallel set of 6-well plates containing uninfected HFF cells that received the same compound concentrations as used for antiviral assays. The cytotoxicity plates were removed from the incubator on the same day as each antiviral assay and the cell monolayer was stained for 6 h with 2 ml of a neutral red solution at a concentration of 0.165 mg / ml in PBS. The dye was then removed, residual dye was rinsed from the cells with PBS, and the cell monolayers were visually inspected for any signs of toxicity. The Vero cell cytotoxicity assay was performed with drug concentrations ranging from 1 pM to 1 mM. Cell viability was determined using CellTiter 96® aqueous solution cell proliferation assay (Promega) according to manufacturer's instructions.

Cell proliferation assays: Inhibition of HFF cell proliferation was used to refine estimates of cytotoxicity for some compounds and was performed according to a standard procedure used in the laboratory. Cells were seeded at low density in six-well plates using 2.5 x 10 4 cells / well and standard culture medium. After 24 h, the medium was aspirated, and a series of compound solutions were prepared in the growth medium starting at 300 pM and added to duplicate wells. The plates were incubated for 72 h at 37 ° C, and the cells were then detached with trypsin and counted on a Beckman Coulter Counter. Compound concentrations that reduced cell proliferation by 50% were interpolated from the experimental data.

Cytotoxicity in lymphocyte assays: Cell viability in all lymphocyte assays was evaluated with the CellTiter-Glo luminescent cell viability assay (Promega). Briefly, assay plates were incubated at room temperature for 30 min, then 50 µl of CellTiter-Glo reagent was added to each well and the plates were mixed for 2 min on an orbital shaker to lyse the cells. The plates were then incubated for an additional 10 min at room temperature and luminescence was quantified on a luminometer. Standard methods were used to calculate drug concentrations that inhibited Akata, HSB-2, BCLB-1 or Molt-3 cell proliferation by 50% (CC ⁵⁰ ).

Table 1: Activity of compound 1 against mouse norovirus in RAW cells

Table 2: Activity of compound 1 against various DNA RNA viruses

Mouse norovirus (MNV) assay: RAW cells (mouse macrophages, ATCC TIV-71) were received from the American Type Culture Collection (ATCC). Cells were incubated at 37 ° C with 5% CO ² in cell culture medium consisting of Dulbecco's minimal essential medium (DMEM) (ATCC) supplemented with 10% fetal calf serum (FBS) (Hyclone, Inc. , Logan UT), 100 U / ml penicillin and 100 ug / ml streptomycin (Hyclone), MEM NEAA 1% (Gibco), GlutaMAX 1% (Gibco) and HEPES 1% (Hyclone). The mouse norovirus isolate used in this assay was isolated from a wild-type mouse, adapted to cell culture, plaque purified, and its genetic sequence confirmed by Chimerix, Inc.

100 µl of serial dilutions of test compounds in DMEM were added to histocultured 96-well Costar plates. 100 µl of cell suspension containing 50,000 RAW cells / well and MNV (MOI = 0.0005) were added to the compound dilutions in the 96-well plate, and incubated at 37 ° C with 5% CO ² for 2 days. Each assay plate included uninfected cell controls and untreated virus control wells. After 2 days of incubation, the untreated virus control wells showed 100% ECP. After 2 days of incubation, 40 µl of Cell Titer 96® aqueous MTS reagent (Promega, G111) was added as indicated by the manufacturer to 200 µl of medium in each well and incubated at 37 ° C until cell controls Untreated revealed an absorbance at 490 nm between 1.1 and 1.8. Final absorbance readings were read using a BioTek Synergy 2, and Gen 5 computer program (BioTek Instruments, Inc.) was used to calculate the concentration that protected MNV-infected RAW cells by 50% compared to cell controls. uninfected (EC50).

Human Norovirus Assays: Antiviral activity against a human norovirus (NoV) was evaluated in a 3-day assay using the stable expression human norovirus replicon cell line, HG23 (genogroup I, genomic length; precursor cell line, HuH7) (Chang, et al., 2006, Virol. 353: 463) maintained as subconfluent cultures in 96-well plates. Typically, 4 doses (factor 10 or factor 3 steps) are used, in triplicate. Antiviral activity was determined by intracellular NoV RNA blot hybridization analysis (normalized to the level of cellular p-actin RNA in each culture sample). Cytotoxicity was evaluated by neutral red dye uptake in cultures maintained in parallel plates (Korba and Gerin, 1992, Antivir. Res. 19:55).

EC ⁵⁰ and CC ⁵⁰ values are calculated by linear regression analysis (MS EXCEL®, QuattroPro®) using pooled data from all treated cultures (Korba and Gerin, 1992, Antivir. Res. 19:55; Okuse, et al. ., 2005, Antivir. Res.

65:23). The standard deviations for the EC ⁵⁰ and EC ⁹⁰ values were calculated from the standard errors generated by the regression analyzes. EC ⁵⁰ and EC ⁹⁰ are drug concentrations at which a factor 2 or factor 10 decrease in intracellular NoV RNA was observed (relative to average levels in untreated cultures), respectively. The CC ⁵⁰ is the drug concentration at which two times less level of neutral red dye uptake is observed (relative to average levels in untreated cultures). The selectivity index (IS) was calculated as CC ⁵⁰ / EC ⁵⁰ . Recombinant human interferon 2b (PBL laboratories, Inc.) is used as an assay control.

EC ⁵⁰ of BKV in VERO cells: Costar 96 well histoculture plates were seeded with 10,000 Vero cells / well in DMEM containing 2% Hyclone standard fetal bovine serum (FBS, Cat SH30088.03) and Hyclone penicillin and streptomycin 1 %. The outer wells were not used to minimize the edge effect produced by prolonged incubations. Cells were inoculated with 115 copies of BKV DNA / cell (ATCC, Gardner strain). Serial dilutions of test compounds were added to the cells and the plates were incubated for 10 days at 37 ° C in 5% CO ² . After the 10 day incubation, 50 µl of supernatant was mixed with 50 µl of 2X lysis buffer that gave a final concentration of 0.5 mg / ml of protease K, 50 mM KCl, 10 mM T ris-Cl pH 8 , 0, MgCl22.5 mM, 0.45% IGEPAL and 0.45% Tween-20 dissolved in DEPC-treated water. Each plate was incubated for 2 hours at 55 ° C. BKV DNA from the supernatant was measured by quantitative polymerase chain reaction (qPCR) using the BKV PCR forward and reverse primers, and a FAM-labeled probe. Absolute quantification of virus copy number was performed using a standard curve with dilutions of BKV DNA amplicon containing sequences homologous to the amplified fragment. The following qPCR amplification conditions were used: 1 cycle at 95 ° C for 10 minutes, followed by 45 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds. The qPCR reactions were performed using an Applied Biosystems 7500 ultra-fast PCR system. The Gen 5 computer program (BioTek Instruments, Inc.) was used to calculate the concentration that inhibited viral DNA levels of BKV-infected Vero cells by 50 % (EC50).

Plaque Reduction Assays for HSV-1, HSV-2, VZV, and HCMV: HFF cell monolayers were prepared in six-well plates and incubated at 37 ° C for 2 days to allow the cells to reach confluence. The medium was then aspirated from the wells and 0.2 ml of virus was added to each of the three wells to produce 20-30 plaques in each well. The virus was allowed to adsorb on the cells for 1 h and the plates were gently rocked every 15 min to redistribute the medium. Compounds were diluted in maintenance cell culture medium consisting of MEM with Earle's salts supplemented with 2% FBS, L-glutamine, penicillin, and gentamicin. Solutions ranging from 300 pM to 0.1 pM were added to duplicate wells and the plates were incubated for various times, depending on the virus used. The plaque reduction assay with HSV-1 strain F was performed in a similar manner, but with infected Vero cells one day after plating. The final concentration of FBS in this assay was 5%. For HSV-1 and 2, monolayers were stained with 1% gentian violet in 20% methanol and unbound dye was washed away with dH ² O. For HCMV and VZV assays, the cell monolayer was stained with 1% neutral red solution for 4 h, then the dye was aspirated and the cells were washed with PBS. For all tests, the plaques were enumerated using a stereomicroscope and the concentration of compound that reduced plaque formation by 50% (EC50) was interpolated from the experimental data.

Plaque reduction assays for MCMV: Mouse embryo fibroblasts were prepared using a procedure similar to that outlined above for HFF cells and suspended in histoculture medium as described above and seeded in 12-well plates and incubated at 37 ° C for 24 h. The medium was aspirated and the cell monolayers were infected with the Smith strain of MCMV in a final volume of 0.2 ml in each of the triplicate wells. The infected cells were then incubated at 37 ° C for 1 h and the plates were occasionally rocked to ensure that the medium covered the entire monolayer. Compounds were serially diluted 1: 5 in tissue maintenance cell culture medium described above and the solutions were added to the infected monolayers. The infected monolayers were incubated for 7 days and then stained with 2 ml of a 1% neutral red solution as described above. Plates were numbered and EC ⁵⁰ values interpolated from experimental data by standard methods.

DNA Quantification Assays for EBV and HHV-6B: The EBV assays were performed on Akata cells that were induced to undergo lytic infection with 50 pg / ml of a goat anti-human IgG antibody by standard methods. The experimental compounds were diluted in round bottom 96 well plates to produce concentrations ranging from 20 to 0.016 pM. Akata cells were added to the plates at a concentration of 4 * 10 4 cells per well and incubated for 72 h. For HHV-6, compounds were serially diluted in 96-well plates, then 1 x 104 uninfected HSB-2 or Molt-3 cells were added to each well. The infection was initiated by adding HHV-6A infected HSB-2 cells, or HHV-6B infected Molt-3 cells, at a ratio of approximately 1 infected cell for every 10 uninfected HSB-2 cells or Molt-3 cells respectively and they were incubated for 7 days at 37 ° C. For all assays, 100 µl Denaturation Buffer (1.2 M NaOH, 80 4.5 M NaCl) was added to each well to denature DNA and a 50 µl aliquot was aspirated through an Immobilon nylon membrane. (Millipore, Bedford, MA) using a Biodot apparatus (Bio-Rad, Hercules, CA). The membranes were then allowed to dry before equilibration in DIG Easy Hyb (Roche Diagnostics, Indianapolis, IN) at 56 ° C for 30 min. Virus-specific digoxigenin-labeled (DIG) probes were prepared according to the manufacturer's protocol (Roche Diagnostics). For EBV, the primers 5'-CCC AGG AGT CCC AGT AGT CA-3 'and 5'-CAG TTC CTC GCC TTA GGT TG-3 amplified a fragment corresponding to coordinates 96802-97234 in the EBV genome (AJ507799). A specific HHV-6 DIG-labeled probe was prepared using the primers 5'-CCT TGA TCA t Tc GAC c Gt TT-3 'and 5'-TGG GAT TGG GAT TAG AGC TG-3' to amplify a segment of ORF2 (coordinates 37820-38418 in X83413). The EBV DNA membranes were hybridized overnight at 56 ° C followed by sequential washes in 0.2 * SSC with 0.1% SDS and 0.1 * SSC with 0.1% SDS at the same temperature. For the HHV-6A and HHV-6B blots, the probe was allowed to hybridize overnight at 42 ° C and the blots were rinsed at the same temperature with 0.2 * SSC with 0.1% SDS and 0 SSC , 1 * with 0.1% SDS. Detection of the specifically bound DIG probe was performed with anti-DIG antibody using manufacturer's protocol (Roche Diagnostics). An image of the photographic film was captured and quantified with QuantityOne software (Bio-Rad) and compound concentrations sufficient to reduce viral DNA accumulation by 50% (EC50) were interpolated from the experimental data.

DNA quantification assays for HHV-8: Test compounds were diluted in duplicate wells of a 96-well plate to the maximum final concentration of 20 pM. BCBL-1 cells were induced to undergo lytic infection by the addition of phorbol 12-myristate 13-acetate (Promega, Madison WI) to a final concentration of 100 ng / ml and 2 * 104 cells were added to each well. on the plate. Cells were incubated for 7 days at 37 ° C in a ^{humidified CO 2} incubator, then total DNA was prepared with a Wizard SV 96-well purification kit (Promega). Viral DNA was quantified by flash PCR using the forward primer 5'-TTC CCC AGA TAC ACGA ^c A GAA TC-3 ', the reverse primer 5'-CG GAGCG CA GGC TAC CT-3' and the probe 5 '- ( FAM) CCTACG TG TTCGTCG AC (TAMRA) -3 '. Plasmid pMP218 containing DNA sequences corresponding to nucleotides 14120-14182 (AF148805.2) was used to provide absolute quantification of viral DNA. Compound concentrations sufficient to reduce genome copy number by 50% were calculated from the experimental data.

Influenza Cell Assays: For dose response curves, single drugs were added to MDCK cells in 96-well microplates (8 x 104 cells / well) using three wells for each concentration used. Compounds were added at the following concentrations: oseltamivir carboxylate at 0.000032, 0.0001, 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0 and 100 pg / ml; amantadine and ribavirin at 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10, 32 and 100 pg / ml. Untreated wells of infected cells (virus controls) and uninfected cells (cell controls) were included in each assay plate. At three days after infection, the virus control wells showed 100% cytopathology. The degree of viral cytopathology in each well was determined microscopically by inspection and by neutral red (NR) staining. Briefly, cells were stained with 0.011% NR diluted in MEM to determine cell viability. Two hours later, the plates were processed for the quantification of NR uptake in viable cells. The amount of NR collected by the cells was determined spectrophotometrically.

QPCR Assays for BKV and JCV: Primary assays for BK viruses were performed in 96-well plates containing HFF cell monolayers. Compound dilutions were prepared on plates containing cells that were subsequently infected with the Gardner strain of BK virus. After a 7 day incubation, total DNA was prepared with a Wizard SV 96-well purification kit and genome copy number was quantified by flash PCR using primers 5'- A ^g TG GA TGGG CA GCC TAT GTA -3 ', 5'- TCA TAT CTGGGTCCCC ^t G GA-3' and the probe 5'-6-FAM AGG TAG AAG AGG TTA GGG TG T TTG ATG G CA CA G TAMRA-3 '. Plasmid pMP526 served as a DNA template for quantification purposes. Compounds that were positive in this assay were confirmed in a similar assay in 96-well plates with compounds added 1 hr after infection to identify compounds that inhibit early stages of replication, including adsorption and penetration. The copy number of the genome was determined by methods described above.

Compounds were also screened against JC virus by methods similar to BK virus core assays, but they were done on 293TT cells and used JC V strain 1-4 on 293TT cells. The viral DNA was quantified using the primers 5'-CTG G TC ATG TGGA TG CTG TCA-3 'and 5'-G CC AGCAGGCTG TTG ATA CTG-3' and the probe 5'-6-FAM -CCC TTT G TT TG GCTG CT-TAM RA-3 together with plasmid pMP508 to provide a standard curve for absolute quantification. Secondary assays against JC V were performed in CO S7 cells by methods similar to those of the BK virus to identify compounds that inhibited virus uptake or penetration.

Hepatitis C virus assays: Luciferase (replicon) / CytoTox-1 (toxicity) indicator. Compounds were screened for anti-HCV activity using a luciferase reporter gene (Luc) endpoint in the primary HCV assay. The Luc indicator was used as an indirect measure of HCV replication since its activity was directly proportional to the levels of HCV RNA. Cytotoxicity evaluation is performed in parallel. Drug stocks were prepared in DMSO unless otherwise noted and diluted with histoculture medium to the desired high assay concentration. For each assay, the compounds were then further diluted in histoculture medium as necessary. After incubation, cells were processed to derive, where applicable, the EC50 and EC90 (concentration of compound that reduces replicon replication by 50% and 90% respectively). The values of CC50 (concentration that decreases cell viability by 50%) and IS50 (CC50 / EC50) were determined and presented. The anti-HCV activity was evaluated with the Luc activity derived from the replicon (genotype 1b or 2a) or the HCVcc virus as reading; while cytotoxic drug concentrations that reduce cell numbers were evaluated by the CytoTox-1 cell proliferation assay (Promega, Madison, WI) according to the manufacturer's protocol. Recombinant alpha interferon was used as a positive control drug to validate the performance of the assay.

Assays for influenza virus, respiratory syncytial virus (RSV) and SARS CoV: The main viruses and cells used were influenza virus strain A / California / 7/2009 (H1N1) in MDCK cells, respiratory syncytial virus strain A -2 in Hep2 cells and SA RS CoV strain Toronto-2 in VeroE6 cells.

Assays for influenza virus, RSV, and SARS CoV were cytopathic effect / toxicity assay using CelITiter-Glo. Antiviral cytoprotection assays examined the effects of compounds at indicated dose-response concentrations in specific cell types to test the efficacy of compounds in preventing virus-induced cytopathic effect. Ribavirin was included as a positive control drug for influenza and RSV viruses, while the IV calpain inhibitor was used for SARS antiviral assays. Subconfluent cell cultures were seeded in 96-well plates for analysis of cell viability (cytotoxicity) and antiviral activity (ECP). For the standard assay, the drugs were added to the cells 24 hours later. The ECP wells also received 100 doses histoculture infections (100 TCID50) of assayed virus. At 72 hours later, cell viability was determined.

Measurement of virus-induced CPE was based on quantification of ATP, an indicator of metabolically active cells. The ECP assay employed a commercially available CellTiter-Glo ™ luminescent cell viability kit (Promega, Madison, WI), and was a reliable method for determining cytotoxicity and cell proliferation in culture. The procedure involved adding the single reagent (CellTiter-Glo ™ reagent) directly to subconfluent cells previously cultured in the medium. This induced cell lysis and the production of a bioluminescent signal (half-life greater than 5 hours, depending on the cell type) that was proportional to the amount of ATP present (which is a biomarker of viability).

Assays for Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus (YFV), Great Falla Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV):

Primary Cytopathic Effect Reduction (CPE) Trial. Four concentration ECP inhibition assays were performed. Confluent or near confluent cell culture monolayers were prepared in disposable 96-well microplates. Cells were kept in MEM or DMEM supplemented with FBS as needed for each cell line. For antiviral assays, the same medium was used, but with FBS reduced to 2% or less and supplemented with 50 pg / ml gentamicin. The test compound was prepared at four log 10 final concentrations, usually 0.1, 1.0, 10 and 100 pg / ml or pM. Virus control and cell control wells were on each microplate. In parallel, an active drug known as a positive control drug is tested using the same method as that applied for the test compounds. The positive control was tested with each assay run. The assay was established by first removing the growth medium from the 96-well plates of cells. The test compound was then applied in a volume of 0.1 ml to the wells at 2X concentration. Virus, typically at <100 50% cell culture infectious doses (CCID50) in 0.1 ml volume, was placed in the wells indicated for viral infection. Medium devoid of virus was placed in the toxicity control wells and cell control wells. Virus control wells were similarly treated with virus. The plates were incubated at 37 ° C with 5% CO ² until a maximum CPE was observed in the virus control wells. The plates were then stained with 0.011% neutral red for approximately two hours at 37 ° C in a 5% CO ² incubator. The neutral red medium was removed by complete aspiration, and cells can be rinsed 1X with phosphate buffered saline (PBS) to remove residual dye. The PBS was removed completely and the incorporated neutral red was eluted with 50% Sorensen citrate / 50% ethanol buffer (pH 4.2) for at least 30 minutes. The neutral red dye penetrates living cells, therefore, the more intense the red color, the greater the number of viable cells present in the wells. The content of dye in each well was quantified using a 96-well spectrophotometer at 540 nm wavelength. The content of dye in each set of wells was converted to a percentage of dye present in untreated control wells using a Microsoft Excel-based computer spreadsheet. The 50% effective concentrations (EC50) and the 50% cytotoxic concentrations (CC50) were then calculated by linear regression analysis. The quotient of CC50 divided by EC50 gives the Selectivity Index (IS) value.

Assays for adenovirus (AdV), measles virus (MEV), poliovirus (POV), and enterovirus (ENTV): The main screening was a cytopathic effect reduction (CPE) assay. Briefly, 96-well cell cultures were infected with virus in the presence of test compounds and incubated for 4-7 days (depending on the specific virus / cells). Each virus was pre-scored so that control wells showed approximately 95% loss of cell viability due to virus replication. Therefore, an antiviral effect, or cytoprotection, was observed when the compounds prevented virus replication. Each assay plate contained cell control wells (cells only), virus control wells (cells plus virus), compound toxicity control wells (cells plus compound only), compound colorimetric control wells (compound only, without cells or virus), as well as experimental wells (composed plus cells plus virus). The cytoprotection and cytotoxicity of the compound was evaluated by reduction of MTS dye (CellTiter®96 reagent, Promega, Madison WI). The percentage reduction in the CPE of the virus (antiviral activity) and the percentage of cell viability (cytotoxicity) were determined and presented.

Vaccine variovirus (VACV) assays: The main trial was a cytopathic effect reduction (CPE) assay. Low passage HFF cells (3-10) were trypsinized, counted, and seeded in 96-well histoculture plates in 0.1 ml MEM supplemented with 10% FBS. The cells were then incubated for 24 h at 37 ° C. The medium was then removed and 100 µl of MEM containing 2% FBS was added to all but the first row. In the first row, 125 µl of medium containing the test drug (ie, compound 1) was added in triplicate wells. Medium alone was added to both cell and virus control wells. The drug in the first row of wells was then serially diluted 1: 5 in all remaining wells. The Plates were then incubated for 60 minutes and 100 µl of a virus suspension was added to each well, excluding cell control wells that received 100 µl MEM. The plates were then incubated at 37 ° C in a CO ² incubator for three days for VACV. After the incubation period, the medium was aspirated and the cells were stained with gentian violet in formalin for 4 h. The dye was then removed and the plates were rinsed until all excess dye was removed. Plates were allowed to dry for 24 h and the amount of EC P in each row was determined using a BioTek automated multi-plate reader. EC ⁵⁰ and CC ⁵⁰ values were determined by comparing drug-treated and untreated cells using a computer program.

Example 2 - Determination of the efficacy of compound 1 against murine norovirus in mice

Methodology

Two studies (Study # 1 and Study # 2) examined the ability of Compound 1 to protect mice against or reduce murine norovirus infection:

Study # 1 The efficacy of twice daily dosing of Compound 1 was evaluated over a range of 30 mg / kg to 300 mg / kg, initiated prior to infection of mice with 106 plaque-forming units (PFU) of norovirus murine (MNV) CR3. A control group of vehicle-treated mice was also included. All doses were started 2 days (39 hours) before infection. The study groups are shown in Table 3.

The compound was administered twice daily in the indicated doses by oral gavage. A control group of mice treated with vehicle only was also included. Mice were infected with murine norovirus by pipetting the virus into the mouth 2 days after the first dose. The mice used were ~ 20 gram female BALB / c mice, 8-12 weeks old in groups of 5 (study groups are shown in Table 3). Mice were housed in metal racks and pooled fecal output was collected every 24 hours. Tissues (distal ileum and cecum) and individual fecal granules were collected on day 3 after inoculation. All samples were titrated by plaque assay.

Table 3: Study # 1: Study Design for Preliminary Evaluation of Efficacy of Compound 1 Against Infection or Murine Norovirus in Mice

Study # 2 The efficacy of twice daily dosing of Compound 1 at 150 mg / kg or 300 mg / kg, initiated prior to infection of mice with 104 pfu of MNV CR3, was evaluated. A control group of vehicle-treated mice was also included. The 150 mg / kg dose was started 2 days (39 hours) before inoculation, 1 day (15 hours) before inoculation, or on the day of (3 hours before) inoculation; the 300 mg / kg dose was started 2 days (39 hours) before infection. The study groups are shown in Table 4.

This study tested the ability of Compound 1 to protect mice against or reduce infection by murine norovirus. The compound was administered twice daily in the indicated doses by oral gavage starting 2 days before inoculation, 1 day before inoculation or at the time of inoculation. A control group of mice treated with vehicle only was also included. Mice were infected with 104 PFU of murine norovirus by pipetting virus into the mouth 3 hours after the day 0 dose. Mice used were ~ 20 gram female BALB / c mice, 8-12 weeks old in groups of 5 (The study groups are shown in table 4). All samples were titrated by plaque assay.

Table 4: Study # 2: Study Design to Evaluate the Efficacy of Compound 1 Against Murine Norovirus Infection in Mice

For both studies, the compound was administered by oral gavage at the indicated doses and times before infection. Mice were infected with murine norovirus CR3 by pipetting the virus into the mouth 3 hours after the first day 0 dose of compound 1 was administered. After infection, the compound was administered twice daily until day 3 after the infection.

The mice used were ~ 20 gram female BALB / c mice, 8-12 weeks old in groups of 5. Mice were housed in metal racks and pooled fecal output was collected every 24 hours, starting on day -1. Tissues (~ 1 cm from distal ileum and cecum) and individual fecal granules were collected on day 3 post infection and weighed. All samples were titrated by plaque assay (qRT-PCR as backup when concentrations are too low); concentrations were normalized to gram of tissue or feces. On day 3, duplicate tissue samples (~ 1 cm from distal ileum and cecum) were collected, rinsed in PBS, and snap frozen; serum and a duplicate stool sample were also collected on day 3 and snap frozen; This sample set was submitted for MS analysis to assess the bioavailability of the drug.

Results

The results of study # 1 show that twice daily treatment with Compound 1, administered beginning 2 days before infection, was effective in reducing the concentration of murine norovirus in both tissue and stool, with a greater reduction in the viral concentration observed with increasing drug concentration as shown in Figure 1A and Figure 1B. The data also demonstrate a significant reduction in viral concentration for animals treated twice daily with 300 mg / kg of Compound 1 in both tissue as in feces compared to vehicle.

As shown in Figures 1A and 1B, mice were orally gavaged with the indicated doses of Compound 1 administered twice daily, beginning 2 days prior to infection with 106 pfu of murine norovirus as set forth in the "study # 1 ".

The results of Study # 2 demonstrate that treating mice twice daily with 300 mg / kg of Compound 1, starting 2 days before infection, significantly reduces the concentration of murine norovirus in both tissue and feces as shown in Figure 2A and Figure 2B. These data confirm the findings of Study # 1 and demonstrate that Compound 1 is effective in reducing murine norovirus infection.

As shown in Figures 2A and 2B, mice were orally gavaged with the indicated doses of Compound 1 administered twice daily, beginning on the indicated days prior to infection with 104 PFU of murine norovirus as set forth in "Study # 1".

Figures 3A and 3B show the number of plaque-forming units per gram from Study 1 on a linear scale rather than a logarithmic scale. As shown in Figure 3A, Compound 1 dosed orally BID (twice daily) starting on day -2 before infection (n = 5 / group) showed a reduction in PFU / gram with increasing dose. . As shown in Figure 3A and 3B, Compound 1 reduces mouse norovirus in tissues and feces.

Example 3 - Norovirus polymerase inhibition assay

Polymerase reactions (10 µl) were performed for 60 minutes at 37 ° C. Nucleoside triphosphates (NTPs) were present at 100 pM each, with 0.05 pCi of a32P-U TP (800 Ci / mmol). Compounds were incubated with polymerase (Pol) or pro-polymerase (ProPol), with or without linked viral protein genome (VPg) in reaction buffer in the absence of NTP for 10 minutes on ice. Reactions were initiated by the addition of NTP, and were terminated by the addition of an equal volume of 2x Tris loading dye / borate / EDTA (TBE) / buffer (Invitrogen, Inc.). RNA products (100 nt) were resolved by electrophoresis on 6% TBE -urea gels (Invitrogen, Inc.). Semi-quantitative analysis of RNA products was performed by exposure of dried gels to GE Healthcare Phosphor sieves, followed by measurement of relative band intensities using GelQuant.NET software (Biochemlab Solutions, Inc.). IC ⁵⁰ and IC ⁹⁰ calculations were obtained using linear regression. The results are given below in Table 5. A comparison with 2'-C-methylcytidine triphosphate (2'CmeC TP) is given by reference. Table 5. In lim rnr go

Example 4 - Conversion of compound 1 to triphosphate in RAW cells

RAW cells were incubated with Compound 1 at the concentrations shown in Table 6a (ng / cell) and Table 6b (pmol / cell).

Table 6a. Conversion of compound 1 to triphosphate in n / cell

Table 6b. n r i n m 1 n rif f n m l l l

RAW cells were incubated with four different concentrations of Compound 1 for 48 hours in T75 flasks at a density of 1.2 * 107 cells / flask. After the incubation period, cells were rinsed twice with cold PBS and counted. The cell pellet was suspended in 1000 µl of cold methanol: distilled water (70:30), vortexed and frozen at -80 ° C until analysis. As shown in Tables 6a and 6b, Compound 1 and Compound 1-T P could be detected in RAW cells treated with Compound 1 at concentrations from 0.5 pM to 10 pM. The concentration of compound 1-T P was 12 to 23 times higher than that of compound 1.

Example 5 - Efficacy of compound 1 against human norovirus

The efficacy of Compound 1 to inhibit norovirus was compared to DMSO (as a control), Compound 2, and 2'-C-methylcytidine triphosphate (2'CmeC TP). Cells were pretreated with 25 pM of experimental compound for two hours. The virus inoculum was added for two hours, and the unbound virus was washed away and fresh medium and fresh test compound were added.

The experiment was performed in duplicate and the results are shown in Figure 4 (first duplicate) and Figure 5 (second duplicate). Figure 6 shows an overlay of the results of the first and second duplicates of the experiment. As shown in Figures 4, 5 and 6, "A" is DMSO, "B" is compound 2, "C" is 2'-C-methylcytidine triphosphate (2'CmeC TP) and "D" is compound 1 Figures 4-6 demonstrate that the viral concentration increased by almost two orders of magnitude when treated with only DMSO or 2'CmeC TP. However, the viral concentration increased by less than an order of magnitude in the presence of compound 1.

Example 6 - Effective concentration of compounds of the disclosure and analogs thereof

Table 7 below shows the EC ⁵⁰ and CC ⁵⁰ values of some compounds of the disclosure, as well as analogs thereof for murine norovirus. In cases where the compounds were tested, but did not measure the value of EC ^50, EC ⁵⁰ value is given as N / A.

Table 7. l r E r m f rm l II

Table 8 - l r E r m l i l i n n l l mi m

Example 7 - Synthesis of compound 1

Step 1 (protocol # 1): 4-amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile (3.00 kg), (3R, 4R, 5R) -2-acetoxy-5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl dibenzoate (6.60 kg) and D CE (18.89 kg). Stirring was started and DBU (3.61 kg) was added. Over a period of 03 h and 14 min, TMSOTf (8.01 kg) was added between 30.6 ° C and 37.3 ° C. The CPI after 01 h and 30 min at approx. 32 ° C showed 4% 4-amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile (3.00 kg), dibenzoate (3R, 4R, 5R ) -2-acetoxy-5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl remaining. The CPI after 03h and 16 min at approx. 32 ° C showed 2% 4-amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile (3.00 kg), dibenzoate (3R, 4R, 5R ) -2-acetoxy-5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl remaining (spec: <3%). The reaction mixture was diluted with DCM (39.81 kg) and quenched with potable water (15.02 kg) for a period of 11 min between 9.5 ° C and 15.6 ° C. The extractive pretreatment (at approx. 22 ° C) was completed by a back extraction of the aqueous phase with DCM (19.90 kg), a wash with sat. (1.3 kg of NaHCO3 in 14.9 kg of drinking water), a back extraction of the bicarbonate phase with DCM (19.71 kg) and a brine wash (4.5 kg of NaCl in 14.9 kg of drinking water). Note: The reactor was cleaned with potable water, acetone and DCM after each wash / back extraction.

The drum stirred organic phase containing the product was charged into the 100 L jacketed reactor through an in-line filter followed by a DCM rinse of the drum and the filter with DCM (2.48 kg). The reactor contents were distilled at 31 L with the aid of vacuum for a period of 06 h and 04 min with a maximum temperature of 50.1 ° C. At this point a thick suspension had formed. Then, over a period of 39 min, IPAc (41.88 kg) was added between 44.5 ° C and 49.5 ° C and the reactor contents were heated to 76.9 ° C over a period of 01 h and 25 min. The reactor contents were then cooled to 9.9 ° C over a period of 04 h and 21 min and stirred for 12 h and 26 min with a minimum temperature of 1.6 ° C.

Step 1 (Protocol # 2): 4-amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile (3.00 kg), (3R, 4R, 5R) -2-acetoxy-5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl dibenzoate (6.60 kg) and D CE (18.80 kg). Stirring was started and DBU (3.59 kg) was added. Over a period of 01 h and 46 min, TMSOTf (7.90 kg) was added between 30.4 ° C and 34.2 ° C. The CPI after 02 h and 49 min at approx. 34 ° C showed 1% of 4-amino-6-bromo-2-methyl-7H-pyrrolo [2,3-d] pyrimidine-5-carbonitrile remaining (spec: <3%). The reaction mixture was diluted with DCM (40/70 kg) and quenched with potable water (14.97 kg) for a period of 04 min between 9.9 ° C and 18.0 ° C. The extractive pretreatment (at approx. 22 ° C) was completed by a back extraction of the aqueous phase with DCM (20.34 kg), a wash with sat. NaHCO3. (1.30 kg of NaHCO3 in 14.90 kg of drinking water), a back extraction of the bicarbonate phase with DCM (20.65 kg) and a brine wash (4.50 kg of NaCl in 14.96 kg of drinking water). Note: The reactor was cleaned with potable water, acetone and DCM after each wash / back extraction.

The drum stirred organic phase containing the product was charged into the 100 L jacketed reactor through an in-line filter followed by a DCM rinse of the drum and the filter with DCM (1.49 kg). The contents of the reactor were distilled with the aid of vacuum for a period of 04 h and 49 min with a maximum temperature of 45.6 ° C. At this point a thick suspension had formed. Then, over a period of 27 min, IPAc (41.70 kg) was added between 45.6 ° C and 48.2 ° C and the reactor contents were heated to 75.7 ° C over a period of 01 h and 20 min. The reactor contents were then cooled to 9.4 ° C over a period of 04 h and 15 min and stirred overnight with a minimum temperature of 2.3 ° C.

Stage 2: (2R, 3R, 4R, 5R) -2- (4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidine dibenzoate -7-yl) -5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl (10.0 kg), 10% Pd on C (Degussa, type E101NE / W), trimethylamine (7.3 kg) and THF (44.5 kg). Hydrogen was sent to the reactor and the mixture was stirred for 03 hr and 54 min between 24.7 ° C and 19.6 ° C at approx. 212.35 kPa (30.8 psig). IPC (HPLC) showed that (2R, 3R, 4R, 5R) -2- (4-amino-6-bromo-5-cyano-2-methyl-7H-pyrrolo [2,3- d] pyrimidin-7-yl) -5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl.

The reaction mixture was filtered over Celite (7.2 kg) and a filter polish and the filter residue was washed with THF (5.2 kg). The combined filtrate and wash were transferred to a 100 L jacketed reactor with the aid of a THF wash (2.12 kg). The reactor contents were vacuum distilled with a maximum batch temperature of 30.0 ° C for a period of 05 h and 38 min to a final volume of 27 L. IPA (31.48 kg) was charged over a 40 min period to the reactor between 39.7 ° C and 53.2 ° C. The reactor contents were vacuum distilled with a maximum batch temperature of 53.2 ° C for a period of 03 h and 02 min to a final volume of 33 l. IPA (48.99 kg) was charged over a 43 min period to the reactor between 53.1 ° C and 57.1 ° C. The reactor contents were heated to 60.2 ° C, stirred for 12 min and cooled over a period of 04 and 28 min to 5.4 ° C. Cold stirring was continued for a period of 08 hr and 55 min with a minimum temperature of 1.1 ° C. The suspension was filtered and washed with IPA (9.41 kg, ca 4.5 ° C). The residue was dried under vacuum with a nitrogen purge for a period of 11 hr and 44 min at a maximum temperature of 44.0 ° C to provide a LOD of 0.36%. Yield: 6.58 kg (73.9%). 1H NMR confirms the structure. Purity: 97.78% (HPLC, ABC).

Stage 3:

Materials PM Equiv./vol Quantity mmol Lot no.

1100 g of NaOH dissolved in drinking water to a total volume of 1 l; 2500 ml diluted

of conc. in 2 l in total with drinking water

A dibenzoate solution of (2R, 3R, 4R, 5R) -2- (4-amino-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -5- ( (benzoyloxy) methyl) tetrahydrofuran-3,4-diyl and THF was heated to 54 ° C and the addition of 2.5 M NaOH was started. The initial addition gave a biphasic mixture and endothermic response (the temperature dropped to 50 ° C ), but as the addition continued a clear single phase solution formed which was accompanied by a rapid exotherm to 61 ° C; the reaction temperature was maintained at 60 ° C to 61 ° C for the remainder of the addition and for an additional 2 h. IPC showed that no (2R, 3R, 4R, 5R) -2- (4-amino-5-cyano-2-methyl-7H-pyrrolo [2,3-d] pyrimidin-7-yl) - dibenzoate remained 5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyl.

The reaction mixture was cooled to 21 ° C and neutralized with 3N HCl with external cooling to pH = 7.06 (Denver Instrument UB-10 pehachimeter equipped with a Sartorius P-P11 pH electrode, the electrode was checked with solutions buffers of pH = 4.00 and pH = 7.00); the mixture continued to cool to 8 ° C. The resulting neutralized mixture was vacuum distilled at a pot temperature of 45 ° C to 50 ° C until solids were observed in the pot. The suspension was cooled and stirred for 2 h at 2 ° C. The beige suspension was filtered to produce a dark filtrate; the off-white residue was washed once with cold water (500 ml, 5 ° C). A first LOD after 16 h gave a value of 18.73%. HPLC of the drying material showed the presence of 1.6% benzoate.

A short review study was run for Compound 1 (containing 1.6% benzoic acid by ABC, HPLC) in 10 vol of water (1 g in 10 ml):

3 h suspension at room temperature

3 h suspension at 50 ° C

24 h suspension at room temperature

All three experiments gave compound 1 with less than 0.1% benzoic acid (ABC, HPLC). The suspensions were flowable, easily shaken, and filtration was rapid. Brief drying on the filter gave a powdery solid indicating that a displacement wash with an organic solvent is not required. Without wishing to be bound by theory, an NMT loss of less than 1% (1 mg / ml solubility) is expected. HPLC data for compound 1 was obtained with a method suitable for polar compounds using a Zorbax Eclipse Plus C18 column (water / ACN / TFA, 97.5 / 2.5 / 0.05). This is the same column used for stages 1 and 2.

The cold product suspension was filtered and the reactor and residue were washed with cold IPAc (ca 7.5 ° C, 13.16 kg and 13.62 kg) until a colorless filtrate had been obtained. The residue was dried under vacuum and a nitrogen purge <45 ° C for a period of 65 hr and 19 min to a LOD of 0%. Yield: 5.87 kg (70.7%), 1H NMR confirmed the identity; purity by Hp Lc of 98.84% (ABC).

The disclosure can be embodied in other specific forms without departing from the essential characteristics of the same. The above embodiments, therefore, are to be considered in all respects illustrative rather than limiting of the disclosure described herein. The scope of the disclosure, therefore, is indicated by the appended claims rather than by the foregoing description.

Claims (18)

1. A compound of formula IB:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, wherein: A is:

X ¹ is -CR ¹¹ R ¹² - or -OCH ² CH ² -, wherein the oxygen atom is distal to the RIB moiety in A; R ¹¹ and R ¹² are independently hydrogen or C ¹ -C ⁴ alkyl, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH, or -NH2; X2 is absent, it is -O-, -C (O) O-, or -OCH2-, wherein the oxygen atom is distal to the RIB moiety in A; each RIB is independently hydrogen, -C ^{1 -C 6} alkyl,

or RIB is an amino acid residue linked via the carbonyl group, wherein the alkyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2; v is 0 or 1; n is 0, 1,2 or 3 and when X ² is -C (O) O-, n is 0; p is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; what is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; Rz is hydrogen, halogen, alkylthio C ¹ -C ⁴ -alkoxy C ¹ -C ⁴ -alkyl C ¹ -C ⁴ -alkenyl , C ² -C ⁴ -alkynyl , C ² -C ^4, aryl, heteroaryl, -C ^{3 -C 8} cycloalkyl, -C ^{4 -C 8} cycloalkenyl or non-aromatic 3- to 5-membered heterocycle, wherein each alkylthio, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, or heterocycle is optionally substituted with one or more of halogen, -OH, -SH, or -NH ² ; Ra, Rb, Rx, and Ry are each independently selected from the group consisting of hydrogen, halogen, -OH, -SH, -C ^{1 -C 6} alkoxy, aryloxy, -C ^{1 -C 6} alkylthio, arylthio, -OC ( O) C ¹ -C ^6, -OC (O) aryl, -C ¹ -C ⁶ -alkenyl , C ² -C ⁶ -alkynyl , C ² -C ^6, aryl, heteroaryl, cycloalkyl C ³ -C ⁸ and -C ^{4 -C 8} -cycloalkenyl, wherein each alkoxy, aryloxy, alkylthio, arylthio, alkyl, aryl, alkenyl, alkynyl, heteroaryl, cycloalkyl, or cycloalkenyl is optionally substituted with one or more of halogen, -OH, -SH or -NH2; or any two Ra or Rb, together with the atom to which they are both attached, may combine to form a C ³ -C ⁸ spirocycloalkyl or 3- to 8-membered spiroheterocycle; or any two Ra or Rb, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond; or any two Ra or Rb, when on adjacent atoms, can combine to form an aryl, heteroaryl, -C ^{3 -C 10} cycloalkyl, -C ^{4 -C 10} cycloalkenyl or 5 to 10 membered ring heterocycle; or any CRaRb can be replaced by -O-, -S-, -S (O) - or -SO ² -; or two Rx or Ry any, together with the atom to which they are attached, can combine to form a C ³ -C ⁸ -espirocicloalquilo or espiroheterociclo 3 to 8 members; or any two Rx or Ry, when on adjacent atoms, can combine to form a carbon-carbon double bond or a cis or trans carbon-carbon triple bond; or any two Rx or Ry, when on adjacent atoms, may combine to form an aryl, heteroaryl, -C ^{3 -C 10} cycloalkyl, -C ^{4 -C 10} cycloalkenyl or 5 to 10 membered ring heterocycle; or any CRxRy can be replaced by -O-, -S-, -S (O) - or -SO ² -; R ¹ and R ⁴⁵ are each independently hydrogen, halogen, -N3, -OH, -NH2, -SH, -C ¹ -C ⁶ -cycloalkyl C ³ -C 6, alkenyl , C ² -C ⁶ -cycloalkenyl C ⁴ -C ^8, alkynyl C ² -C ⁶ -cycloalkynyl C ⁸ -C ¹² -alkoxy or C ¹ -C ⁶ alkylthio ^C1 - ^C6 wherein each alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl , cycloalkynyl, alkoxy, or alkylthio is independently substituted with one or more of halogen, -N3, -OH, -NH2, or -SH; R ^2, R ^3, R ⁴ and R ⁴⁴ are each independently hydrogen, halogen, -N ^3, -OH, -NH ^2, -SH, -C ¹ -C ⁶ -alkoxy or C ¹ -C 6 -alkylthio C ¹ -C ⁶ , wherein each alkyl, alkoxy, or alkylthio is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH; or R ³ and one of R ⁴ and R ⁴⁴ , together with the atoms to which they are attached can form a carbon-carbon double bond; R ⁵ is independently hydrogen, -RIB, M +, aryl, aralkyl, -C ¹ -C ⁶ -heteroalquilo C ¹ -C ⁶ cycloalkyl, heteroaryl or nonaromatic heterocyclic ring, wherein M + is a cation and the that each aryl, aralkyl, alkyl, heteroalkyl, cycloalkyl, heterocycle, or heteroaryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH, and wherein R ⁵ is not an amino acid; Y Rc is -C ¹ -C ⁶ -cycloalkyl , C ³ -C ⁶ -alkenyl , C ² -C ⁶ -cycloalkenyl C ⁴ -C ^8, alkynyl C ² -C ⁶ -cicloalqui or aryl, wherein Each alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aryl is optionally substituted with one or more of halogen, -N3, -OH, -NH2, or -SH.

3. The compound of claim 1, wherein A is

4. The compound of claim 1 or 2, wherein A is

5. The compound of claim 1 or 2, wherein A is

6. The compound of claim 1,2 or 4, wherein A is

7. The compound of any preceding claim, wherein: (a) Ri is -H; me (b) R ² is -OH; me (c) R ⁴ is -OH; me (d) R ³ is -H; me (e) R ⁴⁴ is -H; me (f) RIB is -H; me (g) Rc is -CH ³ ; me (h) v is 1, X ² is -O-, n is 0, and RIB is -H; me (i) R ⁵ is -H or M +, where M + is Na +,

, Mg2 + or NRgRdReRf +, and wherein Rg, independently hydrogen or -C ^{1 -C 5} alkyl. 8. The compound of any preceding claim, wherein RIB is: -H;

Ċ

9. The compound of any of the preceding claims, wherein the compound is of formula l-a:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or wherein the compound is of formula l-b:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or wherein the compound is of formula l-c:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or wherein the compound is of formula l-d:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. 10. The compound of any preceding claim, wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. 11. A compound of formula II:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, wherein: Y is -C O) -, or

wherein X 1 is independently O, NH or S, X 2 is independently a bond, -O-, -S- or -NH-, and X 3 is independently -OR, -NHR or -SR; each R is independently -H, C ¹ -C ^20, alkenyl C ² -C ²⁰ -alkynyl , C ² -C ^{2 0,} -cycloalkyl , C ³ -C ⁸ cycloalkenyl C ⁴ -C ^8, aryl, heteroaryl or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, R1, -O R1, -NR 1R2, -SR 1, - OC (O) R1, -C (O) OR1, -NHC (O) OR1 or -NHC (O) R1; Ra and Rb are each independently at each occurrence -H, C ¹ -C ^20, alkenyl C ² -C ²⁰ -alkynyl , C ² -C ²⁰ alkynyl , C ³ -C ⁸ -cycloalkenyl C ⁴ -C ⁸ , aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, -OR 1, -NR 1R2 , -SR 1, -OC (O) R1, -C (O) OR1, -NHC (O) OR1 or -NHC (O) R1; R1 and R2 are each independently at each occurrence -H, C ¹ -C ^20, alkenyl C ² -C ²⁰ -alkynyl , C ² -C ²⁰ alkynyl , C ³ -C ⁸ -cycloalkenyl C ⁴ -C ⁸ , aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, -R 3, -R 4 , -O R3, -N R3R4, -SR 3, -OC (O) R3, -C (O) OR3, -NHC (O) OR3 or -NHC (O) R3; R3 and R4 are each independently at each occurrence -H, C ¹ -C ^20, alkenyl C ² -C ²⁰ -alkynyl , C ² -C ²⁰ alkynyl , C ³ -C ⁸ -cycloalkenyl C ⁴ -C ⁸ , aryl, heteroaryl, or heterocyclyl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl is optionally substituted with one or more of halogen, oxo, aryl, heteroaryl, -OH, -NH ² , -SH, -OC (O) H, -C (O) OH, -NHC (O) OH or -NHC (O) H; Rd is independently -H or -D; Y n is independently 0, 1,2 or 3. 12. The compound of claim 11, wherein: (a) Ra is -H; me (b) Rb is -H; me (c) Rd is -H; me (d) n is 0; me (e) Ra, Rb and Rd are -H; me (f) Ra, Rb, and Rd are -H and n is 0. The compound of any of claims 11-12, wherein the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof; or in which the compound is:

or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate, or mixture thereof. 14. A pharmaceutical composition comprising a compound of any preceding claim, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereoisomer, racemate or mixture thereof, and a pharmaceutically acceptable carrier. 15. A compound of any one of claims 1-13, for use in treating a disease. 16. A compound of any one of claims 1-13 or a pharmaceutical composition of claim 14, for use in treating a viral infection or a disease or disorder associated with viral infection. 17. A compound of any one of claims 1-13 or a composition of claim 14, for use in the prevention of a viral infection or a disease or disorder associated with viral infection. 18. The compound or pharmaceutical composition for use of claim 16 or claim 17, wherein the viral infection is norovirus infection.