# Patent

## Title
Influenza A virus inhibition

## Abstract

Provided are compounds that are capable of modulating the activity of the influenza A virus via interaction with the M2 transmembrane protein. Also provided are methods for treating an influenza A-affected disease state or infection comprising administering a composition comprising one or more compounds that have been identified as being capable of interaction with the M2 protein.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application No. PCT/US2009/054608, filed Aug. 21, 2009, which claims the benefit of U.S. Provisional Application No. 61/097,474, filed Sep. 16, 2008, the disclosures of which are incorporated herein by reference in their entireties.


STATEMENT OF GOVERNMENT RIGHTS
Research leading to the disclosed invention was funded in part by the U.S. National Institutes of Health, grant numbers GM56423 (William F. DeGrado) and AI 74571 (William F. DeGrado). Accordingly, the United States Government may have certain rights in the invention described herein.


TECHNICAL FIELD
The present invention pertains to, among other things, compounds and methods for modulating the activity of the influenza virus.
BACKGROUND
The M2 protein is found in the viral envelope of influenza A virus and functions as a highly selective, pH-regulated proton channel important for the life cycle of the virus. Unlike neuraminidase inhibitors, rimantadine and amantadine are anti-viral agents capable of blocking the tetrameric M2 channel. In 2006, the CDC issued an alert instructing clinicians to avoid using M2 ion-channel inhibitors during influenza season due to the extraordinarily high frequency of amantadine resistance in influenza A isolates associated with a single point mutation in the M2 protein, S31N (Hayden F. G., Antiviral Resistance in Influenza Viruses—Implications for Management and Pandemic Response, N Enj J Med, 2006, 354; 8). The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Grambas, S., Bennett, M S. & Hay, A. J. Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Virology 191, 541-549 (1992); Bright, R. A., Shay, D. K., Shu, B., Cox, N. J. & Klimov, A. I. Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States. J. Am. Med. Assoc. 295, 891-894 (2006). Recently, it has been reported that resistance to rimantadine and amantadine in humans, birds and pigs has reached more than 90%, casting into doubt the continued ability of these drugs alone to satisfy the need for treatment of influenza (Deyde, V. M. et al. Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J. Infect. Dis. 196, 249-257 (2007)).
Previous studies have suggested that BL-1743 (3-(4,5-Dihydro-1H-imidazol-2-yl)-3-aza-spiro[5.5]undecane) interacts differently with the M2 proton channel as compared with amantadine, but have found that the majority of isolated influenza viruses that are amantadine-resistant are also resistant to BL-1743. Tu Q, et al., Characterization of inhibition of M2 ion channel activity by BL-1743, an inhibitor of influenza A virus, J. Virol. 1996 July; 70(7):4246-52. For example, Tu Q, et al. found that mutations known to confer amantadine resistance at M2 residues 27, 30, 31, and 34, all within the M2 transmembrane domain, also induce “complete” resistance to BL-1743. Id. The publication by Tu Q, et al. concluded that “the overlapping spectra of amantadine and BL-1743 resistance mutations and the higher apparent Ki . . . do not indicate that BL-1743 should replace the use of amantadine (or rimantadine) for the prophylaxis or treatment of influenza virus infections in humans.” Id. See also Kurtz, et al., Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus. Antimicrob Agents Chemother. 1995 October; 39(10):2204-9 (“BL-1743 does not produce an additive effect on M2 inhibition, suggesting that these two compounds interact with similar sites in the M2 protein . . . . Thus, BL-1743 appears to represent a novel structure with an antiviral profile similar to that of amantadine.”).
SUMMARY
In one aspect of the present invention, provided are compounds having the formula (I):




wherein
A is carbon or nitrogen;
a1 and a3 are each independently (CH2)1-2, C═O, or —(CH2)C(═O)—;
a2 and a4 are each independently (CH2)1-2 or S;
R1 is amino, imidazolyl, —NH(R4), or —CH(OH)(R5);
R2 and R3 are each independently C1-C3 alkyl,
or,
R2 and R3 taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring;
R4 is (CH2)1-3NHR6, amino, formamidinyl, aryl, or aralkyl;
R5 is —(R7)imidazolyl;
R6 is hydrogen, aryl, or aralkyl;
R7 is alkyl or aralkyl;
Y is hydrogen, or Y and R1 taken together along with the atoms to which they are respectively attached form a 3- to 6-membered carbocyclic or heterocyclic ring optionally substituted with up to three substituents independently selected from alkyl, aryl, aralkyl, hydroxyl, nitro, amino, and carbonyl; and,
Z is hydrogen or halo;
or a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.
In other aspects, provided are methods treating an influenza A virus-affected disease state or infection comprising the step of administering to a subject in need thereof a composition comprising a compound of formula (I) as described above.


DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention.
The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.
As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings
In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a certain chemical moiety “may be” X, Y, or Z, it is not intended by such usage to exclude other choices for the moiety; for example, a statement to the effect that R1 “may be alkyl, aryl, or amino” does not exclude other choices for R1, such as halo, aralkyl, and the like.
When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” In another example, when a listing of possible substituents including “hydrogen, alkyl, and aryl” is provided, the recited listing may be construed as including situations whereby any of “hydrogen, alkyl, and aryl” is negatively excluded; thus, a recitation of “hydrogen, alkyl, and aryl” may be construed as “hydrogen and aryl, but not alkyl”, or simply “wherein the substituent is not alkyl”.
Protective groups are abbreviated according to the system disclosed in Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons, 1991, which is incorporated in its entirety herein. For example, “CBZ” or “Cbz” or “Z” stands for carbobenzyloxy or benzyloxycarbonyl, “Boc” or “BOC” represents t-butoxycarbonyl, “Alloc” denotes allyloxycarbonyl, Bz means benzoyl, and “Fmoc” stands for 9-fluorenylmethoxycarbonyl.
As used herein, the terms “component,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
The abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: “min” means minute(s), “g” means gram(s), “mg” means milligram(s), “μg” means microgram(s), “eq” means equivalent(s), “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “mM” means millimolar, “M” means molar, “mmol” or “mmole” means millimole(s), “cm” means centimeters, “SEM” means standard error of the mean, and “IU” means International Units. “IC50 value” or “IC50” means dose of the compound which results in 50% alleviation or inhibition of the observed condition or effect.
As used herein, “alkyl” refers to an optionally substituted, saturated straight, or branched, hydrocarbon radical having from about 1 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein). Where appropriate, “alkyl” can mean “alkylene”; for example, if X is —R1R2, and R1 is said to be “alkyl”, then “alkyl” may correctly be interpreted to mean “alkylene”.
“Amino” refers to —NH2 and may include one or more substituents that replace hydrogen.
As used herein, “aryl”, “arene”, and “aromatic” each refer to an optionally substituted, saturated or unsaturated, monocyclic, polycyclic, or other homo- or heterocyclic aromatic ring system having from about 3 to about 50 ring members (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 5 to about 10 ring atom members being preferred. Such moieties encompass (include) “heteroaryl” and “heteroarene” as defined infra. Where appropriate, “aryl” can mean “arene”; for example, if X is —R1R2, and R1 is said to be “aryl”, then “aryl” may correctly be interpreted to mean “arene”.
As used herein, “alkenyl” refers to an alkyl radical having from about 2 to about 20 carbon atoms and one or more double bonds (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), wherein alkyl is as previously defined. In some embodiments, it is preferred that the alkenyl groups have from about 2 to about 6 carbon atoms. Alkenyl groups may be optionally substituted.
As used herein, “aralkyl” refers to alkyl radicals bearing one or more aryl substituents and having from about 4 to about 50 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), wherein aryl and alkyl are as previously defined. In some preferred embodiments, the alkyl moieties of the aralkyl groups have from about 1 to about 4 carbon atoms. In other preferred embodiments, the alkyl moieties have from about 1 to about 3 carbon atoms. Aralkyl groups may be optionally substituted.
“Alkylamino” signifies alkyl-(NH)—, wherein alkyl is as previously described and NH is defined in accordance with the provided definition of amino. “Arylamino” represents aryl-(NH)—, wherein aryl is as defined herein and NH is defined in accordance with the provided definition of amino. Likewise, “aralkylamino” is used to denote aralkyl-(NH)—, wherein aralkyl is as previously defined and NH is defined in accordance with the provided definition of amino. “Alkylamido” refers to alkyl-CH(═O)NH—, wherein alkyl is as previously described. “Alkoxy” as used herein refers to the group R—O— where R is an alkyl group, and alkyl is as previously described. “Aralkoxy” stands for R—O—, wherein R is an aralkyl group as previously defined. “Alkylsulfonyl” means alkyl-SO2—, wherein alkyl is as previously defined.
As used herein, “alkylene” refers to an optionally branched or substituted bivalent alkyl radical having the general formula —(CH2)n—, where n is 1 to 10. Non-limiting examples include methylene, trimethylene, pentamethylene, and hexamethylene.
As used herein, “heteroaryl” or “heteroarene” refers to an aryl radical wherein in at least one of the rings, one or more of the carbon atom ring members is independently replaced by a heteroatom group selected from the group consisting of S, O, N, and NH, wherein aryl is as previously defined. Heteroaryl/heteroarene groups having a total of from about 3 to about 14 carbon atom ring members and heteroatom ring members are preferred. Likewise, a “heterocyclic ring” is an aryl radical wherein one or more of the carbon atom ring members may be (but are not necessarily) independently replaced by a heteroatom group selected from the group consisting of S, O, N, and NH. Heterocyclic rings having a total from about 3 to 14 ring members and heteroatom ring members are preferred, but not necessarily present; for example, “heterocyclohexyl” may be a six-membered aryl radical with or without a heteroatom group.
“Halo” and “halogen” each refers to a fluoro, chloro, bromo, or iodo moiety, with fluoro, chloro, or bromo being preferred.
“Haloalkyl” signifies halo-alkyl- wherein alkyl and halo, respectively, are as previously described.
The phrase reading “[moiety] is absent” means that the substituents to which the moiety is attached may be directly attached to each other.
Typically, substituted chemical moieties include one or more substituents that replace hydrogen. Exemplary substituents include, for example, halo (e.g., F, Cl, Br, I), alkyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aralkyl, aryl, heteroaryl, heteroaralkyl, spiroalkyl, heterocycloalkyl, hydroxyl (—OH), nitro (—NO2), cyano (—CN), amino (—NH2), —N-substituted amino (—NHR′′), —N,N-disubstituted amino (—N(R′′)R′′), oxo (═O), carboxy (—COOH), —O—C(═O)R′′, —C(═O)R′′, —OR′′, —C(═O)OR′′, -(alkylene)-C(═O)—OR′′, —NHC(═O)R′′, aminocarbonyl (—C(═O)NH2), —N-substituted aminocarbonyl (—C(═O)NHR′′), —N,N-disubstituted aminocarbonyl (—C(═O)N(R′′)R′′), thiol, thiolato (—SR′′), sulfonic acid (—SO3H), phosphonic acid (—PO3H), —P(═O)(OR′′)OR′′, —S(═O)R′′, —S(═O)2R′′, —S(═O)2NH2, —S(═O)2NHR′′, —S(═O)2NR′′R′′, —NHS(═O)2R′′, —NR′′S(═O)2R′′, —CF3, —CF2CF3, —NHC(═O)NHR′′, —NHC(═O)NR′′R′′, —NR′′C(═O)NHR′′, —NR′′C(═O)NR′′R′′, —NR′′C(═O)R′′ and the like. In relation to the aforementioned substituents, each moiety R′′ can be, independently, any of H, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, heteroaryl, or heterocycloalkyl, for example.
As used herein, the terms “treatment” or “therapy” (as well as different word forms thereof) includes preventative (e.g., prophylactic), curative or palliative treatment.
As employed above and throughout the disclosure the term “effective amount” refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired result with respect to the treatment of the relevant disorder, condition, or side effect. It will be appreciated that the effective amount of components of the present invention will vary from patient to patient not only with the particular compound, component or composition selected, the route of administration, and the ability of the components to elicit a desired response in the individual, but also with factors such as the disease state or severity of the condition to be alleviated, hormone levels, age, sex, weight of the individual, the state of being of the patient, and the severity of the pathological condition being treated, concurrent medication or special diets then being followed by the particular patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. Dosage regimens may be adjusted to provide the improved therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the components are outweighed by the therapeutically beneficial effects. As an example, the compounds useful in the methods of the present invention are administered at a dosage and for a time such that the level of activation and adhesion activity of platelets is reduced as compared to the level of activity before the start of treatment.
“Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
Within the present invention, the disclosed compounds may be prepared in the form of pharmaceutically acceptable salts. “Pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein 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 acidic residues such as carboxylic acids; and the like. The 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 those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. These physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.
Compounds described herein throughout, can be used or prepared in alternate forms. For example, many amino-containing compounds can be used or prepared as an acid addition salt. Often such salts improve isolation and handling properties of the compound. For example, depending on the reagents, reaction conditions and the like, compounds as described herein can be used or prepared, for example, as their hydrochloride or tosylate salts. Isomorphic crystalline forms, all chiral and racemic forms, N-oxide, hydrates, solvates, and acid salt hydrates, are also contemplated to be within the scope of the present invention.
Certain acidic or basic compounds of the present invention may exist as zwitterions. All forms of the compounds, including free acid, free base and zwitterions, are contemplated to be within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxy groups often exist in equilibrium with their zwitterionic forms. Thus, any of the compounds described herein throughout that contain, for example, both amino and carboxy groups, also include reference to their corresponding zwitterions.
“Hydrate” refers to a compound of the present invention which is associated with water in the molecular form, i.e., in which the H—OH bond is not split, and may be represented, for example, by the formula R.H2O, where R is a compound of the invention. A given compound may form more than one hydrate including, for example, monohydrates (R.H2O) or polyhydrates (R.nH2O wherein n is an integer >1) including, for example, dihydrates (R.2H2O), trihydrates (R.3H2O), and the like, or hemihydrates, such as, for example, R.n/2H2O, R.n/3H2O, R.n/4H2O and the like wherein n is an integer.
“Solvate” refers to a compound of the present invention which is associated with solvent in the molecular form, i.e., in which the solvent is coordinatively bound, and may be represented, for example, by the formula R.(solvent), where R is a compound of the invention. A given compound may form more than one solvate including, for example, monosolvates (R.(solvent)) or polysolvates (R.n(solvent)) wherein n is an integer >1) including, for example, disolvates (R.2(solvent)), trisolvates (R.3(solvent)), and the like, or hemisolvates, such as, for example, R.n/2(solvent), R.n/3(solvent), R.n/4(solvent) and the like wherein n is an integer. Solvents herein include mixed solvents, for example, methanol/water, and as such, the solvates may incorporate one or more solvents within the solvate.
“Acid hydrate” refers to a complex that may be formed through association of a compound having one or more base moieties with at least one compound having one or more acid moieties or through association of a compound having one or more acid moieties with at least one compound having one or more base moieties, said complex being further associated with water molecules so as to form a hydrate, wherein said hydrate is as previously defined and R represents the complex herein described above.
The term “stereoisomers” refers to compounds that have identical chemical constitution, but differ as regards the arrangement of the atoms or groups in space.
“Racemic” means having the capacity for resolution into forms of opposed optical activity.
As used herein, the term “partial stereoisomer” refers to stereoisomers having two or more chiral centers wherein at least one of the chiral centers has defined stereochemistry (i.e., R or S) and at least one has undefined stereochemistry (i.e., R or S). When the term “partial stereoisomers thereof” is used herein, it refers to any compound within the described genus whose configuration at chiral centers with defined stereochemistry centers is maintained and the configuration of each undefined chiral center is independently selected from R or S. For example, if a stereoisomer has three chiral centers and the stereochemical configuration of the first center is defined as having “S” stereochemistry, the term “or partial stereoisomer thereof” refers to stereoisomers having SRR, SRS, SSR, or SSS configurations at the three chiral centers, and mixtures thereof.
“Prodrug” refers to compounds which are themselves inactive or minimally active for the activity desired, but through biotransformation can be converted into biologically active metabolites. For example, a prodrug of the present invention would include, inter alia, any compound which is convertible in vivo by metabolic means to a compound claimed or described in the present disclosure.
“N-oxide” refers to compounds wherein the basic nitrogen atom of either a heteroaromatic ring or tertiary amine is oxidized to give a quaternary nitrogen bearing a positive formal charge and an attached oxygen atom bearing a negative formal charge.
When any variable occurs more than one time in any constituent or in any formula, its definition in each occurrence is independent of its definition at every other occurrence. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
The term “administering” means either directly administering a compound or composition of the present invention, or administering a prodrug, derivative or analog which will form an equivalent amount of the active compound or substance within the body.
“Dosage unit” refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit may contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention may be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).
“Subject” or “patient” refers to an embryonic, immature, or adult animal, including the human species, that is treatable with the compositions, and/or methods of the present invention.
Accordingly, in one aspect there are provided compounds having the formula (I):




wherein
A is carbon or nitrogen;
a1 and a3 are each independently (CH2)1-2, C═O, or —(CH2)C(═O)—;
a2 and a4 are each independently (CH2)1-2 or S;
R1 is amino, imidazolyl, —NH(R4), or —CH(OH)(R5);
R2 and R3 are each independently C1-C3 alkyl,
or,
R2 and R3 taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring;
R4 is (CH2)1-3NHR6, amino, formamidinyl, aryl, or aralkyl;
R5 is —(R7)imidazolyl;
R6 is hydrogen, aryl, or aralkyl;
R7 is alkyl or aralkyl;
Y is hydrogen, or Y and R1 taken together along with the atoms to which they are respectively attached form a 3- to 6-membered carbocyclic or heterocyclic ring optionally substituted with up to three substituents independently selected from alkyl, aryl, aralkyl, hydroxyl, nitro, amino, and carbonyl; and,
Z is hydrogen or halo;
or a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.
In some embodiments of the present compounds, A may be carbon. With respect to such embodiments, Z and Y may both be hydrogen. When Z and Y are both hydrogen, R1 may be —NH(R4) or aryl. In such embodiments, R4 may be formamidinyl or (CH2)1-3NHR6.
In other embodiments, R1 may be amino. With respect to such embodiments, R2 and R3 may be taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring. In other embodiments wherein R1 is amino, A may be nitrogen. In yet other embodiments wherein R1 is amino, a2 and a4 may each be S. In still other embodiments wherein R1 is amino, R2 and R3 may each independently be C1-C3 alkyl.
A may be nitrogen. In some embodiments of this variety, R1 may hydrogen, and R2 and R3 may be taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring. When these conditions apply, a1 and a3 may each independently be (CH2)1-2.
Exemplary compounds according to the present invention include, among others:

    3-Amino-3-aza-spiro[5.5]undecane-2,4-dione;
    3-Aza-spiro[5.5]undec-3-yl-ammonium chloride;
    9-Azonia-spiro[5.6]dodecane chloride;
    N′-Spiro[5.5]undec-3-yl-hydrazinium chloride;
    6,6-Diethyl-4,5,6,7-tetrahydro-benzothiazol-2-ylamine;
    1,5-Dithia-spiro[5.5]undec-3-yl-ammonium chloride;
    1,5-Dithia-spiro[5.6]dodec-3-yl-ammonium chloride;
    N-Spiro[5.5]undec-3-yl-guanidine;
    4,4-dimethyl-cyclohexyl-ammonium chloride;
    4-Ethyl-4-methyl-cyclohexyl-ammonium chloride;
    4-(3-Fluoro-spiro[5.5]undec-3-yl)-1H-imidazole;
    Spiro[5.5]undec-3-yl-(1-tricyclohexylmethyl-3H-imidazol-4-yl)-methanol;
    Spiro[5.5]undec-3-yl-(1H-[1,2,4]triazol-3-yl)-amine;
    N1-Spiro[5.5]undec-3-yl-propane-1,3-diamine;
    N1-Spiro[5.5]undec-3-yl-ethane-1,2-diamine;
    N-(1H-Imidazol-4-ylmethyl)-N′-spiro[5.5]undec-3-yl-ethane-1,2-diamine;


and a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, and N-oxide thereof.
The compounds employed in the present invention may exist in prodrug form. As used herein, “prodrug” is intended to include any covalently bonded carriers which release the active parent drug, for example, as according to the formulas or compounds employed in the methods of the present invention in vivo when such prodrug is administered to a subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) the compounds of the present invention may, if desired, be delivered in prodrug form. Thus, the present invention contemplates methods of delivering prodrugs. Prodrugs of the compounds employed in the present invention, for example, according to formula (I), may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Examples include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.
As will be readily understood, functional groups present may contain protecting groups during the course of synthesis. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention. Protecting groups that may be employed in accordance with the present invention may be described in Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons, 1991.
In a further aspect, the invention relates to pharmaceutical compositions comprising a compound according to formula (I) and a pharmaceutically acceptable carrier, diluent, or excipient. The applicable carrier, diluent, or excipient may be selected on the basis of the chosen route of administration and standard pharmaceutical practice as described, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1985), the disclosure of which is hereby incorporated by reference in its entirety.
Also provided are methods for treating an influenza A virus-affected disease state or infection comprising the step of administering to a subject in need thereof a composition comprising a compound of formula (I):




wherein
A is carbon or nitrogen;
a1 and a3 are each independently (CH2)1-2, C═O, or —(CH2)C(═O)—;
a2 and a4 are each independently (CH2)1-2 or S;
R1 is amino, imidazolyl, —NH(R4), or —CH(OH)(R5);
R2 and R3 are each independently C1-C3 alkyl,
or,
R2 and R3 taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring;
R4 is (CH2)1-3NHR6, amino, formamidinyl, aryl, or aralkyl;
R5 is —(R7)imidazolyl;
R6 is hydrogen, aryl, or aralkyl;
R7 is alkyl or aralkyl;
Y is hydrogen, or Y and R1 taken together along with the atoms to which they are respectively attached form a 3- to 6-membered carbocyclic or heterocyclic ring optionally substituted with up to three substituents independently selected from alkyl, aryl, aralkyl, hydroxyl, nitro, amino, and carbonyl; and,
Z is hydrogen or halo;
or a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.
The influenza A virus-affected disease state or infection may comprise any condition that arises as a direct or indirect result of the presence of influenza A virus. For example, the influenza A virus-affected disease state may comprise influenza (flu), pneumonia, bronchitis, sinus infection, or ear infection, among other conditions. The disease state or infection may arise as a direct or indirect result of the presence of wild-type influenza A virus, or may arise as a direct or indirect result of the presence of a mutant version of the influenza A virus, or may arise as a direct or indirect result of the presence of both a wild-type influenza A virus and a mutant version of the influenza A virus. Thus, in accordance with the present methods, the influenza A virus may be wild-type or may be a mutant virus. The mutant virus may comprise an influenza A virus having the V27G mutation, the V27I mutation, the V27T mutation, the V27S mutation, or the V27A mutation; may comprise an influenza virus having the A30T mutation; may comprise an influenza virus having the S31A mutation or the S31N mutation; may an influenza virus having the G34E mutation or the G34A mutation; may comprise an influenza virus having the W41L mutation or the W41Y mutation; may comprise an influenza virus having the D44N mutation or the D44H mutation; and/or may comprise an influenza virus having the R45K mutation or the R45H mutation.
In some embodiments of the present methods, A may be carbon. With respect to such embodiments, Z and Y may both be hydrogen. When Z and Y are both hydrogen, R1 may be —NH(R4) or aryl. In such embodiments, R4 may be formamidinyl or (CH2)1-3NHR6.
In other embodiments, R1 may be amino. With respect to such embodiments, R2 and R3 may be taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring. In other embodiments wherein R1 is amino, A may be nitrogen. In yet other embodiments wherein R1 is amino, a2 and a4 may each be S. In still other embodiments wherein R1 is amino, R2 and R3 may each independently be C1-C3 alkyl.
A may be nitrogen. In some embodiments of this variety, R1 may hydrogen, and R2 and R3 may be taken together along with the carbon atom to which they are both attached form a 6- to 8-membered optionally substituted ring. When these conditions apply, a1 and a3 may each independently be (CH2)1-2.
Exemplary compounds according to the present methods include, among others:

    3-Amino-3-aza-spiro[5.5]undecane-2,4-dione;
    3-Aza-spiro[5.5]undec-3-yl-ammonium chloride;
    9-Azonia-spiro[5.6]dodecane chloride;
    N′-Spiro[5.5]undec-3-yl-hydrazinium chloride;
    6,6-Diethyl-4,5,6,7-tetrahydro-benzothiazol-2-ylamine;
    1,5-Dithia-spiro[5.5]undec-3-yl-ammonium chloride;
    1,5-Dithia-spiro[5.6]dodec-3-yl-ammonium chloride;
    N-Spiro[5.5]undec-3-yl-guanidine;
    4,4-dimethyl-cyclohexyl-ammonium chloride;
    4-Ethyl-4-methyl-cyclohexyl-ammonium chloride;
    4-(3-Fluoro-spiro[5.5]undec-3-yl)-1H-imidazole;
    Spiro[5.5]undec-3-yl-(1-tricyclohexylmethyl-3H-imidazol-4-yl)-methanol;
    Spiro[5.5]undec-3-yl-(1H-[1,2,4]triazol-3-yl)-amine;
    N1-Spiro[5.5]undec-3-yl-propane-1,3-diamine;
    N1-Spiro[5.5]undec-3-yl-ethane-1,2-diamine;
    N-(1H-Imidazol-4-ylmethyl)-N′-spiro[5.5]undec-3-yl-ethane-1,2-diamine;


and a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, and N-oxide thereof,
or any mixture thereof.
The compounds of this invention may be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing/dispersing agent, tablet-disintegrating agent, encapsulating material, film former or coating, flavors, or printing ink. Of course, any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. Parenteral administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.
In powders, the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the active compound may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound(s) in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained. The therapeutic compositions preferably contain up to about 99% of the active ingredient.
Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators.
Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.
Suitable examples of liquid carriers, diluents and excipients for oral and parenteral administration include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.
For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration. Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the active compounds in the required amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and the freeze drying technique that yields a powder of the active ingredient or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof.
The compounds of the invention may be administered in an effective amount by any of the conventional techniques well-established in the medical field. The compounds employed in the methods of the present invention including, for example, the compounds of formula I or II may be administered by any means that results in the contact of the active agents with the agents' site or sites of action in the body of a patient. The compounds may be administered by any conventional means available.
Preferably the pharmaceutical composition is in unit dosage form, e.g. as tablets, buccal tablets, troches, capsules, elixirs, powders, solutions, suspensions, emulsions, syrups, wafers, granules, suppositories, or the like. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. In addition, dosage forms of the present invention can be in the form of capsules wherein one active ingredient is compressed into a tablet or in the form of a plurality of microtablets, particles, granules or non-perils. These microtablets, particles, granules or non-perils are then placed into a capsule or compressed into a capsule, possibly along with a granulation of the another active ingredient.
The dosage of the compounds of the present invention that will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages may be used initially and, if necessary, increased by small increments until the desired effect under the circumstances is reached. Generally speaking, oral administration may require higher dosages.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The dose may also be provided by controlled release of the compound, by techniques well known to those in the art.
The compounds useful in the methods of the present invention may be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by the methods as described below, or variations thereon as appreciated by the skilled artisan. The reagents used in the preparation of the compounds of this invention can be either commercially obtained or can be prepared by standard procedures described in the literature. All processes disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram or commercial industrial scale.
The present invention is further defined in the following Examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and should not be construed as limiting the appended claims From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
EXAMPLES
All chemicals for use in preparing the inventive compounds were purchased from commercial vendors and used without further purification, unless otherwise noted.
Example 1
Synthesis and Inhibition Activity of Exemplary Influenza A M2 Proton Channel Inhibitors
Synthesis of some embodiments (e.g., compound 10) was accomplished as illustrated in the following generalized schematic and as described below:




The syntheses of the primary amine analog (8) of spiropiperidine-azaspiro[5,5]undecane and the methyl substituted secondary amine 9 are shown in Scheme 1. Intermediate spiro[5.5]undec-1-en-3-one 1 was prepared from both acid catalyzed one-pot Robinson annulation reaction and through Diels-Alder adduct followed by acid hydrolysis and aldol ring formation. The acid-catalyzed annulation often led to low yields (62% or lower) due to acid catalyzed polymerization of methyl vinyl ketone as evidenced by black oily substance formed in the reaction flask. While catalysis with proline derivatives might allow circumvention of these problems, we found the alternative Diels-Alder route provided better overall yields (75%). Hydrogenesis of enone 1 with Pd/C with an H2 balloon gave spiro[5.5]undecan-3-one 2. Conversion of ketone 2 to amine 8 was achieved by treatment with hydroxylamine followed by LiAlH4 reduction. Methylamine 9 was prepared by reductive amination of 8 with formaldehyde as reported.
Syntheses of spiran triazole 11 and spiran amine 12-14 with extended linkers in scheme 2 were accomplished by reductive amination as described before.




Compound 15, with an imidazole head group, was synthesized by nucleophilic attack of imidazol-4-yl anion (generated by treatment of N-trityl 4-iodoimidazole) onto ketone 2, followed by deprotection in TFA/DCM as in scheme 3. The hydroxyl group in 15 was either reduced by Et3SiH/BF3*OEt2 to give 16 or fluorinated by DAST to give 17 after deprotection. Ketone 2 was converted to aldehyde 6 by the Wittig reaction, followed by acid hydrolysis. Compounds 18 and 19 were then synthesized from compound 6 in the same manner as described for 15 and 17.







Molecular Biology, In Vitro cRNA Transcription—
cDNA encoding the Influenza virus A/Udorn/72 A/M2 protein and the A/M2 amantadine insensitive mutants were inserted into pGEMHJ (a gift from N. Dascal Tel-Aviv University, Israel) for the expression on Xenopus oocytes. cRNA was prepared using known procedures.
Heterologous Expression and Electrophysiological Recordings—
Stage V-VI Xenopus laevis oocytes were prepared using known procedures. Oocytes injection and TEVC electrophysiological measurements were done using known procedures. Amantadine (Sigma, St. Louis, Mo.) was applied to inhibit A/M2 induced currents. Data were analyzed using ORIGIN 8.0 software (OriginLab, Northampton, Mass.).
Cells, Viruses and Plasmids—
293-T and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, Calif., Valencia, Calif.) supplemented with 10% FBS. Influenza A/Udorn/72 virus (wt) and mutant viruses were propagated in MDCK cells overlaid with serum-free DMEM containing 3.0 μg/ml N-acetyl trypsin (NAT; Sigma-Aldrich, St. Louis, Mo.) at 37° C. WT and mutant virus (V27A/L38F) were generated by using reverse genetics from cDNAs using known procedures. The eight genome-sense (pHH21) plasmids and four protein-expressing (pcDNA3.1) plasmids used to generate influenza virus by reverse genetics have been described previously. Mutation into the M2 gene in pHH21 vector was generated using Quick Change mutagenesis (Stratagene, La Jolla, Calif.). 293T cells were transfected using TransIT-LT1 (Mirus, Madison, Wis.) according to the manufacturer's protocols. Virus stocks were propagated in MDCK cells, and the virus titers were determined by plaque assay on MDCK cells. For determination of viral genome sequences viral RNA was extracted by using the QIAamp viral RNA kit (Qiagen, Valencia, Calif.), followed by Super Reverse Transcriptase (Molecular Genetic Resources, Tampa, Fla.) and using genome-specific primers, and amplified with AmpliTaq DNA polymerase (Applied Biosystems, Foster City, Calif.). The complete nucleotide sequences of the M genes were determined using a 3100-Avant genetic analyzer (Applied Biosystems).
Plaque Reduction Assays—
Confluent monolayers of MDCK cells were incubated with 10-fold-serially-diluted virus samples in DMEM-1% bovine serum albumin for 1 h at 37° C. The inoculums were removed, and the cells were washed with phosphate-buffered saline (PBS). The cells were then overlaid with DMEM-containing 0.6% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, Pa.) and NAT (1.0 μg/ml). To examine the effect of drug (BL-1743, spiran amines and amantadine) on plaque formation, monolayers were preincubated with DMEM supplemented with drug at 37° C. for 30 min, and virus samples were preincubated with DMEM-1% BSA with drug at 4° C. for 30 min before infection. At 2 to 3 days after infection, the monolayers were fixed and stained with naphthalene black dye solution (0.1% naphthalene black, 6% glacial acetic acid, 1.36% anhydrous sodium acetate).
Structure-Activity Relationship (SAR) of 3-Substituted Spiro-[5,5]-Undecanes.
A previous SAR study of 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (BL-1743) revealed a very potent spiro-piperidine compound with IC50 of 0.9 μM (Wang, J., Cady, S. D., Balannik, V., Pinto, L. H., Degrado, Wf, and Hong, M. (2009) Discovery of Spiro-Piperidine Inhibitors and Their Modulation of the Dynamics of the M2 Proton Channel from Influenza A Virus, J Am Chem. Soc. 2009, 131, 8066-8076). However, compounds in this series failed to inhibit amantadine-resistant variants of A/M2, which prompted the testing alternative structures. In particular, a consideration of the overlay of the parent compound on derivatives of amantadine suggested that conversion of the piperidine in spiro piperidine 20 to a 3-amino-cyclohexyl amine, while maintaining the second spiro-6-member ring. A family of spiro-[5,5]-undecanes was synthesized, in which the 3-position was substituted with amines or other substituents.
The compounds were tested on A/M2 channels expressed in Xenopus oocytes using TEVC technique. The inhibitory effect of the compounds was confirmed by in-vivo plaque reduction assays of influenza A virus (A/Udorn/72). The simple amino-derivative, 8, showed an activity on par with that of amantadine. However, further substitutions to the amine tended to cause a loss in activity. N-methylation led to a slightly less potent compound, while the guanidine derivative, 10, had similar potency as methylamine 9. Modifying the amine by addition of additional polar substituents and extended linkers (compounds 11-14) led to a marked decrease in activity. On the other hand, replacing the amine with an imidazole group caused a slight decrease in activity (compound 16). Additional substitutions at the 3-methylene of 16 were also examined, through the introduction of a hydroxyl and fluoro-substituent in 15 and 17, respectively. These substitutions gave rise to compounds with lower potency. Furthermore, the similarly substituted compounds 18 and 19 had decreased activity compared to the primary amine 8.
Table 1, below depicts the inhibition efficiency of certain synthesized compounds on A/M2 channels. aKd was obtained by global fitting of Circular Dichroism (CD) data of ligand titration to A/M2TM (22-46) using Igor Pro (wavemetrics). The variation of Kd values is ±25% based on different fitting values obtained from three repeats of Amantadine titration and two repeats of compound #20 titration.







TABLE 1













 















AM2 channel activity after




Compound
R1
R2
100 μM compound inhibition
IC50 (μM)
Kd (μM)a





Amantadine


 6%
15.76 ± 1.24
 15.17


BL-1743


 25%
45.31 ± 3.92
193.54


 8
H
NH3+Cl−
 11%
12.59 ± 1.11
 9.16


 9
H
NH2+Cl−CH3
 8%
15.72 ± 1.89
 46.36


 


10
H




 8%
14.60 ± 1.70
 11.50


 


11
H




 25%
n.d



 


12
H
NH2+Cl−(CH3)2NH3+Cl−
100%
n.d



13
H
NH2+Cl−(CH3)3NH3+Cl−
100%
n.d
>500


 


14
H




 91%
n.d



 


15
OH




 40%
n.d



 


16
H




 13%
12.54 ± 1.24



 


17
F




 31%
57.57 ± 2.24



 


18
H




 30%
n.d



 


19
H




 25%
29.19 ± 1.46



 


20


 5%
 0.92 ± 0.11
 12.87









In summary, these data show that the primary amino group is likely to be a nearly optimal substituent for the spiro-[5.5]-undecane scaffold. It was then examined how the piperidine for cyclohexylamine substitution in 20 vs. 8 affected the ability of these compounds to inhibit amantadine-resistant forms of A/M2.
Inhibitory Effect of Spiran Amine Compound 8 on Wild-Type and Amantadine Insensitive A/M2 Channels.
Amantadine resistant mutants carry naturally occurring point mutations of the pore lining residues of the A/M2 channel. Extensive structural, electrophysiological and in-silico investigations suggest that these residues form the binding pocket for the drug. The effect of spiran amine 8 on wild-type A/M2 channels and A/M2 channels with altered amantadine sensitivity was tested by the present inventors and compared the inhibition by compound 8 to that of amantadine and BL-1743. It was found that spiran amine 8 efficiently inhibits the activity of A/M2 wild-type channels and of A/M2-V27A mutants, with IC50 values of 12.6 μM and 84.9 μM respectively. The inhibition of V27A mutants is particularly interesting, given that amantadine, BL-1743, and spiro piperidine 20 (3-Aza-spiro[5.5]undecane) gave less than 10% inhibition of the mutant, when applied at 100 μM concentration. It is also interesting to note that the mutant V27G, which is also naturally occurring, is highly resistant to all compounds tested (Table 2).
Table 2, below shows the inhibitory effect of amantadine, BL-1743, spiran amine 8 and spiran amine 9 on amantadine insensitive A/M2 mutant channels. Data presented as a percent of remaining A/M2 activity after application of an inhibitor (100 μM) for 2 min. The experimental data are the average of three independent experiments. Each point is a mean (±SD) of 5-7 oocytes.











TABLE 2









SPIRAN
SPIRAN




BL-1743
AMINE 8
AMINE 9
AMANTADINE









% remaining activity after application of 100 μM compound














A/M2-L26F
89.2 ± 4.8
32.4 ± 3.3
72.6 ± 8.4
 51.8 ± 3.5


A/M2-V27A
97.4 ± 1.8
46.6 ± 6.6
 70.2 ± 1.55
93.07 ± 1.9


A/M2-V27G
82.9 ± 9.3
87.9 ± 6.1
89.6 ± 4.1
  95 ± 4.9


A/M2-A30T
 102 ± 2.3
98.1 ± 7.0
101. ± 3.2
104.6 ± 4.3


A/M2-S31N
  100 ± 0.67
99.1 ± 0.9
 100 ± 4.1
 65.3 ± 2.7


A/M2-G34E
89.8 ± 6.9
 96. ± 5.9
101.7 ± 2.8 
100.4 ± 6.7









The ability of compound 8 to inhibit other pore-lining mutants was examined next. S31N is a highly frequent mutation, which gives rise to decreased sensitivity to amantadine, and complete resistance to rimantadine (IC50>10 mM). Similarly, compound 8 showed little inhibition of this mutant. Other mutations deeper in the pore than V27 (A30T and G34E) completely eliminated the ability of all drugs tested to inhibit the channel (Table 2).
By contrast to the other mutations considered here, L26F does not involve a pore-lining residue, and instead involves a partially largely lipid-exposed residue that packs at the interface between the helices adjacent to the Val27. Thus, this residue is expected to play a more subtle and less direct role in defining the steric properties of the drug-binding site. Indeed, amantadine inhibits this mutant with reduced affinity (IC50=164.5 μM), versus the much larger decreases seen for other variants. Compound 8 is an even more potent inhibitor of A/M2 L26F (IC50=30.6 μM).
The inhibitory effect of compounds 8 and 9 on the recombinantly expressed A/M2 wild-type and mutant channels was confirmed by in-vivo plaque reduction assays of influenza A viruses. Plaque formation of wild-type influenza virus (A/Udorn/72) was efficiently inhibited by amantadine, 8 and 9 at concentrations ranging from 0.5 to 2 μM, with compound 8 showing more potent activity. BL-1743 inhibited wild-type virus plaque formation only at concentrations as high as 50-100 μM. On the other hand, plaque formation of influenza virus, containing the A/M2-V27A mutation was reduced by 5 μM spiran amine 8, consistent with the electrophysiological finding (data not shown). In comparison, amantadine and BL-1743 at the same concentration range failed to inhibit plaque formation of A/M2-V27A viruses (not shown).
Thus, spiran amine 8 is capable of efficiently inhibiting not only wild-type A/M2 channels and A/M2-L26F and A/M2-V27A mutant channels expressed in oocytes, but it also prevents replication of wild-type virus and these mutant recombinant viruses. As discussed below, we attribute to the greater potency of compound 8 in the plaque versus the electrophysiological studies to kinetic effects arising from the slow kinetics of binding of this class of inhibitors. Compounds are incubated with oocytes for brief periods in the electrophysiological experiments, but much longer periods in the plaque-binding studies.
Also measured was the binding of selected drugs to the transmembrane domain of wild-type A/M2 protein (M2TM, residues 22-46), using a spectroscopic assay that relies upon changes in the CD spectrum of M2 induced by binding of drugs (Table 1). All drugs bound in a stoichiometry of approximately one drug/tetramer. At pH 7.4, potent compounds were found to bind to the transmembrane tetrameric form of M2TM with low μM binding constants. Compounds 8 and 10 were found to be approximately equipotent with amantadine, displaying dissociation constants in the range of 7-8 μM (Table 1). In agreement with the plaque assay, compound 9 was approximately four-fold less potent than amantadine or compound 8. These data support the expectation that the drugs inhibit the channel activity by binding directly to the A/M2 proton channel.
Example 2
Synthesis and Structure Activity Relationship of Additional Exemplary Influenza A M2 Proton Channel Inhibitors
Additional compounds were synthesized as provided below.







Table 3, below, shows the results of studies that were conducted to determine the activity and IC50 for certain inventive compounds. AM2 is the strain of influenza A virus on which the compounds were tested, and AM2(V27A) refers to AM2 virus that possesses the valine→alanine mutation at residue 27 in the M2 protein.









TABLE 3








AM2
AM2 V27A












% inhibition at 
IC50 
% inhibition at 
IC50 


Compounds
100 μM
(μM)
100 μM
(μM)









28  (29%)





 






29 (86%)





 






30  (94%)





 






31  (78.8%)

31  (7.1%)



 






32  (32%)





 






33  (84.2%) 34  (84.1%)

33  (0%) 34  (46.9%)



 






35  (92%)

35  (14.6%)









Additional information regarding the present invention can be found in Design and Pharmacological Characterization of Inhibitors of Amantadine-Resistant Mutants of the M2 Ion Channel of Influenza A Virus, Victoria Balannik, Jun Wang, Xianghong Jing, Emma Magavern, Robert A Lamb, William F DeGrado, Lawrence H Pinto, submitted to Journal of the American Chemical Society, which is hereby incorporated herein by reference in its entirety.

## Claims

1. A method for treating an influenza A virus infection comprising the step of administering to a subject in need thereof a composition comprising a compound, wherein said compound is of formula (I):



and wherein R1 and R2 are as follows:














R1
R2








H
—NH3+Cl−



H
—NH2+Cl−CH3



H
—NH(═NH)NH2


 



H






 



H






 



OH






 



H






 



F






 



H






 



H












or, wherein said compound is





wherein n is 1 or 2,
or a stereoisomer, partial stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid hydrate, or N-oxide thereof.

2. The method according to claim 1 said composition additionally comprises a pharmaceutically acceptable carrier, diluent, or excipient.

3. The method according to claim 1 wherein said influenza A virus is a wild-type virus.

4. The method according to claim 1 wherein said influenza A virus is a mutant.