Aminopyridine compounds and methods for the preparation and use thereof

The present invention relates generally to therapeutics targeting the bacterium Porphyromonas gingivalis, including its proteases arginine gingipain A/B (Rgp), and their use for the treatment of disorders associated with P. gingivalis infection, including brain disorders such as Alzheimer's disease. In certain embodiments, the invention provides compounds according to Formula I and Formula III, as described herein, and pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION

P. gingivalisproduces proteases called gingipains, including Arginine Gingipain A (RgpA), Arginine Gingipain B (RgpB) and Lysine Gingipain (Kgp). Gingipains contribute to many functions of the organism including its survival and virulence. Gingipains can be secreted, transported to outer membrane surfaces ofP. gingivalis, or released in outer membrane vesicles by the bacterium. Gingipains degrade a broad range of proteins (e.g., immunoglobulins, proteinase inhibitors, actin, and collagen) which can lead to cytoskeleton collapse and apoptosis in many types of cells. Recent research has demonstrated that inhibitors of gingipains can preventP. gingivalis-induced cell death. See: Travis, et al.,Adv Exp Med Biol,2000. 477: 455-65; Sheets, et al.,Infect Immun,2005. 73(3): 1543-52; Sheets, et al.,Infect Immun,2006. 74(10): 5667-78; Stathopoulou, et al.,BMC Microbiol,2009. 9: 107. New compounds for the inhibition of gingipain activity and the treatment of diseases associated with gingipain activity andP. gingivalisinfection are needed. The present invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention provides a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, whereinW is selected from the group consisting of a bond, CH2, and O;R1aand R1bare independently selected from the group consisting of H and C1-6alkyl;R2is selected from the group consisting of C1-6alkyl and halogen;subscript n is 0 or 1;R3is selected from the group consisting of C3-8alkyl, C3-8cycloalkyl, C3-12heterocyclyl, C6-10aryl, and C5-12heteroaryl wherein R3is optionally substituted with one or more R3asubstituents;each R3ais independently selected from the group consisting of halogen, —CN, —NO2, —N3, —OH, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, —N(Rc)2, —(CH2)kC(O)Rb, —NR(CH2)uC(O)Rb, —O(CH2)C(O)Rb, —(CH2)kCONRcRc, —(CH2)kNRcC(O)Rb, —NRc(CH2)uCONRcRc, —NRc(CH2)uNRc—(O)Rb, —O(CH2)uCONRcRc, and —O(CH2)uNRcC(O)Rb, and optionally substituted triazolyl;each Rbis independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4deuteroalkyl;each Rcis independently selected from the group consisting of hydrogen and Cis alkyl;each subscript k is independently selected from 0, 1, 2, 3, 4, 5, and 6;each subscript u is independently selected from 1, 2, 3, 4, 5, and 6;R4is selected from the group consisting of —CH2R4aand C1-6haloalkyl;R4ais selected from the group consisting of —O—R5, —S—R6, —SO—R6, —SO2—R6, —N(R7)2, and C5-12heteroaryl;R5is selected from the group consisting of phenyl, C1-6alkyl, C1-6haloalkyl, and C5-12heteroaryl, wherein phenyl is substituted with 1-5 halogens, and wherein C5-12heteroaryl is optionally substituted with halogen or C1-3haloalkyl;R6is selected from the group consisting of phenyl, C1-6alkyl, C1-6haloalkyl, and C5-12heteroaryl, wherein phenyl is optionally substituted with 1-5 halogens, and wherein C5-12heteroaryl is optionally substituted with halogen or C1-3haloalkyl; and each R7is independently selected C1-6alkyl.

In one embodiment, the invention provides a compound according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein:R11is selected from C1-6alkyl and C3-8cycloalkyl;R12aand R12bare independently selected from H, C1-6alkyl, and C6-10aryl, orR12aand R12bare taken together to form C3-6cycloalkyl, orR12aand R11are taken together to form 4- to 10-membered heterocyclyl which is optionally substituted with one or more R17;each R13aand each R13bis independently selected from H, —OH, and C1-6alkyl, orone R13aand R11are taken together to form 4- to 10-membered heterocyclyl, orone R13band R12bare taken together to form a 5- or 6-membered ring;R14is selected from H and halogen, orR14, R12a, and R12bare taken together to form a 6- to 8-membered ring, which is optionally substituted with one or more R18, orR14and one R13aare taken together to form a 5- to 8-membered ring, which is optionally substituted with one or more R18, orR14is taken together with one R13aand one R13bon the same carbon atom to form a 5- to 8-membered ring, which is optionally substituted with one or more R18, orR14, R11, and R12aare taken together to form a 6- to 10-membered bicyclic ring, which is optionally substituted with one or more R18;R15aand R15bare independently selected from H and C1-6alkyl;R16is independently selected from C1-6alkyl and halogen;each R17is independently selected from C1-6alkyl, C1-6alkoxy, C1-6haloalkyl, C1-6haloalkoxy, —OH, and —N(R17a)2, wherein each R17ais independently selected from H and C1-6alkyl;each R18is independently selected from C1-6alkyl and halogen;Y is selected from O, S, C(R19a)2, and NR19b;each R19ais selected from H and C1-6alkyl, orone R19aand one R13bon adjacent atoms are taken together to form a double bond;R19bis selected from H and C1-6alkyl, orR19band R11are taken together to form a 4- to 6-membered ring;subscript m is 0, 1, 2, or 3; andsubscript q is 0 or 1.

In a related embodiment, the invention provides a pharmaceutical composition containing a compound as described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

In another embodiment, the invention provides a method of inhibiting a gingipain. The method includes contacting the gingipain with an effective amount of a compound as described herein.

In another embodiment, the invention provides a method of treating a disease or condition associated withP. gingivalisinfection. The method includes administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Inhibition of gingipains has been shown to protect cells, prevent bacterial growth, increase immune system surveillance ofP. gingivalis, and protect against bacterial reinfection. The present invention provides potent nonpeptidic compounds for inhibition of arginine gingipains. The compounds can be used to prevent cell death, inflammation, and other pathology in a variety of diseases associated withP. gingivalisinfection, including aging-related conditions such as Alzheimer's disease.

As used herein, the term “alkyl,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C1-2, C1-3, C1-4, C1-5, C1-6, C1-7, C1-8, C1-9, C1-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6and C5-6. For example, C1-6alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. For example, “substituted alkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “alkoxy,” by itself or as part of another substituent, refers to a group having the formula —OR, wherein R is alkyl.

As used herein, the term “cycloalkyl,” by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbomene, and norbomadiene. When cycloalkyl is a saturated monocyclic C3-8cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. For example, “substituted cycloalkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “alkylene” refers to an alkyl group, as defined above, linking at least two other groups (i.e., a divalent alkyl radical). The two moieties linked to the alkylene group can be linked to the same carbon atom or different carbon atoms of the alkylene group.

As used herein, the term “alkylthio,” by itself or as part of another substituent, refers to a group having the formula —SR, wherein R is alkyl.

As used herein, the term “heteroalkyl,” by itself or as part of another substituent, refers to an alkyl group of any suitable length and having from 1 to 3 heteroatoms such as N, O and S. For example, heteroalkyl can include ethers, thioethers and alkyl-amines. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, —S(O)— and —S(O)2—. The heteroatom portion of the heteroalkyl can replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion can be the connecting atom, or be inserted between two carbon atoms.

As used herein, the terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

As used herein, the term “haloalkyl,” by itself or as part of another substituent, refers to an alkyl group where some or all of the hydrogen atoms are replaced with halogen atoms. As for alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, such as C1-6. For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some instances, the term “perfluoro” can be used to define a compound or radical where all the hydrogens are replaced with fluorine. For example, perfluoromethyl refers to 1,1,1-trifluoromethyl.

As used herein, the term “haloalkoxy,” by itself or as part of another substituent, refers to an alkoxy group where some or all of the hydrogen atoms are replaced with halogen atoms.

As used herein, the term “halocycloalkyl,” by itself or as part of another substituent, refers to a cycloalkyl group where some or all of the hydrogen atoms are replaced with halogen atoms.

As used herein, the term “deuteroalkyl,” by itself or as part of another substituent, refers to an alkyl group where some or all of the hydrogen atoms are replaced with deuterium atoms. As for alkyl groups, deuteroalkyl groups can have any suitable number of carbon atoms, such as C1-6. In some instances, the term “perdeutero” can be used to define a compound or radical where all the hydrogens are replaced with deuterium.

As used herein, the term “aryl,” by itself or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as C6, C7, C8, C9, C10, C11, C12, C13, C14, C15or C16, as well as C6-10, C6-12, or C6-14. Aryl groups can be monocyclic, fused to form bicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be substituted or unsubstituted. For example, “substituted aryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

As used herein, the term “heteroaryl,” by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, —S(O)— and —S(O)2—. Heteroaryl groups can include any number of ring atoms, such as C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4; or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. For example, heteroaryl groups can be C5-8heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-8heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms; or C5-6heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-6heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms. The heteroaryl group can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. For example, “substituted heteroaryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

Some heteroaryl groups include from 5 to 10 ring members and only nitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, and cinnoline. Other heteroaryl groups include from 5 to 10 ring members and only oxygen heteroatoms, such as furan and benzofuran. Some other heteroaryl groups include from 5 to 10 ring members and only sulfur heteroatoms, such as thiophene and benzothiophene. Still other heteroaryl groups include from 5 to 10 ring members and at least two heteroatoms, such as imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline, quinazoline, phthalazine, and cinnoline.

As used herein, the term “heterocyclyl,” by itself or as part of another substituent, refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms of N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, —S(O)— and —S(O)2—. Heterocyclyl groups can include any number of ring atoms, such as, C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, C6-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms can be replaced with heteroatoms in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocyclyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. The heterocyclyl groups can also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocyclyl groups can be unsubstituted or substituted. For example, “substituted heterocyclyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

The heterocyclyl groups can be linked via any position on the ring. For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine can be 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or 4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine, piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1- or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine, isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be 2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or 5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

As used herein, the term “protecting group” refers to a chemical moiety that renders a functional group (e.g., an amino group) unreactive, but is also removable so as to restore the amino group. Examples of protecting groups include, but are not limited to, benzyloxycarbonyl (Z or Cbz); 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluene sulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamido; phthalimido; and the like. Other protecting groups are known to those of skill in the art including, for example, those described by Green and Wuts (Protective Groups in Organic Synthesis,4thEd. 2007, Wiley-Interscience, New York).

As used herein, the term “carbonyl,” by itself or as part of another substituent, refers to —C(O)—, i.e., a carbon atom double-bonded to oxygen and bound to two other groups in the moiety having the carbonyl.

As used herein, the term “amino” refers to a moiety —NR2, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation. “Dialkylamino” refers to an amino moiety wherein each R group is alkyl.

As used herein, the term “sulfonyl” refers to a moiety —SO2R, wherein the R group is alkyl, haloalkyl, or aryl. An amino moiety can be ionized to form the corresponding ammonium cation. “Alkylsulfonyl” refers to an amino moiety wherein the R group is alkyl.

As used herein, the term “hydroxy” refers to the moiety —OH.

As used herein, the term “cyano” refers to a carbon atom triple-bonded to a nitrogen atom (i.e., the moiety —C≡N).

As used herein, the term “carboxy” refers to the moiety —C(O)OH. A carboxy moiety can be ionized to form the corresponding carboxylate anion.

As used herein, the term “amido” refers to a moiety —NRc(O)R or —C(O)NR2, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO2.

As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (i.e., O═).

In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of 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 either the same or different at every position. Combinations of substituents are generally those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In general, “substituted,” as used herein, does not encompass replacement and/or alteration of a key functional group by which a molecule is identified, e.g., such that the “substituted” functional group becomes, through substitution, a different functional group. For example, a “substituted phenyl” group must still comprise the phenyl moiety and cannot be modified by substitution, in this definition, to become, e.g., a cyclohexyl group.

Examples of suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O; ═S; ═NNRγ2; ═NNHC(O)Rγ; ═NNHC(O)ORγ; ═NNHS(O)2Rγ; ═NRγ; ═NORγ; —O(C(Rγ2))2-3O—; or —S(C(Rγ2))2-3S—; wherein each independent occurrence of Rγis selected from hydrogen; C1-6alkyl, which may be substituted as defined below; C3-8cycloalkyl; C6-10aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CRβ2)2-3O—; wherein each independent occurrence of RR is selected from hydrogen; C1-6alkyl which may be substituted as defined below; C3-8cycloalkyl; C6-10aryl; 4- to 10-membered heterocyclyl; or 6- to 10-membered heteroaryl.

As used herein, the term “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to a subject. By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof. Pharmaceutical excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, glidants, coatings, sweeteners, flavors and colors.

As used herein, the term “salt” refers to acid or base salts of the compounds of the invention. Illustrative examples of pharmaceutically acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that the pharmaceutically acceptable salts are non-toxic.

Pharmaceutically acceptable salts of the acidic compounds of the present invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts (such as sodium, lithium, potassium, calcium, and magnesium salts), as well as ammonium salts (such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts).

Similarly acid addition salts, such as of mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure.

As used herein, the terms “Porphyromonas gingivalis” and “P. gingivalis” refer to the gram-negative asaccharolytic bacterium that is recognized as a key causative microbe in the pathogenesis of periodontitis and related conditions. “P. gingivalisinfection” refers to the invasion and colonization ofP. gingivalisin a bodily tissue such as the gums or the brain.P. gingivalisinfection is frequently characterized by subsequent tissue injury and disease.

As used herein, the term “gingipain” refers to cysteine proteases expressed byP. gingivalishaving trypsin-like specificity (i.e., Lys-Xaa and Arg-Xaa). Gingipains are recognized as the major virulence factors ofP. gingivalisand contribute to bacterial attachment and colonization, nutrient acquisition, evasion of host defenses, and tissue invasion. The terms “arginine gingipain” and “Rgp” are used interchangeably to refer to theP. gingivalisarginine-specific gingipains RgpA and RgpB, classified under EC number EC 3.4.22.37. The rgpA and rgpB gene-translation products, RgpA and RgpB, share a caspase-like protease domain (specific for Arg-Xaa peptide bonds) and an immunoglobulin-like domain. In RgpA, the protease and immunoglobulin-like domains are followed by a large C-terminal extension containing hemagglutinin-adhesin domains.

As used herein, the term “inhibiting” refers to reducing the level of activity (e.g., proteolytic activity) of an enzyme such as a gingipain which can be assessed, for example, using an in vitro assay or other suitable assay. Inhibition of enzyme activity caused by a particular substance (e.g., a gingipain inhibitor as described herein) can be expressed as the percentage of the enzyme activity measured in the absence of the substance under similar conditions. The ability of a particular substance to inhibit an enzyme can be expressed as an IC50value, i.e., the concentration of the compound required to reduce the activity of the enzyme to 50% of its maximum activity.

As used herein, the terms “treat,” “treatment,” and “treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, e.g., the result of a physical examination.

As used herein, the terms “effective amount” and “therapeutically effective amount” refer to a dose of a compound such as an Rgp inhibitor that inhibits the activity of a gingipain and/or produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding(1999); Pickar, Dosage Calculations (1999);Goodman&Gilman's The Pharmacological Basis of Therapeutics,11thEdition, 2006, Brunton, Ed., McGraw-Hill; andRemington: The Science and Practice of Pharmacy,21stEdition, 2005, Hendrickson, Ed., Lippincott, Williams & Wilkins).

As used herein, the term “Alzheimer's disease” refers to a progressive disease of the central nervous system in humans and other mammals. It is manifested by dementia (especially in the elderly); disorientation; loss of memory; difficulty with language, calculation, or visual-spatial skills; and psychiatric manifestations. Alzheimer's disease is associated with progressive neurodegeneration and characteristic pathology, namely beta amyloid plaques and tau tangles.

As used herein, the term “osteoarthritis” refers to a chronic degenerative joint disease that results from breakdown of joint cartilage, synovial tissue, and underlying bone.

In one embodiment, the invention provides a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, whereinW is selected from a bond, CH2, and O;R1aand R1bare independently selected from H and C1-6alkyl;R2is selected from C1-6alkyl and halogen;subscript n is 0 or 1;R3is selected from C3-8alkyl, C3-8cycloalkyl, C3-12heterocyclyl, C6-10aryl, and C5-12heteroaryl wherein R3is optionally substituted with one or more R3asubstituents;each R3ais independently selected from halogen, —CN, —NO2, —N3, —OH, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, —N(Rc)2, —(CH2)kC(O)Rb, —NR(CH2)uC(O)Rb, —O(CH2)C(O)Rb, —(CH2)kCONRcRc, —(CH2)kNRcC(O)Rb, —NRc(CH2)uCONRcRc,13NRc(CH2)NRc—(O)Rb, —O(CH2)uCONRcRc, and —O(CH2)NRcC(O)Rb, and optionally substituted triazolyl;each Rbis independently selected from C1-4alkyl, C14haloalkyl, and C1-4deuteroalkyl;each R is independently selected from hydrogen and C1-8alkyl;each subscript k is independently selected from 0, 1, 2, 3, 4, 5, and 6;each subscript u is independently selected from 1, 2, 3, 4, 5, and 6;R4is selected from —CH2R4aand C1-6haloalkyl;R4ais selected from —O—R5, —S—R6, —SO—R6, —SO2—R6, —N(R7)2, and C5-12heteroaryl;R5is selected from phenyl, C1-6alkyl, C1-6haloalkyl, and C5-12heteroaryl,wherein phenyl is substituted with 1-5 halogens, andwherein C5-12heteroaryl is optionally substituted with halogen or C1-3haloalkyl;R6is selected from phenyl, C1-6alkyl, C1-6haloalkyl, and C5-12heteroaryl,wherein phenyl is optionally substituted with 1-5 halogens, andwherein C5-2heteroaryl is optionally substituted with halogen or C1-3haloalkyl; andeach R7is independently selected C1-6alkyl.

Compounds of the invention can be prepared in protected form (e.g., protected compounds wherein at least one of R1aand R1bis an amine protecting group). A number of suitable protecting groups—as described, for example, by Green and Wuts (Protective Groups in Organic Synthesis,4thEd. 2007, Wiley-Interscience, New York)—can be used. In some embodiments, R1ais H and Rbis selected from benzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxycarbonyl. In some embodiments, R1aand R1bare selected from benzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxycarbonyl. In some embodiments, R1ais H and Rbis tert-butyloxycarbonyl. Compounds can also be prepared in alkylated form (i.e., compounds wherein at least one of R1aand Rbis an alkyl group). One or both of R1aand R1bcan be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl.

In some embodiments, subscript n is 0. In some embodiments, subscript p is 1. In some embodiments, subscript n is 1 and R2is selected from fluoro, chloro, bromo, iodo, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branched hexyl. In some embodiments, subscript n is 1 and R2is selected from fluoro, chloro, and methyl. In some embodiments, subscript n is 2 or 3.

In some embodiments, W is a bond. In some embodiments, W is selected from CH2and O.

In some embodiments, the compound has a structure according to Formula IIa:

In some embodiments, the compound has a structure according to Formula IIb:

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R3is selected from C3-8cycloalkyl, C6-10aryl, C5-12heteroaryl, and C3-12heterocyclyl, each of which is optionally substituted with one or more R3asubstituents. For example, R3can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl. In some embodiments, R3is selected from cyclobutyl, cyclopentyl, and cyclohexyl. In some embodiments, R3is selected from phenyl and naphthyl. In some embodiments, R3is selected from pyrrolyl, pyridinyl, imidazolyl, pyrazolyl, triazolyl, pyrazinyl, triazinyl, indolyl, isoindolyl, and quinolinyl. In some such embodiments, R3is selected from cyclopentyl and phenyl, each of which is optionally substituted with one or more R3asubstituents. In some such embodiments, each R3ais independently selected from halogen, —N3, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, and —NRcC(O)Rb. In some embodiments, R3is cyclopentyl.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R3is selected from C3-8cycloalkyl, C6-10aryl, C5-12heteroaryl, and C3-12heterocyclyl, each of which is optionally substituted with one or more R3asubstituents. In some such embodiments, R3is selected from cyclopentyl and phenyl, each of which is optionally substituted with one or more R3asubstituents. In some such embodiments, each R3ais independently selected from halogen, —N3, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, and —NRcC(O)Rb. In some embodiments, R3is cyclopentyl.

One of skill in the art will appreciate that compounds containing azide groups (e.g., compounds wherein R3ais —N3) can be modified with further functional groups via reaction with a complementary reaction partner such as an alkyne-bearing compound or a phosphine-bearing compound. Reaction of azides and alkynes via [3+2] cycloaddition, commonly referred to as “click chemistry,” can be used to install a variety of substituted triazole groups in the compounds of the invention. Accordingly, some embodiments of the invention provide compounds wherein R3ais an optionally substituted triazolyl moiety according to the formula:

wherein R3bis a functional moiety and L3is a linking moiety.

In some embodiments, the linking moiety L3has a structure —L3a-L3b-, wherein L3aand L3bare independently selected from a bond, a divalent polymer moiety, and linear or branched, saturated or unsaturated C1-30alkyl; wherein:one or more carbon atoms in the C1-30alkyl is optionally and independently replaced by O, S, NRa;two or more groupings of adjacent carbon atoms in the C1-30alkyl are optionally and independently replaced by —NRa(CO)— or —(CO)NRa—; andtwo or more groupings of adjacent carbon atoms in the C1-30alkyl are optionally and independently replaced by a 4- to 8-membered, divalent carbocycle or a 4- to 8-membered, divalent heterocycle having one to four heteroatoms selected from O, S, and N; andeach Rais independently selected from H and C1-6alkyl.

In some embodiments, the functional group R3bis selected from a chromophore, a fluorophore, and a binding moiety (e.g., biotin, glutathione, and the like).

In some embodiments, R3is selected from C3-8alkyl and C3-8cycloalkyl, each of which is optionally substituted with one or more R3asubstituents. In some embodiments, R3is selected from cyclopentyl and isopropyl. In some embodiments, R3is unsubstituted cyclopentyl. In some embodiments, R3is isopropyl and R3ais methoxy.

In certain embodiments, R3and the carbonyl to which it is bonded form a moiety other than a naturally-occurring amino acid residue (an L amino acid residue) or an isomer of a naturally-occurring amino acid residue (a D amino acid residue). In some embodiments, R3and the carbonyl to which it is bonded form a moiety other than asparaginyl, substituted asparaginyl, glutaminyl (i.e., a glutamine residue), substituted glutaminyl (i.e., a substituted glutamine residue), glutamyl (i.e., a glutamic acid residue), substituted glutamyl (i.e., a substituted glutamic acid residue), isoleucinyl, substituted isoleucinyl, leucinyl, substituted leucinyl, lysinyl, substituted lysinyl, methioninyl, substituted methioninyl, prolinyl, substituted prolinyl, threoninyl, substituted threoninyl, valinyl, or substituted valinyl. The substituted amino acid residues may be present in larger peptide groups having two or more amino acid residues linked via amine bonds.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from —S—R6, —SO—R6, —SO2—R6, C5-12heteroaryl, and —N—R7. In some embodiments, R4ais selected from —O—R5, C5-12heteroaryl, and —N—(R7)2. In some embodiments, R4ais selected from —O—R5, —S—R6, —SO—R6, —SO2—R6, and —N—(R7)2. In some embodiments, R4ais selected from —O—R5, —S—R6, —SO—R6, —SO2—R6, and C5-12heteroaryl.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, (C1-6dialkyl)amino, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, C5-12heteroaryl, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, —O-phenyl wherein phenyl is substituted with 1-5 halogens, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, and —S-phenyl wherein phenyl is optionally substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais selected from C1-6alkoxy, C1-6haloalkoxy, C1-6alkylthio, C1-6haloalkythio, C1-6alkylsufonyl, (C1-6dialkyl)amino, C5-12heteroaryl, and —O-phenyl wherein phenyl is substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4is —CH2—O—R5and R5is selected from 1,1,1,3,3,3-hexafluoroprop-2-yl, isoxazolyl, and phenyl, wherein phenyl is substituted with 1-5 halogens.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R5is phenyl substituted with 1-5 halogens. In some embodiments, each halogen in R5is selected from F and Cl. In some embodiments, each halogen in R5is F.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R4ais —OR5, such that R4is a moiety having the structure:

wherein R5a, R5b, R5c, R5d, and R5eare independently selected from hydrogen and halogen, and the wavy line represents the point of connection to the compound.

In certain embodiments, R4is not 2,3,5,6-tetrafluorophenoxymethyl.

In some embodiments, the invention provides compounds of Formula I, Formula IIa, and/or Formula IIb, and pharmaceutically acceptable salts thereof, wherein R5is selected from 2,6-difluorophenyl; 2,3,6-trifluorophenyl; and 2,3,5,6-tetrafluorophenyl.

The compounds of the invention can be further substituted; a compound according to Formula I may contain, for example, an optionally substituted R1aand/or R1bgroup, one or more optionally substituted R2groups, an optionally substituted R3group, and/or an optionally substituted R4group (including, an optionally substituted R5group, an optionally substituted R6group, and/or one or more optionally substituted R7groups).

In some embodiments, the aminopyridyl ketone compound is selected from:

and pharmaceutically acceptable salts thereof.

Aminopyridyl ketones may be prepared by the following approaches, summarized in Scheme 1 and described below. As shown in Scheme 1, aminopyridines (e.g., 2-amino-4-formylpyridine and 2-amino-5-formylpyridine (xi)) can be reacted with a suitable protecting reagent (e.g., di-tert-butyl dicarbonate) to prepare the corresponding mono- and/or di-protected formylpyridines (xii), wherein P1is H or a protecting group and P2is a protecting group. Any of these aldehydes (xii) may be reacted with a protected phosphonate (xiii) (wherein Q1is H or a protecting group; Q2is a protecting group; and each R is independently C1-6alkyl; e.g., ZHNCH(CO2Me)PO3Me2) and a strong base to prepare dehydro-aminopyridylalanine methyl esters (xiv).

When Q1and/or Q2is a Z group (or another protecting group which can be removed by hydrogenation), dehydro-aminopyridylalanines (xiv) may be hydrogenated in the presence of palladium on carbon, providing aminoesters (xv) by simultaneously saturating the olefins and deprotecting the α-amino group. The free amino groups may be reacted with carboxylic acids (R3C(O)OH) and dehydrating agents to form amidoesters (xvi). The dehydrating agent may be HATU or any of many other reagents suitable for carboxamide formation. The amidoesters may be converted to protected products (xvii) by various routes. In one non-limiting example, the amidoesters are hydrolyzed using a strong based such as NaOH. The resulting carboxylic acids are then reacted with CICO2Et, a tertiary amine, and diazo methane to form diazomethyl ketones, which can then be treated with HBr to give bromomethyl ketones. In another sequence, the methyl esters may be treated with CICH2I and LiN(iPr)2to provide chloromethyl ketones in one step. The bromomethyl ketones or chloromethyl ketones can be heated with substituted phenols and KF in DMF to give aryloxymethyl ketones. In another non-limiting example, the bromomethyl or chloromethyl ketones are treated with isoxazole-5-one and KF in DMF to give isoxazolyloxymethyl ketones. Lastly, the protecting groups P1and P2(e.g., Boc groups) may be removed under suitable conditions (e.g., via treatment with trifluoroacetic acid), to afford the desired final products.

The starting materials and reagents used in preparing the compounds of the invention are either available from commercial suppliers or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Vol. 1-28 (Wiley, 2016);March's Advanced Organic Chemistry,7thEd. (Wiley, 2013); and Larock's Comprehensive Organic Transformations, 2ndEd. (Wiley, 1999). The starting materials and the intermediates of the reaction can be isolated and purified if desired using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatography and the like. Such materials can be characterized using conventional means, including measuring physical constants and obtaining spectral data.

Unless specified to the contrary, the reactions described herein take place at atmospheric pressure over a temperature range of from about −78° C. to about 250° C. For example, reactions can be conducted at from about 0° C. to about 125° C., or at about room (or ambient) temperature, e.g., about 20° C. In some embodiments, reactions are conducted at about 0° C., 20° C., 25° C., 90° C., 10° C., 110° C., 125° C., 150° C., 175° C., or 200° C. In some embodiments, reactions are conducted starting at a first temperature (e.g., about −78° C. or about 0° C.), and allowed to warm to a higher second temperature (e.g., about 20° C. or about 25° C.). One of skill in the art will appreciate that various modifications to the procedures described herein can be made.

The invention provides a number of useful aminopyridinecyanamide compounds having various aminopyridine moieties such as aminopyridines (e.g., 2-aminopyridines), aminoisoquinolines (e.g., 1-aminoisoquinolines and 1-amino-6,7,8,9-tetrahydrobenzo[g]iso-quinolines), aminofuropyridines (e.g., 7-aminofuro[2,3-c]pyridines and 4-aminofuro[3,2-c]pyridines), and aminonapthyridines (e.g., 3-amino-1,7-naphthyridines and 8-amino-2,7-naphthyridines).

In one embodiment, the invention provides a compound according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein:R11is selected from C1-6alkyl and C3-8cycloalkyl;R12aand R12bare independently selected from H, C1-6alkyl, and C6-10aryl, orR12aand R12bare taken together to form C3-6cycloalkyl, orR12aand R11are taken together to form 4- to 10-membered heterocyclyl which is optionally substituted with one or more R17;each R13aand each R13bis independently selected from H, —OH, and C1-6alkyl, or one R13aand R11are taken together to form 4- to 10-membered heterocyclyl, or one R13band R12bare taken together to form a 5- or 6-membered ring;R14is selected from H and halogen, orR14, R12a, and R12bare taken together to form a 6- to 8-membered ring, which is optionally substituted with one or more R18, orR14and one R13aare taken together to form a 5- to 8-membered ring, which is optionally substituted with one or more R18, orR14is taken together with one R13aand one R13bon the same carbon atom to form a 5- to 8-membered ring, which is optionally substituted with one or more R18, orR14, R11, and R12aare taken together to form a 6- to 10-membered bicyclic ring, which is optionally substituted with one or more R18;R15aand R15bare independently selected from H and C1-6alkyl;R16is independently selected from C1-6alkyl and halogen;each R17is independently selected from C1-6alkyl, C1-6alkoxy, C1-6haloalkyl, C1-6haloalkoxy, —OH, and —N(R17a)2, wherein each R17ais independently selected from H and C1-6alkyl;each R18is independently selected from C1-6alkyl and halogen;Y is selected from O, S, C(R19a)2, and NR19b;each R19ais selected from H and C1-6alkyl, orone R19aand one R13bon adjacent atoms are taken together to form a double bond;R19bis selected from H and C1-6alkyl, orR19band R11are taken together to form a 4- to 6-membered ring;subscript m is 0, 1, 2, or 3; andsubscript q is 0 or 1.

In some embodiments, the invention provides compounds according to Formula

In some embodiments, the invention provides compounds according to Formula IVb:

In some embodiments, the invention provides compounds according to Formula

In some embodiments, the invention provides compounds according to Formula V:

In some embodiments, the invention provides compounds according to Formula III or Formula V wherein Y is O or S. In some embodiments, the invention provides compounds according to Formula III or Formula V wherein Y is CH2. In some embodiments, W is selected from O, S, and CH2, and each R13aand each R13bis selected from H and C1-6alkyl.

In some embodiments, Y is selected from O, S, and CH2, and one of R13aand R13bis selected from H and —OH, and the remaining 133aand R13bgroups are selected from H and C1-6alkyl.

In some embodiments, Y is CH(R19a), and R19aand one R13bare taken together to form a double bond. In some embodiments, the grouping —CH(R19a)(CR13aR13b)m— is selected from ethen-diyl, prop-1-en-1,3-diyl, and but-1-en-1,4-diyl.

In some embodiments, Y is NR19b, and R19band R11are taken together to form a 5- or 6-membered ring. For example, R19band R11can form imidazolidin-diyl or piperazin-diyl. In some embodiments, R19band R11are taken together to form 1,4-piperazin-diyl.

In some embodiments, the compound has a structure according to Formula III, Formula IVa, Formula IVb, Formula IVc, or Formula V, and R12aand R11are taken together to form pyrrolidin-1,2-diyl or piperidin-1,2-diyl. In some embodiments, the pyrrolidin-1,2-diyl or piperidin-1,2-diyl is subsisted with one or two R17. In some embodiments, the pyrrolidin-1,2-diyl or piperidin-1,2-diyl is subsisted with one R17. In some embodiments, R17is selected from C1-6alkoxy, C1-6haloalkoxy, —OH, and —N(R17a)2. In some embodiments, R17is selected from —OH, —NH2, methoxy, and dimethylamino.

In some embodiments, the compound has a structure according to Formula V, and R12aand R11are taken together to form azetidin-1,2-diyl, pyrrolidin-1,2-diyl, piperidin-1,2-diyl, indolin-1,2-diyl, or isoindolin-1,2-diyl. In some embodiments, the azetidin-1,2-diyl, pyrrolidin-1,2-diyl, piperidin-1,2-diyl, indolin-1,2-diyl, or isoindolin-1,2-diyl is substituted with one or two R7. In some embodiments, the azetidin-1,2-diyl, pyrrolidin-1,2-diyl, piperidin-1,2-diyl, indolin-1,2-diyl, or isoindolin-1,2-diyl is substituted with one R17. In some embodiments, R17is C1-6alkoxy, C1-6haloalkoxy, —OH, or —N(R17a)2. In some embodiments, R17is selected from —OH, —NH2, methoxy, and dimethylamino.

The aminopyridinecyanamide compounds of the invention can be prepared in protected form (e.g., protected compounds wherein at least one of R15a, R15b, R17a, and R19bis an amine protecting group). A number of suitable protecting groups—as described, for example, by Green and Wuts (Protective Groups in Organic Synthesis,4thEd. 2007, Wiley-Interscience, New York)—can be used. In some embodiments, R15ais H and R15bis selected from benzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxycarbonyl. In some embodiments, R15aand R15bare selected from benzyloxycarbonyl; 9-fluorenylmethyl-oxycarbonyl; tert-butyloxycarbonyl; and allyloxycarbonyl. In some embodiments, R15ais H and R15bis tert-butyloxycarbonyl. Compounds can also be prepared in alkylated form (i.e., compounds wherein at least one of R15a, R15b, R17a, and R19bis an alkyl group). One or both of R17aand R19bcan be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, or t-butyl.

In some embodiments, the invention provides compounds according to Formula III, Formula IVa, Formula IVb, Formula IVc, and/or Formula V, wherein subscript q is 0. In some embodiments, the invention provides compounds according to Formula III, Formula IVa, Formula IVb, Formula IVc, and/or Formula V, wherein subscript q is 1. In some such embodiments, subscript q is 1 and R16is selected from fluoro, chloro, bromo, iodo, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, branched pentyl, n-hexyl, and branched hexyl. In some embodiments, subscript q is 1 and R16is selected from fluoro, chloro, and methyl. In some embodiments, subscript q is 2.

The compounds of the invention can be further substituted; a compound according to Formula III may contain, for example, an optionally substituted R11group, an optionally substituted R12aand/or R12bgroup, one or more optionally substituted R13aand/or R13bgroups, an optionally substituted R14group, an optionally substituted R15aand/or R15bgroup, one or more optionally substituted R16groups, one or more optionally substituted R17groups, one or more optionally substituted R18groups, one or more optionally substituted R19agroups, and/or an optionally substituted R19bgroup.

In some embodiments, the aminopyridinecyanamide compound is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the aminopyridinecyanamide compound is selected from:

and pharmaceutically acceptable salts thereof.

In some embodiments, the aminopyridinecyanamide compound is selected from:

and pharmaceutically acceptable salts thereof.

The aminopyridinecyanamide compounds of the invention may be prepared by the approaches summarized in Scheme 2 and described below. As shown in Scheme 2, an amine (i), bearing a reactive group G1, can be reacted with an amino-heterocycle (ii), bearing a reactive group G2. This reaction involves transformation of reactive groups G1and G2to form intermediate (iii) containing a linkage Y between the amine and the amino-heterocycle structures. Prior to this reaction, the amine is usually protected and the amino-heterocycle is sometimes protected. In general, P11is selected from R11as described above, hydrogen, and a protecting group; P12is selected from hydrogen and a protecting group; and Q11and Q12are independently selected from hydrogen, R15aas described above, R15bas described above, and a protecting group. Any suitable protecting group (e.g., tert-butoxy carbonyl (Boc), benzyloxycarbonyl (Z), trifluoroacetyl, or another easily removed group) can be used for protecting the amine and the amino-heterocycle, and the protecting groups are typically chosen so that either may be removed without removing the other. For example, Boc may be removed by treatment with trifluoroacetic acid, which does not remove Z. Meanwhile, Z may be removed by hydrogenolysis in the presence of palladium on carbon, which does not remove Boc.

A wide variety of reactive groups G1and G2are suitable for forming the linkage Y between the amine and amino-heterocycle structures. A halide G1group or a sulfonate ester G1group can undergo a Williamson reaction with a phenol G2group or thiophenol G2group on the amino-heterocycle to form a linking ether or thioether. Employing a phosphine (e.g., Ph3P) and an azodicarboxylate (e.g., DEAD) as co-reagents, an alcohol G1group can undergo a Mitsunobu reaction with a phenol G2group or thiophenol G2group to form a linking ether

or thioether. An unprotected primary amine G1group or unprotected secondary amine G1group can undergo an Ullmann reaction with a halide G2group to form a secondary or tertiary linking amine. The Ullmann reaction can be catalyzed with a homogeneous copper or palladium catalyst, as described by Buchwald, Hartwig, Fu, and others.

An olefin G1group can undergo a Heck reaction with a halide G2group to form a linking olefin. An alkyne G1group can undergo a Sonagashira reaction with a halide G2group to form a linking alkyne. A boronic acid G1group can undergo a Suzuki reaction with a halide G2group or triflate G2group to form a biaryl linkage. A stannane G1group can undergo a Stille reaction with a halide G2group or triflate G2group to form a biaryl linkage. After a Heck, Sonagashira, Suzuki, or Stille reaction, certain intermediates (iii) (e.g., interemdiates wherein P11and R12aare taken together to form unsaturated heterocyclyl) can be hydrogenated to achieve partial or full saturation.

An aldehyde G1group on the amine may undergo a Grignard or “Grignard-like” reaction with an organomagnesium or organolithium G2group on the amino-heterocycle, to form a linkage bearing a secondary alcohol. A carboxylic acid G1group on the amine may undergo a decarboxylating photoreaction with a halide G2group on the amino-heterocycle to form a carbon-carbon bond between the decarboxylated amine and the amino-heterocycle.

After the reaction involving reactive groups G1and G2, and formation of the linkage Y between the amine and amino-heterocycle, the protecting group on the amine can be removed to provide an amine (iv), and the amine can be reacted with cyanogen bromide to form a cyanamide (v). After formation of the cyanamide, any protecting group on the amino-heterocycle can be removed to provide products according to Formula III.

Other methods can also be used for preparing an amine (iv) for the corresponding cyanamide (v). In some instances, the amine precursor can be obtained via reductive amination of a ketone or aldehyde on the amino-heterocycle. In some instances, the amine precursor can be obtained via reduction of a nitro group on the amino-heterocycle and subsequent mono-alkylation of the resulting amino G2group. In some instances, the amine precursor can be obtained via reduction of a cyano group on the amino-heterocycle and subsequent monoalkylation of the resulting amino G2group. In some instances, the amine precursor can be obtained via simultaneous reduction of a nitro group and an olefin on the amino-heterocycle, and subsequent mono-alkylation of the resulting amino G2group. In some instances, the amine precursor can be obtained via simultaneous reduction of a cyano group and an olefin on the amino-heterocycle, and subsequent mono-alkylation of the resulting amino G2group. In some instances, the amine precursor can be obtained via a Schmidt reaction of a carboxylic acid on the amino-heterocycle, and subsequent mono-alkylation of the resulting amino G2group.

The compounds of the invention are highly active Rgp inhibitors, typically exhibiting Rgp IC50values in the nanomolar and micromolar range.

The term “IC50” indicates how much of a compound is needed to inhibit a given biological process (or component of a process, e.g., an enzyme, cell, cell receptor, or microorganism) by one half (50%). The IC50value for a particular test compound can be measured as follows. Fifty microliters (μL) of an enzyme such as RgpA or RgpB (1 nM in 50 mM bis-Tris propane [pH 8.0] containing 1% [vol/vol] Triton X-100 and 5 mM 2-mercaptoethanol) is added to columns 1 to 11 of a 96-well plate, and 100 μL is added to column 12. Two μL of the test compound (100 μL in 100% DMSO) is added to column 12, and the sample is mixed three times by pipetting. Then, a doubling dilution is prepared across the plate by serial transfer into adjacent wells. 50 μL of Z-Arg-7-amido-4-methylcoumarin (“Z-Arg-AMC;” 40 μM in buffer) is added to all wells, and the contents are mixed. The reaction is monitored for AMC fluorescence for 15 min at 25° C., and the progress curves are automatically converted to rates by the Fluoroskan Ascent software. The IC50of a compound can then be determined by constructing a dose-response curve and examining the effect of different concentrations of the compound on reversing the activity of the enzyme. From the dose-response curve, IC50values can be calculated for a given compound by determining the concentration needed to inhibit half of the maximum biological response of the enzyme.

The method can also be used to assay enzymes including Kgp, trypsin, and cathepsin B. For Kgp, the substrate can be succinyl-Ala-Phe-Lys-AMC. For trypsin, the buffer can contain 10 mM Tris and 10 mM CaCl2(pH 8.0), and the substrate can be Z-Gly-Gly-Arg-AMC. For cathepsin B, the buffer can contain 50 mM sodium phosphate, 1 mM EDTA, and 10 mM 2-mercaptoethanol (pH 6.25), and the substrate can be Z-Arg-Arg-AMC.

In general, the Rgp IC50values for compounds of the invention range from about 0.01 nM to about 100 μM. The Rgp IC50value for a compound of the invention can range, for example, from about 0.01 nM to about 0.1 nM, or from about 0.1 nM to about 1 nM, or from about 1 nM to about 100 nM, or from about 100 nM to about 250 nM, or from about 250 nM to about 500 nM, or from about 500 nM to about 750 nM, or from about 750 nM to about 1 μM, or from about 1 μM to about 10 μM, or from about 10 μM to about 25 μM, or from about 25 μM to about 50 μM, or from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. The Rgp IC50value for a compound of the invention can range from about 0.01 nM to about 1 nM, or from about 0.05 nM to about 0.75 nM, or from about 0.1 nM to about 0.5 nM, from about 1 nM to about 100 nM, or from about 20 nM to about 80 nM, or from about 40 nM to about 60 nM, or from about 1 μM to about 100 μM, or from about 20 μM to about 80 μM, or from about 40 μM to about 60 μM.

In some embodiments, an Rgp inhibitor according to the invention has an RgpB IC50of 75 nM or less. In some embodiments, the Rgp inhibitor has an RgpB IC50of 50 nM or less. In some embodiments, the Rgp inhibitor has an RgpB IC50of 25 nM or less. In some embodiments, the Rgp inhibitor has an RgpB IC50of 10 nM or less. In some embodiments, the Rgp inhibitor has an RgpB IC50of 1 nM or less.

In certain embodiments, Rgp inhibitors according to the invention are selective for Rgp. As used herein, a “selective” Rgp inhibitor is a compound that does not substantially affect the activity of proteases other than RgpA and RgpB when administered at a therapeutically effective dose for treating a disease or condition associated withP. gingivalisinfection. Typically, a protease that is not substantially affected by a particular compound exhibits at least 90% of its normal enzymatic activity in the presence of the compound under physiological conditions. Selective Rgp inhibitors include those compounds that do not affect the activity of proteases other than Rgp when administered at a therapeutically effective dose for treating a brain disorder, periodontal disease, diabetes, a cardiovascular disease, arthritis, rheumatoid arthritis, osteoarthritis, infectious arthritis, psoriatic arthritis, preterm birth, pneumonia, cancer, a kidney disease, a liver disease, a retinal disorder, or glaucoma associated withP. gingivalisinfection. Preferably, selective Rgp inhibitors do not adversely affect the coagulation cascade when administered at therapeutically effective levels.

IV. Pharmaceutical Compositions and Administration of Gingipain Inhibitors

In a related embodiment, the invention provides a pharmaceutical composition comprising a compound of Formula I, Formula IIa, Formula IIb, Formula III, Formula IVa, Formula IVb, Formula IVc, or Formula V, and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be prepared by any of the methods well known in the art of pharmacy and drug delivery. In general, methods of preparing the compositions include the step of bringing the active ingredient into association with a carrier containing one or more accessory ingredients. The pharmaceutical compositions are typically prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. The compositions can be conveniently prepared and/or packaged in unit dosage form.

Pharmaceutical compositions containing compounds of the invention can be formulated for oral use. Suitable compositions for oral administration include, but are not limited to, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs, solutions, buccal patches, oral gels, chewing gums, chewable tablets, effervescent powders, and effervescent tablets. Compositions for oral administration can be formulated according to any method known to those of skill in the art. Such compositions can contain one or more agents selected from sweetening agents, flavoring agents, coloring agents, antioxidants, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Tablets generally contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, including: inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as corn starch and alginic acid; binding agents, such as polyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG), starch, gelatin, and acacia; and lubricating agents such as magnesium stearate, stearic acid, and talc. The tablets can be uncoated or coated, enterically or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Tablets can also be coated with a semi-permeable membrane and optional polymeric osmogents according to known techniques to form osmotic pump compositions for controlled release.

Compositions for oral administration can be formulated as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium (such as peanut oil, liquid paraffin, or olive oil).

Rgp inhibitors can also be administered topically as a solution, ointment, cream, gel, or suspension, as well as in mouth washes, eye-drops, and the like. Still further, transdermal delivery of Rgp inhibitors can be accomplished by means of iontophoretic patches and the like.

Pharmaceutical compositions containing Rgp inhibitors can also be in the form of a sterile injectable aqueous or oleaginous solutions and suspensions. Sterile injectable preparations can be formulated using non-toxic parenterally-acceptable vehicles including water, Ringer's solution, and isotonic sodium chloride solution, and acceptable solvents such as 1,3-butane diol. In addition, sterile, fixed oils can be used as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic monoglycerides, diglycerides, or triglycerides.

In some embodiments, an Rgp inhibitor can be formulated with a polymer such as Pluronic F127 and delivered subcutaneously. Pluronic is a hydrogel that solidifies at body temperature and can provide extended drug delivery over periods of time lasting from days to weeks.

Aqueous suspensions can contain one or more Rgp inhibitors in admixture with excipients including, but not limited to: suspending agents such as sodium carboxymethylcellulose, methylcellulose, oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin, polyoxyethylene stearate, and polyethylene sorbitan monooleate; and preservatives such as ethyl, n-propyl, and p-hydroxybenzoate. Dispersible powders and granules (suitable for preparation of an aqueous suspension by the addition of water) can contain one or more Rgp inhibitors in admixture with a dispersing agent, wetting agent, suspending agent, or combinations thereof. Oily suspensions can be formulated by suspending an Rgp inhibitor in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil), or in a mineral oil (e.g., liquid paraffin). Oily suspensions can contain one or more thickening agents, for example beeswax, hard paraffin, or cetyl alcohol. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, such as gum acacia or gum tragacanth; naturally-occurring phospholipids, such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

The use of hybrid molecules to promote active transport or nanoparticles can be used in certain embodiments to increase blood brain barrier transport. For example liposomes, proteins, engineered peptide compounds or antibodies that bind to the receptors that transport proteins across the blood brain barrier including LPR-1 receptor, transferrin receptor, EGF-like growth factor or glutathione transporter can be used to increase penetration into the brain. Physical techniques including osmotic opening, ultrasound, lasers, sphenopalantine ganglion stimulation, direct intracranial, intrathecal, or intraventricular delivery via a pump can be used.

Pharmaceutical compositions according to the invention can also include one or more additional active agents useful in the treatment of conditions associated withP. gingivalisinfection. In certain embodiments, the invention provides a pharmaceutical composition comprising one or more Rgp inhibitors as described herein in combination with one or more additional active agents for treatment of Alzheimer's disease. Several therapeutics are in development and in clinical use for treatment of Alzheimer's disease. Therapeutic strategies include lowering circulating levels of β-amyloid and tau (as described in more detail below), stabilizing microtubules, removing atherosclerotic plaques, modulating autophagy, modulating neurotransmitter levels, and inhibiting GABA(A) α5 receptors. Such therapeutics can maintain and/or restore cognitive function in subjects with Alzheimer's disease; slow the decline of cognitive function; and promote neuroplasticity and recovery of the brain.

Any suitable antibiotic can be combined with one or more Rgp inhibitors in the pharmaceutical compositions of the invention. In certain embodiments, the invention provides a pharmaceutical composition containing one more Rgp inhibitors and an antibiotic having aP. gingivalisMIC50of less than 25 μg/ml. For example, theP. gingivalisMIC50of the antibiotic can be less than 20 μg/ml, less than 15 μg/ml, less than 10 μg/ml, less than 8 μg/ml, less than 6 μg/ml, or less than 5 μg/ml. In some embodiments, theP. gingivalisMIC50of the antibiotic is less than 1 μg/ml. In some embodiments, theP. gingivalisMIC50of the antibiotic is less than 0.2 μg/ml.

Examples of bacteriocidal and bacteriostatic compounds include, but are not limited to: quinolones (e.g., moxifloxacin, gemifloxacin, ciprofloxacin, of laxacin, trovafloxacin, sitafloxacin, and the like), β-lactams (e.g., penicillins such as amoxicillin, amoxacilin-clavulanate, piperacillin-tazobactam, penicillin G, and the like; and cephalosporins such as ceftriaxone and the like), macrolides (e.g., erythromycin, azithromycin, clarithromycin, and the like), carbapenems (e.g., doripenem, imipenem, meropinem, ertapenem, and the like), thiazolides (e.g., tizoxanidine, nitazoxanidine, RM 4807, RM 4809, and the like), tetracyclines (e.g., tetracycline, minocycline, doxycycline, eravacycline, and the like), clindamycin, metronidazole, and satranidazole. Bacteriocidal and bacteriostatic compounds also include agents that inhibit or otherwise interfere with formation of biofilms by anaerobic, gram-negative bacteria; such agents include oxantel, morantel, thiabendazole, and the like. Compositions of the invention can contain one or more Rgp inhibitors with one or more (e.g., two, three, four, five, six, or more) bacteriocidal/bacteriostatic compounds. Compositions containing bacteriocidal/bacteriostatic compounds can further contain a chlorhexidine (e.g., chlorhexidine digluconate) alone or in combination with a zinc compound (e.g., zinc acetate), can also be used in combination with the administered antibiotics.

In some embodiments, a combination of a penicillin (e.g., amoxicillin) and metronidazole or a combination of penicillin (e.g., amoxicillin), metronidazole and a tetracycline is used. In some embodiments, the antibiotic is selected from minocycline, doxycycline, metronidazole, amoxicillin, clindamycin, augmentin, satranidazole, and combinations thereof.

Examples of suitable cholinesterase inhibitors include, but are not limited to, donepezil, donepezil/memantine, galantamine, rivastigmine, and tacrine, as well as pharmaceutically acceptable salts thereof. Examples of suitable serotonin modulators include, but are not limited to, idalopirdine, RVT-101, citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline, as well as pharmaceutically acceptable salts thereof. Examples of suitable alpha-7 nicotinic receptor modulators include, but are not limited to, alpha-7 agonists such as encenicline and APN1125. Suitable NMDA modulators include, but are not limited to, NMDA receptor antagonists such as memantine and derivatives thereof.

Pharmaceutical compositions of the invention can also contain active agents that are directed to biomolecular targets associated with neurological diseases. Such targets include beta amyloid peptides (also referred to as beta amyloid, abeta, or Aβ), apolipoprotein E (also referred to as ApoE), and microtubule-associated tau (also referred to as tau proteins, or simply as tau).

Examples of ApoE-targeted therapies include, but are not limited to retinoid X receptor agonists (see, Cramer, et al.,Science2012. 335(6075): 1503-1506) and others described by Liu et al. (Nat Rev Neurol.2013. 9(2): 106-118). Tau-targeted therapies include, but are not limited to, methylthioninium, leuco-methylthioninium, antibodies and those described by Lee, et al. (Cold Spring Harb Perspect Med2011; 1:a006437).

Pharmaceutical compositions of the invention can also contain complement-targeted therapies. Such therapies target components of the complement system involved in the innate immune response. Complement targeted therapies include, but are not limited to, those described by Ricklin and Lambris (Nat. Biotechnology2007. 25(11): 1265-1275).

Examples of suitable anti-inflammatories include, but are not limited to, NSAIDs such as apazone, diclofenac, ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, piroxicam, and sulindac, as well as pharmaceutically acceptable salts thereof.

V. Methods for Inhibiting Gingipains and Treating Conditions Associated with P.GingivalisInfection

In another embodiment, the invention provides a method of inhibiting a gingipain. The method includes contacting the gingipain with an effective amount of a compound as described herein. In certain embodiments, the gingipain is an arginine gingipain (e.g., RgpA, RgpB, or a variant containing one or more amino acid substitutions, deletions, and/or other peptide sequence variations). Inhibiting the gingipain generally includes contacting the gingipain with an amount of the compound sufficient to reduce the activity of the gingipain as compared to the gingipain activity in the absence of the compound. For example, contacting the gingipain with the gingipain inhibitor can result in from about 1% to about 99% gingipain inhibition (i.e., the activity of the inhibited gingipain ranges from 99% to 1% of the gingipain activity in the absence of the compound). The level of gingipain inhibition can range from about 1% to about 10%, or from about 10% to about 20%, or from about 20% to about 30%, or from about 30% to about 40%, or from about 40% to about 50%, or from about 50% to about 60%, or from about 60% to about 70%, or from about 70% to about 80%, or from about 80% to about 90%, or from about 90% to about 99%. The level of gingipain inhibition can range from about 5% to about 95%, or from about 10% to about 90%, or from about 20% to about 80%, or from about 30% to about 70%, or from about 40% to about 60%. In some embodiments, contacting the gingipain with a compound as described herein will result in complete (i.e., 100%) gingipain inhibition.

Extracellular proteases produced byP. gingivalis, including Arginine Gingipain A (RgpA), Arginine Gingipain B (RgpB), and Lysine Gingipain (Kgp), can also degrade a broad range of proteins in connective tissue and plasma (e.g., collagen, immunoglobulins, and proteinase inhibitors, etc.). Gingipains can enter systemic circulation and/or synoviocytes and chondrocytes, and they can also cause disruption to the kallikrein-kinin cascade, blood coagulation, and host defense systems. Patients with gingipains in their joints and circulatory system may be subject to gingipain-induced death of synovial cells and/or chondrocytes, contributing to osteoarthritis.

It has recently been discovered that RgpB and Kgp can infiltrate human and dog joints, contributing to the development of osteoarthritis. It is believed thatP. gingivalisand gingipains can infiltrate joint tissues via a number of routes. Gingipains can be secreted, transported to outer membrane surfaces ofP. gingivalis, or released in outer membrane vesicles by the bacterium.P. gingivalishas previously been identified in periodontal tissues, coronary arteries, aorta, and recently, the liver-release ofP. gingivalisand/or gingipains from any of these niches into the systemic circulation could result in translocation ofP. gingivalisand/or gingipains to the joints. See: Travis, et al.Adv Exp Med Biol,2000. 477: 455-65; Byme, et al.Oral Microbiol Immunol,2009. 24(6): 469-77; Mahendra, et al.J Maxillofac Oral Surg,2009. 8(2): 108-13; Stelzel. Periodontol, 2002. 73(8): 868-70; Ishikawa, et al.Biochim Biophys Acta,2013. 1832(12): 2035-2043.

P. gingivalisand/or gingipains may also enter joints by degrading the endothelial cells protecting the blood/joint barrier, or by a traumatic event to the joint, such as a meniscus injury, which permanently or transiently reduces the integrity of the joint tissues. Such a disruption in traumatic joint injury for example, may contribute to the infiltration ofP. gingivalisand/or gingipains in infected individuals and subsequent development of chronic osteoarthritis. People who are at a high risk of traumatic joint injury, including athletes in contact sports like football, could be preventatively treated with gingipain inhibitors to reduce the risk of trauma-related osteoarthritis.

P. gingivalisand gingipains may also reach the joint through other mechanisms including active transport, passive transport or macrophage delivery. Osteoarthritis resulting from any of these mechanisms can be limited to a single joint or present in multiple joints.

Similar to humans,P. gingivalisinfection and periodontal disease is one of the most common infectious diseases affecting adult dogs and cats. Dogs and cats withP. gingivalisinfection and gingipains in their joints and circulatory system may experience periodontal disease and osteoarthritis due to gingipain-induced cell death, which could be treated or prevented according to the methods of the invention. Aged dogs spontaneously develop many features of osteoarthritis, including a common inflammatory knee arthritis associated with degeneration of the anterior cruciate ligament (ACL). A study by Muir et al. of dogs with inflammatory knee arthritis and ACL degeneration detected DNA from a range of bacterial species in 37% of knee joints from affected dogs. Muir et al. hypothesized that bacteria may be an important causative factor in the pathogenesis of inflammatory arthritis in dogs. In the Muir et al. study, DNA fromP. gingivaliswas not detected in the dog joints. See, Muir, et al.Microb Pathog,2007. 42(2-3): 47-55. However, similar to humans,P. gingivalisis a common oral pathogen affecting adult dogs, and could potentially translocate from the oral cavity to joint tissues as a result of bacteremia.P. gingivalishas been demonstrated to infect chondrocytes in vitro causing chondrocyte apoptosis, indicating a pathway for cartilage loss in osteoarthritis of both dogs and humans. See: Rohner, et al.Calcif Tissue Int,2010. 87(4): p. 333-40; Houle, et al.FEMSMicrobiol Lett,2003. 221(2): p. 181-5; Kataoka, et al.FASEB J,2014. 28: 3564-3578; Pischon, et al.Ann Rheum Dis,2009. 68(12): p. 1902-7.

Rgp inhibitors can therefore be used to treat diseases and conditions, such as brain disorders, caused by or otherwise affected byP. gingivalis. Accordingly, another aspect of the invention provides a method of treating a disease or condition associated withP. gingivalisinfection. The method includes administering an effective amount of a compound or a composition of the invention, as described above, to a subject in need thereof.

In certain embodiments, compounds of the invention inhibit active Rgp in the brain of a mammal, e.g., a human or an animal (e.g., a dog), and are cytoprotective or neuroprotective. By “neuroprotective,” it is meant that the compounds prevent aberrant changes to neurons or death of neurons. Compounds of the invention are therefore useful, e.g., in treatment of a brain disorder (e.g., a neurodegenerative disease (e.g., Alzheimer's disease, Down's syndrome, epilepsy, autism, Parkinson's disease, essential tremor, fronto-temporal dementia, progressive supranuclear palsy, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, mild cognitive impairment, age associated memory impairment, chronic traumatic encephalopathy, stroke, cerebrovascular disease, Lewy Body disease, multiple system atrophy, schizophrenia and depression, etc.), diabetes, cardiovascular disease, arthritis, rheumatoid arthritis, osteoarthritis, infectious arthritis, psoriatic arthritis, retinal disorders (e.g., age related macular degeneration) and glaucoma.

In some embodiments, the disease or condition is selected from a brain disorder, periodontal disease, diabetes, a cardiovascular disease, arthritis, rheumatoid arthritis, osteoarthritis, preterm birth, pneumonia, cancer, a kidney disease, a liver disease, a retinal disorder, and glaucoma.

In some embodiments, the disease or condition is a brain disorder.

In some embodiments, the brain disorder is Alzheimer's disease.

In some embodiments, the method further includes administering to the subject one or more active agents selected from a cholinesterase inhibitor, a serotonin modulator, an NMDA modulator, an Aβ targeted therapy, an ApoE targeted therapy, a microglia targeted therapy, a blood brain barrier targeted therapy, a tau targeted therapy, a complement targeted therapy, and an anti-inflammatory.

In some embodiments, the disease or condition is periodontal disease. In some embodiments, the disease or condition is a liver disease. In some embodiments, the liver disease is non-alcoholic steatohepatitis. In some embodiments, the disease or condition is a retinal disorder. In some embodiments, the retinal disorder is age-related macular degeneration.

In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is breast cancer, oral cancer, pancreatic cancer, or glioblastoma multiforme.

Rgp inhibitors as described herein can be administered at any suitable dose in the methods of the invention. In general, an Rgp inhibitor is administered at a dose ranging from about 0.1 milligrams to about 1000 milligrams per kilogram of a subject's body weight (i.e., about 0.1-1000 mg/kg). The dose of Rgp inhibitor can be, for example, about 0.1-1000 mg/kg, or about 1-500 mg/kg, or about 25-250 mg/kg, or about 50-100 mg/kg. The dose of Rgp inhibitor can be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg/kg. The dosages can be varied depending upon the requirements of the patient, the severity of the disorder being treated, and the particular formulation being administered. The dose administered to a patient should be sufficient to result in a beneficial therapeutic response in the patient. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of the drug in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the typical practitioner. The total dosage can be divided and administered in portions over a period of time suitable to treat to the disease or condition.

Rgp inhibitors can be administered for periods of time which will vary depending upon the nature of the particular disorder, its severity, and the overall condition of the subject to whom the Rgp inhibitor is administered. Administration can be conducted, for example, hourly, every 2 hours, three hours, four hours, six hours, eight hours, or twice daily including every 12 hours, or any intervening interval thereof. Administration can be conducted once daily, or once every 36 hours or 48 hours, or once every month or several months. Following treatment, a subject can be monitored for changes in his or her condition and for alleviation of the symptoms of the disorder. The dosage of the Rgp inhibitor can either be increased in the event the subject does not respond significantly to a particular dosage level, or the dose can be decreased if an alleviation of the symptoms of the disorder is observed, or if the disorder has been remedied, or if unacceptable side effects are seen with a particular dosage.

A therapeutically effective amount of an Rgp inhibitor can be administered to the subject in a treatment regimen comprising intervals of at least 1 hour, or 6 hours, or 12 hours, or 24 hours, or 36 hours, or 48 hours between dosages. Administration can be conducted at intervals of at least 72, 96, 120, 144, 168, 192, 216, or 240 hours (i.e., 3, 4, 5, 6, 7, 8, 9, or 10 days). In certain embodiments, administration of one or more Rgp inhibitors is conducted in a chronic fashion over periods ranging from several months to several years. Accordingly, some embodiments of the invention provide a method of treating a disease or condition associated withP. gingivalisinfection as described above, wherein the compound is administered to the subject for at least one year. In some embodiments, the compound is administered to the subject for at least 10 years. In some embodiments, the compound is administered to the subject for at least 60 years.

Administration of Rgp inhibitors according to the methods of the invention typically results in the reduction of circulating levels of active Rgp in a subject and/or the reduction of active Rgp in the brain. In certain embodiments, administration of an Rgp inhibitor according to the methods of the invention results in at least a 20% reduction of circulating levels of active Rgp and/or at least a 20% reduction of active Rgp in the brain. For example, the circulating levels of Rgp and/or the levels of Rgp in the brain are preferably reduced by from about 25% to about 95%, or from about 35% to about 95%, or from about 40% to about 85%, or from about 40% to about 80% as compared to the corresponding levels of Rgp 24 hours prior to the first administration of the Rgp inhibitor.

Rgp inhibitors can be administered alone or in combination with one or more additional therapeutically active agents, as described above. The one or more additional therapeutically effective agents include, e.g.; (i) a pharmaceutically acceptable agent which inhibits RgpA, RgpB, and/or Kgp production, translocation of RgpA, RgpB, and/or Kgp into systemic circulation or brain, and/or pathological (e.g., neurotoxic effects) of RgpA, RgpB, and/or Kgp in a mammal; (ii) an antibacterial agent which is bacteriostatic or bacteriocidal with respect toP. gingivalis; (iii) one or more antibodies which bind to RgpA, RgpB and/or Kgp (e.g., 18E6, which binds to the first half of the immunoglobulin domain of RgpB; Kgp-specific monoclonal antibody, 7B9, which recognizes an epitope within the Kgp catalytic domain; the RgpA antibody 61Bg 1.3, humanized versions of any of the foregoing, etc.); (iv) epitopes of antibodies which bind to RgpA, RgpB and/or Kgp or other proteins expressed byP. gingivalis; and (v) combinations of any of the foregoing.

The additional therapeutically active agents also include Aβ peptides level reducers, pathogenic level tau reducers, microtubule stabilizers, agents capable or removing atherosclerotic plaques, agents that lower circulating levels of β-amyloid and tau, modulators of autophagy, neurotransmitter level regulators, GABA(A) a5 receptors inhibitors, and additional agents that help maintain and/or restore cognitive function and functional deficits of Alzheimer's disease, and/or slow down decline in cognitive functions and functional deficits in Alzheimer's disease.

Pharmaceutical compositions of the invention can contain one or more Rgp inhibitors as described herein in combination with ritonavir (RTV), which can increase bioavailability and increase blood brain barrier penetration. For example, ritonavir is commonly combined with oral peptidic HIV protease inhibitors to increase plasma levels by inhibiting the P450 3A4 enzyme and thus decreasing first-pass metabolism (see, Walmsley, et al.,N Engl J Med,2002. 346(26): 2039-46). In addition, RTV binds to P-glycoprotein, a transmembrane efflux pump that is found in many tissues, including the blood brain barrier, allowing co-administered compounds better access to the brain (see, Marzolini, et al.,Mol Pharm,2013. 10(6): 2340-9). Therefore, a combination of RTV and Rgp inhibitors can be used to increase plasma concentrations and brain levels of the gingipain inhibitors. As described in U.S. patent application Ser. No. 14/875,416, oral administration of RTV 15 minutes prior to the Kgp inhibitor Kyt-36 increases the half-life therefore it is expected that RTV will also increase the half-life of other gingipain inhibitors.

In some embodiments, compounds of the invention can be administered with natural gingipain inhibitors including melabaricone C, isolated from nutmeg or polyphenolic compounds derived from plants, such as cranberry, green tea, apple, and hops can be administered in conjunction for treatment or prevention of brain disorders. Naturally and unnaturally occurring antimicrobial peptides including: K-casein peptide (109-137) 34, histatin 5, and CL(14-25), CL(K25A) and CL(R24A, K25A), can also be administered in conjunction with the Rgp inhibitors of the invention. (see, e.g., Taniguchi et al.,Biopolymers,2014. 102(5): 379-89).

Rgp inhibitors as described herein can be administered with antibodies targeting gingipains or otherP. gingivalisproteins. Antibodies may rely on damage to the blood brain barrier for access to the brain or peripheral interference with gingipains andP. gingivalispropagation. Antibodies can also help to stimulate the efficacy of the immune system in clearing the bacteria. New or existing antibodies to RgpA, RgpB, or Kgp can be utilized including 18E6 and 7B9. An RgpA antibody 61BG 1.3 has previously demonstrated efficacy topically in prevention of recolonization byP. gingivalisafter periodontal treatment. See, Booth et al.,Infect Immun,1996. 64(2): 422-7. Antibodies would preferably be humanized for use in humans. Methods known to those in the field for delivery of biologics to improve half-life and brain penetration can be used including, but not limited to, intravenous delivery, subcutaneous delivery, intranasal delivery, intrathecal delivery, intra-articular delivery, vector transport, and direct brain delivery.

The methods of the invention also encompass administration of Rgp inhibitors as described herein with one or more of the following additional therapeutically active agents or pharmaceutically acceptable salts thereof: an arginine derivative; histatin 5; baculovirus p35; a single point mutant of cowpox viral cytokine-response modifier (CrmA (Asp>Lys)); phenylalanyl-ureido-citrullinyl-valyl-cycloarginal (FA-70C1); (acycloxy)methyl ketone (Cbz-Phe-Lys-CH2OCO-2,4,6-Me3Ph); peptidyl chloro-methyl ketones (e.g., chloromethyl ketone derivatives of arginine, chloromethyl ketone derivatives of lysine, and the like); fluoro-methyl ketones; bromo-methyl ketones; ketopeptides; 1-(3-phenylpropionyl)piperidine-3(R,S)-carboxylic acid [4-amino-1 (S)-(benzothiazole-2-carbonyl)butyl]amide (A71561); azapeptide fumaramide; aza-peptide Michael acceptors; benzamidine compounds; acyclomethylketone; activated factor X inhibitors (e.g., DX-9065a); cranberry nondialyzable fraction; cranberry polyphenol fraction; pancreatic trypsin inhibitor; Cbz-Phe-Lys-CH2O—CO-2,4,6-Me3-Ph; E-64; chlorhexidine; zinc (e.g., zinc acetate); or a combination of two, three or more of any of foregoing. In some of these embodiments, Zn can enhance potency and selectivity of the compounds (e.g., chlorhexidine, benzamidine, etc.) used in the methods of the invention.

An Rgp inhibitor of the invention can be administered in the same composition as an additional therapeutically active agent. Alternatively, the additional therapeutically active agent can be administered separately before, concurrently with, or after administration of the Rgp inhibitor.

To a solution of compound 1.5 (3 g, 12.4 mmol, 1 eq) in NMP (30 mL) was added NH3.H2O (30 mL). The mixture was stirred 150° C. for 15 hours. The reaction mixture was quenched by addition H2O 100 mL at 25° C., and then extracted with EtOAC (100 mL×3). The combined organic layers were washed with saturated brines (15 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1). Compound 1.4 (2.0 g, 9 mmol, 72.42% yield) was obtained as a brown oil. LCMS (ESI): m/z: [M+H] calcd for C9H7N2Br: 223; found 223; RT=1.049 min.

To a mixture of compound 3.5 (700 mg, 3.48 mmol, 1 eq) and 4-methylbenzenesulfonyl chloride (796.15 mg, 4.18 mmol, 1.20 eq) in DCM (15 mL) was added DMAP (68.02 mg, 556.80 μmol, 0.16 eq) and TEA (528.21 mg, 5.22 mmol, 723.58 L, 1.50 eq) in one portion at 0° C. under N2. The mixture was then heated to 25° C. and stirred for 10 hours. The reaction mixture was diluted with H2O 20 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layers were washed with brine 20 mL (20 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 3.4

To a solution of compound 3.3 (443.30 mg, 4.03 mmol, 1.10 eq) in DMF (20 mL) was added NaH (131.76 mg, 5.49 mmol, 1.50 eq) portionwise at 25° C. under N2. The mixture was stirred at 25° C. for 30 mins, then was added compound 3.4 (1.30 g, 3.66 mmol, 1 eq). The mixture was heated to 60° C. and stirred for 9.5 hours. The reaction mixture was diluted with H2O 20 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layers were washed with brine 20 mL (20 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH=20:1) to give compound 3.2 (200 mg, 681.76 μmol, 18.63% yield) as a colorless oil. LCMS (ESI): m/z: [M+H] calcd for C15H23N3O3: 293; found 294; RT=0.663 min.

To a solution of compound 4.7 (500 mg, 3.26 mmol, 1 eq) in PhOH (2.45 g, 26.05 mmol, 2.29 mL, 8 eq) was added KOH (365.37 mg, 6.51 mmol, 2 eq) at 25° C. under N2. The resulting mixture was stirred at 140° C. for 16 hrs. TLC (PE:EtOAc=5:1, Rf=0.49) showed the reaction was successful. The reaction mixture was added water (10 mL), extracted with EtOAc (15 mL×3). The organic phase was separated, washed with saturated NaCl (15 mL) and dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue of compound 4.6 (0.5 g, crude) as a yellow oil which was combined with a second batch for a total of 1.5 g.

To a mixture of compound 4.6 (1.5 g, 7.10 mmol, 1 eq) in NH4OAc (12.50 g, 162.20 mmol, 22.84 eq) in one portion under N2. The mixture was stirred at 140° C. for 12 hours, then heated to 180° C. and stirred for 12 hours. The mixture was allowed to cool to ambient temperature, after which 3 N sodium hydroxide (20 ml) was added with stirring. The thus obtained solution was extracted with ethyl acetate (2×10 ml) and the combined organic layers were extracted with 2 N hydrochloric acid (20 ml). Subsequently, the pH of the aqueous layer was adjusted to 12 with 2 N sodium hydroxide. Extraction with ethyl acetate (20 ml) then afforded an organic layer, which was washed with brine (10 ml), dried and concentrated under reduced pressure to give compound 4.5 (900 mg, 6.71 mmol, 94.48% yield) was obtained as light yellow solid. LCMS (ESI): m/z: [M+H] calcd for C7H6N20: 135; found 135; RT=0.199 min.

To a mixture of compound 4.4 (500 mg, 2.10 mmol, 1 eq) in THF (3 mL) was added n-BuLi (2.5 M, 2.10 mL, 2.5 eq) in one portion at −78° C. under N2. The mixture was stirred at −78° C. for 1 h, then was added DMF (613.58 mg, 8.39 mmol, 645.87 μL, 4 eq) dropwise and stirred at −78° C. for 1 h. TLC (PE:EtOAc=3:1, Rf=0.4) showed the reaction was successful. The reaction mixture was quenched by addition H2O (20 mL), and extracted with EtOAc (10 mL×3). The combined organic layers were washed with saturated brines (10 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product of compound 4.3 (0.24 g, crude) as white solid.

To a solution of compound 5.6 (24 g, 63.42 mmol, 1 eq) in MeOH (1.50 L) was added Pd—C under Ar2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 24 hours. Pd/C was filtered off by filtration. The filtrate was concentrated to dry. Compound 5.5 (22 g, 57.83 mmol, 91.19% yield) was obtained as a white solid.

To a mixture of compound 5.4 (2 g, 7.51 mmol, 1 eq) and DPPA (2.48 g, 9.01 mmol, 1.95 mL, 1.20 eq) in dioxane (20 mL) was added TEA (1.52 g, 15.02 mmol, 2.08 mL, 2 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 min, then heated to 90° C. and stirred for 2 hours. Then the mixture was added dropwise into a solution of NaOH (1.20 g, 30.04 mmol, 4 eq) and stirred for 18 hours at 25° C. The pH was adjusted to around 6 by progessively adding 1M HCl, then the mixture was partitioned between EtOAc (30) and water (30). The combined organic layers were washed with brine 30 mL (30 mL×2), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 5.3 (800 mg, 3.37 mmol, 44.89% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C12H19N3O2: 237; found 294; RT=1.547 min.

To a solution of compound 6.7 (24 g, 63.42 mmol, 1 eq) in MeOH (1.50 L) was added Pd—C under Ar2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 24 hours. Pd/C was filtered off by filtration. The filtrate was concentrated to dry. Compound 6.6 (22 g, 57.83 mmol, 91.19% yield) was obtained as a white solid.

To a solution of compound 6.3 (670 mg, 2.18 mmol, 1 eq) in THF (10 mL) was added a solution of BH3-Me2S (10 M, 872 μL, 4 eq) dropwise with at 0° C. over a period of 1 mins under N2. Then stirring at 25° C. 4 hours. The reaction was quenched by addition of 6 ml methanol, then the reaction mixture was concentrated under reduced pressure to remove solvent. The mixture was further purification by pre-HPLC to give compound 6.2 (70 mg, 238.58 μmol, 10.94% yield) (70 mg, 238.58 μmol, 10.94% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C16H27N3O2: 294; found 294; RT=0.735 min.

To a mixture of compound 6.2 (70 mg, 177.88 μmol, 1 eq) in HCl/EtOAc (2 mL) at 25° C. for 13 hours. The reaction mixture was concentrated under reduced pressure to give compound 6.1 (65 mg, crude) as light yellow oil.

Compound 12.7 (2 g, 9.29 mmol, 1 eq) was added into HCl/EtOAc (20 mL), the reaction was stirred for 15 hours at 18° C. The reaction mixture was concentrated under reduced pressure to give compound 12.6 (2.10 g, crude) as a white solid.

To a mixture of compound 12.6 (2.10 g, 11.16 mmol, 1 eq, 2HCl) and TEA (3.39 g, 33.48 mmol, 4.64 mL, 3 eq) in DCM (25 mL) was added compound 12.5 (5.19 g, 23.44 mmol, 2.10 eq) in one portion at 0° C. The mixture was then heated to 18° C. and stirred for 15 hours. The reaction mixture was quenched by addition H2O 20 mL and then diluted with DCM 10 mL and extracted with DCM 20 mL (20 mL×3). The combined organic layers were washed with brine 40 mL (40 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 12.4 (4.50 g, crude) as a yellow solid. LCMS (ESI): m/z: [M−H] calcd for C18H19N3O9S2: 485; found 486; RT=0.869 min.

To a solution of compound 14.6 (1.50 g, 3.65 mmol, 1 eq) in MeOH (20 mL) was added NaOH (438 mg, 10.95 mmol, 3 eq) at 25° C. under N2. The resulting mixture was stirred at 25° C. for 16 hours. The reaction mixture was added water (30 mL), extracted with EtOAc (50 mL×3). The organic phase was separated, washed with saturated NaCl (30 mL) and dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=20:1 to 3:1). Compound 14.5 (550 mg, 2.62 mmol, 71.68% yield) was obtained as a white solid.

To a mixture of compound 16.6 (2 g, 8.72 mmol, 1 eq) in THF (20 mL) was added LAH (661.85 mg, 17.44 mmol, 2 eq) in one portion at 0° C. under N2. The mixture was stirred at 0° C. for 2 hours. After the reaction mixture was cooled to 0° C., the reaction mixture was quenched by addition of 5 mL of H2O, followed by 2 mL of 15% aqueous NaOH. After being stirred at room temperature for 10 mins, the solid was removed by filtration. The filtrate was concentrated to dryness to give crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/1 to 2:1). Compound 16.5 (1.38 g, 6.41 mmol, 73.51% yield) was obtained as a colorless oil.

To a mixture of compound 16.4 (300 mg, 1.08 mmol, 1 eq) and compound 16.3 (118.92 mg, 1.08 mmol, 1 eq) in DMF (10 mL) was added K2CO3(298.53 mg, 2.16 mmol, 2 eq) in one portion at 15° C. under N2. The mixture was stirred at 80° C. for 2 hours. The aqueous phase was extracted with ethyl acetate (10 mL×3). The combined organic phase was washed with brine (5 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. Compound 16.2 (70 mg, crude) was obtained as black brown oil. It was combined with a second batch for a total of 140 mg, crude. LCMS (ESI): m/z: [M+H] called for C16H25N3O3: 308; found 308; RT=0.689 min.

To a mixture of compound 17.6 (3 g, 11.75 mmol, 1 eq) in THF (60 mL) was added LAH (2.23 g, 58.75 mmol, 5 eq) in one portion at 0° C. under N2. The mixture was stirred at 0° C. for 2 hours. After the reaction mixture was cooled to 0° C., the reaction mixture was quenched by addition of 50 mL of H2O, followed by 10 mL of 15% aqueous NaOH. After being stirred at room temperature for 10 mins, the solid was removed by filtration. The filtrate was concentrated to dryness to give crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10:1 to 4:1). Compound 17.5 (1.07 g, 5.02 mmol, 42.70% yield) was obtained as a colorless oil.

To a solution of compound 17.5 (670 mg, 3.14 mmol, 1 eq) in MeOH (10 mL) was added Pd/C (200 mg, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(15 psi) at 15° C. for 3 hours. The reaction mixture was filtered and the filter was concentrated. Compound 17.4 (500 mg, 2.32 mmol, 73.96% yield) was obtained as a colorless oil.

To a solution of compound 18.10 (10 g, 38.71 mmol, 1 eq) in THF (200 mL) was added DIBAL-H (6.01 g, 42.58 mmol, 1.10 eq) in one portion at −78° C. under N2. The mixture was stirred at −78° C. for 2 hours. After the reaction mixture was cooled to 0° C., the reaction mixture was quenched by addition of 100 mL of H2O, followed by 15 mL of 15% aqueous NaOH. After being stirred at room temperature for 10 min, the solid was removed by filtration. The filtrate was concentrated to dryness to give crude product. Compound 18.9 (6.70 g, crude) was obtained as colorless oil.

To a solution of compound 18.7 (2 g, 7.43 mmol, 1 eq) in THF (30 mL) was added LAH (1.41 g, 37.15 mmol, 5 eq) in one portion at 0° C. under N2. The mixture was stirred at 0° C. for 15 min. After the reaction mixture was cooled to 0° C., the reaction mixture was quenched by addition of 5 mL of H2O, followed by 2 mL of 15% aqueous NaOH. After being stirred at room temperature for 10 min, the solid was removed by filtration. The filtrate was concentrated to dryness to give crude product. The residue was purified by prep-TLC (SiO2, PE:ethyl acetate=2:1). Compound 18.6 (1.08 g, 4.71 mmol, 63.39% yield) was obtained as rless oil. LCMS (ESI): m/z: [M+H] called for C12H23NO3: 230; found 230; RT=0.741 min.

To a solution of compound 19.5 (579.28 mg, 5.26 mmol, 1.10 eq) in DMF (20 mL) was added NaH (172.18 mg, 7.17 mmol, 1.50 eq) portionwise at 25° C. under N2. The mixture was stirred at 25° C. for 30 mins, then was added compound 19.3 (1.70 g, 4.78 mmol, 1 eq). The mixture was heated to 60° C. and stirred for 9.5 hours. The reaction mixture was diluted with H2O 20 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layers were washed with brine 20 mL (20 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH=20:1) to give compound 19.2 (700 mg, 2.39 mmol, 49.92% yield) as a colorless oil. LCMS (ESI): m/z: [M+H] calcd for C15H23N3O3: 293; found 294; RT=0.667 min.

To a solution of compound 21.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L) was added LiAlH4(12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. under N2. The mixture was stirred at 0° C. for 1 hours, then heated to 18° C. and stirred at 18° C. for 14 hours. The reaction mixture was quenched by addition 8% NaOH (15 ml), filtered and then diluted with H2O 1000 mL and extracted with EtOAc 1500 mL (500 mL×3). The combined organic layers were washed with brine 1000 mL (1000 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1) to give compound 21.9 (15 g, 66.89 mmol, 39.28% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C11H16N2O3: 293; found 294; RT=0.313 min.

To a mixture of compound 21.8 (2.50 g, 11.25 mmol, 1 eq) and methyl 2-dimethoxyphosphorylacetate 21.7 (2.05 g, 11.25 mmol, 1.63 mL, 1 eq) in THF (30 mL) was added K2CO3(3.11 g, 22.50 mmol, 2 eq) in one portion at 50° C. under N2. The mixture was stirred at 50° C. for 15 hours. The reaction mixture was diluted with H2O 30 mL and extracted with EtOAc 90 mL (30 mL×3). The combined organic layers were washed with brine 50 mL (50 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5:1) to give compound 21.6 (2.40 g, 8.62 mmol, 76.62% yield) as a white solid.

To a solution of DIPA (505.95 mg, 5 mmol, 702.71 μL, 2 eq) in THF (10 mL) was added n-BuLi (2.5 M, 1.50 mL, 1.50 eq) dropwise at −78° C. under N2. The mixture was then added compound 21.4 (589.84 mg, 3.75 mmol, 601.88 μL, 1.50 eq) in one portion at −78° C., the mixture was stirred at −78° C. for 30 mins, then was added compound 21.5 (700 mg, 2.50 mmol, 1 eq) in one portion at −78° C., the mixture was heated to 18° C. and stirred for 14.5 hours. The reaction mixture was quenched by addition H2O 10 mL and then diluted with EtOAc 5 mL and extracted with EtOAc 15 mL (5 mL×3). The combined organic layers were washed with brine 10 mL (10 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO2, PE:EA=2:1) to give compound 21.3 (180 mg, 539.92 μmol, 21.60% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C17H23N3O4: 333; found 334; RT=0.597 min.

Compound 21.3 (180 mg, 539.92 μmol, 1 eq) was mixed with 6 N HCl (5 mL), heated at 100° C. for 15 hours. The mixture was concentrated at reduced pressure to a syrup and then basified with 10% KOH (10 ml), the resulting two phase mixture was extracted with EtOAc 15 ml (5 ml×3) and the extract dry Na2SO4and concentrated to give compound 21.2 (90 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C16H23N3O2: 189; found 190; RT=0.097 min.

To a solution of compound 24.6 (950 mg, 3.61 mmol, 1 eq) in THF (15 mL) was added BH3-Me2S (10 M, 721.64 μL, 2 eq) at 0° C. The mixture was stirred at 25° C. for 15 hours. The reaction mixture was quenched by addition H2O 30 mL at 25° C. and extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with saturated brines (5 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. Compound 24.5 (750 mg, 3.01 mmol, 83.34% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C14H19NO3: 250; found 250; RT=0.800 min.

To a solution of compound 25.6 (800 mg, 3.04 mmol, 1 eq) in THF (8 mL) was added BH3-Me2S (10 M, 608 μL, 2 eq) at 0° C. The mixture was stirred at 25° C. for 15 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with saturated brines (10 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. Compound 25.5 (600 mg, 2.41 mmol, 79.17% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C14H19NO3: 250; found 250; RT=0.752 min.

To a solution of compound 25.4 (230 mg, 570.03 μmol, 1 eq) in DMF (3 mL) was added K2CO3(157.57 mg, 1.14 mmol, 2 eq) and compound 25.3 (75.32 mg, 684.04 μmol, 1.20 eq). The mixture was stirred at 80° C. for 15 hours. The reaction mixture was quenched by addition H2O 10 mL at 25° C. and extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with saturated brines (5 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1). Compound 25.2 (50 mg, 146.46 μmol, 25.69% yield) was obtained as a black brown oil. LCMS (ESI): m/z: [M+H] called for C19H23N3O3: 342; found 342; RT=0.742 min.

To a solution of compound 26.7 (2 g, 8.25 mmol, 1 eq) in NMP (20 mL) was added NH3.H2O (18.20 g, 519.26 mmol, 20 mL, 62.94 eq). The mixture was stirred at 150° C. for 15 hours. The reaction mixture was quenched by addition H2O 50 mL at 25° C. and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with saturated brines (10 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced

To a solution of compound 26.2 (65 mg, 323.01 μmol, 1 eq) in THF (5 mL) was added BH3-Me2S (10 M, 323.01 μL, 10 eq) at 0° C. The mixture was stirred at 70° C. for 4 hours. The reaction mixture was quenched by addition MeOH 20 mL at 25° C., then concentrated under reduced pressure to give a crude product, to the crude product was added HCl (15 mL), the mixture was stirred at 25° C. for 2 hours and extracted with DCM (25 mL×5). The combined organic layers were washed with saturated brines (5 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by prep-HPLC (neutral condition). Compound 26.1 (40 mg, 213.63 μmol, 66.14% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C11H13N3: 188; found 188; RT=0.506 min.

extracted with DCM (20 mL×3). The combined organic phase was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford compound 28.6 (1.80 g, crude) was obtained as a black brown oil. The crude was used for next step directly. But it didn't work. The crude was purified by by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10:1 to 2:1) to afford pure compound 28.6 (1.67 g, yield 59.87%).

A solution of compound 28.2 (140 mg crude) in HCl/EtOAc (5 mL) was stirred at 20° C. for 15 hours under N2. The reaction was monitored by TLC (SiO2, PE:EtOAc=1:1). After the reaction was completed, the solution was concentrated in vacuum to give compound 28.1 (50 mg, 156.89 μmol, 79.96% yield, 3HCl) was obtained as a light yellow solid.

3). The combined organic layers were washed with brine 60 mL (60 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 29.7 (8.90 g, crude) as a yellow oil. LCMS (ESI): m/z: [M+H] calcd for C17H25NO5S: 355; found 300,256; RT=0.890 min.

Compound 29.2 (800 mg, 1.58 mmol, 1 eq) was added into a solution of HCl/MeOH (15 mL). The mixture was stirred at 18° C. for 10 hours. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition) to give compound 29.1 (200 mg, 964.92 μmol, 61.07% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C11H17N3O: 207; found 208; RT=0.173 min.

To a mixture of compound 30.3 (1 g, 3.41 mmol, 1 eq) in DMF (15 mL) was added NBS (606.69 mg, 3.41 mmol, 1 eq) in one portion at 18° C. under N2. The mixture was stirred at 18° C. for 3 hours. The reaction mixture was quenched by addition H2O 15 mL and extracted with EtOAc 30 mL (10 mL×3). The combined organic layers were washed with brine 10 mL (10 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate) to give compound 30.2 (1 g, 2.69 mmol, 78.78% yield) as a colorless oil. LCMS (ESI): m/z: [M+H] calcd for C15H22BrN3O3: 372; found 372, 374; RT=0.763 min.

To a solution of compound 31.4 (1.50 g, 4.86 mmol, 1 eq) in THF (20 mL) was added t-BuLi (1.3 M, 7.48 mL, 2 eq) drop-wise at −78° C. under N2. During which the temperature was maintained below −78° C. The reaction mixture was stirred at −78° C. for 1 h. Then compound 31.3 (969.21 mg, 4.86 mmol, 1 eq) was added to above mixture. The resulting mixture was stirred at 25° C. for 16 hours. The reaction mixture was added water (10 mL), extracted with EtOAc (20 mL×3). The organic phase was separated, washed with saturated NaCl (10 mL) and dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10:1 to 1:1). Compound 31.2 (500 mg, 984.99 μmol, 20.27% yield) was obtained as a yellow oil.

To a solution of compound 31.2 (500 mg, 984.99 μmol, 1 eq) in HCl/EtOAc (10 mL) was stirred at 25° C. for 16 hours. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 31.1 (400 mg, crude) was obtained as a yellow oil.

To a solution of compound 32.6 (5 g, 14.19 mmol, 1 eq) in THF (50 mL) was added LAH (1.35 g, 35.48 mmol, 2.50 eq) at 0° C. and stirred for 1 hour. The mixture was warmed to 25° C. gradually and stirred for 11 hr. The reaction mixture was quenched by addition 8% NaOH solution 2 mL at 25° C., and then diluted with H2O 20 mL and extracted with EtOAc 60 mL (20 mL×3). The combined organic layers were washed with brines 20 mL (20 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 32.5 (1.50 g, 6.69 mmol, 47.14% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C11H16N2O3: 224; found 225; RT=0.404 min.

To a solution of compound 32.4 (400 mg, 1.39 mmol, 1 eq) in toluene (7 mL) was added PPh3(401.04 mg, 1.53 mmol, 1.10 eq) under nitrogen and reflux 120° C. for 3 hours. After cooling to 25° C., the white solid was filtered off, washed with EtOAc and dried under vacuum. Then the mixture was stirred in THF (4 mL) under nitrogen and t-BuOK (171.57 mg, 1.53 mmol, 1.10 eq) was added. The mixture was stirred at 70° C. for 1 hr. After cooling to 25° C., compound 32.3 (415.44 mg, 2.09 mmol, 1.50 eq) in THF (1 mL) was added to the reaction flask and reflux for 30 min. The reaction mixture was diluted with H2O 15 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layers were washed with brine 15 mL (15 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 32.2 (200 mg, 513.49 μmol, 36.94% yield) as a brown oil. LCMS (ESI): m/z: [M+H] calcd for C21H31N3O4: 389; found 390; RT=1.508 min.

To a solution of compound 33.2 (500 mg, 1.04 mmol, 1 eq) in HCl/EtOAc (5 mL) was stirred at 25° C. for 16 hours. The reaction mixture was concentrated under reduced pressure to give a residue. Compound 33.1 (100 mg, 561.07 μmol, 53.95% yield) was obtained as a white solid.

organic layers were washed with saturated brine (25 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. Compound 34.7 (4 g, crude) was obtained as a yellow oil, and the crude product was used into the next step without further purification.

To a solution of compound 34.7 (4 g, 11.71 mmol, 1 eq) in THF (30 mL) was added TEA (4.74 g, 46.84 mmol, 6.49 mL, 4 eq) and Boc2O (3.83 g, 17.57 mmol, 4.04 mL, 1.50 eq). The mixture was stirred at 25° C. for 14 hours. The reaction mixture was quenched by addition H2O 50 mL at 25° C., and extracted with ethyl acetate (50 mL×3). The combined organic layers were washed with saturated brine (25 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5/1). Compound 34.6 (3 g, 6.79 mmol, 93.69% yield) was obtained as a colorless oil.

To a solution of compound 34.6 (1 g, 2.26 mmol, 1 eq) in THF (20 mL) was added NaH (108.48 mg, 4.52 mmol, 2 eq) the mixture was stirred at 25° C. for 1 hr. Then to the mixture was added MeI (384.94 mg, 2.71 mmol, 168.83 μL, 1.20 eq). The mixture was stirred at 0° C. for 12 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1). Compound 34.5 (600 mg, 1.32 mmol, 58.41% yield) was obtained as a yellow oil.

To a solution of compound 34.5 (600 mg, 1.32 mmol, 1 eq) in THF (15 mL) was added TBAF (345.13 mg, 1.32 mmol, 1 eq) the mixture was stirred at 25° C. for 2 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1). Compound 34.4 (250 mg, 1.15 mmol, 87.16% yield) was obtained as a yellow oil.

To a solution of compound 34.3 (400 mg, 1.08 mmol, 1 eq) and compound 34.3a (118.56 mg, 1.08 mmol, 1 eq) in DMF (10 mL) was added K2CO3(595.27 mg, 4.31 mmol, 4 eq). The mixture was stirred at 80° C. for 13 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1). Compound 34.2 (200 mg, 646.41 μmol, 60.03% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C16H27N3O3: 310; found 310; RT=0.591 min.

To a solution of compound 35.7 (1 g, 4.76 mmol, 1 eq) and compound 35.6 (342.98 mg, 4.76 mmol, 413.23 μL, 1 eq) in DMF (25 mL) was added K2CO3(1.97 g, 14.27 mmol, 3 eq). The mixture was stirred at 80° C. for 14 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. Compound 35.5 (2.5 g, crude) was obtained as a yellow oil. It was used into the next step without further purification.

A mixture of compound 35.3 (100 mg, 356.74 μmol, 1 eq) in MeNH2(2 mL) was stirred at 0° C. for 1 hr. To the mixture was added HOAc (32.13 mg, 535.10 μmol, 30.60 μL, 1.50 eq) make the mixture to pH=4 and then added NaBH3CN (89.67 mg, 1.43 mmol, 4 eq). The mixture was stirred at 25° C. for 12 hours. The reaction mixture was quenched by addition saturated NaHCO320 mL at 25° C. make the pH>7 and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1). Compound 35.2 (80 mg, 270.84 μmol, 75.92% yield) was obtained as a yellow oil. It was combined with a second batch for a total of 160 mg. LCMS (ESI): m/z: [M+H] called for C15H25N3O3: 296; found 296; RT=0.520 min.

To a solution of compound 36.6 (3 g, 9.67 mmol, 1 eq) in MeOH (40 mL) and H2O (4 mL) was added NaOH (966.65 mg, 24.17 mmol, 2.50 eq). The mixture was stirred at 25° C. for 16 hours. The rethyl acetatection mixture was quenched by addition H2O 25 mL at 25° C., and extracted with ethyl acetate (25 mL×3). The combined organic layers were washed with saturated brine (25 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=4/1) to give a compound 36.5 (1.50 g, 7.14 mmol, 73.79% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C10H14O3N2: 211; found 211; RT=0.352 min.

To a solution of compound 36.5 (500 mg, 2.38 mmol, 1 eq) in DMF (30 mL) was added K2CO3(986.82 mg, 7.14 mmol, 3 eq) and compound 36.4 (570 mg, 2.86 mmol, 1.2 eq). The mixture was stirred at 25° C. for 14 hours. The reaction mixture was quenched by addition H2O 20 mL at 25° C., and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a compound 36.3 (260 mg, 791.81 μmol, crude) as a white solid, and the crude product was without further purification. LCMS (ESI): m/z: [M+H] calcd for C18H20O4N2: 329; found 329; RT=0.701 min.

A solution of compound 39.3 (2.80 g, 11.05 mmol, 1 eq) in HCl/MeOH (40 mL) was stirred at 15° C. for 2 hrs under N2. The mixture was concentrated in vacuum to give compound 39.2 (2.40 g, crude, HCl salt) was obtained as a white solid.

3). The combined organic layers were washed with brine 60 mL (60 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 41.5 (8.90 g, crude) as a yellow oil. LCMS (ESI): m/z: [M+H] calcd for C17H25NO5S: 356; found 300; RT=0.890 min.

To a mixture of compound 43.7 (2 g, 4.72 mmol, 1 eq), compound 43.6 (996.01 mg, 4.72 mmol, 1 eq), K2CO3(3.91 g, 28.32 mmol, 6 eq) in dioxane (40 mL) and H2O (10 mL) was added Pd(dppf)Cl2(1.04 g, 1.42 mmol, 0.30 eq) in one portion at 15° C. under N2. The mixture was stirred at 80° C. for 8 hours. The reaction mixture was quenched by addition H2O 100 mL at 25° C., and extracted with EtOAc (50 mL×3). The combined organic layers were washed with saturated brines (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20/1, 3/1) to afford compound 43.4

To a mixture of HCl/EtOAc (4 M, 5 mL) in compound 43.2 (300 mg, 584.08 μmol, 1 eq) in one portion at 15° C. The mixture was stirred at 15° C. for 12 hour. The reaction solution was concentrated in vacuum to obtain compound 43.1 (180 mg, crude, 2HCl) was obtained as light yellow solid. LCMS (ESI): m/z: [M+H] calcd for C13H15N3:214; found 214; RT=0.177 min.

To a mixture of compound 44.2 (300 mg, 584.08 μmol, 1 eq) in HCl/EtOAc (4 M, 5 mL) in one portion at 15° C. under N2. The mixture was stirred at 15° C. for 5 hours. The reaction solution was concentrated in vacuum to obtain compound 44.1 (180 mg, crude, 2HCl) was obtained as light yellow solid. LCMS (ESI): m/z: [M+H] calcd for C13H15N3:214; found 214; RT=10 min.

quenched by addition H2O 20 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1). Compound 45.4 (100 mg, 196.24 μmol, 16.63% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C18H19N3O2: 310; found 310; RT=0.734 min.

To a solution of compound 45.3 (160 mg, 313.98 μmol, 1 eq) in EtOH (5 mL) was added PtO2(11 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 12 hours. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1). Compound 45.2 (150 mg, 292.04 μmol, 93.01% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C28H39N3O6: 514; found 514; RT=1.672 min.

A mixture of compound 46.3 (100 mg, 258.77 μmol, 1 eq) in MeNH2(2 mL) was stirred at 0° C. for 1 hour. To the mixture was added NaBH3CN (65.04 mg, 1.04 mmol, 4 eq) and then added HOAc (23.31 mg, 388.16 μmol, 22.20 μL, 1.50 eq) make the mixture to pH=4. The mixture was stirred at 25° C. for 12 hours. The reaction mixture was quenched by addition saturated NaHCO320 mL at 25° C. make the pH>7 and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (20 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1). Compound 46.2 (50 mg, 124.53 μmol, 48.12% yield) was obtained as a yellow oil. LCMS (ESI): m/z: [M+H] called for C22H31N3O4: 302; found 302; RT=0.565 min.

To a solution of compound 48.5 (710 mg, 3.15 mmol, 1 eq) in THF (2 mL) was added LiBH4(102.80 mg, 4.72 mmol, 1.50 eq) and stirred at 0° C. for 12 hours. The reaction mixture was diluted with H2O 15 mL and extracted with EtOAc 45 mL (15 mL×3). The combined organic layers were washed with brine 15 mL (15 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2/1 to 1:1) to give compound 48.4 (300 mg, 1.63 mmol, 51.88% yield) as a colorless solid. LCMS (ESI): m/z: [M+H] calcd for C8H6ClNO2:183; found 184; RT=0.593 min.

To a solution of compound 48.2 (50 mg, 254.28 μmol, 1 eq) in i-PrOH (7 mL) was added NH2NH2.H2O (2.40 g, 48.01 mmol, 2.33 mL, 188.80 eq). The mixture was stirred at 90° C. for 72 hours. The reaction mixture was concentrated under reduced pressure to remove solvent to give compound 48.1 (60 mg, crude) as a yellow oil.

To a solution of compound 49.7 (1.5 g, 6.19 mmol, 1 eq) in NMP (15 mL) was added NH3.H2O (54.58 g, 389.32 mmol, 15 mL, 25% purity, 62.94 eq). The mixture was stirred at 150° C. for 15 hours. The reaction mixture was quenched by addition of H2O (500 mL) at 25° C. and extracted with EtOAc (500 mL×3). The combined organic layers were washed with saturated brine 50 mL (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1) to give compound 49.6 (1.6 g, 7.17 mmol, 38.65% yield) as yellow oil.

Compound 49.3 (100 mg, 188.81 μmol, 1 eq) was dissolved into HCl/EtOAc (10 mL), the mixture was stirred at 15° C. for 10 hours. The reaction mixture was concentrated under reduced pressure to give compound 49.1 (60 mg, crude) as a white solid.

To a solution of compound 50.5 (1.5 g, 6.19 mmol, 1 eq) in NMP (15 mL) was added NH3.H2O (54.58 g, 389.32 mmol, 15 mL, 63 eq). The mixture was stirred at 150° C. for 15 hours. The reaction mixture was quenched by addition H2O (500 mL) at 25° C. and extracted with EtOAc (500 mL×3). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under

reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH=10:1) to give compound 50.4 (1.6 g, 7.17 mmol, 39% yield) as yellow oil.

To a solution of compound 51.8 (2.5 g, 10.31 mmol, 1 eq) in NMP (30 mL) was added NH3.H2O (30 mL, 33% aqueous solution) in one portion at 150° C. under N2. The mixture was stirred at 150° C. for 10 hours. The reaction mixture was diluted with H2O (500 mL) and extracted with EtOAc (500 mL×3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:2) to give compound 51.7 (35 g, 95% yield) as a white solid. LCMS (ESI) m/z: [M+H] calcd for C9H8BrN2: 223; found 223; RT=0.761 min.

To a solution of compound 53.9 (10 g, 83.95 mmol, 1 eq) in THF (150 mL) was added LAH (6.37 g, 167.90 mmol, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1.5 hours. The reaction mixture was quenched by addition saturated sodium sulfate at 0° C. and added 100 ml H2O then extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with saturated brines (30 mL×1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the product compound 53.8 (7.50 g, 61.42 mmol, 73.16% yield) as a yellow oil. LCMS (ESI): m/z: [M+H] called for C6H6N2O: 123; found 123; RT=0.099 min.

To a solution of compound 54.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L) was added LiAlH4(12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. under N2. The mixture was stirred at 0° C. for 1 hours, then heated to 18° C. and stirred at 18° C. for 14 hours. The reaction mixture was quenched by addition 8% NaOH (15 ml), filtered and then diluted with H2O 1000 mL and extracted with EtOAc 1500 mL (500 mL×3). The combined organic layers were washed with brine 1000 mL (1000 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1) to give compound 54.9 (15 g, 66.89 mmol, 39.28% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C11H16N2O3: 293; found 294; RT=0.313 min.

To a solution of compound 54.7 (1.49 g, 4.50 mmol, 1 eq) in DCM (15 mL) was added DBU (1.03 g, 6.75 mmol, 1.02 mL, 1.50 eq) in one portion at 18° C. under N2. The mixture was stirred at 18° C. for 0.5 hour, then to the mixture was added compound 54.8 (1 g, 4.50 mmol, 1 eq) in one portion at 18° C., then was stirred at 18° C. for 0.5 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 54.6 (1.90 g, 4.44 mmol, 98.78% yield) as a white solid.

To a solution compound 54.6 (1 g, 2.34 mmol, 1 eq) in MeOH (50 mL) and THF (50 mL) was added Pd—C (10%, 100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 20° C. for 15 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 54.5 (700 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C14H19N3O4: 293; found 294; RT=0.638 min.

To a solution of compound 54.5 (600 mg, 2.05 mmol, 1 eq) in MeOH (50 mL) and THF (50 mL) was added Pd—C (10%, 100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 20° C. for 15 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 54.4 (600 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C14H21N3O4: 295; found 296; RT=0.239 min.

To a solution of compound 55.10 (60 g, 170.27 mmol, 1 eq) in THF (1 L) was added LiAlH4(12.92 g, 340.54 mmol, 2 eq) portionwise at 0° C. under N2. The mixture was stirred at 0° C. for 1 hour, then heated to 18° C. and stirred at 18° C. for 14 hours. The reaction mixture was quenched by addition 8% NaOH (15 ml), filtered and then diluted with H2O 1000 mL and extracted with EtOAc 1500 mL (500 mL×3). The combined organic layers were washed with brine 1000 mL (1000 mL×1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1) to give compound 55.9 (15 g, 66.89 mmol, 39.28% yield) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C11H16N2O3: 293; found 294; RT=0.313 min.

To a solution of compound 55.7 (1.49 g, 4.50 mmol, 1 eq) in DCM (15 mL) was added DBU (1.03 g, 6.75 mmol, 1.02 mL, 1.50 eq) in one portion at 18° C. under N2. The mixture was stirred at 18° C. for 0.5 hour, and then to the mixture was added compound 55.8 (1 g, 4.50 mmol, 1 eq) in one portion at 18° C., then was stirred at 18° C. for 0.5 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 55.6 (1.90 g, 4.44 mmol, 98.78% yield) as a white solid.

To a solution compound 55.6 (1 g, 2.34 mmol, 1 eq) in MeOH (50 mL) and THF (50 mL) was added Pd—C (10%, 100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 20° C. for 15 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 55.5 (700 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C14H19N3O4: 293; found 294; RT=0.638 min.

To a solution of compound 55.5 (600 mg, 2.05 mmol, 1 eq) in MeOH (50 mL) and THF (50 mL) was added Pd—C (10%, 100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 20° C. for 15 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 55.4 (600 mg, crude) as a white solid. LCMS (ESI): m/z: [M+H] calcd for C14H21N3O4: 295; found 296; RT=0.239 min.

Compounds 56, 57, and 58 were prepared according to the methods described above.

To a solution compound 59.9 (20 g, 167.89 mmol, 1 eq) in THF (500 mL) was added LiAlH4(12.74 g, 335.78 mmol, 2 eq) portions wise at 0° C. under N2. The mixture was stirred at 0° C. for 2 hours. The reaction mixture was quenched by the addition of Na2SO4.10H2O (30 g) then filtered. The filtrate was diluted with H2O (200 mL) and extracted with EtOAc (200 mL×3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3:1) to give compound 59.8 (10 g, 81.88 mmol, 49% yield) as a yellow solid.

To a mixture of compound 59.7 (9.5 g, 29.47 mmol, 1 eq) and 59.6 (9.76 g, 29.47 mmol, 1 eq) in DCM (60 mL) was added DBU (8.97 g, 58.94 mmol, 8.88 mL, 2 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 hours. The reaction mixture was quenched by addition Na2SO4.10H2O (30 g), then filtered, and then the filtrate was diluted with H2O (200 mL) and extracted with EtOAc (200 mL×3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1) to give compound 59.5 (7 g, 13.27 mmol, 45% yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd for C27H34N3O8: 528; found 528; RT=1.60 min.

To a solution of compound 59.5 (7 g, 13.27 mmol, 1 eq) in MeOH (200 mL) was added 10% Pd on carbon catalyst (800 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 10 hours. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3:1) to give compound 59.4 (2.8 g, 7.08 mmol, 53% yield) as a yellow solid. LCMS (ESI) m/z: [M+H]+ calcd for C19H30N6O3: 396; found 396; RT=1.029 min.

To a mixture of compound 59.2 (100 mg, 194.55 μmol, 1 eq) and isoxazol-3-ol (16.55 mg, 194.55 μmol, 1 eq) in DMF (2 mL) was added DIPEA (75.43 mg, 583.65 μmol, 101.66 μL, 3 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 hours. The residue was diluted with H2O (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were washed with H2O (50 mL×3); the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3:1) to give compound 59.1 (150 mg) as a white solid. LCMS (ESI) m/z: [M+H-Boc] called for C22H31N4O7: 563; found 463; RT=0.937 min.

To a solution of compound 60.9 (10 g, 83.95 mmol, 1 eq) in THF (300 mL) was added LAH (6.37 g, 167.9 mmol, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1.5 hour. The reaction mixture was quenched by addition of saturated sodium sulfate at 0° C. and added 300 mL of H2O then extracted with ethyl acetate (300 mL×3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound 60.8 (10 g, crude) as a yellow solid.

To a solution of compound 60.5 (2.6 g, 4.93 mmol, 1 eq) in MeOH (200 mL) was added 10% palladium on carbon (1 g) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 15 hours. The reaction mixture was filtered, and the filter was concentrated to give compound 60.4 (900 mg, 2.28 mmol, 46% yield) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C19H29N3O6: 396; found 396; RT=1.161 min.

To a solution of compound 62.5 (1 g, 2.34 mmol, 1 eq) in MeOH (20 mL) was added Pd—C (10% palladium on carbon, 100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 10 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate) to give compound 62.4 (270 mg, 914.2 μmol, 39.1% yield) as a white solid.

To a solution of compound 66.9 (20 g, 167.9 mmol, 1 eq) in THF (250 mL) was added LiAlH4(12.74 g, 335.8 mmol, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1.5 hour. The reaction mixture was quenched by addition of saturated sodium sulfate at 0° C. and added H2O (200 mL) then extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with saturated brines (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound 66.8 (15 g, crude) as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C6H7N2O: 123; found 123; RT=0.21 min.

To a solution of DIPA (1.03 g, 10.17 mmol, 1.43 mL, 5 eq) in THF (10 mL) was added n-BuLi (2.5 M, 4.07 mL, 5 eq). The mixture was stirred at 0° C. for 0.5 hr under N2. Then the mixture was added to the solution of compound 66.3 (1 g, 2.03 mmol, 1 eq) and chloroiodomethane (1.79 g, 10.17 mmol, 738.3 μL, 5 eq) in THF (10 mL) was stirred at −78° C. for 2.5 hours. The reaction mixture was quenched by addition saturated NH4Cl (20 mL) at 25° C. and extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with saturated Na2SO3(10 mL) and saturated NaHCO3(10 mL) and saturated brines (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound 66.2 (1.6 g) as yellow oil.

To a solution of compound 66.2 (400 mg, 975.83 μmol, 1 eq) in DMF (5 mL) was added K2CO3(404.6 mg, 2.93 mmol, 3 eq) and isoxazol-3-ol (124.51 mg, 1.46 mmol, 1.5 eq). The mixture was stirred at 25° C. for 15 hours. The reaction mixture was quenched by addition saturated NaHCO320 mL at 25° C. and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with saturated brines (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 68.1 (50 mg) as yellow oil.

To a solution of compound 69.5 (5.7 g, 13.33 mmol, 1 eq) in MeOH (20 mL) was added 10% palladium on carbon catalyst (100 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 10 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Ethyl acetate) to give compound 69.4 (2.8 g, 9.48 mmol, 71% yield) as a white solid.

To a mixture of cyclopentanecarboxylic acid (680.21 mg, 5.96 mmol, 647.82 μL, 1 eq) and EDCI (1.26 g, 6.56 mmol, 1.1 eq) in DMF (20 mL) was added HOBt (885.75 mg, 6.56 mmol, 1.1 eq) in one portion at 0° C. under N2. The mixture was stirred at 0° C. for 1 hour, then the mixture was added dropwise a solution of compound 69.4 (1.76 g, 5.96 mmol, 1 eq) in DMF (5 mL), then the mixture was added dropwise DIPEA (2.31 g, 17.88 mmol, 3.12 mL, 3 eq) and stirred at 0° C. for 1 hour. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=3:1) to give compound 69.3 (1.8 g, 4.6 mmol, 77% yield) as a white solid.

To a mixture of compound 71.2 (70 mg, 133.6 μmol, 1 eq) and isoxazol-3-ol (11.36 mg, 133.6 μmol, 1 eq) in DMF (2 mL) was added DIEA (17.26 mg, 133.6 μmol, 23.3 μL, 1 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 hours. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 71.1 (80 mg) as a yellow oil.

To a solution of compound 72.9 (10 g, 83.9 mmol, 1 eq) in THF (250 mL) was added LAH (6.37 g, 167.9 mmol, 2 eq) at 0° C. The mixture was stirred at 0° C. for 1.5 hour. The reaction mixture was quenched by addition saturated sodium sulfate at 0° C. and H2O (200 mL) was added; then this was extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the product compound 72.8 (6 g, crude) as a yellow solid. LCMS (ESI): m/z: [M+H]+ calcd for C6H7N2O: 123; found 123; RT=0.276 min.

To a solution of compound 72.5 (3 g, 5.69 mmol, 1 eq) in MeOH (400 mL) was added 10% Pd on carbon catalyst (1 g) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2(50 psi) at 25° C. for 15 hours. The reaction mixture was filtered, and the filtrate was concentrated. compound 76.4 (1.5 g) was obtained as yellow oil. LCMS (ESI) m/z: [M+H]+ calcd for C19H30N3O6: 396; found 396; RT=1.084 min.

stirred at 0° C. for 1 hours, then stirred for 14 hours. The reaction mixture was quenched by addition 8% NaOH (15 ml), filtered and then diluted with H2O (1000 mL) and extracted with EtOAc (500 mL×3). The combined organic layers were washed with brine (1000 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=2:1) to give compound 73.8 (15 g, 66.89 mmol, 40% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C11H17N2O3: 225; found 225; RT=0.833 min.

To a solution of compound 73.8 (15 g, 66.89 mmol, 1 eq) in DCM (150 mL) was added Dess-Martin periodinane (42.55 g, 100.33 mmol, 31.06 mL, 1.5 eq) portion-wise at 18° C. under N2. The mixture was stirred for 2 hours. The reaction mixture was diluted with H2O (60 mL) and extracted with DCM 150 mL (50 mL×3). The combined organic layers were washed with brine (100 mL) (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5:1) to give compound 73.7 (9 g, 40.5 mmol, 61% yield) as a white solid.

To a solution of compound 73.5 (3 g, 7.02 mmol, 1 eq) in MeOH (300 mL) was added 10% palladium on carbon catalyst Pd/C (300 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was shaken under H2(50 psi) at 25° C. for 20 hours. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by MPLC (SiO2, Petroleum ether/Ethyl acetate=10/1 to 1:1) to give compound 73.4 (1.4 g, 4.74 mmol, 68% yield) as a white solid. LCMS (ESI) m/z: [M+H]+ calcd for C14H22N3O4: 296; found 296; RT=0.654 min.

To a solution of DIPA (633.32 mg, 6.26 mmol, 884.52 μL, 5.5 eq) in THF (20 mL) was added n-BuLi (2.5 M, 2.5 mL, 5.5 eq) at 0° C., the mixture was stirred at 0° C. for 30 mins. To the mixture was added a solution of compound 73.3 (0.45 g, 1.14 mmol, 1 eq) and chloroiodomethane (1.10 g, 6.26 mmol, 454.29 μL, 5.5 eq) in THF (20 mL) at −78° C. The mixture was stirred at −78° C. for 1.5 h. The reaction mixture was quenched by addition of NH4Cl (20 mL) and extracted with EtOAc (20 mL×3), The combined organic layers were washed with saturated brines (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=1:1) to give compound 73.2 (100 mg, 241.61 μmol, 21% yield) as a white solid.

To a mixture of compound 74.8 (5 g, 28.9 mmol, 1 eq) and TEA (11.7 g, 115.6 mmol, 16.09 mL, 4 eq) in DMF (100 mL) was added Boc2O (18.92 g, 86.7 mmol, 19.92 mL, 3 eq) in one portion at 25° C. under N2. The mixture was stirred at 25° C. for 10 hours. The reaction mixture was diluted with H2O (100 mL) then was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue that remained was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5:1) to give compound 74.7

To a solution of compound 74.5 (3.8 g, 6.99 mmol, 1 eq) in MeOH (100 mL) was added 10% Pd on carbon catalyst (400 mg) under N2. The suspension was degassed under vacuum and purged with H2several times. The mixture was stirred under H2atmosphere (50 psi) at 25° C. for 0.5 hour. The reaction mixture was purged with N2gas, filtered through a Celite pad and was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10:1 to 1:1) to give compound 74.4 (2 g, 4.88 mmol, 70% yield) as a yellow solid.

Example 75. Inhibition of Arginine Gingipain by the Aminopyridinecyanamide Compounds of the Invention

The capacities of compounds of the present invention to inhibit the activity of RgpB were measured in a fluorogenic assay similar to those described in BarretBiochemical Journal.1980, 187(3), 909. The specific assay conditions were as follows. Buffer: pH=7.5, 100 mM Tris-HCl, 75 mM NaCl, 2.5 mM CaCl2, 10 mM cysteine, 1% DMSO after all additions. Protein: 0.02 nM RgpB, isolated from culture ofPorphyromonas gingivalis, as described in Pike et al.J. Biol. Chem.1994, 269(1), 406, and Potempa and Nguyen.Current Protocols in Protein Scienc.2007, 21.20.1-21.20.27. Fluorogenic substrate: 10 μM Boc-Phe-Ser-Arg-MCA. Time=90 minutes. Temperature=37° C. Each compound: 10 concentrations, starting at either 100 μM or 100 nM, with lower concentrations generated by serial 3-fold dilutions. By testing a range of concentrations for each compound, the concentration required to inhibit the activity of RgpB by 50% (the “IC50”) was determined. RgpB inhibitory activity is summarized in the following table.

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.