AU2016202040B2

AU2016202040B2 - Certain medical entities, compositions, and methods

Info

Publication number: AU2016202040B2
Application number: AU2016202040A
Authority: AU
Inventors: Jeffrey T. Finer; Pu-Ping Lu; Bradley P. Morgan; David J. Morgans Jr.; Alex Muci; Alan James Russell
Original assignee: Cytokinetics Inc
Current assignee: Cytokinetics Inc
Priority date: 2006-08-02
Filing date: 2016-04-01
Publication date: 2018-08-16
Anticipated expiration: 2027-08-01
Also published as: AU2016202040A1

Abstract

Abstract Provided are certain chemical entities, and methods of use to modulate skeletal myosin, skeletal actin, skeletal tropomyosin, skeletal troponin C, skeletal troponin I, skeletal troponin T, and skeletal muscle, including fragments and isoforms thereof, as well as the skeletal sarcomere, and methods of use in the treatment of obesity, sarcopenia, wasting syndrome, frailty, cachexia, muscle spasm, post-surgical and post-traumatic muscle weakness, neuromuscular disease, and other indications.

Description

The present invention is meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

2016202040 01 Apr 2016 [044] Tautomers are structurally distinct isomers that interconvert by tautomerization. Tautomerization is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. Prototropic tautomerization or proton-shift tautomerization involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g. in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconverision of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconverision of pyridin-4-ol and pyridin-4(1H)one tautomers. Compounds of Formula I and compounds of Formula li are tautomeric. [045] A leaving group or atom is any group or atom that will, under the reaction conditions, cleave from the starting material, thus promoting reaction at a specified site. Suitable examples of such groups unless otherwise specified are halogen atoms, mesyloxy, p-nitrobenzensulphonyloxy and tosyloxy groups.

[046] The term pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active Ingredients can also be incorporated into the compositions.

[047] Protecting group has the meaning conventionally associated with it in organic synthesis, i.e. a group that selectively blocks one or more reactive sites in a multifunctional compound such that a chemical reaction can be carried out selectively on another unprotected reactive site and such that the group can readily be removed after the selective reaction is complete. A variety of protecting groups are disclosed, for example, in T.H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999). For example, a hydroxy protected form is where at least one of the hydroxy groups present in a compound is protected

2016202040 01 Apr 2016 with a hydroxy protecting group. Likewise, amines and other reactive groups may similarly be protected.

[0481 The term pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of the compounds described herein and, which are not biologically or otherwise undesirable. In many cases, the compounds described herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. fn some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[049] The term solvate” refers to a compound (e.g., a compound selected from Formula I and Formula II, or a pharmaceutically acceptable salt thereof) in physical association with one or more molecules of a pharmaceutically acceptable solvent. It will be understood that a compound of Formula I and a compound of Formula II encompass the compound of Formula I and the compound of Formula II, and solvates of those compounds, as well as mixtures thereof.

[050] The terms substituted alkyl, cycloalkyl, aryl, heterocycloalkyl, and

2016202040 01 Apr 2016 heteroaryl, unless otherwise expressly defined, refer respectively to alkyl, cycloalkyl, aryl, heterocycloalkyl, and heteroaryl wherein one or more (such as up to 5, for example, up to 3) hydrogen atoms are replaced by a substituent independently chosen from:

-R^a, -OR^b, optionally substituted amino (including -NR^eCOR^b, -NR^cCO2R^a, -NR^cCONR^bR^c, -NR^bC(NR^c)NR^bR^c, -NR^bC(NCN)NR^bR^c, and -NR^CSO2R⁸), halo, cyano, nitro, oxo (as a substituted for cycloalkyl, heterocycloalkyl, and heteroaryl), optionally substituted acyl (such as-COR^b), optionally substituted alkoxycarbonyl (such as -CO2R^b), aminocarbonyl (such as -CONR^bR^c), -OCOR^b, -0CO2R^a, -OCONR^bR^c, -OCONR^bR^c, -OP(O)(OR^b)OR^c, sulfanyl (such as SR¹⁵), sulfinyl (such as -SOR^a), and sulfonyl (such as -SO2R^a and -SO2NR^bR^c), where

R^a is chosen from optionally substituted Ci-C₆ alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, and optionally substituted heteroaryl;

R^b is chosen from hydrogen, optionally substituted Ci-C_B alkyl, optionally substituted cycioalkyt, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; and

R^c is independently chosen from hydrogen and optionally substituted Ci-C₄ alkyl; or

R^band R^c, and the nitrogen to which they are attached, form an optionally substituted heterocycloalkyl group; and where each optionally substituted group is unsubstituted or independently substituted with one or more, such as one, two, or three, substituents independently selected from CrC₄ alkyl, aryl, heteroaryl, aryl-Ci-C₄ alky!-, heteroaryl-CrCL alkyl-, C-|-C₄ haloalkyl, -OCi-C₄ alkyl, -OC_rC₄ alkylphenyl, -C1-C4 alkyl-OH, -0C_rC₄ haloalkyl, halo, -OH, -NH₂, -C,-C₄ alkyl-NH₂, -N(C_rC₄ alkylXC,-^ alkyl), -NH(Ci-C₄ alkyl), -N(C₁-C₄ alkyl)(Ci-C₄ alkylphenyl), -NH(C_rC₄ alkylphenyl), cyano, nitro, oxo (as a substitutent for cycloalkyl or heterocycloalkyl), -CO2H, -C(O)0CrC₄ alkyl, -ΟΟΝ(Οί-Ο₄

2016202040 01 Apr 2016 a!kyl)(Ct-C₄ alkyl), -CONH(C₁-C₄ alkyl), -CONH₂, -NHC(O)(C,-C₄ alkyl),

-NHC(O)(phenyl), -N(C_rC₄ alkyl)C(O)(C_rC₄ alkyl), -N(C_rC₄ alkyl)C(O)(phenyl), -C(O)Ci-C₄ alkyl, -C(O)CrC₄ alkylphenyl, -C(O)Ci-C₄ hatoalkyl, -OC(O)Ci-C₄ alkyl, SO₂(C_VC₄ alkyl), -SO₂(phenyl), -SO₂(C_t-C₄ haloalkyl), -S0₂NH₂, -SO₂NH(C,-C₄ alkyl), -SO₂NH(phenyl), -NHSO₂(C_rC₄ alkyl), -NHSO₂(phenyl), and -NHSO₂(Ci-C₄ haloalkyl). [051] The term sulfanyl refers to the groups: -S-toptionally substituted alkyl), -S(optionally substituted cycloalkyl), -S-(optionally substituted aryl), -S-(optionally substituted heteroaryl), and -S-(optionally substituted heterocycioalkyl).

[052] The term sulfinyl refers to the groups: -S(O)-H, -S(O)-(optionally substituted alkyl), -S(0)-(optionally substituted cycloalkyl), -S(O)-(optionally substituted amino), S(0)-(optionally substituted aryl), -S(0)-(optionally substituted heteroaryl), and -S(O)(optionaily substituted heterocycioalkyl).

[053] The term sulfonyl refers to the groups: -S(O₂)-H, -S(0₂)-(optionally substituted alkyl), -S{O₂)-(optionally substituted cycloalkyl), -S(O₂)-(optionally substituted amino), -S(O₂)-(optionally substituted aryl), -S(O_z)-(optionally substituted heteroaryl), and -S(0₂)-(optlonally substituted heterocycioalkyl).

[054] The term therapeutically effective amount or effective amount refers to that amount of a compound selected from Formula I and Formula IE that is sufficient to effect treatment, as defined below, when administered to a mammal in need of such treatment. The therapeuticaily effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound selected from Formula I and Formula II, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art.

[055] ATPase refers to an enzyme that hydrolyzes ATP. ATPases include proteins comprising molecular motors such as the myosins.

[056] As used herein, frailty is a syndrome characterized by meeting three of the of the following five attributes: unintentional weight loss, muscle weakness, slow walking speed, exhaustion, and low physical activity.

[057] As used herein, cachexia means a metabolic defect often associated with

2016202040 01 Apr 2016 cancer that is characterized by progressive weight loss due to the deletion of adipose tissue and skeletal muscle.

[058] As used herein, muscle spasm means an involuntary contraction of a muscle. Muscle spasms may lead to cramps.

[059] As used herein, post-surgical muscle weakness refers to a reduction in the strength of one or more muscles following surgical procedure. Weakness may be generalized (i.e. total body weakness) or localized to a specific area, side of the body, limb, or muscle.

[060] As used herein, post-traumatic muscle weakness'¹ refers to a reduction in the strength of one or more muscles following a traumatic episode (e.g. bodily injury). Weakness may be generalized (i.e. total body weakness) or localized to a specific area, side of the body, iimb, or muscle.

[061] As used herein, neuromuscular disease means any disease that affects any part of the nerve and muscle. Neuromuscular disease encompasses critical illness polyneuropathy, prolonged neuromuscular blockade, acute myopathy as well as acute inflammatory demyelinating polyradiculoneuropathy, amyotrophic lateral sclerosis (ALS), autonomic neuropathy, Charcot-Marie-Tooth disease and other hereditary motor and sensory neuropathies, chronic inflammatory demyelinating polyradiculoneuropathy, dermatomyositis/polymyositis, diabetic neuropathy, dystrophinopathies, endocrine myopathies, focal muscular atrophies, hemifacial spasm, hereditary neuropathies of the Charcot-Marie-Tooth disease type, inclusion body myositis, Kennedy disease, LambertEaton myasthenic syndrome, muscular dystrophy (e.g., limb-girdle, Duchenne, Becker, myotonic, facioscapulohumeral, etc.), metabolic myopathies, metabolic neuropathy, multifocal motor neuropathy with conduction blocks, myasthenia gravis, neuropathy of Friedreich Ataxia, neuropathy of leprosy, nutritional neuropathy, periodic paralyses, primary lateral sclerosis, restrictive lung disease, sarcoidosis and neuropathy, Schwartz-Jampel Syndrome, spinal muscle atrophy, stiff person syndrome, thyroid disease, traumatic peripheral nerve lesions, vasculitic neuropathy, among others.

[062] As used herein obesity means having a body mass index (BMI) greater than or equal to 30 kg/m^z. BMI is defined as weight (kg) divided by height (m²). Obesity encompasses hyperplastic obesity, an increase in the number of fat cells, and

2016202040 01 Apr 2016 hypertrophic obesity, an increase in the size of the fat celts. Overweight is defined as having a BMI from 25 up to 30 kg/m²; obesity as a BMI greater than or equal to 30 kg/m², as stated above, and severe (or morbid) obesity is defined as a BMI greater than or quality to 40 kg/m².

[063] As used herein, sarcopenia means a loss of skeletal muscle mass, quality, and strength. Often sarcopenia is attributed to ageing, but is also associated with HIV infection. Sarcopenia may lead to frailty, for example, in the elderly.

[064] As used herein, wasting syndrome means a condition characterized by involuntary weight loss associated with chronic fever and diarrhea. In some instances, patients with wasting syndrome lose 10% of baseline body weight within one month. [065] Compounds of Formula I and compounds of Formula II also include crystalline and amorphous forms of those compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof. Crystalline form, polymorph, and novel form may be used interchangeably herein, and are meant to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to.

[066] Chemical entities include, but are not limited to, compounds of Formula I, compounds of Formula II, and all pharmaceutically acceptable forms thereof. Pharmaceutically acceptable forms of the compounds recited herein include pharmaceutically acceptable salts, chelates, non-covalent complexes, prodrugs, and mixtures thereof. In certain embodiments, the compounds described herein are in the form of pharmaceutically acceptable salts. Hence, the terms chemical entity and chemical entities also encompass pharmaceutically acceptable salts, chelates, noncovalent complexes, prodrugs, and mixtures.

[067] Pharmaceutically acceptable salts include, but are not limited to salts with inorganic acids, such as hydrochlorate, phosphate, diphosphate, hydrobromate, sulfate, sutfinate, nitrate, and like salts; as well as salts with an organic acid, such as malate,

2016202040 01 Apr 2016 maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, ptoluenesulfonate, 2-hydroxyethy (sulfonate, benzoate, salicylate, stearate, and alkanoate such as acetate, HOOC-(CH2)_n-COOH where n ranges from 0 to 4, and like salts. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.

[068] in addition, if the compound of Formula I or the compound of Formula II is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.

[069] As noted above, prodrugs also fall within the scope of chemical entities, for example, ester or amide derivatives of the compounds selected from Formula I and Formula II. The term prodrug includes any compound that becomes a compound of Formula I or a compound of Formula II when administered to a patient, e.g., upon metabolic processing of the prodrug. Examples of prodrugs include, but are not limited to, acetate, formate, benzoate, and like derivatives of functional groups (such as alcohol or amine groups) in the compounds selected from Formula I and Formula II. [070] The term chelate refers to the chemical entity formed by the coordination of a compound to a metal ion at two (or more) points.

[071] The term non-covalent complex refers to the chemical entity formed by the interaction of a compound and another molecule wherein a covalent bond is not formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also called ionic bonding).

[072] The term active agent is used to Indicate a chemical entity which has biological activity. In certain embodiments, an active agent is a compound having pharmaceutical utility.

[073] The term therapeutically effective amount of a chemical entity means an

2016202040 01 Apr 2016 amount effective, when administered to a human or non-human patient, to treat a disease, e.g., a therapeutically effective amount may be an amount sufficient to treat a disease or disorder responsive to myosin activation. The therapeutically effective amount may be ascertained experimentally, for example by assaying blood concentration of the chemical entity, or theoretically, by calculating bioavailability.

[074] By significant is meant any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p <0.05.

[075] Patient refers to an animal, such as a mammal, for example a human, that has been or will be the object of treatment, observation or experiment. The methods described herein can be useful in both human therapy and veterinary applications. In some embodiments, the patient is a mammal, and in some embodiments, the patient is human.

[076] Treatment or treating means any treatment of a disease in a patient, including:

(a) preventing the disease, that is, causing the clinical symptoms of the disease not to develop;

(b) inhibiting the disease;

(c) slowing or arresting the development of clinical symptoms; and/or (d) relieving the disease, that is, causing the regression of clinical symptoms.

[077] As used herein, modulation refers to a change in one or more of skeletal myosin, skeletal actin, skeletal tropomyosin, skeletal troponin C, skeletal troponin I, skeletal troponin T, and skeletal muscle, including fragments and isoforms thereof, as well as the skeletal sarcomere as a direct or indirect response to the presence of at least one chemical entity described herein, relative to the activity of the myosin or sarcomere in the absence of the compound. The change may be an increase in activity (potentiation) or a decrease in activity (inhibition), and may be due to the direct interaction of the compound with myosin or the sarcomere, or due to the interaction of the compound with one or more other factors that in turn effect one or more of skeletal myosin, skeletal actin, skeletal tropomyosin, skeletal troponin C, skeletal troponin I, skeletal troponin T, and skeletal muscle, including fragments and isoforms thereof, as

2016202040 01 Apr 2016 well as the skeletal sarcomere.

[078] Provided is at least one chemical entity chosen from compounds of Formula I and compounds of Formula II:

\

OH

O

Formula I

Formula II and pharmaceutically acceptable salts thereof, wherein

Ri and R4 are independently selected from hydrogen, halo, hydroxy, optionally substituted acyl, optionally substituted alkyl, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted aminocarbonyl, sulfonyl, sulfanyl, sulfinyl, carboxy, optionally substituted alkoxycarbonyl, and cyano; and in the alternative, R4 and Ri, taken together with any intervening atoms, form a fused ring system selected from optionally substituted fused aryl, optionally substituted fused heteroaryl, optionally substituted fused cycloalkyl, and optionally substituted fused heterocycloalkyl; and

Rz is is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl;

provided that

Ri is not hex-1-enyl; and further provided that the compound of Formula I or the compound of Formula II is not (S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one; 1,5,6-trimethyl-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

1-methyl-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-1 -(3-nitrobenzyl)-1 H-imidazo[4,5-b]pyrazln-2(3H)-one;

2016202040 01 Apr 2016

5-(hydroxymethyl)-1,6-dimethyi-1 H-imidazo[4,5-b]pyrazin-2(3H)-one; or 1-(piperidin-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one.

[079] In some embodiments, R₂ is selected from optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted alkoxy, and optionally substituted heterocycloalkyl.

[080] in some embodiments, R₂ is selected from heterocycloalkyl, cycloalkyl, lower alkyl, and lower alkyl substituted with optionally substituted phenyl, hydroxy, optionally substituted alkoxy, optionally substituted amino and optionally substituted heterocycloalkyl.

[081] In some embodiments, R₂ is selected from 1-(R)-phenylethyl, 1-(S)phenylethyl, benzyl, 3-pentyl, 4-heptyl, 4-methyl-1-morpholinopentan-2-yl isobutyl, cyclohexyl, cyclopropyl, sec-butyl, tert-butyl, isopropyl, 1-hydroxybutan-2-yl, tetrahydro2H-pyran-4-yl, 1-methoxybutan-2-yl, 1-aminobutan-2-yl, and 1-morpholinobutan-2-yl. [082] In some embodiments, Ri is selected from hydrogen, halo, acyl, optionally substituted lower alkyl, optionally substituted amino, optionally substituted pyrazolyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted lower alkoxy, and -S-(opti0nally substituted lower alkyl).

[083] In some embodiments, Ri is selected from hydrogen, halo, acyl, optionally substituted lower alkyi, dialkylamino, amino substituted with an aikyl group and with a group chosen from acyl, aminocarbonyl, alkoxycarbonyl, and sulfonyl; optionally substituted pyrazolyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted lower alkoxy, and -S-(optionally substituted lower alkyl).

[084] In some embodiments, Ri is selected from hydrogen, halo, acyl, alkenyl, alkynyl. lower alkoxy, optionally substituted amino, pyrazolyl substituted with lower alkyl, -S-(optionally substituted lower alkyl), lower alkyl, and lower alky! substituted with halo. [085] In some embodiments, Ri is selected from hydrogen, halo, acyl, alkenyl, alkynyl, lower alkoxy, dialkylamino, amino substituted with an alkyi group and with a group chosen from acyi, aminocarbonyl, alkoxycarbonyl, and sulfonyl, pyrazolyl substituted with lower alkyl, -S-(optionally substituted lower alkyl), lower alkyl, and lower alkyl substituted with halo.

[086] In some embodiments, Ri is selected from hydrogen, bromo, chloro, fluoro,

2016202040 01 Apr 2016 methyl, ethyl, propyl, hexenyl, butenyl, propenyl, vinyl, ethynyl, methoxy, ethoxy, methylsulfanyl, dimethylamino, and methyl substituted with up to three fluoro groups. [087] In some embodiments, Ri is selected from hydrogen, bromo, chloro, fluoro, methyl, ethyl, n-propyf, isopropyl, dimethylamino, isobuten-1-yl, (Z)-propen-l-yl, (E)propen-1-yl, propen-2-yl, vinyl, ethynyl, methoxy, ethoxy, methylsulfanyl, and trifluoromethyl.

[088] In some embodiments, R₄ is selected from hydrogen, halo, acyi, optionally substituted alkyl, alkenyl, optionally substituted cycloalkyl, optionally substituted aminocarbonyl, sulfanyl, optionally substituted amino, and optionally substituted alkoxycarbonyl.

[089] In some embodiments, R, is selected from hydrogen, halo, acyl, optionally substituted lower alkyl, lower alkenyl, optionally substituted cycloalkyl, optionally substituted aminocarbonyl, sulfanyl, optionally substituted amino, and optionally substituted lower alkoxycarbonyl.

[090] In some embodiments, R4 is selected from hydrogen, halo, acyl, lower alkyl, lower alkenyl, cycloalkyl, optionally substituted aminocarbonyl, sulfanyl, and lower alkoxycarbonyl.

[091] In some embodiments, R₄ is selected from hydrogen, bromo, chloro, fluoro, acetyl, methyl, ethyl, vinyl, cyclohexen-1-yl, methylcarbamoyl, dimethylcarbamoyl, methylsulfanyl, and methoxycarbonyl.

[092] In some embodiments, R₄ is hydrogen.

[093] in some embodiments, R₄ and R_<f taken together with any intervening atoms, form a fused ring system selected from optionally substituted fused aryl, optionally substituted fused cycloalkyl, and optionally substituted fused heterocycloalkyl.

[094] In some embodiments, R₄ and R₄ are taken together to form an optionally substituted benzo group.

[095] In some embodiments, R4 and R1 are taken together to form a benzo group.

[096] In some embodiments, the compound of Formula I is chosen from

1-((1R)-1-methyl-2-morpholin-4-ylethyl)-6-bromoimidazo[4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-ethynylimidazo[4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-methoxyimidazo[4,5-b]pyrazin-2-ol;

2016202040 01 Apr 2016

1-(1,1-dimethyl-2-morphoHn-4-ylethyl)-6-bromoimFdazo[4,5-bJpyrazin-2-ol;

6-(1H-1,2,3-triazol-4-yl)-1-(ethylpropyl)imidazo[4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-(trifluoromethyl)imidazo[4,6-b]pyrazin-2-ol;

1-[(1R)-1-(morpholin-4-ylmethyl)propyl)-6-ethynylimidazo[4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-{2-[1-(ethylpropyl)-2-hydroxyimi(iazo[4,5-e]pyrazin'6yl]ethynyl)imidazo[4,5-b]pyrazin-2-ol;

6-(dimethylamino)-1-(ethylpropyl)imidazo(4,5-b)pyrazin-2-ol;

6-ethy I-1 -(ethy Ipropyl) im idazo[4,5-b)py razin-2-ol;

(E)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(E)-1-cyclohexyl-6-(prop-1-enyl)-1H-rmidazo[4,5-b]pyrazin-2-ol;

(E)-1 -cyclopropy l-6-(p ro p-1 -eny 1)-1 H-im idazo[4,5-b]pyrazin-2-o i; (E)-1-isopropyl-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol; (E)-6-(prop-1-enyl)-1-(tetrahydro~2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2oi;

(R)-6-(methylthio)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol; (R)-6-bromo-1-(1-hydroxybutan-2-yl)-1H-imidazo[4,5-b]pyrazin-2-ol; (R)-6-bromo-1 -(1 -roorphoiinobutan-2-yJ)-1 H-imidazo[4,5-bJpyrazin-2-ol; (R)-6-bromo-1-(1-morpholinopropan-2-yi)-1H-imidazo[4,5-b]pyrazin-2-ol; (R)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(R) -6-bromo-1 -sec-butyl-1 H imidazo[4,5-b]pyrazin-2-ol;

(S) -(2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-6-yl)(4methylpiperazin-1-yl)methanone;

(S)-(2-hydroxy-1 -(1 -phenylethyl)-1 H-imidazo(4,5-b]pyrazin-6yl)(morpholino)methanone;

(S)-(2-hydroxy-1-(1-phenylethyl)-lH-imidazo[4,5-b]pyrazin-6-yl)(piperidin-1yl)methanone;

(S)-1-(1-phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol;

(S)-1 -(1-phenylethyl)-1 H-imidazo[4,5-b]quinoxalin-2-ol,‘ (S)-1-(1-phenylethyl)-6-(piperidln-1-ylmethyl)-1H’imidazo[4,5-b]pyrazin-2-ol;

(SJ-l-il-phenylethyO-e-propyl-IH-imidazoK.S-bJpyrazin^-ol;

(S)-1 -(1 -phenylethyl)-6-vinyl-1 H-imidazo[4,5-bJpyrazin-2-ol;

2016202040 01 Apr 2016 (S)-1-(2-hydraxy-1-(1-phenylethyl)-1H-imrdazo[4,5-b]pyrazin-6-yl)ethanone; (S)-2-hydroxy-1 -(1 -phenylethyt)-1 H-imidazo[4,5-b]pyrazine-6-carbonitrile; (S)-2-hydroxy-1-(1-phenylethyl)-1H-imidazo(4,5-b]pyrazine-6-carboxamide; (S)-2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6-carboxyiic acid; (S)-2-hydroxy-N,N-dinnethyl-1-(1-phenylethyl)-1H-imidazo[4_<5-b]pyrazine-6carboxamide;

(S)-2-hydroxy-N-methyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6carboxamide;

(S)-6-((4-methylpiperazin-1-yl)methyl)-1-(1-phenylethyl)-1 H-imtdazo^.Sb]pyrazin-2-ol;

(S)-6-((dimethylamino)methyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2ol;

(S)-6-(2-hydroxypropan-2-yl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-(2-methytprop-1-enyl)-1-(1-phenylethyl)-1H-imrdazo[4,5-b]pyrazin-2-ol;

(S)-6-(methylsulfonyl)-1-(1-phenyIethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-(methylthio)-1 -(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-(morpholinomethyl)-1-(1-phenylethyl)-1H-imidazo(4,5-b]pyrazin-2-ol;

(S)-6-bromo-1 -(1 -hydroxybutan-2-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-bromo-1-(1-morpholinobutan-2-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-bromo-1-(1-morpholinopropan-2-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-bromo-1-sec-butyl-lH-imidazo[4,5-b]pyrazin-2-ol;

(S)-6-cyclohexenyt-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol; (S)-6-cyclohexyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol; (S)-6-ethoxy-1 -(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazirt-2-ol;

(S)-6-ethyl-1 -(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol; (S)-6-hexyl-1-(1-phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol; (S)-6-isobutyl-1-(1-phenylethyl)-lH-imidazo[4,5-b]pyrazin-2-ol; (S)-6.-methoxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(S)-methyl 2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6carboxylate;

2016202040 01 Apr 2016 (S)-N,N-diethyl-2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6carboxamide;

(S)-N-benzyl-2-hydroxy-1-(1-phenylethyl)-1 H-imidazo[4,5~b]pyrazine-6carboxamide;

(S, E)-1 -(1 -pheny1ethyl)-6-(prop-1 -any 1)-1 H-imidazo[4,5-b]pyrazin-2-ol;

{S,Z)“1-(1-phenyIethyl)-6-(prop-1-enyl)-1H-imidazo[4,5-bJpyrazin-2-ol;

(S,Z)-6-(hex-2-enyl>-1-(1-phenylethyl)-1H-imidazo[4,5'b]pyrazin-2-ol;

(Z)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

(Z)-1-cyclohexyl-6-(prop-1-eriyl)-1H~imidazo[4,5-b]pyrazin-2-ol;

(Z)-1-cycIopropyl-6-(prop-1-enyl)-1H-rmidazol4,5-b]pyrazin-2-ol;

(Z)-1-isopropyI-6-(prop-1 -enyl)-1 H-imidazo(4,5-b}pyrazin-2-ol;

(Z)-6-(prop-1-enyl)-1-(tetrahydro-2H-pyran~4-yI)'1 H-imidazo[4₍5-bJpyrazin-2 ol;

1-(1~aminobutan-2-yl)-6-bromo-1H-imidazo[4,5-b]pyrazin-2-ol;

1-(1-morpholinobutan-2-yl)-1H-imidazo(4,5-b]pyrazin-2Of;

1-(2-hydroxy-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-5-yl)ethanone;

-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol;

-(pentan-3-yQ-1 H-imidazo[4,5-b]pyrazine'2,6-dioi;

-(pentan-3-yl)-1 H-imidazo[4,5-b]quinoxalin-2-ol; 1-(pentan-3-yl)-5-vinyl-1H-imidazo[4,5-b]pyrazin-2-ol; 1-(pentan-3-yl)-6-(prop-1-ynyl)-1 H-imidazo[4,5-b]pyrazin-2-ol; 1-(pentan-3-yl)-6-(trifluoromethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

-benzyl-6-(methylthio)-1 H-imidazo[4,5-b]pyrazin-2-ol;

1-benzyl-6-bromo-1 H-imidazo[4,5-b]pyrazin-2-ol;

-cyclohexyl-6-(methylthio)-lH-imidazo[4,5-b]pyrazin-2-ol; 1-cyclopropyl-6-(nnethyllhio)-1H“imidazo[4,5-b]pyrazrn-2-ol;

1- isopropyl-6-(rT)ethylthio)-1H-imidazo[4,5-b]pyrazin-2-C]l;

2~(6-bromo-2-hydroxy-1H-imidazo[4,5-b]pyrazin-1-yl)-1-morpholinobutan-1one;

Z-ie-bromo-Z-hydroxy-l H-imidazo[4,5-b]pyrazin-1 -yt)butanoic acid;

2- (6-bromo-2-hydroxy-1H-imidazo[4,5-b]pyrazin-1 -yi)propane-1,3-diol;

2016202040 01 Apr 2016

2-hydroxy-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazire-5-carboxyiic acid; 2-hydroxy-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazine-6-carbonitnle; 2-hydroxy-N,N-dimethyl-1-(pentan-3-yl)-1H4midazo[4,5-b]pyrazine-5carboxamide;

2-hydroxy-N-methyM-{pentan-3-yl)-1H-imidazo[4,5-b]pyrazine-5“ carboxamide;

5-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

5-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

5- ethyi-l-ipentan-S-yO-IH-imidazo^.S-blpyrazin^-ol;

6- (methylsulfinyl)-1-((S)-1-phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol; 6~(methylthio)-1-(pentan-3-yi)-lH-imidazot4,5-b]pyrazin-2-ol; 6-(methylthio)ⁱ1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4₎5-b]pyrazin-2-ol,· 6-bromo-1-(1“(4-(methylsuifonyl)piperazin-1-yl)butan-2-yl)-1H-imidazo[4,5b]pyrazin-2-ol;

6-b romo-1 -(1 -(4-methylpiperazin-1 -yl)buta n-2-y I)-1 H-imidazo[4,5-b]pyrazin-2ol;

6-bromo-1-(1-(dimethylamino)butan-2-yi)-1H-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(1-(methylamino)butan-2-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(1-methoxybutan-2-yl)-1H-imidazo[4_J5-bJpyrazin-2-ol;

6-bromo-4-(2-methyl-1-morphotinopropan-2-yl)-1H4midazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(2-morpholinoethyl)-1H-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(pentan-3-y!)-1H-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2-ol; 6-bromo-1 -cyclohexyl-1 H-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1 -cyclopropyl-1 H-imidazo[4,5-bJpyrazin-2-ol;

6-bromo-1 -isopropyl-1 H-imldazo[4,5-b]pyra zin-2-ol;

6-bromo-1-tert-butyl-1H-imidazo[4,5-b]pyrazin-2-ol;

6-cyclopropyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

6-ethynyl-1-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol;

6-methoxy-1-(pentan-3-yl)-1H-imidazo[4,5'b]pyrazin-2-ol;

6-methyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol;

2016202040 01 Apr 2016 methyl 2-hydroxy-1 -(pentan-3-yl)-1 H-imidazo£4,5-b]pyrazine-5-carboxylate; methyl 4-(2-(6-bromo-2-hydroxy-1 H-imidazo[4,5-b]pyrazin-1y l)buty l)p ipe razine-1 -ca rboxylate;

1-(ethylpropyl)-6-(1-methyJpyrazol-4-yl)imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-(propylbutyl)imidazo£4,5-b]pyrazin-2-ol;

1-[(1R)-3-methyl-1-(morpholin-4-ylmethyl)butyl]-6-bromoimidazot4,5b]pyrazin-2-ol;

1-(ethylpropyf)-6-vinylimidazo[4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-(1-methylvinyl)imidazot4,5-b]pyrazin-2-ol;

1-(ethylpropyl)-6-(methylethyl)imidazo[4,5-b]pyrazin-2-ol;

6-ch loro-1 -(ethylpropyl)imrdazo[4,5-b]pyrazin-2-ol; and 6-(dimethylamino)-1-(ethy1propyl)imldazo[4,5-b]pyrazin-2-ol.

[097] In some embodiments, the compound of Formula II is chosen from the following tautomers of compounds of Formula I;

(R)-6-bromo-1-(1-morpholinopropan-2-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)one;

6-ethynyl-1 -(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one; 6-methoxy-1-(pentan-3-y))-1H-im!dazo[4,5-b]pyrazin-2(3H)-one; 6-bromo-1-(2-methyl-1-morphotinopropan-2-yl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

1-(pentan-3-yl)-6-(1H-1,2,3-triazol-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

1-(pentan-3-yl)-6-(tnfluoromethyl)-1H-imidazo£4,5-b]pyrazin-2(3H)-one;

(R)-6-ethynyl-1-(1-morpholinobutan-2-yl)-1H-imidazo[4,5-b]pyrazin-2{3H)one;

6-((2-hydroxy-1 -(pentan-3-yl)-1 H-imidazo^.S-bJpyrazin^-ylJethynylJ-l (penta n-3-yl)-1 H-imid azo[4,5-b]pyrazin-2(3 H )-one;

6-(dimethylamino)-1-(pentan-3-yl)-1H-imidazo£4,5-b]pyrazin-2(3H)-one;

6-ethyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(E)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(E)-1-cyclohexyl-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(E)-1-cyclopropyI-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

2016202040 01 Apr 2016 (E)-1-isopropyl-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(E)-6-(prop-1-enyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

(R)-6-(methylthio)-1-(1-phenylethyt)-1H-imidazo[4,5-b]pyrazin-2(3H)-one; (R)-6-bromo-1 -{1 -hydroxybutan-2-yl)-1 H-jmidazo[4,5-b]pyrazin-2(3H)-one; (R)-6-bromo-1-(1-morpbolinobutan-2-yl)-1H-imidazc[4,5-b]pyrazin-2(3H)-one; (R)-6-bromo-1-{1-morpho1inopropan-2-yl)-1H-imi£lazo[4,5-b]pyrazin-2(3H)one;

(R)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b)pyrazin-2(3H)-one;

(R) -6-bromo-1 -sec-butyl-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S) -1 -(1 -phenylethyi)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-1-(1-phenylethyl)-1H-imidazo[4,5--b]quinoxalin-2(3H)-one;

(S)-1 -(1 -phenylethyO-e-ipiperidin-l -ylmethyl)-1 H-lmidazo[4,5-b]pyrazin-2(3H)one;

(S)-1-(1 -phenylethyl)-6-(piperidine-1-carbonyl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

(S)-1-(1-phenylethyl)-6-propyl-1 H-imidazo[4,5-b]pyraztn-2(3H)-one; (S)-1-(1-phenylethyl)-6-vinyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one; (S)-2-oxo-3-(1-phenylethyt)-2,3-dihydro-1H-imidazo[4,5'b]pyrazine-5carbonitrile;

(S)-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5carboxamide;

(S)-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imldazo[4,5-b]pyrazine-5carboxylic acid;

(S)-6-((4-methylpiperazin-1-yl)methyl)-1-(1-phenylethyl)-1H-imidazo[4,5b]pyrazin-2(3H)-one;

(S)’6-<(dimethylamino)methyl)-l-(1-phenylethy[)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

($)-6-(2-hydroxypropan-2-yl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

(S)-6-(2-methylprop-1-enyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin33

2016202040 01 Apr 2016

2(3H)-one;

(S)-6-(4-methylpiperazine-1-carbonyl)-1-(1-phenylethyl)-1H-imidazo[4,5bjpy razin-2 (3H)-one;

(S)-Q-(methylsulfonyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

{S)-6-(methylthio)-1-(1-phenylethyl)-1H-irnidazoi4,5-b]pyrazin-2(3H)-one;

(S)-6-(morpholine-4-carbonyl)-1-(1-phenylethyl)-1H-imtdazo[4,5-b]pyrazin2(3H)-one;

(S)-6-(morpholinomethyl)-1-(1“phenylethyl)-1H-ini(dazo[4₎5-b]pyrazin-2(3H)one;

(S)-6-acetyi-1 -(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-bromo-1-(1-hydroxybutan-2-yl)~1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-bromo-1-(1-morpholinobutan-2-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-bromo-1-(1-morpholinopropan-2-yl)-1H-imidazo[4,5-b]pyrazin-2{3H)one;

(S)-6-bromo-1-(1-phenylethyi)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-bromo-1 -sec-butyl-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-cyclohexenyl-1-{1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-cyclohexyl-1-(1~pheny!ethyl)-1H-imidazo[4,5-b)pyrazin-2(3H)-one;

(S)-6-ethoxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-ethyI-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-hexyt-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-6-isobutyl-1-(1-phenylethyl)-1H-imidazo[4,5-bJpyrazin-2(3H)-one;

(S)-6-methoxy-1 -(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S)-methyl 2-oxo-3-(1-pheny(ethyl)-2,3-dihydro-1H-imjdazo[4,5-bJpyrazine-5carboxylate;

(S)-N,N-diethyl-2-oxo-3-(1-phenylethyl)-2,3-dihyd.O-1H-imidazo[4,5b]pyrazine-5-carboxamide;

(S)-N,N-dimethyl-2-oxo-3-(1-phenylethyJ)-2,3-dihydro-1H-imidazo[4,5b]pyrazine-5-carboxamide;

(S)-N-benzyl-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine5-carboxamide;

2016202040 01 Apr 2016 (S)-N-methyl-2-oxo-3-(1-pheriylethyl)-2,3-dihydro-1H-inriidazo[4,5-b]pyrazirie5-carboxamide;

(S,E)-1-(1-phenylethyl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(S,Z)-1-(1-phenylethyi)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

{S,Z)-6-(hex-2-enyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazln-2(3H)-one;

(Z)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(Z)-1-cyclohexyt-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(Z)-1-cyclopropyl-6-(prop-1-enyl)-1H-im!dazo[4,5-b]pyrazin-2(3H)-one;

(Z)-1-isopropyl-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

(Z)-6-(prop-1-enyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

1-(1-aminobutan-2-yl)-6-bromo-1H-imidazo[4,5-b]pyrazin-2(3H)-one; 1-(1-morpholinobiitan-2-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one; .

-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

-(pentan-3-yl)-1 H-imidazo[4,5-b]quinoxalin-2(3H)-one;

1-(pentan-3-yl)-5-vinyHH-imidazo[4,5-b]pyrazin-2(3H)-one;

1-(pentan-3-yi)-6-(prop-1-ynyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

1-(pentan-3-yl)-6-(trifiuoromethyl)-1H-imidazoI4,5-b]pyrazin-2(3H)-one;

-benzyl-6-(methytthio)-1 H-imidazo[4,5-b]pyrazin-2{3H)-one;

-benzyl-6-bromo-1 H-imidazo[4,5-b]pyrazin-2(3H}-one;

1-cyclohexyl-6-(methylthio)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one; l-cydopropyl-e-imethylthioMH-innidazoH.S-bJpyrazin-GiSHJ-one;

1- isopropyl-6-(methytthio)-1 H-imidazo[4,5-bJpyrazin-2(3H)-one;

2- (6-bromo-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyrazin-Lyl)butanoic acid; 2-oxo-1-(pentan-3-yl)-2,3-dihydro-1H-irTiidazo[4,5-b]pyrazine-5-carboxytic acid;

2-oxo-3-(pentan~3-yl)-2,3-drhydro-1H-imidazo[4,5-b]pyrazine-5-carbonitrile;

5-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

5-acetyI-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

5-bromo-1 -(pentan-3-yl)-1 H-irnidazo[4,5-b]pyraziri-2(3H)-one;

5-ethyl-1 -(pentan-3-yl)-1 H-iimidazo[4,5-b]pyrazin’2{3H)-one;

2016202040 01 Apr 2016

6-(methylsulfinyl)-1-((S)-1-phenytethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-(methylthio)-1-(pentan-3-yl)-1H-imidazo(4,5-b]pyrazin-2(3H)-one;

8-(methylthio)-1-{tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)one;

6-bromo-1 -(1-(4-(methylsulfonyl)piperazin-1-yl)butan-2-yl)-1H-lmidazo[4,5b]py razin-2(3 H)-one;

6-bromo-1 -(1 -(4-methy Ipi perazin-1 -y l)butan-2-yl)-1 H-im idazo [4,5-b]py razin2(3H)-one;

6-bromo-1-(1 -(d imethyl amino)butan-2-y 1)-1 H-imidazo[4,5-b)pyrazin-2(3H)one;

6-bromo-1-(1-(methylamino)butan-2-yl)-1H~imidazo(4_I5-b]pyrazin-2(3H)-one; 6-bromo-1 -(1,3-dihydroxypropan-2-yl)-1 H-imidazo[4,5-b]pyrazln-2(3H)-one; 6-bromo-1 -(1 -m ethoxybutan-2-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one; 6-bromo-1-(1-morpholino-1-oxobutan-2-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)one;

6-bromo-1-{2-methyl-1-morpholinopropan-2-yI)-1 H-imidazo[4,5-b]pyrazin2(3H)-one;

6-bromo-1-(2-morpholinoethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-1-(pentan-3-yl)-1H-imidazoE4,5-b]pyrazin-2(3H)-one;

6-bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-1 -cyclohexyl-1 H-imidazo[4,5-b)pyrazin-2(3H)-one;

6-bromo-1-cyclopropyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-1-isopropyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-1-tert-butyl-1H-imidazo[4,5-bJpyrazin-2(3H)-one;

6-cyclopropyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-ethynyl-1 -(pentan-3-yt)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-hydroxy-1 -(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-methoxy-1 -{pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

6-methyl-1 -(pentan-3-yl)-1 H-imidazo[4,5-bJpyrazin-2(3H)-one; methyl 2-oxo-1-(pentan-3-yl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5carboxylate;

2016202040 01 Apr 2016 methyl 4-(2-(6-bromo-2-oxo-2,3-dihydro-1H-imidazo[4,5-b)pyrazin-1yl)butyl)piperazine-1-carboxylate;

N,N-dimethyl-2-oxo-1-(pentan-3-yl)-2,3-dihydro1H-imidazo[4,5-b)pyrazine-5carboxamide;

N-methyl~2-oxo-1-(pentar>-3-yt)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5carboxamide;

6-(1-methyl-1 H-pyrazol-4-yl)-1 -(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)one;

6-bromo-1 -(heptan-4-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one;

(R)-6-bromo-1-(4-methyl-1-morpholinopentan-2-yl)-1H-imidazo[4,5-b]pyrazin2(3H)-one;

1-(pentan-3-yl)-6-vinyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one; 1-(pentan-3-yl)-6-(prop-1-en-2-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one; 6-isopropyl-1-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one; 6-chloro-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one; and 6-(dimethylamino)-1-{pentan-3-yl)-1H-imidazo[4,5-b]pyrazin“2(3H)-one.

[098] The compounds of Formula I can be named and numbered (e.g., using NamExpert™ available from Cheminnovation or the automatic naming feature of ChemDraw Ultra version 10.0 from Cambridge Soft Corporation) as described below. For example, the compound:

i.e., the compound according to Formula I where Ri is (E)-propen-lyl, R₂ is (S)-secphenethyl, and R₄ is H, can be named (S,E)-1-(1-phenylethyl)-6-(prop-1-enyl)-1Himidazo[4,5-b]pyrazin-2-ol.

[099] Likewise the compound;

2016202040 01 Apr 2016

i.e., the compound according to Formula I where Ri is (Z)-propen-l-yi, R2 is 3-pentyl, and R4 is H, can be named (Z)-1-{pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5b]pyrazin-2-ol.

[0100] Similarly, the compounds of Formula II can be named and numbered {e.g., using NamExpert™ available from Cheminnovation or the automatic naming feature of ChemDraw Ultra version 10.0 from Cambridge Soft Corporation) as described below. For example, the compound:

i.e., the compound according to Formula II where R1 is (E)-propen-l-yl, R₂ is {S)-secphenethyl, and R₄ is H, can be named (S_tE)-1-(1-phenylethyl)-6-(prop-1-enyl)-1Himidazo[4,5-b]pyrazin-2(3H)-one.

[0101] Likewise the compound:

i.e., the compound according to Formula II where R1 is (Z)-propen-l-yl, R₂ is 3-pentyl, and R₄ is H, can be named (Z)-1-(pentan-3-yl)-6-{prop-1-enyi)-1H-imidazo[4,5b]pyrazin-2(3H)-one.

[0102] The chemical entities described herein can be synthesized utilizing techniques well known in the art, e.g., as illustrated below with reference to the Reaction Schemes.

[0103] Unless specified to the contrary, the reactions described herein take place at atmospheric pressure, generally within a temperature range from -10 °C to 200 °C. Further, except as employed in the Examples or as otherwise specified, reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about -10 °C to about 110 °C over a period of . about 1 to about 24 hours; reactions left to run overnight average a period of about 16

2016202040 01 Apr 2016 hours.

[0104] The terms solvent, organic solvent, and inert solvent each mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (’’DMF), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, N-methylpyrrolidone (NMP), pyridine and the like]. Unless specified to the contrary, the solvents used in the reactions described herein are inert organic solvents. Unless specified to the contrary, for each gram of the limiting reagent, one cc (or mL) of solvent constitutes a volume equivalent [0105] Isolation and purification of the chemical entities and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thinlayer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can also be used.

[0106] When desired, the (R)- and (S)-isomers may be resolved by methods known to those skilled in the art, for example by formation of dlastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent. Alternatively, a specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

[0107] Many of the optionally substituted starting compounds and other reactants are commercially available, e.g., from Aldrich Chemical Company (Milwaukee, Wl) or can be readily prepared by those skilled in the art using commonly employed synthetic

2016202040 01 Apr 2016 methodology.

Reaction Scheme 1

101 [0108] Referring to Reaction Scheme 1, Step 1, to a compound of Formula 101, wherein X is halo, is added an excess (such as about 2 to 20 equivalents) of a compound of formula R₂-NH₂. The reaction vessel is heated to about 110 °C to 190 °C over about 20 to 40 minutes, optionally with microwave irradiation. The product, a compound of Formula 102, is isolated and optionally purified.

[0109] Referring to Reaction Scheme 1, Step 2, to a solution of a compound of Formula 102 in a suitable solvent (such as THF) is added a di-activated carbonyl equivalent such as carbonyl diimidazole (CDI), phospgene, or triphosgene. The product, a compound of Formula 103, is isolated and optionally purified.

Reaction Scheme 2

201

Step 3 (a-e)

104 [0110] Reaction Scheme 2 illustrates reactions for further converting compounds of Formula 201, wherein X is a leaving group, to compounds of Formula 104 through one of Steps 3(a-e). in some embodiments, X is halo, for example, bromo.

[0111] Referring to Reaction Scheme 2, Step 3(a), a compound of Formula 201 and about 0.05 to 0.15 equivalents of a suitable catalyst such as (Ph₃P)₄Pd in a suitable solvent such as NMP or dioxane, a suitable base, and an excess (such as about 1.5 to 32 molar equivalents) of a suitable tin reagent such as RiSn(butyl)₃ is mixed at from about 0 to about 200°C for about 6 to 48 hours. The product, a compound of Formula 104 wherein Ri is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted cycloalky), is isolated and optionally purified.

[0112] Referring to Reaction Scheme 2, Step 3(b), a compound of Formula 201 and

2016202040 01 Apr 2016 an excess (such as about 4 to 5 equivalents) of a compound of the formula NaSR_xwhere SR_X is R-ι, and a suitable solvent (such as NMP) is heated to about 50 °C to 200 °C over about 10 min to 24 h, optionally with microwave irradiation. The product, a compound of Formula 104 wherein R₄ is sulfanyl, is isolated and optionally purified. [0113] Referring to Reaction Scheme 2, Step 3(c), a compound of Formula 201, an excess (such as about 1.9 to 2.3 equivalents) of a compound of the formula RiB(OH)₂, about 0.10 to 0.15 equivalents of (DPPFJPdCb, a suitable solvent (such as dioxane) and about 2 to 3 equivalents of a suitable base (such as 2N K₂CO₃) is heated to about 90 °C for about 6 to 24 hours. The product, a compound of Formula 104 wherein Ri is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted cycloalkyl, is isolated and optionally purified.

[0114] Referring to Reaction Scheme 2, Step 3(d), about 2 equivalents of R_XOH where OR* is R·) and a suitable solvent (such as NMP), and about 2 equivalents of suitable base (such as NaH) is added followed by a compound of Formula 201. The mixture is heated to about 50 to about 200’C for about 10 minutes to about 48 hours. In some embodiments, the reaction is heated for about 30 minutes, optionally with microwave irradiation. The product, a compound of Formula 104 wherein R-ι is optionally substituted alkoxy, is isolated and optionally purified.

[0115] Referring to Reaction Scheme 2, Step(e), a compound of Formula 201, an excess (such as about 2.0 equivalents) of a compound of the formula KR₄BF3, an excess (such as about 3 equivalents) of a suitable base (such as CS2CO3), a suitable amount of (DPPF)PdCI₂ (such as about 0.2 equivalents), a suitable solvent (such as dioxane and water) is mixed at about room fermperature to about 100 °C for about 8 to 48 hours and the product, a compound of Formula 104 wherein R1 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted cycloalkyl, is Isolated and optionally purified.

[0116] A racemic mixture is optionally placed on a chromatography column and separated into (R)~ and (S)-enantiomers.

[0117] The compounds described herein are optionally contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salts.

2016202040 01 Apr 2016 [0118] Pharmaceutically acceptable acid addition salts of compounds of Formula I or compounds of Formula II are optionally contacted with a base to form the corresponding free base.

[0119] The chemical entities described herein modulate one or more of skeletal myosin, skeletal actin, skeletal tropomyosin, skeletal troponin C, skeletal troponin I, skeletal troponin T, and skeletal muscle, Including fragments and isoforms thereof, as well as the skeletal sarcomere, and are useful to bind to, inhibit and/or potentiate the activity thereof. As used in this context, modulate means either increasing or decreasing myosin activity, whereas potentiate means to increase activity and inhibit means to decrease activity.

[0120] The chemical entities, pharmaceutical compositions and methods described herein are used to treat obesity, sarcopenia, wasting syndrome, frailty, cachexia, muscle spasm, post-surgical and post-traumatic muscle weakness, neuromuscular disease, and other indications in a mamma!.

[0121] Methods to identify the chemciai entities as binding to a protein or as a modulator of the binding characteristics or biological activity of a protein are described in, for example, U.S. Patent No. 6,410,254 and U.S. Patent Application No. 10/987,165. [0122] For example, test compounds can be assayed in a highly parallel fashion by using multiwell plates by placing the compounds either individually in wells or testing them in mixtures. Assay components including the target protein complex, coupling enzymes and substrates, and ATP can then be added to the wells and the absorbance or fluorescence of each well of the plate can be measured with a plate reader.

[0123] In some embodiments, the method uses a 384 well plate format and a 25 pireaction volume. A pyruvate kinase/Iactate dehydrogenase coupled enzyme system (Huang TG and Hackney D D. (1994) J Biol Chem 269(23):16493-501) can be used to measure the rate of ATP hydrolysis in each well. As will be appreciated by those in the art, the assay components are added in buffers and reagents, The incubation periods can be optimized to give adequate detection signals over the background. The assay can be done in real time giving the kinetics of ATP hydrolysis which increases the signal to noise ratio of the assay.

[0124] The compounds can be further tested using skinned muscle fiber

2016202040 01 Apr 2016 preparations. Such assays are known in the art. See, e.g., Cheung et al. (2002) Nature Cell Biol. 4:83 and U.S. Patent Publication No. 20020006962.

[0125] The chemical entitles described herein are administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease states previously described. While human dosage levels have yet to be optimized for the chemical entities described herein, generally, a daily dose ranges from about 0.05 to 100 mg/kg of body weight; in certain embodiments, from about 0.10 to 10.0 mg/kg of body weight, and in certain embodiments, from about 0.15 to 1.0 mg/kg of body weight. Thus, for administration to a 70 kg person, in certain embodiments, the dosage range would be about from 3.5 to 7000 mg per day; In certain embodiments, about from 7.0 to 700.0 mg per day, and in certain embodiments, about from 10.0 to 100.0 mg per day. The amount of the chemical entity administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician; for example, a likely dose range for oral administration would be from about 70 to 700 mg per day, whereas for intravenous administration a likely dose range would be from about 70 to 700 mg per day depending on compound pharmacokinetics.

[0126] Administration of the chemical entities described herein can be via any of the accepted modes of administration for agents that serve similar utilities including, but not limited to, orally, sublingually, subcutaneously, intravenously, intranasally, topically, transdermally, intraperitoneatly, intramuscularly, intrapulmonarilly, vaginally, rectally, or intraocularly. In some embodiments, oral or parenteral administration is used.

[0127] Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as, e.g., tablets, capsules, powders, liquids, suspensions, suppositories, aerosols or the like. The chemical entities can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate. In certain embodiments, the compositions are provided in unit dosage forms suitable for single administration of a precise dose.

[0128] The chemical entities described herein can be administered either alone or

2016202040 01 Apr 2016 more typically in combination with a conventional pharmaceutical carrier, excipient or the like (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbttan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). Generally, depending on the intended mode of administration, the pharmaceutical composition will contain about 0.005% to 95%; in certain embodiments, about 0.5% to 50% by weight of a chemical entity. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for exampie, see Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0129] In addition, the chemical entities described herein can be co-adminfstered with, and the pharmaceutical compositions can include, other medicinal agents, pharmaceutical agents, adjuvants, and the like. Suitable medicinal and pharmaceutical agents include modulators of one or more of skeletal myosin, skeletal actin, skeletal tropomyosin, skeletal troponin C, skeletal troponin I, skeletal troponin T, and skeletal muscle, including fragments and isoforms thereof, and the skeletal sarcomere and other suitable therapeutic agents useful in the treatment of the aforementioned disorders including: anti-obesity agents, anti-sarcopenia agents, anti-wasting syndrome agents, anti-frailty agents, anti-cachexia agents, anti-muscle spasm agents, agents against post-surgicai and post-traumatic muscle weakness, and anti-neuromuscular disease agents, as well as the agents described in U.S. Patent Application No. 2005/0197367.

[0130] Suitable additional medicinal and pharmaceutical agents include, for example: orlistat, sibramine, diethylpropion, phentermine, benzaphetamine, phendimetrazine, estrogen, estradiol, levonorgestrel, norethindrone acetate, estradiol valerate, ethinyl estradiol, norgestimate, conjugated estrogens, esterified estrogens, medroxyprogesterone acetate, testosterone, insulin-derived growth factor, human growth hormone, riluzole, cannabidiol, prednisone, albuterol, non-steroidal anti44

2016202040 01 Apr 2016 inflammatory drugs, and botulinum toxin, [0131] Other suitable medicinal and pharmaceutical agents include TRH, diethylstilbesterol, theophylline, enkephalins, E series prostaglandins, compounds disclosed in U.S. Patent No. 3,239,345 (e.g., zeranol), compounds disclosed in U.S. Patent No. 4,036,979 (e.g., sulbenox), peptides disclosed in U.S. Patent No. 4,411,890 growth hormone secretagogues such as GHRP-6, GHRP-1 (disclosed in U.S. Patent No. 4,411,890 and publications WO 89/07110 and WO 89/D7111), GHRP-2 (disclosed in WO 93/04081), NN703 (Novo Nordisk), LY444711 (Lilly), MK-677 (Merck),

CP424391 (Pfizer) and B-HT920, growth hormone releasing factor and its analogs, growth hormone and its analogs and somatomedins including IGF-1 and IGF-2, alphaadrenergic agonists, such as clonidine or serotonin 5-HTd agonists, such as sumatriptan, agents which inhibit somatostatin or its release, such as physostigmine, pyridostigmine, parathyroid hormone, PTH(1-34), and bisphosphonates, such as MK217 (alendronate).

[0132] Still other suitable medicinal and pharmaceutical agents include estrogen, testosterone, selective estrogen receptor modulators, such as tamoxifen or raloxifene, other androgen receptor modulators, such as those disclosed in Edwards, J. P. et. al., Bio. Med. Chem. Let., 9, 1003-1008 (1999) and Hamann, L. Θ. et. al., J. Med. Chem., 42, 210-212 (1999), and progesterone receptor agonists (”PRA”), such as levonorgestrel, medroxyprogesterone acetate (MPA).

[0133] Still other suitable medicinal and pharmaceutical agents include aP2 inhibitors, such as those disclosed in U.S. Ser. No. 09/519,079 filed Mar. 6, 2000, PPAR gamma antagonists, PPAR delta agonists, beta 3 adrenergic agonists, such as AJ9677 (Takeda/Dainippon), L750355 (Merck), orCP331648 (Pfizer), other beta 3 agonists as disclosed in U.S. Patent Nos. 5,541,204, 5,770,615, 5,491,134, 5,776,983 and 5,488,064, a lipase inhibitor, such as oriistat or ATL-962 (Alizyme), a serotonin (and dopamine) reuptake inhibitor, such as sibutramine, topiramate (Johnson & Johnson) or axokine (Regeneron), a thyroid receptor beta drug, such as a thyroid receptor ligand as disclosed in WO 97/21993, WO 99/00353, and GB98/284425, and anorectic agents, such as dexamphetamine, phentermine, phenylpropanolamine or mazindol.

2016202040 01 Apr 2016 [0134] Still other suitable medicinal and pharmaceutical agents include HIV and AIDS therapies, such as indinavir sulfate, saquinavir, saquinavir mesylate, ritonavir, lamivudine, zidovudine, lamivudine/zidovudine combinations, zalcitabine, didanosine, stavudine, and megestrol acetate.

[0135] Still other suitable medicinal and pharmaceutical agents include antiresorptive agents, hormone replacement therapies, vitamin D analogues, elemental calcium and calcium supplements, cathepsin K inhibitors, MMP inhibitors, vitronectin receptor antagonists, SrcSH.sub.2 antagonists, vacular -H⁺~ATPase inhibitors, ipriflavone, fluoride, Tibo lone, pro stanoids, 17-beta hydroxysteroid dehydrogenase inhibitors and Src kinase inhibitors.

[0136] The above other therapeutic agents, when employed in combination with the chemical entities described herein, may be used, for example, in those amounts indicated in the Physicians’ Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[0137] In certain embodiments, the compositions will take the form of a pill or tablet and thus the composition will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polywylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like. In another solid dosage form, a powder, marume, solution or suspension (e.g., in propylene carbonate, vegetable oils or triglycerides) is encapsulated in a gelatin capsule.

[0138] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. at least one chemical entity and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of chemical entities contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the chemical entities and the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and will be higher if the composition is a solid which

2016202040 01 Apr 2016 will be subsequently diluted to the above percentages. In certain embodiments, the composition will comprise from about 0.2 to 2% of the active agent in solution.

[0139] Pharmaceutical compositions of the chemical entities described herein may also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the pharmaceutical composition have diameters of less than 50 microns, in certain embodiments, less than 10 microns. [0140] The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

Example 1

Synthesis of (S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol

NH_a

Me' Ph

XX nh₂

Br

MW, 180 °C [0141] Step 1: (S)-6-bromo-N²-(1-phenylethyl)pyrazine-2,3-diamine. A thickwalled microwave bottle equipped with a stirbar was charged with 1.0 equiv of 3,5dibromopyrazin-2-amine and 6.6 equiv of (S)-sec-phenethylamine. The bottle was fitted with a septum and cap and heated to 180 ”C in a microwave for 30 min. The resulting solution was adsorbed onto 20 g of silica; flash chromatography (10% - 50% EtOAc/Hexanes) provided the title compound (60%) as an off-white foam. LCMS m/z (APCI) = 293.0, 295.0 (M+H).

n^nh₂

XX

ΒΓ N NH

Me Ph excess CD I THF,67°C

[0142] Step 2: (S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol. To a

2016202040 01 Apr 2016 solution of (S)-6-bromo-N^z-(1-phenylethyl)pyrazine-2,3-diamine (1.0 equiv) in refluxing anhydrous THF (5 volume equivalents) was added carbonyldiimidazole (CDI). Successive portions of CDI were added until the starting material was consumed (approx. 3.6 equiv total) as judged by TLC (50% EtOAc/Hexanes). After complete reaction, the mixture was cooled to room temperature and quenched by the careful addition of water until gas evolution had ceased. The mixture was diluted with 25 volume equivalents of EtOAc and washed with 3 x 7.5 volume equivalents of water and 1 x 7.5 volume equivalents of brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Flash chromatography (Biotage MPLC 5% - 40% EtOAc/Hexanes) provided the title compound (66%) as a white solid. LCMS m/z (APCI) = 319.0, 321.0 (M+H).

Example 1(a)

Jl X

Me

Ύ

OEt

Sn{n-Bu)₃

Et₃N, Dioxane 80 °C

[0143] (S)-1-(2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-6yljethanorte. An oven-dried scintillation vial equipped with a stirbar was charged with (S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv), and (PhaPfiPd (0.07 equiv). The bottle was covered with a sheet of Parafilm and purged with nitrogen for 5 min, and anhydous dioxane (13 volume equivalents), triethylamine (3.0 equiv), and tributyl(1-ethoxyvinyl)stannane (1.5 equiv) were added by syringe. The resulting mixture was heated to 80 ’C overnight. The reaction was quenched with 1 N KHSO₄ and stirred for 30 min. The mixture was then diluted with EtOAc, washed twice with NaHCOa and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Flash chromatography (Biotage MPLC 10% - 66% EtOAc/Hexanes) provided the title compound (33%) as a white solid, LCMS m/z (APCI) = 283.1 (M+H). '

2016202040 01 Apr 2016

Example 1(b)

Aph Et₃N, MW

Et₃N, MW NMP, 120 °C

[0144] (S,E)-1-(1-phenylethyl)-€-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol. A thick-walled microwave bottle equipped with a stirbar was charged with (S)-6-bromo-1(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv), and (PtbP^Pd (0.12 equiv). The bottle was covered with a sheet of Parafilm and purged with nitrogen for 5 min, and W-methylpyrrolidone (14 volume equivalents), triethyt amine (2.0 equiv), and (E)tributy!(prop-1-enyl)stannane (3.0 equiv) were added by syringe. The resulting mixture was immediately fitted with a septum and cap and heated to 120 ’C in a microwave for 20 min. The reaction was then diluted with EtOAc, washed four times with saturated aq, NaHCO₃ and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Reverse-phase preparative HPLC provided the title compound (54%) as a foam. LCMS m/z (APCi) = 281.1 (ΜΗΊ).

Example 1(c)

[0145] (S)-6-(methytthio)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-oL A thick-wailed microwave bottle equipped with a stirbar was charged with (S)-6-bromo-1(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv), sodium thiomethoxide (4,5 equiv), and A/-methylpyrrolidone (10 volume equivalents). The bottle was fitted with a septum and cap and heated to 180 °C in a microwave for 30 min. The reaction mixture was then diluted with 100 volume equivalents of EtOAc and washed with 4 x 100 volume equivalents of water and 1 x 100 volume equivalents of brine. The organic layer

2016202040 01 Apr 2016 was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Reversephase preparative HPLC (20% - 80% MeCN/H₂O) provided the title compound (24%) as a white solid. LCMS m/z (APCI) = 287.1 (M+H).

Example 1(d) _CrX„X/^OH %^B(0Hh , νζ,Χζ™

A~Ph DPPFPdCIz, aq. K₂CO₃ ,X-Ph ^Me Dioxane, 90 °C ^Me [0146] {S,Z)-1-(1-phenylethyl)-64prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol. A scintillation vial equipped with a stirbar was charged with (S)-6-bromo-1-(1-phenylethyl)1 H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv), (Z)-prop-1-enyl boronic acid (2.0 equiv) and (DPPF)PdCI₂ (0.10 equiv). The vial was fitted with a septum-lined cap and purged with nitrogen for 5-10 min. To this mixture was added dioxane (16.6 volume equivalents) and degassed 2 N K₂CO₃ (4.2 volume equivalents) by syringe. The resulting mixture was heated to 90 °C overnight. The mixture was cooled to room temperature, diluted with EtOAc, washed with twice with saturated aq. NaHCO3, and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Flash chromatography (Biotage MPLC 5% - 40% EtOAc/Hexanes) provided the title compound (63%) as an off-white foam. LCMS m/z (APCI) = 281.1 (M+H).

Example 1(e)

T OH ^B(OH)₂ fl X /'—OH /-Ph aq- K₂CO₃ ,X-Ph

Me Dioxane, 90 °C Me [0147] (S)-1 -(1-pheny lethyl)-6-vinyl-1H-imidazo[4,5-b]pyrazin-2-ol. A scintillation vial equipped with a stirbar was charged with (S)-6-bromo-1-(1-phenylethy1)-1Himidazo[4,5-b]pyrazin-2-ol (1.0 equiv), vinyl boronic acid (2.2 equiv), and (DPPF)PdCI₂

2016202040 01 Apr 2016 {0.10 equiv). The vial was fitted with a septum-lined cap and purged with nitrogen for .510 min. To this mixture was added dioxane (16.6 volume equivalents) and degassed 2 N K₂CO₃ (4.2 volume equivalents) by syringe. The resulting mixture was heated to 90 'C overnight. The mixture was cooled to room temperature, diluted with 12 volume equivalents EtOAc, washed with twice with saturated aq. NaHCC>3, and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Flash chromatography (Blotage MPLC 5% - 40% EtOAc/Hexanes) provided the title compound as an off-white solid (54%). LCMS m/z (APCl) = 267.0 (M+H).

Example 1(f)

Md'

Me' [0148] (S)-6-methoxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol. A thickwalled microwave bottle equipped with a stirbar was charged with MeOH (2.0 equiv) and NMP (20 volume equivalents). To the resulting solution was added NaH (2.0 equiv), resulting in gas evolution. (S)-6-Bromo-1-(1-phenylethy))-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv) was then added to the reaction, and the resulting mixture was immediately fitted with a septum and cap and heated to 200 *C in a microwave for 30 min. The reaction was then diluted with EtOAc, washed twice with saturated aq. NaHCO3 and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Reverse-phase preparative HPLC provided the title compound (29%) as a white solid. LCMS m/z (APCl) = 271.1 (M+H).

2016202040 01 Apr 2016

Example 1(g)

H_a(1 atm), Pd/C MeOH, 23 °C

[0149] (S)-1-(1-phenylethyl)-6-ethyl-1H-imidazo[4,5-b]pyrazln-2-ol. A scintillation vial equipped with a stirbar was charged with (S)-6-vinyl-1-(1-phenylethyt)-1Himidazo[4,5-b]pyrazin-2-ol (1.0 equtv), methanol (280 volume equivalents), and catalytic Pd/C. The resulting mixture was purged with hydrogen for 45 minutes; the reaction was complete as judged my LCMS. The mixture was then filtered through a pad of diatomaceous earth and the pad of diatomaceous earth was washed twice with MeOH. The solution was concentrated in vacuo. Flash chromatography (Biotage MPLC 5% 40% EtOAc/Hexanes) provided the title compound (86%) as a foam. LCMS m/z (APCI) = 269.1 (M+H).

Example 2

Synthesis of 6-bromo-1-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol

[0150] Step 1: 6-bromo-N²-(pentan-3-yl)pyrazine-2,3-diamine. The title compound was prepared in a manner analogous to Example 1 Step 1 except that 3-aminopentane (2.6 volume equivalents) was substituted for (S)-sec-phenethylamine. LCMS m/z (APCI) = 259.0, 260.0 (M+H).

Sr

excess CDI

THF, 67 °C

2016202040 01 Apr 2016

[0151] Step 2: 6-bromo-1-(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol. The title compound was prepared by treating 6-bromo-N^z-(pentan-3-yl)pyrazine-2,3-diamine in a manner analogous to Example 1 Step 2 LCMS m/z (APCI) = 285.0, 287.0 (M+H).

Example 2(a)

Vo„ ^Sn.n-Bu,, _M^X;_XV-OH _c>-et BsN, MW ^Et NMP, 120 °C ^Et [0152] (E)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-ol. A thickwailed microwave bottle equipped with a stirbar was charged with 6-bromo-1-(pentan-3yl)-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv) and (PhjPfcPd (0.15equiv). The bottle was covered with a sheet of Parafilm and purged with nitrogen for 5-10 min, and A/methylpyrrofidone (11.7 volume equivalents), triethyl amine (2.0 equiv), and (E)tributyl(prop-1-enyl)stannane (2.0 equiv) were added by syringe. The resulting mixture was immediately fitted with a septum and cap and heated to 120 ’C in a microwave for 20 min. The reaction was then diluted with EtOAc, washed with three times with water and once with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. Reverse-phase preparative HPLC provided the title compound (15%) as a foam. LCMS m/z (APCI) = 247.1 (M+H).

Example 2(b)

X X?~^{oh NasMe} X Xx°^H

- MeS N N /Et MW, NMP, 180 °C /Et

Et Et [0153] 6-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol. The title compound was prepared by reacting 6-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin2-ol (1.0 equiv), sodium thiomethoxide (1.4 equiv), and W-methylpyrrolidone (5.9 volume

2016202040 01 Apr 2016 equivalents) in a manner analagous to Example 1{c). LCMS m/z (APCI) = 253.0 (M+H).

Example 2{c)

Me aq, K₂CO₃Dioxane, 90 °C

[0154] (Z)-1-(pentan'3~yl)-6-(prop-1-enyl)-1H-imldazo[4,5-b]pyrazin-2-ol. The title compound was prepared by reacting 6-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin· 2-ol (1.0 equiv), (Z)-prop-1-enyl boronic acid (1.5 equiv) and (DPPF)PdCl2 (0.15 equiv) in a manner analagous to Exmple 1(d), LCMS m/z (APCI) = 247.0 (M+H).

Example 3

Synthesis of 1*(pentan-3-yt)-1H-imidazo[4,5-b]quinoxa1in-2-oI

[0155] Step 1: N²-(pentan-3-yl)quinoxaline-2,3-diamine. A thick-walled microwave bottle equipped with a stirbar was charged with 3-chloroquinoxalin-2-amine (1.0 equiv) and 20 volume equivalents of 3-aminopentane. The bottle was fitted with a septum and cap and heated to 120 ’C in a microwave for 30 min. The resulting solution was concentrated in vacuo. Flash chromatography (20% - 60% EtOAc/Hexanes) provided the title compound (78%) as a yellow solid. LCMS m/z (APCl)= 231.1 (M+H).

2016202040 01 Apr 2016

excess CDI THF, 67 °C

[0156] Step 2:1 -(pentan-3-yl)-1 H-imidazo[4,5-b]quir»oxalin-2-ol. The title compound was prepared by treating N²-(pentan-3-yl)quinoxaline-2,3-diamine in a manner analogous to Example 1 Step 2 LCMS m/z (APCI) = 257.2 (M+H).

Example 3(a)

KMeBF₃aq. Cs₂CO₃Dioxane, 90 °C

[0157] Synthesis of (Z)-1-(pentan-3-yl)-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin· 2-ol. A scintillation vial equipped with a stirbar was charged with 6-bromo-1-(pentan-3yl)-1H-imidazo[4,5-b]pyrazin-2-ol (1.0 equiv), KF₃BMe (2.0 equiv), Cs₂CO₃ (3.0 equiv) and (DPPF)PdCI₂ {0.20 equiv). The vial was fitted with a septum-lined cap and purged with nitrogen for 5-10 min. To this mixture was added dioxane (25 volume equivalents) and degassed water (5 volume equivalents) by syringe. The resulting mixture was heated to 90 “C overnight. The mixture was cooled to room temperature, diluted with 15 mL EtOAc, washed twice with saturated aq. NaHCO₃ and once with brine. The organic layer was dried over sodium sulfate, Filtered, and concentrated in vacuo. Flash chromatography (Biotage MPLC 5% - 40% EtOAc/Hexanes) provided the title compound (53%) as a white solid. LCMS m/z (APCI) = 221.1 (M+H).

Example 4

Synthesis of 6-ethynyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol

Example 4(a)

2016202040 01 Apr 2016

X >-OH ₊

-TMS

PdCI₂(PPh₃)₂

Cul, NEt₃, DMF, 80 °C

TMS^-'

X>-OH

1-(pentan-3-yl)-6-((trimethylsiIyl)ethynyl)-1K-imidazo[4,5-b]pyrazin-2-ol. A mixture of 6-bromo-1-(pentan-3-yt)-1H-imidazo[4,5-b]pyrazin-2-ol (3.75 g, 13.2 mmol)), trimethytsilylacetylene (2.8 mL, 19.8 mmol), PdCI₂(PPh)₂ (O.93g, 1.32 mmol), Cul (0.5 g, 2.64 mmol) and triethylamine (5.5 mL, 39.5 mmol) in DMF (50 mL) was purged with N₂for 30 seconds followed by stirring at 80 °C. After 3 hours the reaction mixture was diluted with EtOAc, washed with NH₄Ct solution, dried over Na₂SO₄, and concentrated in vacuo. Purification with over silica gel (Biotage MPLC 10 - 80% EtOAc/hexanes) provided the title compound (3.2 g, 80%) as a brown solid.

KF

Me0H/THF/H₂O

I T'Jhoh

6-ethynyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-ol. A mixture of 1-(pentan-3-yl)6-((trimethylsilyl)ethynyt)-1H-imidazo(4,5-b]pyrazin-2-ol (3.2 g, 10.6 mmol), KF (1.6 g, 27.5 mmol) in MeOH/THF/H₂O (50 mL, 2/2/1) was stirred at room temperature. Upon complete conversion by LCMS, the solution was concentrated in vacuo, diluted with EtOAc, washed with NH₄CI solution, dried over Na₂SO₄, and concentrated in vacuo. Purification over silica gel (Biotage MPLC 10 - 80% EtOAc/Heaxanes) gave a brown solid, which was washed with 15% EtOAc/hexanes to give product the title compound as a white solid (0.9 g, 37%). .

Example 5

2016202040 01 Apr 2016

Synthesis of 6-(dimethylannino)-1-{pentan-3-y!)-1H-imidazo[4,5-b]pyrazin-2-ol

Example 5(a)

Boc_aO

NEt₃, DCM

Boc /xv° tert-butyl 5-bromo-2-oxo-3-(pentan-3-yl)-2,3-dihydroimidazo[4,5-b]pyrazine-1carboxylate. To a solution of 6-bromo-1-(pentan-3-yl)-1H-imidazo(4,5-b]pyrazin-2-ol (5.0 g, 17.6 mmol) in DCM (80 mL) containing triethylamine (7.34 mL, 52.8 mmol) was added Boc₂O (7.0 g, 35 mmol). The reaction mixture was stirred at room temperature overnight, washed with NaHCCh solution, dried over Na₂S0₄ and concentrated in vacuo. Purification over silica gel (Biotage MPLC 10 - 50% EtOAc/hexanes) gave the desired product as a white solid (1.7 g, 25%).

Pd₃(dba)₂, NaOtBu HCl

BiPhP(Cy)₂, Toluene. 120 °C

6-(dimethylamino)-1-(pentan-3-yl)-1H-imIdazo[4,5-b]pyrazin-2-ol. A mixture of tertbutyl 5-bromo-2-oxO’3-(pentan-3-y1)-2,3-dihydroimidazo[4,5-bJpyrazine-1-carboxylate (200 mg, 0.52 mmol), dimethylamine (0.5 mL, 2M in THF, 1.0 mmol), Pd3(dba)₂ (48 mg, 0.052 mmol), BlPhP(Cy)₂ (37 mg, 0.10 mmol) and NaOtBu (150 mg, 1.56 mmol) in anhydrous toluene (4 mL) was purged with N₂ for 15 second and then stirred at 120 °C for 20 minutes in microwave. The mixture was diluted with EtOAc, washed with NaHCO3 solution, dried over Na₂SO₄, and then concentrated in vacuo to give a dark muddy residue. The residue was dissolved in MeOH (10 mL) and treated with 1 mL 4M HCl in dioxane. After one hour, the reaction was concentrated, diluted with EtOAc,

2016202040 01 Apr 2016 washed with NaHCO_3l dried over NaaSO₄, and concentrated in vacuo. Purification over silica gel (Biotage MPLC 10-70% EtOAc/hexanes) gave the title compound as a light yellow solid (42 mg, 32%).

Example 6

Preparation of Sarcomeric Proteins from Skeletal Muscle [0158] Actin was purified by first preparing an ether powder of cardiac muscle (Zot HG and Potter J D. (1981) Preparative Biochemistry 11:381-395) as described below. Subsequently, actin was cycled between the Filamentous and soluble state through rounds of centrifugation and dialysis (Spudich J A and Watt S. (1971) J. Biol. Chem. 246:4866-4871). It was stored in the filamentous state at 4 ’C.

[0159] Tropomyosin was extracted from the ether powder and separated from the other proteins based on pH dependent precipitations followed by successive ammonium sulfate cuts at 53% and 65% (Smtllie LB. (1981) Methods Enzymol 85 Pt B:234-41). The troponins were isolated as an intact complex of TnC, TnT, and Tnl.

Ether powder is extracted in a high salt buffer. Successive ammonium sulfate cuts of 30% and 45% were done; the precipitate was solubilized by dialysis into a low salt buffer and then further purified on a DEAE Toyopearl column with a 25-350 mM KCI gradient. There was no measurable ATPase in any of the components except for myosin which naturally had a very low basal ATPase in the absence of actin.

[0160] Just prior to screening, the actin, tropomyosin, and troponin complex are mixed together in the desired ratio (e.g., 7:1:1) to achieve maximal calcium regulation of the actin filament. The screen is conducted at a pCa=6.5. This calcium concentration is in the physiological range during muscle contraction.

[0161] To measure the generation of ADP during the reaction, a pyruvate kinase/lactate dehydrogenase/NADH coupled enzyme system (PK/LDH) is added to the actin. The myosin is kept separately. The plates are read in real time so that kinetic curves are obtained. These compounds are in DMSQ and were already spotted onto the bottoms of 384 well plates at 10 to 40 pg/ml Final concentration.

2016202040 01 Apr 2016

Example 7 Actin Preparation

1. Extract powder (as prepared in Example 6 or 7 below) with 20 ml buffer A (see below, add BME and ATP just prior to use in each of the following steps) ' per gram of powder (200 ml per 10 g). Use a large 4 L beaker for 150 g of powder. Mix vigorously to dissolve powder. Stir at 4 °C. for 30 min.

2. Separate extract from the hydrated powder by squeezing through several layers of cheesecloth. Cheesecloth should be pre-sterilized by microwaving damp for 1-2 min.

3. Re-extract the residue with the same volume of buffer A and combine extracts.

4. Spin in JLA10 rotor(s) for 1 hr at 10K rpm (4 °C.). Collect supernatant through 2 layers of cheesecloth.

5. Add ATP to 0.2 mM and MgCI₂ to 50 mM. Stir on stir piate at 4 °C for 60 minutes to allow actin to polymerize/fomn para-crystals.

6. Slowly add solid KCI to 0.6 M (45 g/i). Stir at 4 °C for 30 min.

7. Spin in JLA10 rotor(s) at 10K rpm for 1 hr.

8. Depolymerization: Quickly rinse surface of pellets with buffer A and dispose of wash. Soften the pellets by pre-incubation on ice with small amount of buffer A in each tube (use less than half of final resuspension volume total in all tubes). Resuspend by hand first with cell scraper and combine pellets. Wash tubes with extra buffer using a 25 ml pipette and motorized pipettor, aggressively removing actin from sides of tubes. Homogenize in large dounce in cold buffer A on ice. Use 3 ml per gram of powder originally extracted.

9. Dialyze against buffer A with 4 changes over 48 hour period.

10. Collect dialyzed actin and spin in the 45Ti rotor at40K rpm for 1.5 hr (4 °C.).

11. Collect supernatant (G-Actin). Save a sample for gel analysis and determination of protein concentration.

[0162] To polymerize G-actin for storage add KCI to 50 mM (from 3 M stock), MgCI₂to 1 mM, and NaN₃ to 0.02% (from 10% stock). Store at 4 °C. Do not freeze.

2016202040 01 Apr 2016 [0163] Buffer A: 2 mM tris/HCl, 0.2 mM CaCI_2l 0.5 mM (36 μΙ/L) 2-mercaptoethanol, 0.2 mM Na₂ ATP (added fresh), and 0.005% Na-azide; pH 8.0,

Example 8 Powder Preparation

1. Volumes are given per about 1000 g of the minced muscle.

2. Pre-cut and boil cheesecloth for 10 min in water. Drain and dry.

3. Mince chicken breast in a prechilled meat grinder.

4. Extract with stirring in 2 L of 0.1 M KCI, 0.15 M K-phosphate, pH 6,5 for 10 min at 4 °C. Spin 5000 rpm, 10 min, 4 °C in JLA. Collect the pellet,

5. Extract pellets with stirring with 2 L of 0.05 M NaHCO3 for 5 min. Spin 5000 rpm, 10min, 4 °C in JLA. Collect the pellet. Repeat the extraction once more.

6. Extract the filtered residue with 2 L of 1 mM EDTA, pH 7.0 for 10 min with stirring.

7. Extract with 2 L of H₂O for 5 min with stirring. Spin 10000 rpm, 15min, 4 °C in JLA. Carefully collect the pellet, part of which will be loose and gelatinous.

8. Extract 5 times with acetone (2 L of acetone for 10 min each with stirring). Squeeze through cheesecloth gently. Alt acetone extractions are performed at room temperature. Acetone should be prechilled to 4 °C.

9. Drying: Place the filtered residue spread on a cheesecloth in a large glass tray and leave in a hood overnight. When the residue is dry, put in a wide mouth plastic bottle and store at 20 °C.

Example 9

Alternate Powder Preparation

Based on Zot & Potter (1981) Prep. Biochem. 11(4) pp.381-395.

1. Dissect left ventricles of the cardiac muscle. Remove as much of the pericardial tissue and fat as possible. Grind in a prechilled meat grinder.

2016202040 01 Apr 2016

Weigh.

2. Prepare 5 volumes of Extract buffer (see below). Be sure the pH=8.0. Then, homogenize the meat in a blender, 4 times 15 sec on blend with 15 secs in between. Do this with 1 volume weight/volume) of buffer taken from the 5 volumes already prepared. Add the homogenate back to the extract buffer and stir until well mixed (5 minutes).

3. Filter through one layer of cheesecloth in large polypropylene strainer. Resuspend back into 5 volumes of extract buffer as above.

4. Repeat Step 3 four more times. At the end, do not resuspend in extraction buffer but proceed to Step 5. The pellets should be yellow white.

5. Resuspend in 3 volumes (according to original weight) of 95% cold ethanol. Stir for 5 min and squeeze through cheesecloth as above, repeat two more times.

6. Weigh squeezed residue and then resuspend in 3 volumes (new weight/volume) of cold diethyl ether.

7. Repeat Step 6 a total of three times.

8. Leave overnight in a single layer on a cheesecloth in a glass tray.

9. When dry, collect the powder, weigh and store in a wide-mouth jar at 4 °C.

EXTRACT BUFFER: 50 mM KCI, 5 mM Tris pH 8.0 Prepare as 50 times concentrate:

For2L

250 mM Tris pH 8.0. Tris Base (121.14 g/mol, 60.6 g) pH to 8.0 with cone. HCI, then add:

2.5 M KCI (74.55 g/mol, 372 g)

Example 10

Purification of Skeletal Muscle Myosin [0164] See, Margosstan, S.S. and Lowey, S. (1982) Methods Enzymol. 85, 55-123 and Goldmann, W.H. and Geeves, M.A. (1991) Anal. Biocbem. 192, 55-58.

2016202040 01 Apr 2016

Solution A: 0.3 1VJ KCI, 0.15 M potassium phosphate, 0.02 M EDTA, 0.005 M MgClz, 0.001 MATP, pH 6.5,

Solution B: 1 M KCI, 0.025 M EDTA, 0.06 M potassium phosphate, pH 6.5.

Solution C: 0.6 M KCI, 0.025 M potassium phosphate, pH 6.5.

Solution D: 0.6 M KCI, 0.05 M potassium phosphate, pH 6.5.

Solution E: 0.15 M potassium phosphate, 0.01 M EDTA, pH 7.5.

Solution F: 0.04 M KCI, 0.01 M potassium phosphate, 0.001 M DTT, pH 6.5.

Solution G: 3 M KCI, 0.01 M potassium phosphate, pH 6.5.

All procedures are carried out at 4 °C.

1. Obtain -1000 g skeletal muscle, such as rabbit skeletal muscle.

2. Grind twice; extract with 2 L solution A for 15 min white stirring; add 4 L cold H₂O, filter through gauze; dilute with cold H₂O to ionic strength of 0.04, (about 10-fold); let settle for 3 h; collect precipitate at 7,000 rpm in GSA rotor for 15 min.

3. Disperse pellet in 220 ml solution B; dialyze overnight against 6 L solution C; slowly add -400 ml equal volume cold distilled H₂O; stir for 30 min; centrifuge at 10,000 rpm for 10 min in GSA rotor.

4. Centrifuge supernatant at 19,000 rpm for 1 h.

5. Dilute supernatant to ionic strength of 0.04 (~8-fold); let myosin settle overnight; collect about 5-6 L fluffy myosin precipitate by centrifuging at 10,000 rpm for 10 min in GSA rotor.

6. Resuspend pellet in minimal volume of solution G; dialyze overnight against 2 L solution D; centrifuge at 19,000 rpm for 2 h, in cellulose nitrate tubes; puncture tubes and separate myosin from fat and insoluble pellet.

7. Dilute supernatant to 5-10 mg/ml and dialyze against solution E extensively, load onto DEAE-sephadex column.

8. Preequilibrate with solution E; apply 500-600 g myosin at 30 ml/h; wash with 350 ml solution E; elute with linear gradient of 0-0.5 M KCI in solution E (2 x 1

2016202040 01 Apr 2016 liter); collect 10 ml fractions; pool myosin fractions {>0.1 M KCI); concentrate by overnight dialysis against solution F; centrifuge at 25,000 rpm for 30 min; store as above,

9. The myosin is then cut with chymotrypsin or papain in the presence of EDTA to generate the S1 fragment which is soluble at the low salt conditions optimal for ATPase activity (Margossian supra).

Example 11 [0165] Using procedures similar to those described herein, the compounds in the following table were synthesized and tested.

Name	m/z	Ion	SKM MF AC1.4 (μΜ)
(S)-6-bromo-1-(1-phenylethyl)-1H-imidazo[4,5- b]pyrazin-2-ol	319.0; 321.0	[M+Hf; [M+Hf	0.45
(S)'6-bromo-1 -{1 -phenylethyl)-1 H-imidazo[4,5b]pyrazin-2{3H)-one	319.0; 321.0	^— [M+Hf; [M+Hf	0.45
(R)-6-bromo-1-(1“phenylethyl)-1H-imidazo[4,5- b]pyrazin-2-ol	319.0; 321.0	[M+Hf; [M+Hf	2.4
(R)-6-bromo-1-(1-phenylethyi)-1H-imidazo[4,5b]pyrazin-2(3 H)-one	319.0; 321.0	[M+Hf; [M+Hf	2.4
1 -benzy l-6-bromo-1 H-imidazo[4,5-b]pyrazin-2-ol	305.1; 307.1	[M+Hf; [M+Hf	23.5
1-benzyl-6-bromo-1 H-imidazo[4,5-b]pyrazin2(3H)-one	305.1; 307.1	[M+Hf; [M+Hf	23.5
(S)-6-(methylthio)-1-(1-phenyiethyl)-1H- imidazo[4,5-b]pyrazin-2-ol	287.1	[M+H]	1.1

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (μΜ)
(S)-6-(methylth io)-1 -(1 -p henylethyl)-1Himidazo[4,5-b]pyrazin-2(3H)-one	287,1	[M+Hf	1.1
1- benzyl-6-(methylthio)-1H-imldazo[4,5-b]pyrazin- 2- ol	273.0	[M+Hf	22.6
l-benzyl-e-O'nethylthfcO-iH-imidazotd.S-blpyrazin- 2(3H)-one	273.0	(M+Hf	22.6
(R)-6-(methylthio)-1-(1-phenyIethyl)-1H- imidazo[4,5-b]pyrazin-2-ol	287.0	[M+Hf	2.9
(R)-6-(methylthio)-1-(1-phenylethyl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	287.0	[M+Hf	2.9
(S)-6-(2-methylprop-1-enyl)-1~(1-phenylethyl)-1H- imidazo[4,5-b]pyrazin-2-ol	295.1	[M+H]*	2.7
(S)-6-(2-methylprop-1 -enyl)-1 -(1 -phenylethyl)-1 Himidazo[4,5-b]pyrazln-2(3H)-one	295.1	[M+Hf	2.7
(S)-6-cyclo h exeny 1-1 -(1 -p henylethyl)-1Himidazo[4,5-b]pyraztn-2-ol	321.1	[M+Hf	31.0
(S)-6-cyc!ohexenyl-1-(1-phenylethyl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	321.1	[M+Hf	31.0
(S,Z)-1-(1-phenylethyl)-6-(prop-1-enyl)-1H- imidazo[4,5-b]pyrazin-2-ol	281.1	[M+Hf	1.0
(S,Z)-1-(1-phenylethyl)-6-(prop-1-enyl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	281.1	[M+Hf	1.0
(S)-1-(1 -phenylethyl)-6-vinyl-1 H-im idazo[4,5b]pyrazin-2-ol	267.0	[M+Hf	2.4
(S)-1-(1-phenylethyl)-6-vinyl-1H-imidazo[4,5- b]pyrazin-2(3H)-one	267.0	[M+Hf	2.4

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 <μΜ)
(S)-6-ethyl-1-(1-phenyIethyi)-1H-imidazo[4,5- b]pyrazin-2-o!	269.1	[M+Hf	17.4
(S)-6-ethyl-1-(1-phenylethyl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	269.1	[M+Hf	17.4
(S)-1-(1 -pheny lethyl)-6-propyl-1 H-im idazo[4,5b]pyrazin-2-ol	283.1	[M+Hf	29.7
(S)-1 -(1-phenylethyl)-6-propyl-1 H-imidazo[4,5b]pyrazin-2(3H)-one	283.1	[M+Hf	29.7
(S)-6-methoxy^1 -(1 -phenyiethyl)-1 H-imidazo[4,5- b]pyrazin-2-ol	271.1	[M+Hf	8.8
(S)-6-me1hoxy-1-(1-phenylethyl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	271.1	[M+Hf	8.8
6-bromo-1 -cydohexyl-1 H-imidazo[4,5-b]pyrazin-2- ol	296.9; 298.9	[M+Hf; [M+Hf	10.4
6-bromo-1-cydohexyl-1 H-imidazo[4,5-b]pyrazin2(3H)-one	296.9; 298.9	[M+Hf; [M+Hf	10.4
l-cyclohexyl-e-imethylthioJ-IH-imidazo^.S- b]pyrazin-2-ol	265.1	[M+Hf	13.9
1-cyclohexy l-6-(m ethylthio) -1 H-im idazo[4,5b]pyrazin-2(3H)-one	265.1	[M+Hf	13.9
(Zj-1-cyclohexyl-6-(prop-1-enyi)-1H-imidazo[4,5- b]pyrazin-2-ol	259.1	[M+Hf	6.0
(Z)-1-cyclohexyl-6-(prop-1-enyl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	259.1	[M+Hf	6.0
(E)-1 -cyclohexy l-6-(prop-1-enyl)-1H-imidazo[4,5b]pyrazin-2-o!	259.1	[M+Hf	6.1

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (μΜ)
(E)-1-cyclohexyl-6-(prop-1-enyl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	259.1	(M+Hf	6.1
(S, E)-1 -(1 -phenylethyl)-6-(prop-1 -enyl)-1 Himidazo[4,5-b]pyrazin-2-ol	281.1	[M+Hf	1.6
(S,E)-1-(1-phenylethyl)-6-(prop-1-enyl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	281.1	[M+Hf	1.6
6-bromo-1 -isopropyl-1 H-imidazo[4,5-b]pyrazin-2-ol	259.0	[M+Hf	6.0
6-bromo-1 -isopropyl-1 H-imidazo[4,5-b]pyrazin2(3H)-one	259.0	[M+Hf	6.0
1 -isopropyl-6-(methylthio)-1 H-imidazo[4,5b]pyrazin-2-ol	225.1	[M+Hf	6.7
1 -isop ropyl-6-(methylth io)-1 H-im idazo[4,5- b]pyrazin-2(3H)-one	225.1	[M+Hf	6.7
(Z)-1 -isopropyI-6-(prop-1 -enyl)-1 H-imid azo[4,5b]pyrazin-2~ol	219.2	[M+Hf	4.6
(Z)-1-isopropyl-6-(prop-1-enyl)-1H-imidazo[4,5- b]pyraztn-2(3H)-one	219.2	[M+Hf	4.6
(S)-6-ethoxy-1-(1-phenytethyI)-1H-imidazo[4,5- b]pyrazin-2-ol	285.2	[M+Hf	10.6
(S)-6-ethoxy-1-(1 -phenylethyl)-1 H-imid azo [4,5- b]pyrazin-2(3H)-one	285.2	[M+Hf	10.6
6-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin- 2-ol ,	286.0; 288.0	[M+2Hf⁺; [M+2H]²*	0.25
6-bromo-1 -{pentan-3-yl)-1 H-imidazo[4,5-bjpyrazin- 2(3H)-one	286.0; 288.0	[M+2Hf⁺; [M+2Hf⁺	0.25
6-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5- b]pyrazin-2-ol	253.1	[M+Hf	0.4

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (pM)
6-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5- bJpyrazin-2(3H)-one	253.1	[M+Hf	0.4
(E)-1 -(pentan-3-ylf 6-(prop-1 -eny If1 Himidazo[4,5-b]pyrazin-2-ol	247.1	[M+Hf	0.4
(E)-1 -(pentan-3-yl)-6-(prop-1 ~enyl)-1 Himidazo[4,5-b]pyrazin-2(3H)-one	247.1	[M+Hf	0.4
(Z)-1-(pentan-3-yl)’6-(prop-1-enyl)-1H- imidazo[4,5-bJpyrazin-2-ol	247.1	[M+Hf	0.6
(Z)-1 -(pentan-3-yl)-6-(prop-1 -enyl)-1 Himidazo[4,5-b]pyrazin-2(3H)-one	247.1	[M+Hf	0.6
6-methyl-1-(pentan-3-yl)-1H-imidazo[4,5- b]pyrazin-2-ol	221.1	[M+Hf	0.53
6-methyl-1 -(pentan-3-yl)-1 H-imidazo[4,5b]pyrazin-2(3H)-one	221.1	[M+Hf	0.53
(S,Z)-6-(hex-2-enyl)-1-(1-phenylethyl)-1H- imidazo[4,5-b]pyrazin-2-ol			3.1
2-hyd roxy-1 -(pentan-3-yl)-1 H-imidazo[4,5- b]pyrazine-6-carbonitnle	232.1	[M+Hf	36.4
2-oxo-3-(pentan-3-yl)-2,3-dihydro-1H-imidazo[4,5- b]pyrazine-5-carbonitrile	232.1	[M+Hf	36.4
(R)-6-bromo-1-sec-butyl-1H-imidazo[4,5- b]pyrazin-2-ol	273.0	[M+Hf	1.5
(R)-6-bromo- 1-sec-butyl-1 H-imidazo[4,5b]pyrazin-2(3H)-one	273.0	[M+Hf	1.5
(S)-6-bromo-1-sec-butyl-1H-imidazo[4,5-b]pyrazin- 2-ol	272.0	[M+2H]²⁺	0.8

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (μΜ)
(S)-6-bromo-1-sec-butyl-1H-imidazo[4,5-b]pyrazin- 2(3H)-one	272.0	[M+2H]²*	0.8
6-bromo-1-tert-butyl-1H-imidazo[4,5-b]pyrazin-2-ol	272.0	[M+2H]^k+	0.8
6-bromo-1-tert-butyl-1 H-imidazo[4,5-b]pyrazin2(3H)-one	272.0	[M+2H]²*	0.8
2-(6-bromo-2-hydroxy-1H-imidazo[4,5-b]pyrazin-1yI)butanoic acid	299.0	[Μ-ΗΓ	>49
2-{6-bromo-2-oxo-2,3-dihydro-1H-imidazot4,5b]pyrazin-1-yl)butanoic acid	299.0	[M-Hy	>49
2-(6-bromo-2-hydroxy-1H-imidazo[4,5-b]pyrazin-1y 1)-1 -morpholinobutan-1 -one	370.0	[M+HJ*	>49
6-bromo-1-(1'morpholinO“1-oxobutan-2-yl)-lH- imidazo[4,5-b]pyrazin-2(3H)-one	370.0	[M+H]*	>49
(R)-6-bromo-1-(1-hydroxybutan-2-yl)-1H- imidazo[4,5-b]pyrazin-2-ol	285.0	[Μ-ΗΓ	1.1
(R)-6-bromo-1-(1 -hydroxybutan-2-yl)-1 Himidazo[4,5-b]pyrazin-2(3H)-one	285.0	[M-Hf	1.1
(S)-6-bromo-1-(1 -hydroxybutan-2-yl)-1 Himidazo[4,5-b]pyrazin-2-ol	285.0	[M-Hy	19.1
(S)-6-bromo-1-(1-hydroxybutan-2-yl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	285.0	[M-Hy	19.1
6-bromo-1 -(1 -m eth oxybuta n-2-y I)-1 H-im idazo{4,5b]pyrazin-2-ol	301.0	[M+H]*	4.9
6-bromo-1-(1-methoxybutan-2-yl)-1 H-imtd azo]4,5b]pyrazin-2(3H)-one	301.0	[M+H]*	4.9
1-(1-amtnobutan-2-yl)-6-bromo-lH-imidazo[4_l5- b]pyrazin-2-ol	287.0	[M+2H]²*	1.1

2016202040 01 Apr 2016

Name	m/z	Ion	SKWI MF AC1.4 (PM)
1-(1-aminobutan-2-yl)-6-bromo-1H-imidazo[4,5- b]pyrazin-2(3H)-on©	287.0	[M+2HJ^a+	1,1
6-bromo-1-(1-(methylamino)butan-2-yl)-1H- imidazo[4,5-b]pyrazin-2-ol	300.0	[M+Hf	45(7
6-bromo-1 -(1 -(methyla mino)butan-2-yl)-1Himidazo[4,5-b]pyrazin-2(3H)-one	300.0	[M+Hf	45.7
6-bromo-1-(1-(dimethylamino)butan-2-yl)-1H- imidazo[4,5-b]pyrazin-2-ol	315.0	[M+2H]²*	43.8
6-bromo-1-(1-(dimethylamino)butan-2-yl)-1H- imtdazo[4,5-b]pyrazin-2(3H)-one	315.0	[M+2H]^Z+	43.8
6-bromo-1-(1-(4-methylprperazin-1-yl)butan-2-yl)1 H-imidazo[4,5-b]pyrazin-2-ol	369.0	[M+Hf	14.9
6-bromo-1-(1-(4-methylpiperazin-1-yl)butan-2-yl)- 1H-imidazo[4,5-b]pyrazin-2(3H)-one	369.0	[M+Hf	14.9
(R)-6-bromo-1-(1-morphotinobutan-2-yi)-1Him id azo[4,5-bJpy ra zin-2-ol	356.0; 358.0	[M+Hf; [M+Hf	0.3
(R)-6-bromo-1-(1-morpholinobutan-2-yi)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	356.0; 358.0	[M+Hf; [M+Hf	0.3
(S)-6-bromo-1-(1-morpholinobutan-2-yl)-1Him id azo[4,5 -b]py razi n-2-oi	356.0; 358.0	[M+Hf; (M+Hf	>49
(S)-6-bromo-1-(1-morpholinobutan-2-yl)-1H- imidazo[4,5-b]pyrazin-2(3H)-one	356.0; 358.0	[M+H]⁺; [M+Hf	>49
(EJ-l-isopropyl-e-tprop-l-enyO-IH-imidazo^.B- b]pyrazin-2-ol	219.1	[M+Hf	7.1
{E)-1-isopropyl-6-(prop-1-enyi)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	219.1	[M+Hf	7.1
1-(pentan-3-y()-1H-innidazo[4,5-b]pyrazin-2-ol	207.1	[M+Hf	16.0^T

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (pM)
1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)- one	207.1	[M+Hf	16.0+
methyl 4-(2-(6-bromo-2-hydroxy-1 H-imldazo[4,5b]pyrazin-1 -yl)butyl)piperazine-1 -carboxylate	414.0	[M+2H]²*	0.9+
methyl 4-(2-(6-bromo-2-oxa-2,3-dihydro-1 H- imidazo[4,5-b]pyrazin-1-yl)butyl)piperazine-1- carboxyfate	414.0	[M+2H]²*	0.9+
6-bromo-1-(1-(4-(methylsulfonyl)piperazin-1- yl)butan-2-yl)-1H-lmidazo[4,5-b]pyrazin-2-ol	434.0	[M+2H]²*	3.2+
6-bromo-1-(1-(4-(methylsulfonyl)piperazin-1- yl)butan-2-yl)-1H-tmidazo[4,5-b]pyrazin-2(3H)-one	434.0	[M+2H]²*	3.2+
5-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin- 2-ol	285.0; 287.0	[M+Hf; [M+Hf	11.5
5-bromo-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazln- 2{3H)-one	285.0; 287.0	[M+Hf; [M+H]*	11.5
i-(pentan-3-yl)-1H-imidazo[4,5-b]qulnoxalin-2-ol	257.1	[M+Hf	19.8
1-(pentan-3-yl)-1H-imidazo[4,5-b]quinoxalin- 2(3H)-one	257.1	[M+Hf	19.8
6-ethynyH-(pentan-3-yl)-1H-lmidazo[4,5- b]pyrazin-2-ol	231,2	[M+Hf	0.1+
6-ethynyl-1-(pentan-3-yl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	231.2	[M+Hf	0.1 +
1 -(pentan-3-yl)-6-(trifluoromethyl)-1 H-imidazo[4,5- b]pyrazin-2-ol	275.2	[M+Hf	10.4*
1 -{pentan-3-yl)-6-(trifluoromethyl)-1 H-imidazo[4,5b]pyrazin-2(3H)-one	275.2	[M+Hf	10.4*

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (yM)
6-bromo-1-(2-methyl-1-morpholinopropan-2-yl)1 H-imidazo[4,5-b]pyrazin-2-ol	356.0; 358.0	[M+Hf; [M+Hf	16.4*
6-bromo-1 -(2-methyl-1 -morpholinop ropan-2-yl)1H-imidazo[4,5-bJpyrazin-2(3H)-one	356.0; 358.0	“[M+Hf; [M+Hf	16.4*
(S)-6-bromo-1-(1-morpholinopropan-2-yl)-1 Himidazo[4,5-b]pyrazin-2-ol	342.0; 344.0	[M+Hf; [M+Hf	>49
(S)-6-bromo-1 -(1 -mo rpholin opropan-2-yl)-1 Himidazo[4,5-b]pyrazin-2(3H)-one	342.0; 344.0	[M+Hf; [M+Hf	>49
6-methoxy-1 -(pentan-3-y ])-1H- imid azo[4,5b]pyrazin-2-ol	237.1	[M+Hf	2.2*
6-methoxy-1-(pentan-3-yl)-1H-imidazo[4,5b] pyrazin-2{3 H )-one	237.1	[M+Hf	2.2*
(R)-6-bromo-1 -(1 -morpholinopropan-2-yl)-1Himidazo[4,5-b]pyrazin-2-ol	342.0; 344.0	[M+Hf; ’ [M+Hf	5.2*
(R)-6-bromo-1-(1-morpholinopropan-2-yl)-1 Himidazo[4,5-b]pyrazin-2(3H)-one	342.0; 344.0	[M+Hf; [M+Hf	5.2*
1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazine-2,6-diol	223.2	[M+Hf	>49
6-hydroxy-1-(pentan-3-yl)-lH-imidazo[4,5- b]pyrazin-2(3H)-one	223.2	[M+Hf	>49
1-(pentan-3-yl)-6-(prop-1-ynyl)-1H-imidazo[4,5- b]pyrazin-2-ol	245.2	[M+Hf	>49
1-(pentan-3-yl)-6-(prop-1-ynyl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	245.2	[M+Hf	>49
1-(1-morpholinobutan-2-yl)-1H-imidazo[4,5- b]pyrazin-2-ol	278.0	[M+Hf	>49
1-(1-morpholinobutan-2-yl)-1H-imidazo[4,5- b]pyrazin-2(3H)-one	278.0		>49

2016202040 01 Apr 2016

Name	m/z	Ion	SKMMF AC1.4 (μΜ)
1 -(ethylpropyl)-6-(1 -m.ethylpyrazol-4- yl)imidazo[4,5-b]pyrazin-2-ol	287.1	[M+Hf	15
6-bromo-1-(propylbutyl)tmidazo[4,5-b]pyrazin-2-ol	313.1	[M+Hf	0.7
1-[(1 R)-3-methyl-1 -(morpholin-4-ylmethyl)butyl]-6bromoimidazo[4,5-b]pyrazin-2-o!	384	[M+Hf	0.5
1-(ethylpropyl)-6-vinyiimidazo[4,5-b)pyrazin-2-ol	233.1	[M+Hf	2.3
1-(ethylpropyl)-6-(1-methylvinyl)imidazo[4,5- b]pyrazin-2-ol	247	[M+Hf	0.6
1’(ethylpropyl)-6-(methyleihyl)imidazo[4,5- b]pyrazin-2-ol	249	[M+Hf	29.6
6-chloro-1-(ethylpropyl)imidazo[4,5-b]pyrazin-2-oi	239.1	[M+Hf	0.7
6-(dimethylamino)-1-(ethylpropyl)imidazo[4,5- b]pyrazln-2-ol	250.1	[M+Hf	2.2
1 -((^ R)-1 -methyl-2-morpholin-4-ylethyl)-6bromoimidazo[4,5-b]pyrazin-2-ol	342, 344	(M+H), (M+2+H)	5.19
1-(ethylpropyl)-6-ethynylimidazo[4,5-b]pyrazin-2-ol	229	(M-H)	0.15
1 -(ethylpropy l)-6-methoxyimidazo[4,5-b]pyrazin-2- ol	237	(M-H)	2.20
1 -(1,1-dimethyl-2-morpholin-4-ylethyl)-6bromoimtdazo[4,5-b]pyrazin-2-o!	356, 358	(M+1), (M+2+H)	19.11
6-(1H-1,2,3-triazol-4-yl)-1- (ethylpropyl)imidazo[4,5-bJpyrazin-2-ol	274	(M+H)+	46.64
1 -(ethyl p ropyl)-6-(trifluoro methyl) imid azo[4,5b]pyrazin-2-ol	275	(M+H)	10.37
1 -[(1 R)-1 -(morpholin -4-ylmethyl)propyl]-6ethynylimidazo[4,5-b]pyrazin-2-ol	302	(M+H)	0.13

2016202040 01 Apr 2016

Name	m/z	Ion	SKM MF AC1.4 (μΜ)
1-(ethy Ipropy 1)-6-(2-( 1-{ethyl propyl )-2- hydroxyiiriidazo[4,5-e]pyrazin-6- yljethynyl}imidazo[4,5-b]pyrazin-2-ol	435	(M+H)	19.32
6-(dimethylamino)-1~(ethylpropyl)imidazo[4,5- b]pyrazin-2-ol	250	(M+H)	2.79
6-ethyl-1-(ethyipropyl)imidazo[4,5-b]pyrazin-2-ol	235	[M+H]	8.58

¹ Mean value.

* Median value.

[0166] Using procedures similar to those described herein, the compounds in the following table were also synthesized and tested.

Name

(S)-1 -(2-hydroxy-1 -(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazin-6-yl)ethanone

(S)-6-acetyl-1-(1-phenylethyl)-1H-imidazo[4,5-bJpyrazin-2(3H)-one

(S)-6-isobutyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol

(S j-6-isobutyl-1 -(1 -phenyl ethyl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-6-hexyl-1-(1 -phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol

(S)-6-hexyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-6-cyclohexy|-1-(1 -phenylethyl)-1 H-im idazo[4,5-b]py razrn-2-ol

(S)-6-cyclohexyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-1 -(1 -pheny)ethyl)-1 H-imidazo[4,5-b]pyrazin-2-ol

(S)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6-carboxylic acid

(S)-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5-carboxylic acid

(S)-methy! 2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6-carboxylate

(S)-m ethyl 2-oxo-3-( 1 -p h en ylethy l)-2,3-d ihyd ro-1 H-imid azo[4,5-b] pyrazine-5-ca rboxyl ate

2016202040 01 Apr 2016

(S)-2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazfne-6-carbonitriie

(S)-2-oxo-3-(1-phenyiethyl)-2,3-dihydro-1H-imidazo(4_l5-b]pyrazine-5-carbonitrile

(S)-2-hydroxy-1-(1-pheny)ethyl)-1H-imidazo[4_t5-b]pyrazine-6-carboxamide

(S)-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5-carboxamide

(S)-2-hydroxy-N-methyl-1 -(1 -phenylethyi)-1 H-imidazo[4,5-b]py razine-6-carboxamide

(S)-N-methyl-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5- carboxamide

(S)-2-hydroxy-N,N-dimethyl-1 -(1 -phenylethyl)-1 H-imldazo[4,5-b]pyrazine-6-carboxamide

(S)-N,N-dimethyl-2-oxo-3-(1-phenylethyl)-2,3-dihydro-1H-imidazoi4,5-b]pyrazine-5- carboxamide

(S)-N_tN-diethyl-2-hydroxy-1-(1-phenylethyt)-1H-imidazo[4,5-bJpyrazine-6-carboxamide

(S)-N,N~diethyl-2-oxo-3-(1-phenylethyl)-2,3-difiydro-1 H-imidazo[4,5-bjpyrazine-5- carboxamide

(S)-(2-hydroxy-1-(i-phenylethyl)-1H-imidazo[4,5-b]pyrazin-6-yl)(morpholino)methanone

(S)-6-(morpholine-4-carbonyl)-1 -(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-(2-hydroxy-l-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-6-y!)(piperidin-1- yl)methanone

(8)-1-(1-phenyiethyl)-6-(piperidine-1-carbonyl)-1H-imidazo[4,5-bjpyrazin-2(3H)-one

(S)-(2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b)pyrazin-6-yi)(4-methylpjperazin-1- yl)methanone

(S)-6-(4-methylpiperazine-1-carbonyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin- 2(3H)-one

(S)-N-benzyl-2-hydroxy-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazine-6-carboxamide

(SJ-N-benzyl^-oxo-S-il-phenylethyO^S-dlhydro-IH-imidazo^.S-bJpyrazine-S- carboxamide

(S)-6-((dimethylamino)methyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-oi

(S)-6-((dimethyiamino)methyl)-1-(1-phenylethyl)-1H-imidazo[4,5~b]pyrazin-2(3H)-one

(S)-6-(morpholinomethyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-oi

(S)-6-(morpholinomethyf)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

2016202040 01 Apr 2016

{S)-1-(1-phenylethyl)-6-(piperidin-1-yImethyl)-lH-irnidazo[4,5-b]pyrazin-2-ol

(S)-1-(1-phenyletbyl)-6-(piperidin-1-ylrnethyl)-1 H-imidazoi4,5~b)pyrazin-2{3H)-one

(S)-6-((4-methylpiperazin-1-yl)methyl)-1-(1-phenylethyl)-t H-imidazo[4,5-blpyrazin-2-ol

(S)-6-((4-methylpiperazin-1 -yl)methyl)-1 -(1-phenylethyl)-1 H-imidazo[4,5-b]pyrazin2(3H)-one

(S)-6-(2-hydroxypropan-2-yl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol

(S)-6-(2-hydroxypropan-2-yl)-1-(1-phenylethyl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one

6-(methylsulfinyl)-'l-((S)-1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-o|

6-(methylsulfinyt)-1-((S)-1-phenylethyl)-1H-imidazo[4,5-bJpyrazin-2(3H)-one

(S)-6-(methylsiilfonyl)-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2-ol

(S)-6-{methylsulfonyl)-1-(1-phenyIethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

6-cyclopropyl-1-(pentan-3-yl)-1H-tmidazo[4,5-b]pyrazin-2-ol

6-cyclopropyl'-1-(pentan-3-y!)-1H-imidazo[4_l5-b]pyrazin-2(3H)-one

6-bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2-ol

6-bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

1-cyclopropyl-6-(methylthio)-1H-imidazo[4,5-b]pyrazin-2-ol

1-cyclopropy1-6-(methylthio)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

6-(methy!thio)-1 -(tetra hyd ro-2 H-pyra π-4-y 1)-1 H-i midazo[4,5-b]py razi n-2-ol

6-(methy1thio)-1-(tetrahydro-2H-pyran-4-yl)-lH-imidazo[4,5-b]pyrazin-2(3H)-one

{E)-1-cycIopropyl-6-(prop-1 -enyl)-lH-imidazo[4,5-b]pyrazin-2-ol

(E)-1-cyclopropy1-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyraz1n-2(3H)-one

(Ej-6-(prop-1-enyl)-1-(tetrahydro-2H-pyran-4-y()-1H-imidazot4₁5-b]pyrazin-2-ol

(E)-6-(prop-5-enyl)-1 -(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(2)-1-cyclopropy(-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2-o[

(2)-1-cyclopropyl-6-(prop-1-enyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

(2)-6-(prop-1-enyi)-1-(tetrahydro-2H-pyran^4-yi)-1H-imidazo[4₁5-b]pyrazin-2-ol

(Z)-6-(prop-i-enyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

6-bromo-1 -cyclopropyi-1 H-im idazo[4,5-b]pyrazin-2-ol

6-bromo-1-cyclopropyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one

2016202040 01 Apr 2016

5-(methylthio)-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2-oS

5-(methylthio)-1-(pentan-3-yl)-1H-imidazoI4,5-b]pyrazin-2(3H)-one

5-ethy I-1 -(pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2-ol

5-ethyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazin-2{3H)-one

1 -{pentan-3-yl)-5-vinyl-1 H-imidazo[4,5-b]pyrazin-2-ol

1-(pentan-3-yl)-5-vinyl-1H-imidazo[4,5-b)pyrazin-2(3H)-one

methyl 2-hydroxy-1 (pentan-3-yl)-1 H-imidazo[4,5-b]pyrazine-5-carboxylate

methyl 2-oxo-1-(pentan-3-yl)-2,3-dihydro-1 H-imidazo[4,5-b]pyrazine-5-carboxylate

2-hydroxy-N,N-dimethyl-1-(pentan-3-yl)-1H-imidazo[4,5-b]pyrazine-5>carboxamide

N,N-dimethyl-2-oxo-1-(pentan-3-yl)“2,3-dihydro-1 H-imidazo[4,5~b]pyrazine-5- carboxamide

2-1^Γθχν-Ν-ΓηΘί(ΐ7ΐ-1-(ρθηΊ3η-3-ν!)-1 H-imidazo[4,5-b]pyrazine-5-carboxamide

N-methyl-2-oxo-1-(pentan-3-yl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5-carboxamide

1-(2-hydroxy-1-(pentan-3-yl)-1H-imidazo[4,5-bJpyrazin-5-yl)ethanone

5-acetyl-1 -{pentan-3-yl)-1 H-imidazo[4,5-b]pyrazin-2(3H)-one

(S)-1-(1-phenylethyl)-1H-imidazo[4,5-blquinoxalin-2-oI

(S)-1-(1-phenylethyl)-1 H-imidazo[4,5-b]quinoxalin-2(3H)-one

2-(6-bromo-2-hydroxy-1 H-tmidazo[4,5-b]pyraztn-1-y!)propane-1,3-diol

6-bromo-1-(1,3-dihydroxypropan-2-yl)-1 H lmidazo[4,5-b]pyrazin-2(3H)-one

6-bromo-1-(2-morpholinoethyl)-1H-imidazo[4,5-b]pyrazin-2-ol

6-bromo-1-(2-morphorinoethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one

2-hydroxy-1-(pentan-3-yl)-1H-imrdazo[4,5-b]pyrazine-5-carboxyh'c acid

2-oxo- 1-(pentan-3-yl)-2,3-dihydro-1H-imidazo[4,5-b]pyrazine-5-carboxylic acid

[0167] While some embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. For example, for claim construction purposes, it is not intended that the claims set forth hereinafter be construed in anyway narrower than the iiteral language thereof, and it is thus not intended that exemplary embodiments from the specification be read into the claims. Accordingly, it is to be understood that the

2016202040 01 Apr 2016 present invention has been described by way of illustration and not limitations on the scope of the claims.

[0168] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0169] Throughout this specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

2016202040 19 Dec 2016

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

E A method of producing a compound of Formula 103, or a pharmaceutically acceptable salt thereof:

R₂

Formula 103 wherein:

Ri is selected from the group consisting of halo, alkyl, alkenyl, alkynyl and cycloalkyl;

R4 is hydrogen; and

R2 is selected from the group consisting of 3-pentyl, 4-heptyl, 4-methyl-lmorpholinopentan-2-yl, isobutyl, cyclohexyl, cyclopropyl, sec-butyl, tert-butyl, isopropyl, l-hydroxybutan-2-yl, tetrahydro-2H-pyran-4-yl, l-methoxybutan-2-yl, l-aminobutan-2-yl and l-morpholinobutan-2-yl;

the method comprising:

(a) treating a compound of Formula 101:

R4.

.N NH₂

Formula 101 wherein X is halo;

wherein Ri and R4 are as defined for the compound of Formula 103;

with an amine of formula R2-NH2, wherein R2 is as defined for the compound of

Formula 103, in a suitable solvent under sufficient conditions to produce a compound of Formula 102:

2016202040 19 Dec 2016 r₄. .nh.

Ri .

N NH

R?

Formula 102 wherein Ri, R₂, and R4 are as defined for the compound of Formula 103; and (b) treating the compound of Formula 102 with a di-activated carbonyl equivalent in a suitable solvent under sufficient conditions to produce the compound of Formula 103.
2. A method according to claim 1, wherein the amine of formula R2-NH2 is present in excess of the compound of Formula 101.
3. A method according to claim 1 or 2, wherein the mixture comprising the compound of Formula 101 and the amine of formula R2-NH2 is heated to a temperature from about 110°C to about 190°C.
4. A method according to any one of claims 1-3, wherein the di-activated carbonyl equivalent is selected from the group consisting of carbonyl diimidazole (CDI), phosgene and triphosgene;
5. A method according to any one of claims 1-4, wherein the di-activated carbonyl equivalent is carbonyl diimidazole (CDI).
6. A method according to any one of claims 1-5, wherein Ri is halo;
7. A method according to any one of claims 1-6, wherein Ri is bromo.
8. A method according to any one of claims 1-5, wherein Ri is ethynyl.

2016202040 19 Dec 2016
9.

A method according to any one of claims 1-8, wherein R2 is 3-pentyl.
10. A method of producing a compound of Formula 104, or a pharmaceutically acceptable salt thereof:

R₂

Formula 104 wherein

Ri is selected from the group consisting of alkyl, alkenyl, alkynyl and cycloalkyl;

R4 is hydrogen; and

R2 is selected from the group consisting of 3-pentyl, 4-heptyl, 4-methyl-lmorpholinopentan-2-yl, isobutyl, cyclohexyl, cyclopropyl, sec-butyl, tert-butyl, isopropyl, l-hydroxybutan-2-yl, tetrahydro-2H-pyran-4-yl, l-methoxybutan-2-yl, l-aminobutan-2-yl and l-morpholinobutan-2-yl;

the method comprising treating a compound of Formula 201:

R₂

Formula 201 wherein

X is a leaving group; and

R2 and R4 are as defined for the compound of Formula 104;

with a palladium catalyst in a suitable solvent under sufficient conditions to produce the compound, or pharmaceutically acceptable salt thereof, of Formula 104.

2016202040 19 Dec 2016
11. A method according to claim 10, further comprising treating a compound of

Formula 102:

NH

Formula 102 wherein

X is as defined for the compound of Formula 201; and

R2 and R4 are as defined for the compound of Formula 104;

with a di-activated carbonyl equivalent in a suitable solvent under sufficient conditions to produce the compound of Formula 201.
12. A method according to claim 11, wherein the di-activated carbonyl equivalent is selected from the group consisting of carbonyl diimidazole (CDI), phosgene and triphosgene;
13. A method according to claim 11 or 12, wherein the di-activated carbonyl equivalent is carbonyl diimidazole (CDI).
14. A method according to any one of claims 11-13, further comprising treating a compound of Formula 101:

X

Formula 101 wherein X is as defined for the compound of Formula 201; and R4 are as defined for the compound of Formula 104;

Y is halo;

2016202040 19 Dec 2016 with an amine of formula R2-NH2, wherein R2 is as defined for the compound of Formula 104, in a suitable solvent under sufficient conditions to produce the compound of Formula 102.
15. A method according to any one of claims 10-14, wherein X is selected from the group consisting of halo, mesyloxy, p-nitrobenzenesulfonyloxy and tosyloxy;
16. A method according to any one of claims 10-15, wherein X is halo;
17. A method according to any one of claims 10-16, wherein X is bromo.
18. A method according to any one of claims 10-17, wherein Ri is ethynyl.
19. A method according to any one of claims 10-18, wherein R2 is 3-pentyl.
20. A method according to any one of claims 10-19, wherein the compound of Formula

201 is treated with the palladium catalyst in the presence of i) a suitable tin reagent, ii) a compound of the formula RiBfOHfi, wherein Ri is as defined for the compound of Formula 104, or iii) a compound of the formula KR1BF3, wherein Ri is as defined for the compound of Formula 104.
21. A method according to claim 20, wherein the suitable tin reagent is RiSnfbutylfi, wherein Ri is as defined for the compound of Formula 104.
22. A method according to any one of claims 10-19, wherein the compound of Formula 201 is treated with the palladium catalyst in the presence of trimethylsilylacetylene.
23. A compound of Formula I or Formula II:

2016202040 19 Dec 2016

Formula I Formula II wherein

Ri is halo;

R4 is hydrogen; and

R2 is selected from the group consisting of 3-pentyl, 4-heptyl, 4-methyl-lmorpholinopentan-2-yl, isobutyl, cyclohexyl, cyclopropyl, sec-butyl, tert-butyl, isopropyl, l-hydroxybutan-2-yl, tetrahydro-2H-pyran-4-yl, l-methoxybutan-2-yl, l-aminobutan-2-yl and l-morpholinobutan-2-yl, or a pharmaceutically acceptable salt thereof.
24. A compound according to claim 23, or a pharmaceutically acceptable salt thereof, wherein R2 is 3-pentyl.
25. A compound according to claim 23 or 24, or a pharmaceutically acceptable salt thereof, wherein Ri is bromo.
26. A compound selected from the group consisting of (R) -6-bromo-l-sec -butyl- lH-imidazo[4,5-b]pyrazin-2-ol;

(S) -6-bromo-l-sec-butyl- lH-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-l-(pentan-3-yl)-lH-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-l-cyclohexyl-lH-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-1-cyclopropyl- lH-imidazo[4,5-b]pyrazin-2-ol;

6-bromo- 1-tert-butyl- lH-imidazo[4,5-b]pyrazin-2-ol;

6-bromo-l-(propylbutyl)imidazo[4,5-b]pyrazin-2-ol; and

6-chloro-l-(ethylpropyl)imidazo[4,5-b]pyrazin-2-ol;

or a pharmaceutically acceptable salt thereof.

2016202040 19 Dec 2016
27. A compound selected from the group consisting of (R) -6-bromo-l-sec -butyl- lH-imidazo[4,5-b]pyrazin-2(3H)-one;

(S) -6-bromo-l-sec-butyl- lH-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-l-(pentan-3-yl)-lH-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-l-cyclohexyl-lH-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-l-cyclopropyl-lH-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo- 1-tert-butyl- lH-imidazo[4,5-b]pyrazin-2(3H)-one;

6-bromo-l-(heptan-4-yl)-lH-imidazo[4,5-b]pyrazin-2(3H)-one; and

6-chloro-l-(pentan-3-yl)-lH-imidazo[4,5-b]pyrazin-2(3H)-one;

or a pharmaceutically acceptable salt thereof.
28. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 23-27.
29. A compound, or a pharmaceutically acceptable salt thereof, which is 6-bromo-N(pentan-3-yl)pyrazine-2,3-diamine.
30. A compound, or a pharmaceutically acceptable salt thereof, which is 6-bromo-1(pentan-3 - yl) -1 H-imidazo [4,5 -b] pyrazin-2-ol.
31. A compound, or a pharmaceutically acceptable salt thereof, which is 6-bromo-1(pentan-3-yl)-lH-imidazo[4,5-b]pyrazin-2(3H)-one.
32. A compound, or a pharmaceutically acceptable salt thereof, which is l-(pentan-3yl)-6-((trimethylsilyl)ethynyl)-lH-imidazo[4,5-b]pyrazin-2-ol.