Inhibitors of smooth muscle myosins and uses thereof

Abstract

Provided are novel chiral aromatic diazatricyclo-compounds and pharmaceutically acceptable salts thereof, that are smooth muscle myosin 2 specific inhibitors useful for the treatment of ischemic events and conditions associated with capillary no-reflow, or other smooth-muscle related symptoms.

Description

INHIBITORS OF SMOOTH MUSCLE MYOSINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 673,469 filed on July 19, 2024, the contents of which are hereby incorporated by reference in their entirety for any and all purposes.FIELD

[0002] The present technology is generally related to inhibitors of myosin for treating smooth muscle-related conditions.BACKGROUND

[0003] Smooth muscle myosin is present all over the body and is responsible for movements, tone of internal organs, and involuntarily controlled contractions. It is an important effector protein in several physiological processes, including peristaltic movement of the gastrointestinal tract, regulated excretion of digestive solutions, bladder emptying, stiffness of the womb during pregnancy and contraction during labour, regulation of broncho-tracheal functions and the contraction of alveoli in the lungs. Also, smooth muscle myosin is responsible for regulating blood circulation by controlling the diameter of blood vessels and the opening / closing of capillaries.

[0004] No-reflow phenomenon is defined by reduced blood flow upon reperfusion after an ischemic event. It is suggested that smooth muscle myosin-mediated, hypoxia- induced permanent contraction of capillary pericytes is responsible for the no-reflow phenomenon. Although recently emerging reperfusion techniques restore blood flow in the large arteries with high efficacy, capillaries remain permanently constricted thereby preventing the restart of oxygen and nutrient exchange, the removal of accumulated inflammatory factors, consequently resulting in large-scale tissue damage and poor functional outcome. No-reflow phenomenon accompanies many serious, life-threatening conditions including stroke in the brain, myocardial infarction in the heart, renal artery occlusion in the kidney, retinal artery occlusion in the eye, spinal cord injuries, and otherischemic events. No-reflow can also impair function of allografts after organ transplantation. Treatment that restores blood flow at the capillary level, reversing or preventing no-reflow is an urgent unmet medical need.

[0005] So far upstream regulators of capillary contraction were tested to treat noreflow. Calcium channel blockers and nitroprusside have not demonstrated sufficient data for significant beneficial effects, and adenosine has safety concerns (Annibali 2022 JCM). Hydroxyfasudil, an inhibitor of the central hub Rho-kinase, was shown to be effective on AKI animal model (Freitas 2022 eLife). Pharmacological treatment of capillary no-reflow remains an urgent unmet medical need.

[0006] Smooth muscle myosin is a member of the myosin 2 family, which also includes cardiac myosin, skeletal myosin and non-muscle myosin 2s. Several small molecule inhibitors have been developed and co-crystallized with myosin 2s, unveiling several binding sites (ref DOI: 10.1016 / j .tibs.2018.06.006), and these binding sites have very high sequence similarity among the myosin 2 family members / isoforms. Myosin 2 isoforms all have very important and distinct physiological roles, therefore for therapeutic purposes their selective targeting would be desirable. However, selective targeting is extremely challenging, due to the high similarity between the myosin 2 isoforms.SUMMARY

[0007] In one aspect, compounds of formula (IR) or (IIR) are provided, including a pharmaceutically acceptable salt or tautomer thereof:In some embodiments, in formula (IR) and (UR), Q1, Q2, Q3, and Q4are each independently N or CR20, wherein at least one of Q1, Q2, Q3, and Q4is CR20;Q5, Q6, and Q7are each independently S, O, N, NR30, or CR31, provided that the aromatic nature of the ring is retained, wherein at least one of Q5, Q6, and Q7is S, O, N, or NR30;R20is H, or C1-C6 alkyl;R30is H, or C1-C6 alkyl;R31is H, or C1-C6 alkyl;R2, R3, R4, and R5are each independently H, F, Cl, Br, I, or C1-C6 alkyl;R1is H; orR1is a group of formula:wherein: n = 0, 1, 2, 3, 4, 5, or 6; and“D ring” is a saturated, partially saturated, unsaturated, or aromatic, 5- or 6- membered cyclic, or 9- or 10-membered fused bicyclic group, which may be deuterated or tritiated, which may contain one or more N, O, or S atoms, and which may be substituted by: F; Cl; Br; I;C1-C6 straight chain alkyl;C1-C6 haloalkyl;-OH;C1-C6 hydroxyalkyl;C2-C6 hydroxy alkenyl;C2-C6 hydroxyalkynyl;C1-C6 alkoxy or C1-C6 alkoxyalkyl, wherein the alkyl group may be substituted by any number of F, Cl, Br, or I atoms;C1-C6 carboxylic acid; azido;C1-C6 alkylamino or C1-C6 dialkylamino, wherein the alkyl groups may be deuterated or tritiated, and may be substituted by any number of F, Cl, Br, or I atoms; sulfonylamino; a 4-7 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, or CO group; or a C4-C12 fused bicycle, which may be substituted by C1-C6 alkyl, or C1-C6 carboxylic acid; andR6is:H;F; Cl; Br; I;-OH;C1-C6 alkoxy;-CN;-NH2;C1-C6 alkylamino;C1-C6 dialkylamino; azido; amido; or a 5 or 6 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, which may be substituted by:C1-C6 alkyl;-OH;C1-C6 hydroxyalkyl;C1-C6 carboxylic acid; orC1-C6 ester of a carboxylic acid and a saturated alcohol, unsaturated alcohol, or aryl alcohol.

[0008] In another aspect, provided is a pharmaceutical composition containing a compound of Formula (IR) or Formula (IIR) or a pharmaceutically acceptable salt described herein, and a pharmaceutically acceptable excipient.

[0009] In certain aspects, the disclosure provides a method of treating or preventing a smooth-muscle myosin related disorder, condition, or phenomenon comprising administering to a subject in need thereof a compound or salt of any one of Formulas (IR) or (IIR). In some embodiments, the disorder, condition, or phenomenon is no-reflow. In some embodiments, the disorder or condition may be accompanied by stenosis, stricture, or motility-related dysfunction. In some embodiments, the disorder or condition may be accompanied by hypertension. In some embodiments, the disorder or condition is accompanied by smooth muscle myosin mutation.

[0010] In certain aspects, the disclosure provides a method of treatment related to organ or tissue transplantation, comprising administering to a subject in need thereof or the organ / tissue to be transplanted a compound or salt of any one of Formulas (IR) or (IIR).

[0011] In some embodiments, a compound or salt of Formula (IR) or (HR) is an inhibitor of smooth muscle contraction. In some embodiments, a compound or salt of Formula (IR) or (IIR) is an inhibitor of smooth muscle myosin 2. In some embodiments, a compound or salt of Formula (IR) or (IIR) binds to the relay-SHl drug binding cleft (see omecamtiv mecarbil binding site in ref: DOI: 10.1016 / j .tibs.2018.06.006) on myosin 2.

[0012] In another aspect, provided is a compound of formulaor a pharmaceutically acceptable salt or tautomer thereof.

[0013] In various embodiments provided herein, the compounds as inhibitors of smooth muscle myosin 2. This includes, in some embodiments, using the compounds to target the relay-SHl drug binding cleft on smooth muscle myosin 2. In some embodiments, the compound is of formula:

[0014] In other embodiments, the compounds disclosed herein are for the treatment of smooth muscle myosin 2 related diseases. Some of those diseases include those with no-reflow. In some embodiments, the compounds are intended for treatment of myocardial infarction, acute kidney injury, stroke, hypertension, or gastrointestinal motility disorder. In some embodiments, the compound is of formula:

[0015] In another aspect, a method of inhibiting smooth muscle myosin 2 is provided, where the method includes contacting any of the compounds disclosed herein with smooth muscle myosin 2. In some embodiments, the compound is of formula:

[0016] In another aspect, a method of treating a disease mediated by smooth muscle myosin 2 is provided, where the method includes administering a compound disclosed herein to an individual in need thereof. In some embodiments, the compound is of formula:

[0017] In another aspect, a method of treating myocardial infarction, acute kidney injury, stroke, hypertension, or gastrointestinal motility disorder in an individual, where the method includes administering a compound (or pharmaceutically acceptable formulation thereof) disclosed herein to an individual in need thereof. In some embodiments, the compound is of formula:

[0018] In another aspect, a method is provided for preparing a compound of formulaThe method includes adding lithium bis(trimethylsilyl)amide to a solution of a compound of formulaadding aqueous ammonium chloride; and isolating the compound of formulaAdditional details of an illustrative method of making such a compound are provided in the examples. The method may also include preparing the compound of formulaadding a solution of a base in an ether solvent to a solution of a compound of formulafollowed by quenching with acetic acid; isolating the compound of formulaThe method(s) may further include preparing the compound of formulaadding phosphorus oxychloride to a solutionmixture of 1,4- dioxane and dichloromethane; adding methyl 2-amino-5-methylthiophene-3-carboxylate and heating at reflux; and collecting the compound of formulaBRIEF DESCRIPTION OF THE DRAWINGS

[0019] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0020] FIGs. 1 A and IB depict pharmacokinetics and tissue distribution of compound 2501. FIG. 1A shows a change of concentration of compound 2501 as a function of time in blood plasma of rats, where 12 mg / kg compound 2501 was administered to each rat at t = 0. FIG. IB shows the concentration of compound 2501 in various tissues 120 min after i.v. administration of 5 (white), 10 (striped), or 40 (black) mg / kg of said compound.

[0021] FIGs. 2A-2C depict relaxation of ex vivo trachea (FIG. 2A), bronchus (FIG. 2B), or artery (FIG. 2C) isolated from rat in a dose dependent manner upon administration of compound 2501. Points of administration are marked by arrows. (D) Half-life of artery relaxation as a function of vessel diameter. Data for basilar (BA), femoral (FA), and common carotid (CCA) arteries are presented.

[0022] FIGs. 3A-3C depicts effect of different doses of compound 2501 and various additives on the survival (FIG. 3A and 3B) and kidney function (FIG. 3C) of rats after bilateral kidney ischemia.

[0023] FIGs. 4A-4F depict the effect of compound 2501 on in vivo capillary blood flow (flux and velocity of red blood cells) after ischemia in the kidney. Flux and velocity, respectively, are presented as a function of time (FIGs. 4A and 4B), dose (FIGs. 4C and 4D), or duration of the ischaemia (FIGs. 4E and 4F).

[0024] FIGs. 5A-5E depict the effect of LADO and subsequent administration of 10 mg / kg compound 2501 bolus injection, followed by infusion (2 mg / ml, 1 ml / min, 1 hour) on ECG (FIG. 5 A), heart capillary diameter (FIG. 5B), ratio of ischemic tissue (FIG. 5C), 2-hour survival (FIG. 5D), and dose-dependent rescue of ischemic tissue (bolus injection only) (FIG. 5E).

[0025] FIG. 6 depicts the dose-dependent effect for compound 2501 on the blood flow measured in the LAD of landrace pig as a function of time.

[0026] FIG. 7 depicts the dose-dependent effect for compound 2501 on blood flow increase in the LAD of landrace pig, during systole and diastole, separately.

[0027] FIGs. 8A-8C depict a cardiac cycle determined in landrace pig model, showing the LAD blood flow as a function of arterial pressure after the administration of the vehicle, HPBCD (FIG. 8 A), nitroglycerine (FIG. 8B), or compound 2501 (Fig: 8C).

[0028] FIG. 9 depicts the amino acid residue in the relay-SHl drug binding site which can covalently attach to the R enantiomer, i.e. (+) azidoblebbistatin upon photoactivation.

[0029] FIG. 10 is an X-ray structure projection of Compound 2501, where the hydrogen atoms are not shown, the carbon atoms are unlabeled, and other heteroatoms are labelled.DETAILED DESCRIPTION

[0030] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s).DEFINITIONS

[0031] As utilized herein with respect to numerical ranges, the terms “approximately,” “about,” “substantially,” and similar terms will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the terms that are not clear to persons of ordinary skill in the art, given the context in which it is used, the terms will be plus or minus 10% of the disclosed values. When “approximately,” “about,” “substantially,” and similar terms are applied to a structural feature (e.g., to describe its shape, size, orientation, direction, etc.), these terms are meant to cover minor variations in structure that may result from, for example, the manufacturing or assembly process and are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the disclosure as recited in the appended claims.

[0032] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0033] In general, “substituted” refers to an alkyl, alkenyl, alkynyl, aryl, or ether group, as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group will be substituted by one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted by 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include: halogens (i.e., F, Cl, Br, and I); alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl; hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N- oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN); and the like.

[0034] As used herein, “alkyl” groups include straight chain and branched alkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 6 carbon atoms. As employed herein, “alkyl groups” include cycloalkyl groups as defined below. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, sec-butyl, t-butyl, neopentyl, and isopentyl groups. Representative substituted alkyl groups may be substituted one or more times with, for example, amino, thio, hydroxy, cyano, alkoxy, and / or halo groups such as F, Cl,Br, and I groups. As used herein the term haloalkyl is an alkyl group having one or more halo groups. In some embodiments, haloalkyl refers to a per-haloalkyl group.

[0035] Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 6, or 7. Cycloalkyl groups may be substituted or unsubstituted. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted by straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to: 2,2-; 2,3-; 2,4-; 2,5-; or 2,6-disubstituted cyclohexyl groups or mono-, di-, or trisubstituted norbornyl or cycloheptyl groups, which may be substituted by, for example, alkyl, alkoxy, amino, thio, hydroxy, cyano, and / or halo groups.

[0036] Alkenyl groups are straight chain, branched or cyclic alkyl groups having 2 to about 20 carbon atoms, and further including at least one double bond. In some embodiments alkenyl groups have from 1 to 12 carbons, or, typically, from 1 to 8 carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups include, for instance, vinyl, propenyl, 2-butenyl, 3-butenyl, isobutenyl, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl groups among others. Alkenyl groups may be substituted similarly to alkyl groups. Divalent alkenyl groups, i.e., alkenyl groups with two points of attachment, include, but are not limited to, CH-CH=CH2, C=CH2, or C=CH-CH3.

[0037] As used herein, “aryl,” or “aromatic,” groups are cyclic aromatic hydrocarbons. Aryl groups include monocyclic, bicyclic and polycyclic ring systems. Thus, aryl groups include, but are not limited to phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. The phrase “aryl groups” includesgroups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Aryl groups may be substituted or unsubstituted.

[0038] The term “haloalkyl” refers to an alkyl group as defined above, that is substituted by one or more F, Cl, Br, or I atoms. Haloalkyl groups include, but are not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 1 -chi oro-2-fluorom ethyl, and the like. The alkyl part of the haloalkyl group is optionally further substituted as described herein.

[0039] The term “haloalkoxy” refers to an alkoxy group, in which the alkyl part of the group is substituted by one or more F, Cl, Br, or I atoms. Some non-limiting examples are trifluoromethoxy, 2,2-dichloroethoxy, l,l-dichloro-2-iodoethoxy, and the like. The alkyl part of the haloalkoxy group is optionally further substituted as described herein.

[0040] In general, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for administration to human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0041] As used herein, “RT,” “rt” or “room temperature” means 22.5 °C + / - 3 °C.

[0042] The following abbreviations stand for various organic solvents:AcOH: acetic acidACN: AcetonitrileDCM: DichloromethaneDMA or DMAc: DimethylacetamideEtOAc: Ethyl acetateFA: Formic acidIPA: Isopropyl alcoholKOtBu: Potassium tert-butoxideLiHMDS: Lithium bis(trimethylsilyl)amideMeOH: MethanolMTBE: Methyl tert-Butyl EtherTEA, Et3N: Tri ethylamineTHF: Tetrahydrofuran

[0043] The following abbreviations are used for various reagents:R-Davis reagent: (+)-(4S)-7,7-dimethoxy-l 1,1 l-dimethyl-5-oxa-3X6-thia-4- azatetracyclo[6.2.1.O’^.O4,6]undecane-3,3-dioneNaH: Sodium hydride

[0044] Further abbreviations used: min: minutesL: litres ml: millilitres pl: microlitresAmm Bic: Ammonium bicarbonateBCA: Bicinchoninic acidBSA: Bovine serum albuminDPBS: Dulbecco’s phosphate buffered salineLAD: Left anterior descending coronary arteryLADO: Left anterior descending coronary artery occlusionLC-MS / MS: Liquid chromatography -tandem mass spectrometryMS: Mass spectrometry m / r. Mass to charge ratioPBS: Phosphate buffered salinePRM: Parallel reaction monitoringRBC: Red blood cellRef: Relative centrifugal forceSIL: Stable isotope labeledTCEP: Tris (2-carboxyethyl) phosphineTFA: Trifluoroacetic acid w / w: weight to weight z: charge stateCOMPOUNDS

[0045] In one aspect, compounds of formula (IR) or (IIR) are provided, including pharmaceutically acceptable salts or tautomers thereof:wherein:Q1, Q2, Q3, and Q4are each independently N or CR20, wherein at least one of Q1, Q2, Q3, and Q4is CR20;Q5, Q6, and Q7are each independently S, O, N, NR30or CR31, provided that the aromatic nature of the ring is retained, and at least one of Q5, Q6, and Q7is S, O, N, or NR30;R20is H, or C1-C6 alkyl;R30is H, or C1-C6 alkyl;R31is H, or C1-C6 alkyl;R2, R3, R4, and R5are each independently H, F, Cl, Br, I, or C1-C6 alkyl; andR1is H; orR1is a group of formula:wherein:n = 0, 1, 2, 3, 4, 5, or 6; and“D ring” is a saturated, partially saturated, unsaturated, or aromatic, 5- or 6- membered cyclic or 9- or 10-membered fused bicyclic group, which may be deuterated or tritiated, which may contain one or more N, O, or S atoms, and which may be substituted by:F; Cl; Br; I;C1-C6 straight chain alkyl;C1-C6 haloalkyl;-OH;C1-C6 hydroxyalkyl;C2-C6 hydroxy alkenyl;C2-C6 hydroxyalkynyl;C1-C6 alkoxy or C1-C6 alkoxyalkyl, wherein the alkyl group may be substituted by any number of F, Cl, Br, or I atoms;C1-C6 carboxylic acid; azido;-NH2;C1-C6 alkylamino or Cl -6 dialkylamino, wherein the alkyl groups may be deuterated or tritiated, and may be substituted by any number of F, Cl, Br, or I atoms; sulfonylamino; a 4-7 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, or CO group; or a C4-C12 fused bicycle, which may be substituted by C1-C6 alkyl or C1-C6 carboxylic acid; andR6is:H;F; Cl; Br; I;-OH;C1-C6 alkoxy;-CN;-NH2;C1-C6 alkylamino;C1-C6 dialkylamino; azido; amido; or a 5 or 6 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, which may be substituted by:C1-C6 alkyl;-OH;C1-C6 hydroxyalkyl;C1-C6 carboxylic acid; orC1-C6 ester of a carboxylic acid and a saturated alcohol, unsaturated alcohol, or aryl alcohol.

[0046] In one embodiment, the compounds of Formula (IIIR) or (IVR) are provided, including pharmaceutically acceptable salts and tautomers thereof:(IIIR) (IVR)wherein:Q5and Q7are each independently S, O, N, or CH, and Q6is S, O, N, or CR31, provided that the aromatic nature of the ring is retained, and at least one of Q5, Q6, and Q7is S, O, or N; andR1and R6are as described above; andR7is H, or C1-C6 alkyl; andR31is H, or C1-C6 alkyl.

[0047] In one embodiment, the compounds of Formula (IIIR) or (IVR) are provided, including pharmaceutically acceptable salts and tautomers thereof:wherein:Q5and Q7are CH or S; and Q6is S or CR31, provided that the aromatic nature of the ring is retained, and at least one of Q5, Q6, and Q7is S; andR1is: a 5 membered aromatic heterocycle containing one or more N; a 6 membered aromatic heterocycle containing one or more N, which may be substituted by C1-C6 alkyl, C1-C6 alkylamino, or C1-C6 dialkylamino; a 9 membered partially saturated or aromatic fused bicycle containing one or more N or O, which may be substituted by C1-C6 alkyl, F, Cl, Br, or I; a 10 membered aromatic heterocycle containing one or more N; naphthyl; orR1is a group of formula:wherein R10is a bond or C1-C6 alkyl, and R14, R15, R16, R17, and R18are each independently H or D; orR1is a group of formula:wherein:R11is H, and R13is H or a 5 membered aromatic heterocycle containing one or more N, which may be substituted by C1-C6 alkyl; andR12is:H;F; Cl; Br; I;C1-C6 alkyl;C1-C6 haloalkyl;C1-C6 alkoxy;C1-C6 haloalkoxy;C1-C6 alkoxyalkyl, C2-C6 alkoxyalkenyl, or C2-C6 alkoxy alkynyl;NH2;C1-C6 alkylamino or C1-C6 dialkyalkylamino, which may be deuterated, and which may be substituted by any number of F, Cl, Br, or I atoms;S(O)2NH2; or a 4, 5, 6, or 7 membered heterocycle containing one or more N or O atoms or CO groups; andR6is:F; Cl; Br; I;OH;NH2;C1-C6 alkylamino;C1-C6 dialkylamino;C1-C6 alkoxy;CN;C(O)NH2; orR6is a 5 membered aromatic heterocycle containing one or more N, which may be substituted by C1-C6 alkyl, C1-C6 hydroxyalkyl, or C1-C6 ester of a carboxylic acid and a saturated alcohol, unsaturated alcohol, or aryl alcohol; orR6is a 6 membered saturated heterocycle containing one or more N, or O; andR7is H or C1-C6 alkyl; andR31is H or C1-C6 alkyl.

[0048] In one embodiment, the compounds of Formula (IIIR) or (IVR) are provided, including pharmaceutically acceptable salts and tautomers thereof:wherein:Q5and Q7are CH or S; and Q6is S or CR31, provided that the aromatic nature of the ring is retained, and at least one of Q5, Q6, and Q7is S; andR6is:F; Cl; Br; I;OH;NH2;C1-C6 alkylamine;C1-C6 dialkylamine;C1-C6 alkoxy;CN;C(O)NH2; orR6is a 5 membered aromatic heterocycle containing one or more N, which may be substituted by C1-C6 alkyl, C1-C6 hydroxyalkyl, or C1-C6 ester of a carboxylic acid and a saturated alcohol, unsaturated alcohol, or aryl alcohol; orR6is a 6 membered saturated heterocycle containing one or more N or O; andR7is H or C1-C6 alkyl; andR31is: H, or C1-C6 alkyl; andR1is selected from:

[0049] In one embodiment, the compounds of Formula (IIIR) or (IVR) are provided, including pharmaceutically acceptable salts and tautomers thereof:(IIIR) (IVR)wherein:Q5and Q7are CH or S; and Q6is S or CR31, provided that the aromatic nature of the ring is retained, and at least one of Q5, Q6, and Q7is S; andR7is H, or C1-C6 alkyl; andR31is: H, or C1-C6 alkyl; andR1is a group of formula:wherein R12is:F; Cl; Br; I; methyl, ethyl, or propyl, which may be substituted by any number of F,Cl, Br, or I atoms;isopropyl; methoxy or ethoxy, which may be substituted by any number of F, Cl, Br, or I atoms; methoxymethyl;NH2; methylamino, ethylamino, or dimethylamino, which may be deuterated, and which may be substituted by any number of F, Cl, Br, or I atoms; orS(O)2NH2; andR6is:F; Cl; Br; I;OH;NH2; methylamino, ethylamino, or dimethylamino; methoxy, or ethoxy;CN; orC(O)NH2.

[0050] In some embodiments, at least one compound is selected fromand pharmaceutically accepted salts thereof.

[0051] Compounds of present invention include all pharmaceutically acceptable forms and formulations, such as crystalline and amorphous forms, pharmaceutically acceptable salts, and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates,unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, all diastereomeric and epimeric forms synthesized or separated.

[0052] In one embodiment, the sulphate salt in 30% HPBCD is used for direct administration into the bloodstream (intravenous or intraarterial) in doses of 0.02-20 mg / kg.

[0053] In another aspect, disclosed herein are pharmaceutical compositions comprising, consisting essentially of, or consisting of a compound as described herein, and one or more pharmaceutically acceptable excipients.

[0054] In another aspect, disclosed herein are pharmaceutical compositions comprising, consisting essentially of, or consisting of a compound of Formulas IR, IIR, IIIR or IVR, as described herein, and one or more pharmaceutically acceptable excipients.

[0055] The compounds may be formulated for administration by any suitable route of administration, such as for example, oral, topical (including transdermal), rectal, vaginal, transmucosal, or intestinal administration, parenteral delivery, including by intramuscular, subcutaneous, intravenous, intraarterial, intracoronary, intracardiac, intrathecal, epidural, or intraorbital injection, as well as inhalation, direct intraperitoneal, or intranasal delivery.

[0056] Pharmaceutical compositions as disclosed herein may comprise, as one or more pharmaceutically acceptable excipients, a pharmaceutically acceptable carrier, diluent, disintegrant, sweetening agent, glidant (such as magnesium stearate), flavoring agent, emulsifying agent, suspending agent, stabilizer, isotonic agent, etc. Pharmaceutical compositions as disclosed herein may be formulated into an oral dosage form such as tablets, capsules, powders, granules, suspensions, emulsions, or syrups; or a topical (including transdermal) or transmucosal dosage form such as liquids, suspensions, emulsions, gels (ointments or the like), or a parenteral dosage form such as liquids, suspensions, emulsions, and freeze-dried powders. Said dosage forms may be formulated in various forms, e.g., a dosage form for single administration or for multiple administrations.

[0057] Illustrative excipients include, without limitation, lactose, polyethylene glycol (PEG), hydrogenated castor oil (HCO), cremophors, carbohydrates, starches (e.g., corn starch), inorganic salts, antimicrobial agents, antioxidants, binders / fillers, surfactants, lubricants (e.g., calcium or magnesium stearate), glidants such as talc, disintegrants, diluents, buffers, acids, bases, film coats, solubilizers (e.g. alcohols, including polyols, cyclodextrins, including Hydroxypropyl-P-cyclodextrin, ethers of polyethylene glycols, amides, esters, and water), combinations thereof, and the like.

[0058] The amount of any individual excipient in the composition will vary depending on the role of the excipient, the dosage requirements of the active agent, and particular needs of the composition. Generally, however, the excipient will be present in the composition in an amount of from about 1% to about 99% by weight, such as from about 5% to about 98% by weight, including from about 15 to about 95% by weight of the composition. In general, the amount of excipient present in a composition of the disclosure is selected from the following: about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by weight.

[0059] A pharmaceutical composition as disclosed herein may include a compound disclosed herein as the only active agent, or may be formulated with other active agents.

[0060] Techniques for formulation and administration of the compounds disclosed herein may be found in “Remington: The Science and Practice of Pharmacy,” Academic Press, London, United Kingdom, 23rd edition, 2020.

[0061] Pharmaceutical compositions as disclosed herein may be formulated to provide a therapeutically effective amount of a compound as disclosed herein in a reasonable volume or mass of the composition. Although the exact dosage may be determined on a drug-by-drug (compound-by-compound) basis, for most compounds, some generalizations regarding the dosage can be made. For example, the dosage regimen for an adult human patient may be between 0.001 mg and 1000 mg, such as between 0.01 mg and 500 mg, for example from 5 to 500 mg of the compound or pharmaceutically acceptable salt thereof, calculated as the free base or free acid.TREATMENT

[0062] Abnormal functioning of smooth muscles lead to serious acute and chronic diseases. Gastrointestinal motility disorders lead to irritable bowel syndrome (IBS), visceral myopathy, ulcerative colitis or Crohn’s disease (Can. J. Gastroenterol 10(4) 1996), gallbladder leiomyopathy (DOI: 10.1007 / sl 1894-004-0043-0), and cholesterol gallstone disease (DOI: 10.1111 / j.1365-2982.2012.01935.x). Perturbed function of bladder detrusor muscle (major muscle component of the urinary bladder wall) during micturition leads to lower urinary tract symptoms such as Underactive Bladder Syndrome with inadequate magnitude or duration of bladder emptying, or Overactive Bladder Syndrome due to involuntary contractions of the smooth muscle of the bladder leading to urgency, frequency, and urge incontinence, and also leading to bladder obstruction (DOI: 10.1152 / physrev.00038.2003). Preterm contractions of uterine myometrium smooth musculature due to hormonal, physical, neurological, or other reasons lead to preterm birth, which is life-threatening for the foetus (DOI: 10.1203 / 00006450-199811000-00001). Preterm birth is also associated with not fully differentiated smooth muscle cell contractions in human umbilical artery (DOI: 10.1016 / j.placenta.2012.03.005). Uncontrolled growth of uterine smooth muscle cells leads to contractile and painful tumours or uterine fibroids categorized as benign leiomyomas or malignant leiomyosarcomas (PMID: 25550862). Dysfunction of airway smooth muscle leads to asthma and chronic obstructive pulmonary disease (COPD) associated with declined lung functions due to contractile abnormalities and / or hypertrophy in airway smooth muscle (DOI: 10.1016 / S0140-6736(22)02125-0) (DOI: 10.1016 / s0140-6736(03)14416-9 ).

[0063] When ischaemia occurs, tissues suffer from deprivation of oxygen, glucose, and other nutrients required for healthy metabolism. Perturbed metabolic functions lead to anaerobic metabolism, decrease in tissue pH, and reduced ATP production and increased cytosolic calcium levels. These processes result in the permanent contraction of smooth muscles in vessels and in capillary pericytes. (Cowled P, Fitridge R. Pathophysiology of Reperfusion Injury. In: Fitridge R, Thompson M, editors. Mechanisms of Vascular Disease: A Reference Book for Vascular Specialists [Internet], Adelaide (AU): University of Adelaide Press; 2011. 18.) Such ischemia induced events lead to infarction and dysfunction of the affected area, as in several diseases and symptoms including acute kidney injury (AKI), ischemic heart attack, stroke, limb ischemia. Ischaemia is also artificially induced during certain medical procedures, such as hypothermic circulatory arrest or low-flow cardiopulmonary bypass (DOI: 10.1016 / 0003-4975(93)90731-v).

[0064] In one aspect, compounds of Formula (IR) and (HR) are useful in treatment of smooth-muscle myosin related diseases and symptoms including those listed above.

[0065] In certain aspects, the disclosure provides a method of treating or preventing a smooth-muscle myosin related disorder, condition or phenomenon comprising administering to a subject in need thereof a compound or salt of any one of Formulas (IR) or (IIR). In some embodiments, the disorder, condition or phenomenon is no-reflow or hypoperfusion, including but not limited to microangiopathy, microvascular disease, small vessel disease, or microvascular dysfunction. No-reflow phenomenon is the failure of blood to reperfuse an ischemic area after the physical obstruction has been removed, resolved, or bypassed. No-reflow or hypoperfusion may accompany infarction or other ischemic events including but not limited to stroke, spinal cord injuries, retinal artery occlusion, cerebral vasospasm, myocardial infarction (i.e. ischemic heart attack), coronary artery disease, angina (including stable and unstable angina, microvascular angina, vasospastic angina, refractory angina), Takotsubo disease, cocaine vasoconstriction, renal artery occlusion, acute kidney injury (AKI), cardiorenal syndrome, intestinal ischemia (i.e. bowel infarction / mesenteric ischemia / ischemic enteropathy), testicular torsion / detorsion, pulmonary embolism or infarction, bone marrow ischemia or infarction of bone, peripheral ischemia, limb ischemia, Raynaud’s phenomenon, splenic infarction, CADASIL syndrome, hemorrhagic stroke, ischemic stroke, subarachnoidal hemorrhage, hemodynamic stroke, post-resuscitation shock, post-cardiac arrest syndrome, cardiogenic shock. In some embodiments, the disorder or condition is no-reflow or ischemia accompanying medical interventions including but not limited to hypothermic circulatory arrest, low-flow cardiopulmonary bypass (in infant heart surgery), thrombectomy, thrombolysis, percutaneous coronary intervention, angioplasty. In some embodiments, the disorder or condition may be accompanied by stenosis, stricture, or motility-related dysfunction e.g. migraine, reversible cerebral vasoconstriction syndrome (including but not limited to migrainous vasospasm, migraine angiitis, Call-Fleming syndrome or Call syndrome), atherosclerosis, gastrointestinal motility disorders including irritable bowel syndrome (IBS), Hirschsprung’s disease, chronic intestinal pseudo-obstruction, Chagas disease, scleroderma, visceral myopathy, ulcerative colitis or Crohn’s disease, gallbladder, and cholesterol gallstone disease, gallbladder attack, spasm of gastro-intestinal (or alimentary canal) smooth muscle, including esophagus, stomach, intestine and colons, biliary and pancreatic duct and sphincters, such as sphincter of Oddi dysfunction, gastricspasm, pylorospasm, and spasms of the biliary tract or pancreatic tract; esophageal dysmotility, achalasia, preterm contractions of uterine myometrium smooth musculature, preterm labor, spontaneous or induced e.g. surgically during transuterine fetal surgery, menstrual and premenstrual cramps, primary dysmenorrhea, spasm of uterus or Fallopian tube, erectile dysfunction, underactive bladder syndrome and overactive bladder syndrome, incontinence, stress urinary incontinence, spasm of urinary tract muscle, ureterolithiasis, ureteric / renal colic, dysfunction of airway smooth muscle leading to asthma or chronic obstructive pulmonary disease, PAH (pulmonary arterial hypertension), bronchospasm, bronchoconstrictive disease, spasm of tracheobronchial tree smooth muscle, breathlessness, dyspnea, airway wall remodeling, which is a condition associated with diseases or conditions characterized by airway wall thickening and air obstruction, peripheral vascular disease, glaucoma, increased intraocular pressure, intraocular hypertension, reduced flow of intraocular aqueous humor. In some embodiments, the disorder or condition is accompanied by hypertension e.g. primary or secondary hypertension, resistant hypertension, malignant hypertension, isolated systolic hypertension, gestational hypertension, glaucoma. In some embodiments, the disorder or condition is accompanied by smooth muscle myosin mutation, e.g. Loeys-Dietz syndrome, Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP), human intestinal neoplasia (linked with colorectal cancer), pyloric stenosis, visceral myopathy, megacystis- microcolon-intestinal hypoperistalsis syndrome, thoracic aortic aneurysms and dissections, increased ER stress and autophagy, acute aortic dissections, acute myeloid leukemia, human colorectal cancer, breast or prostate cancers.

[0066] In certain aspects, the disclosure provides a method for controlling smooth muscle tone of visceral organs and vascular dilation during medical procedures, comprising administering to a subject in need thereof a compound or salt of any one of Formulas (IR) or (IIR). In some embodiments, the medical procedure is endoscopy, surgery, or control of labor during pregnancy.

[0067] In certain aspects, the disclosure provides a method of treatment related to organ or tissue transplantation, comprising administering to a subject in need thereof or the organ / tissue to be transplanted a compound or salt of any one of Formulas (IR) or (IIR). In some embodiments the transplanted organ or tissue is from live donor. In some embodiments the transplanted organ or tissue is from deceased donor. In someembodiments the transplanted organ or tissue is a xenograft. In some embodiments the tissue or organ is lung, uterus, liver, pancreas, small bowel, kidney, heart, heart valve, cornea, thymus, skin or bone.

[0068] Smooth muscle myosin is a member of the myosin 2 family, which also includes cardiac myosin, skeletal myosin and non-muscle myosin 2s. As myosin 2 family members are very similar, their selective targeting is extremely challenging. Several small molecule inhibitors have been developed and co-crystallized with myosin 2s, unveiling a few potentially targetable binding sites (ref DOI: 10.1016 / j .tibs.2018.06.006) in various deeper or shallower pockets or clefts. In one aspect, compounds of present invention target the drug binding cleft between the relay and SHI helices and at the N-terminal domain of myosin heads.

[0069] In one aspect, compounds provided are direct inhibitors of smooth muscle myosin. In certain aspects, compounds provided are useful for achieving smooth muscle relaxation, smooth muscle myosin ATPase inhibition, selective inhibition of smooth muscle myosin, detachment of smooth muscle myosin from actin, or pericyte relaxation. In certain aspects, compounds provided are useful for targeting the relay-SHl drug binding cleft of myosin 2, opening of capillaries, reopening of capillaries, elimination of no-reflow, saving tissue affected by ischemia, or the treatment of smooth-muscle myosin related diseases and symptoms. In one aspect, compounds of the invention are smooth muscle myosin specific relative to cardiac or skeletal myosins.

[0070] In one aspect, compounds of the invention have suitable pharmacokinetic properties for the treatment of no-reflow phenomenon or smooth muscle related diseases.SYNTHESIS

[0071] In another aspect, the present disclosure provides methods of producing the above-defined compounds. Compounds of the present invention may be synthesized via the following general Schemes 1-6, where A and D rings may be exchanged to any rings, which are in agreement with Formula (IR) and Formula (IIR).Scheme 1 general total synthesis strategy starting from D ringScheme 2 general strategy for synthesis of tricyclic core, which can be functionalized and linked to D-ringScheme 3 Synthesis of a tricyclic core with C6 A ringScheme 4 General strategy for variations for substitutions at the chiral centreScheme 5 General scheme for substitutions on the A-ringScheme 6 general scheme for D-ring substitutions

[0072] The compounds may be synthesized using conventional techniques. Synthetic chemistry transformations and methodologies useful in synthesizing the compounds described herein are known in the art and include, for example, references: Lawson et al. 2011, DOI: 10.1039 / c0cc03624b, US11746112B2, Bart I. Roman et al. DOI: 10.1021 / acs.jmedchem.8b00503

[0073] The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.EXAMPLESExample 1. In vitro efficacy: smooth muscle myosin inhibition and selectivity

[0074] ATPase activity measurements were carried out using i) rabbit actin, ii) skeletal muscle myosin-2 subfragment-1 (SkSl) isolated from rabbit m. psoas and digested by a-chymotrypsin (0.05 mg / ml a-chymotrypsin, 10 min, 25°C; reaction was stopped with 3 mM PMSF), iii) cardiac muscle myosin (CM) isolated from left ventricle of porcine heart, digested by a-chymotrypsin (0.25 mg / ml a-chymotrypsin, 10 min, 25 °C, reaction was stopped with 3 mM PMSF) iv) smooth muscle myosin subfragment- 1(SmSl) from freshly slaughtered chicken gizzard digested by activated papain (0.2 mg / ml papain, 12 min, 25°C; reaction was stopped with 5 mM sodium iodoacetate).

[0075] Steady-state ATPase measurements were carried out in 50 pl volume in a flat-bottom 384-well plate (Nunc-Thermo Fischer) using an NADH-PK / LDH coupled assay at 25°C in the presence of 0.5 mM ATP and F-actin (20 pM for SkSl; 11.5 pM for CM; and 33 pM for SmSl) in a low ionic strength buffer (10 mM MOPS pH 7.0, 4 mM MgCh, 2 mM P-mercaptoethanol) for 15 min. Compounds were added to the reaction as a solution in DMSO, in 0.5 pl (1% of total volume), and three parallels were measured for each point. Controls containing DMSO with myosin, but no inhibitor, and actin-control containing actin and DMSO, but no myosin were measured in all measurement sets. ATPase activity was calculated from the slope of the linear fit to the time-dependent absorbance data collected at 340 nm. Relative ATPase activity is normalized to the ATPase activity of the DMSO control, taken to be 100%. Applied myosin concentrations (100 nM SkSl, 500 nM CM and SmSl) were used to fit quadratic function to determine IC50 values.

[0076] Results are summarized in Tables 1-5.Table 1. Ratio of ATPase inhibition parameters of R(+) enantiomers on smooth muscle and cardiac myosin 2aInitial slope (% / pM, INI) of the quadratic fits / to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (% / pM, SmINI) and SI subfragment of cardiac myosin-2 (% / pM, CmlNI) calculated from the maximal inhibition (%, Imax) and the inhibitory constant (pM, IC50) based on the following formula: INI = Imax / IC50. Labeled categories are as follows: A: INI < 1 % / pM; B: 1 % / pM < INI < 3 % / pM; C: 3 % / pM < INI < 10 % / pM; D: 10 % / pM < INI < 30 % / pM; E: 30 % / pM < INI.bRatio of the measured ATPase inhibitions at 50 11M inhibitor concentration on SI subfragment of smooth muscle myosin-2 (%, Sm50) and on SI subfragment of cardiac myosin-2 (%, Cm50). Labeled categories are as follows: A: Sm50 / Cm50 < 1; B: 1 < Sm50 / Cm50 < 3; C: 3 < Sm50 / Cm50 < 10; D: 10 < Sm50 / Cm50 < 30; E: 30 < Sm50 / Cm50.cRatio of the maximal inhibition (%, Imax) calculated from the quadratic fits / to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (%, Smlmax) and SI subfragment of cardiac myosin-2 (%, Cmlmax). Labeled categories are as follows: A: Smlmax / Cmlmax < 1; B: 1 < Smlmax / Cmlmax < 3; C: 3 < Smlmax / Cmlmax < 10; D: 10 < Smlmax / Cmlmax < 30; E: 30 < Smlmax / Cmlmax.dRatio of the inhibitory constant iiiM. IC50) calculated from the quadratic fits! to the measured ATPase data points for SI subfragment of cardiac myosin-2 (pM, CmIC) and SI subfragment of smooth muscle myosin-2 (pM, SmIC). Labeled categories are as follows: A: CmIC / SmIC < 1; B: 1 < CmIC / SmIC < 3; C: 3 < CmIC / SmIC < 10; D: 10 < CmIC / SmIC < 30; E: 30 < CmIC / SmIC.eSpecificity (Spec, S) towards SI subfragment of smooth muscle myosin-2 over SI subfragment of cardiac myosin-2 calculated as follows: S = Sm50 / Cm50 * CmIC / SmIC; Labeled categories are as follows: A: S < 1; B: 1 < S < 3; C: 3S < 10; D: 10 < S < 30; E: 30 < S. t Quadratic equation used to fit the measured ATPase data points: Relative ATPase activity = R0 + Imax *((M+I+IC50)- sqrt(sqr(M+I+IC50)-4*M*I)) / (2*M); where R0: relative ATPase activity in the absence of inhibitor and in the presence of DMSO as a vehicle control, Imax: maximal inhibition, M: concentration of myosin, IC50: inhibitory constant corresponding to 50% inhibition, I: inhibitor concentration.Table 2. Ratio of ATPase inhibition parameters of S(-) enantiomers on smooth muscle and cardiac myosin 2aInitial slope (% / pM, INI) of the quadratic fits / to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (% / pM, SmINI) and SI subfragment of cardiac myosin-2 (% / pM, CmlNI) calculated from the maximal inhibition (%, Imax) and the inhibitory constant iiiM. IC50) based on the following formula: INI = Imax / IC50. Labeled categories are as follows: A: INI < 1 % / pM; B: 1 % / pM < INI < 3 % / pM; C: 3 % / pM < INI < 10 % / pM; D: 10 % / pM < INI < 30 % / pM; E: 30 % / pM < INI.bRatio of the measured ATPase inhibitions at 50 11M inhibitor concentration on SI subfragment of smooth muscle myosin-2 (%, Sm50) and on SI subfragment of cardiac myosin-2 (%, Cm50). Labeled categories are as follows: A: Sm50 / Cm50 < 1; B: 1 < Sm50 / Cm50 < 3; C: 3 < Sm50 / Cm50 < 10; D: 10 < Sm50 / Cm50 < 30; E: 30 < Sm50 / Cm50.cRatio of the maximal inhibition (%, Imax) calculated from the quadratic fits / to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (%, Smlmax) and SI subfragment of cardiac myosin-2 (%, Cmlmax). Labeled categories are as follows: A: Smlmax / Cmlmax < 1; B: 1 < Smlmax / Cmlmax < 3; C: 3 < Smlmax / Cmlmax < 10; D: 10 < Smlmax / Cmlmax < 30; E: 30 < Smlmax / Cmlmax.dRatio of the inhibitory constant iiiM. IC50) calculated from the quadratic fits! to the measured ATPase data points for SI subfragment of cardiac myosin-2 iiiM. CmIC) and SI subfragment of smooth muscle myosin-2 iiiM. SmIC). Labeled categories are as follows: A: CmIC / SmIC < 1; B: 1 < CmIC / SmIC < 3; C: 3 < CmIC / SmIC < 10; D: 10 < CmIC / SmIC < 30; E: 30 < CmIC / SmIC.eSpecificity (Spec, S) towards SI subfragment of smooth muscle myosin-2 over SI subfragment of cardiac myosin-2 calculated as follows: S = Sm50 / Cm50 * CmIC / SmIC; Labeled categories are as follows: A: S < 1; B:S < 10; D: 10 < S < 30; E: 30 < S. t Quadratic equation used to fit the measured ATPase data points: Relative ATPase activity = RO + Imax *((M+I+IC50)- sqrt(sqr(M+I+IC50)-4*M*I)) / (2*M); where RO: relative ATPase activity in the absence of inhibitor and in the presence of DMSO as a vehicle control, Imax: maximal inhibition, M: concentration of myosin, IC50: inhibitory constant corresponding to 50% inhibition, I: inhibitor concentration.Table 3. Ratio of ATPase inhibition parameters of R(+) enantiomers on smooth muscle and fast skeletal myosin 2aInitial slope (% / pM, INI) of the quadratic fits! to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (% / pM, SmINI) and SI subfragment of fast skeletal muscle myosin-2 (% / pM, SkINI) calculated from the maximal inhibition (%, Imax) and the inhibitory constant (pM, IC50) based on the following formula: INI = Imax / IC50. Labeled categories are as follows: A: INI < 1 % / pM; B: 1 % / pM < INI < 3 % / pM; C: 3 % / pM < INI < 10 % / pM; D: 10 % / pM < INI < 30 % / pM; E: 30 % / pM < INI.bRatio of the measured ATPase inhibitions at 50 11M inhibitor concentration on SI subfragment of smooth muscle myosin-2 (%, Sm50) and on SI subfragment of fast skeletal muscle myosin-2 (%, Sk50). Labeled categories are as follows: A: Sm50 / Sk50 < 1; B: 1 < Sm50 / Sk50 < 3; C: 3 < Sm50 / Sk50 < 10; D: 10 < Sm50 / Sk50 < 30; E: 30 <Sm50 / Sk50.cRatio of the maximal inhibition (%, Imax) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (%, Smlmax) and SI subfragment of fast skeletal muscle myosin-2 (%, Skimax). Labeled categories are as follows: A: Smlmax / Sklmax < 1; B: 1 < Smlmax / Sklmax < 3; C: 3 < Smlmax / Sklmax < 10; D: 10 < Smlmax / Sklmax < 30; E: 30 < Smlmax / Sklmax.dRatio of the inhibitory constant iiiM. IC50) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of fast skeletal muscle myosin-2 iiiM. SkIC) and SI subfragment of smooth muscle myosin-2 iiiM. SmIC). Labeled categories are as follows: A: SkIC / SmIC < 1; B: 1 < SkIC / SmIC < 3; C: 3 < SkIC / SmIC < 10; D: 10 < SkIC / SmIC < 30; E: 30 < SkIC / SmIC.eSpecificity (Spec, S) towards SI subfragment of smooth muscle myosin-2 over SI subfragment of fast skeletal muscle myosin-2 calculated as follows: S = Sm50 / Sk50 * SkIC / SmIC; Labeled categories are as follows: A: S < 1; B: 1 < S < 3; C: 3 < S < 10; D: 10 < S < 30; E: 30 < S. t Quadratic equation used to fit the measured ATPase data points: Relative ATPase activity = RO + Imax *((M+I+IC50)- sqrt(sqr(M+I+IC50)-4*M*I)) / (2*M); where RO: relative ATPase activity in the absence of inhibitor and in the presence of DMSO as a vehicle control, Imax: maximal inhibition, M: concentration of myosin, IC50: inhibitory constant corresponding to 50% inhibition, I: inhibitor concentration.Table 4. Ratio of ATPase inhibition parameters of S(-) enantiomers on smooth muscle and fast skeletal myosin 2aInitial slope (% / pM, INI) of the quadratic fits* to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (% / pM, SmINI) and SI subfragment of fast skeletal muscle myosin-2 (% / pM, SkINI) calculated fromthe maximal inhibition (%, Imax) and the inhibitory constant (pM, IC50) based on the following formula: INI = Imax / IC50. Labeled categories are as follows: A: INI < 1 % / pM; B: 1 % / pM < INI < 3 % / pM; C: 3 % / pM < INI < 10 % / pM; D: 10 % / pM < INI < 30 % / pM; E: 30 % / pM < INI.bRatio of the measured ATPase inhibitions at 50 11M inhibitor concentration on SI subfragment of smooth muscle myosin-2 (%, Sm50) and on SI subfragment of fast skeletal muscle myosin-2 (%, Sk50). Labeled categories are as follows: A: Sm50 / Sk50 < 1; B: 1 < Sm50 / Sk50 < 3; C: 3 < Sm50 / Sk50 < 10; D: 10 < Sm50 / Sk50 < 30; E: 30 <Sm50 / Sk50.cRatio of the maximal inhibition (%, Imax) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of smooth muscle myosin-2 (%, Smlmax) and SI subfragment of fast skeletal muscle myosin-2 (%, Skimax). Labeled categories are as follows: A: Smlmax / Sklmax < 1; B: 1 < Smlmax / Sklmax < 3; C: 3 < Smlmax / Sklmax < 10; D: 10 < Smlmax / Sklmax < 30; E: 30 < Smlmax / Sklmax.dRatio of the inhibitory constant (TiM. IC50) calculated from the quadratic fits / to the measured ATPase data points for SI subfragment of fast skeletal muscle myosin-2 (pM, SkIC) and SI subfragment of smooth muscle myosin-2 (pM, SmIC). Labeled categories are as follows: A: SkIC / SmIC < 1; B: 1 < SkIC / SmIC < 3; C: 3 < SkIC / SmIC < 10; D: 10 < SkIC / SmIC < 30; E: 30 < SkIC / SmIC.eSpecificity (Spec, S) towards SI subfragment of smooth muscle myosin-2 over SI subfragment of fast skeletal muscle myosin-2 calculated as follows: S = Sm50 / Sk50 * SkIC / SmIC; Labeled categories are as follows: A: S < 1; B: 1 < S < 3; C: 3 < S < 10; D: 10 < S < 30; E: 30 < S. t Quadratic equation used to fit the measured ATPase data points: Relative ATPase activity = R0 + Imax *((M+I+IC50)- sqrt(sqr(M+I+IC50)-4*M*I)) / (2*M); where R0: relative ATPase activity in the absence of inhibitor and in the presence of DMSO as a vehicle control, Imax: maximal inhibition, M: concentration of myosin, IC50: inhibitory constant corresponding to 50% inhibition, I: inhibitor concentration.Table 5. Ratio of smooth muscle myosin specificity of R(+) enantiomers over the smooth muscle myosin specificity of S(-)aRatio of the smooth muscle myosin specificity (Spec) of the R(+) enantiomer (SpecR) over the smooth muscle specificity of the S(-) enantiomer (SpecS) on cardiac myosin calculated as in Tables 1-2 as follows: Spec = Sm50 / Cm50 * CmIC / SmIC, where Sm50: measured ATPase inhibitions at 50 pM inhibitor concentration on SI subfragment of smooth muscle myosin-2, Cm50: measured ATPase inhibitions at 50 pM inhibitor concentration on SI subfragment of cardiac myosin-2, CmIC: inhibitory constant (pM, IC50) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of cardiac myosin-2, SmIC: inhibitory constant (pM, IC50) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of smooth muscle myosin-2; Labeled categories are as follows: A: SpecR / SpecS < 1; B: 1 < SpecR / SpecS < 3; C: 3 < SpecR / SpecS < 10; D: 10 < SpecR / SpecS < 30; E: 30 < SpecR / SpecS.bRatio of the smooth muscle myosin specificity (Spec) of the R(+) enantiomer (SpecR) over the smooth muscle specificity of the S(-) enantiomer (SpecS) on fast skeletal muscle myosin calculated as in Tables 3-4 as follows: Spec = Sm50 / Sk50 * SkIC / SmIC, where Sm50: measured ATPase inhibitions at 50 pM inhibitor concentration on SI subfragment of smooth muscle myosin-2, Sk50: measured ATPase inhibitions at 50 pM inhibitor concentration on SI subfragment of fast skeletal muscle myosin-2, SkIC: inhibitory constant (pM, IC50) calculated from the quadratic fits! to the measured ATPase data points for SI subfragment of fast skeletal muscle myosin-2, SmIC: inhibitory constant (pM, IC50) calculated from the quadratic fitst to the measured ATPase data points for SI subfragment of smooth muscle myosin-2; Labeled categories are as follows: A: SpecR / SpecS < 1; B: 1 < SpecR / SpecS < 3; C: 3 < SpecR / SpecS < 10; D: 10 < SpecR / SpecS < 30; E: 30 < SpecR / SpecS. t Quadratic equation used to fit the measured ATPase data points: Relative ATPase activity = R0 + Imax *((M+I+IC50)- sqrt(sqr(M+I+IC50)-4*M*I)) / (2*M); where R0: relative ATPase activity in the absence of inhibitor and in the presence of DMSO as a vehicle control, Imax: maximal inhibition, M: concentration of myosin, IC50: inhibitory constant corresponding to 50% inhibition, I: inhibitor concentration.Example 2. Pharmacokinetics and tissue distribution

[0077] Pharmacokinetics (PK) of compound 2501 in rat blood plasma was determined in 12 male Wistar rats (weights between 220 g - 270 g) treated intravenously (i.v.) with 12 mg / kg compound 2501 dissolved in 30% 2-hydroxypropyl-P-cyclodexrin (HPBCD, Cyclolab, Hungary) in Dulbecco's phosphate buffered saline without calcium and magnesium (DPBS, Lonza). The animals were divided into 4 cohorts (3 rats in each cohort), and blood samples were obtained twice from each animal. Blood samples were collected at 0.5, 1, 2, 5, 15, 45, 90 and 180 min after the i.v. injection as follows: Cohort 1 :0.5 min and 180 min; Cohort 2: 1 min and 90 min; Cohort 3: 2 and 45 min; Cohort 4: 5 and 15 min. Blood samples were centrifuged at 12,300xg for 10 min at 4°C. 100 pl of the supernatant was mixed with 300 pl acetonitrile (AcN, HiPerSolv CHROMANORM® for LC-MS, VWR Chemicals) containing 267 ng / ml / / ra-nitroblebbistatin (pNbleb) as an internal standard (IS), and centrifuged at 270,000xg for 5 min at 4°C. 50 pl of the supernatant was transferred to HPLC vials (ND9, Short thread vial, VWR) for injection.

[0078] Compound 2501 concentration in rat tissues was determined in male Wistar rats (weights between 220 g - 270 g) treated with 5 mg / kg (n=5), 10 mg / kg (n=2), 40 mg / kg (n=2), compound 2501 i.v. 120 min after i.v. injection, the animals were sacrificed and samples from the heart, kidney, liver, spleen, lung, esophagus, m. vastus lateralis, m. gastrocnemius, m. latissimus dor si, m. biceps brachii, m. triceps brachii, m. abdominalis, diaphragm, trachea, small intestine, colon, stomach and brain were collected. Each sample (sample weights between 0.7-1.5 g) was weighed (with 0.01 g accuracy) and stored in a - 20°C freezer until further processing. Frozen tissue samples were thawed in 2 ml DPBS (Lonza) and cut into smaller pieces using surgical scissors. The samples were further homogenized to a gel-like consistency using a bladed homogenizer and mixed with 2 ml chloroform (HiPerSolv CHROMANORM® for HPLC, VWR Chemicals). The samples were thoroughly vortexed, sonicated for a further 10 min and centrifuged at 12,300xg for 10 min at 4°C. The aqueous phase was discarded, the organic phase containing compound 2501 was collected and the tissues in the centrifuge tubes were mixed with a further 2 ml of chloroform and the extraction process was repeated two more times. Chloroform was evaporated from the samples overnight under a chemical hood at room temperature. To remove any remaining fat and organic contaminants, the samples were redissolved in 100% AcN, centrifuged at 12,300xg for 10 min and the supernatant was saved to a clean tube (Eppendorf 5424 R). This procedure was repeated 2 more times. AcN was evaporated overnight under a chemical hood at room temperature. Dried and purified compound 2501 was dissolved in 50 pl DMSO (Molecular Biology Grade, D8418, Sigma-Aldrich, Merck), centrifuged at 270,000xg for 5 min at room temperature, and 2.5 pl of each sample was mixed into a 3 :1 AcN: water mixture containing 267 ng / ml pNbleb as internal standard, and transferred to HPLC vials.

[0079] The compound 2501 content of the plasma and tissue samples was analyzed using a Waters Acquity uHPLC-MS system (Acquity Arc System HPLC equipped with anAcquity QDa mass detector (Waters Co.)) with a Cl 8 reversed-phase column (CORTECS® Cl 8, 2.7 pm, 4.6 mm X 50 mm). The mobile phase of the system was a mixture of AcN and water (HiPerSolv CHROMANORM® for HPLC, VWR Chemicals), supplemented with 0.5% formic acid (FA, 99.0+%, Optima™ LC / MS Grade, Fisher Chemical). A gradient elution was used with an initial AcN: water ratio of 5%-100% in 4.5 min, plateaued for 0.1 min and re-equilibrated to the original ratio for 1.4 min. Ionization of the analyte was achieved by electrospray, producing positively charged ions. Single ion recording (SIR) was used to monitor the following masses: compound 2501 at 341 Da, pNbleb (IS) at 337 Da. For quantitative determination of the concentration of compound 2501 in plasma, a compound 2501 concentration standard was also analyzed in parallel with the unknown analytes. The standard consisted of the following concentrations of compound 2501 : 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 250 ng / ml, 500 ng / ml, 750 ng / ml, 1500 ng / ml and 3000 ng / ml compound 2501. The compound 2501 standards were dissolved in DMSO and 2.5 pl were added to 97.5 pl of untreated plasma and then mixed with 300 pl AcN containing the 267 ng / ml pNbleb as internal standard. The Area Under the Curve (AUC) of the standard points were divided by the AUC of the corresponding IS peaks and plotted as a function of the nominal concentrations. Linear fitting with 1 / x2weighting was used to find the equation of the fitted line, which was used to determine the exact concentration of compound 2501 in the plasma samples.

[0080] All chromatographic data are collected and analyzed by the MassLynx 4.2 software. The peak areas of analyte are calculated by the computing software and its ratio to the area of IS was calculated by MS Excel. The calibration curves are constructed on the basis of the response ratio of analyte to the internal standard versus the ratio of the concentration of analyte to the concentration of the internal standard. The weighting type is set to 1 / x2. Slope, y-intercept and correlation coefficient was calculated by Graphpad Prism® software. The equations of the calibration curves are then used to calculate the back-calculated concentrations for each calibrator, as well as the concentration of the analyte in the test samples from their measured peak area ratios. The accuracy of calibrators was also calculated as follows: Accuracy %= (measured cone. -nominal conc.) / nominal cone. *100. Measurements were accepted if deviation from the nominal concentration at each calibrator concentration was below 10%.

[0081] Results: half-life of compound 2501 in the plasma of healthy rats was found to be 5-20 min (Fig 1 A). Tissue distribution of compound 2501 is shown on Fig IB.Example 3. Ex vivo efficacy

[0082] Brain and respiratory system was quickly removed from over-anesthetized male Wistar rats (weights between 220 g - 270 g) and immersed in cold Krebs solution (NaCl: 119 mM, KC1: 4.7 mM, KH2PO4: 1.2 mM, NaHCCh: 25 mM, Mg2SO4: 1.2 mM, CaChx2 H2O: 1.6 mM, EDTA: 0.026 mM , and glucose: 11.1 mM). Basilar arteries were isolated from the brain. Bronchi, and trachea were isolated from the respiratory system. After isolation of basilar arteries, bronchi, and trachea, connective tissue was removed, and the tissue samples were cut into cylindrical segments of 2 mm in length. A four- chamber wire myograph system (DMT 610M, Danish Myo Technology A / S, Aarhus, Denmark) was used for the study. Tissue segments were mounted to the micrometer and then to the transducer part of the myograph using 2 cm long 15 pm diameter Tungsten stainless steel wires. The transducer was connected to an analogue-to-digital converter unit (ADInstruments, Hastings, UK) and the isometric force developed by the mounted tissues was recorded. Each chamber of the myograph was filled with 5 ml of prewarmed Krebs solution and continuously perfused with a mixture of 95%:5% oxygemcarbon dioxide. The chamber temperature was kept constant at 37.0 ± 0.1 °C throughout the procedure. Before starting each measurement, normalization procedure was carried out according to the manufacturer's instructions using a commercial software (LabChart Pro-DMT Normalization module, AD Instruments, Hastings, UK). After normalization, tissues were allowed to stabilize for 30-60 min before starting the experimental protocol. For measuring the vasodilatory effect of the drug, tissue samples were preincubated with 60 mM KC1 solution until the maximum contraction was reached. Compounds were added as a cumulative dose to the 5 ml volume of the organ bath.

[0083] Results: compounds induced 20-100% relaxation within 2-20 min in trachea (Fig 2A), bronchi (Fig 2B) as well as in arteries (Fig 2C). Fig 2D presents the relaxation half-life measured in the basilar, femoral and common carotid arteries at 40 pM of compound 2501.Example 4. In vivo efficacy - survival and renal function

[0084] Male Wistar rats (weights between 250 g - 300 g) underwent surgery under general inhalation anesthesia 4% isoflurane mixed with medical oxygen (1 L / min) in an isoflurane vaporizer, and then the animals were kept under 2% isoflurane inhalation anesthesia until the end of surgery. Body temperature of the rats was kept at 37°C with an adjustable heating pad. The upper retroperitoneal region of the abdomen was shaved, and a 1-cm incision was made. The left or both kidneys were localized and the soft tissues surrounding the kidneys were carefully removed. Renal ischemia was induced by bilateral cross-clamping of both renal pedicles with a vascular clamp for 90 min.

[0085] After vascular clamp removal, rats were untreated or intravenously treated with i.v. injection of either 30% 2-hydroxypropyl-P-cyclodextrin (HPBCD, Cyclolab, Hungary) dissolved in DPBS as vehicle control, compound 2501 (2-20 mg / kg) dissolved in 30% HPBCD in DPBS, or 10 mg / kg compound 2501 supplemented with 100 mg / kg vitamin C, 100 mg / kg Allopurinol or 10 mg / kg Rimadyl, also dissolved in 30% HPBCD in DPBS.

[0086] Serum carbamide and creatinine levels were determined from male Wistar rats undergone bilateral renal artery occlusion several times over a 9-day period. Briefly, anesthesia was performed as described above which was followed by blood sampling from the tail vein of the animals using a 25Gx5 / 8 needle (Braun Sterican 0.5x16 mm BL / LB) and a serum separator blood collection tube (BD Vacutainer SST II Advance). Blood samples were centrifuged at 3,000xg for 4 min to separate the serum, which was then collected in new Eppendorf tubes. The separated sera were analyzed by a diagnostic center for determination of serum carbamide and creatinine levels. Kidney function expressed as glomerular filtration rate (eGFR) was calculated from the serum carbamide and creatinine levels using the following formulas: eGFR = 5862*((rat weight(g))A0.695)*((concentration of creatinine (pmol / L))A(-1.15))*((concentration of carbamide (mmol / L)A(-0.391)) if concentration of creatinine was equal to or more than 52 pmol / L, and eGFR = 880*((rat weight (g))A0.695)*((concentration of creatinine (pmol / L))A(- 0.66))*((concentration of carbamide (mmol / L)A(-0.391)) if concentration of creatinine was less than 52 pmol / L.

[0087] Results: Effect of compounds on survival and renal function are illustrated on Fig 3 A, B and C, respectively.Example 5. In vivo efficacy - renal ischemia

[0088] RBC labeling: Blood was obtained from the hearts of intact anaesthetized male Wistar Rats using a heparinized sterile syringe (2 ml blood / rat). Blood samples were centrifuged for 7 min at 2,000xg at room temperature, supernatant plasma was removed. Remaining red blood cells were washed with DPBS (Lonza) solution containing 1 g / L EDTA (pH 7.0) and centrifuged for 4 min at 2,000xg at room temperature. After centrifugation, the supernatant was removed and cells were suspended and incubated for 2 hours at room temperature in DPBS solution containing 1 g / L EDTA (pH 7.0) supplemented with 10 mg / ml fluorescein isothiocyanate (FITC ‘Isomer F, Invitrogen, F1097) previously dissolved in 1 g / L EDTA-containing DPBS solution (pH 8.0). To remove unbound FITC, samples were washed four times with and resuspended in 0.6 ml 1 g / L EDTA-containing DPBS solution (pH 7.0). Samples were pooled.

[0089] Surgery was performed as in Example 4, except that renal ischemia was induced by unilateral cross-clamping of the left renal pedicle with a vascular clamp for 10, 30, 60 or 120 min and i.v. treatment (dose of 2-20 mg / kg of compound 2501 dissolved in either 30% HPBCD dissolved in DPBS, or DPBS, or the vehicle only) was administered 30 min after removing the clamp. For quantification / visualization of capillary blood flow, left kidney was elevated through the 1 cm incision, fixed to avoid movement artefacts. Rats were i.v. injected with 1.0 ml FITC-labelled red blood cell suspension, and labeled red blood cells were visualized at an excitation wavelength of 900 nm. Blood flow parameters, i.e. red blood cell velocities and red blood cell flux (defined as the number of red blood cells passing through the cross-section of a vessel during a given time), were determined from kymograms created by line scanning (scanning frequency of 500 Hz for 10 seconds) parallel or perpendicular to the direction of blood flow, respectively.Kymograms were recorded before and up to 50 min after treatment.

[0090] Kymograms were analyzed using FIJI software. . Velocities of the individual RBCs were determined by dividing the distance travelled by the time. Red blood cell flux was expressed as the number of red blood cells passing through the scanning area divided by the time elapsed during the acquisition of a trace (10 seconds).Red blood cell flux values were corrected for the labelling efficiency: At the end of the microscopic observations, 1-5 pl of blood was collected from the tail vein of the animals using a 27Gxl / 2 needle and the red blood cell labelling ratio was determined by counting the labeled and unlabeled RBCs under a fluorescence microscope.

[0091] Results: Effect of compound 2501 on capillary dilation is presented as a change in the flux (Fig 4A, C, E) and average velocity (Fig 4B, D, F) of red blood cells.Example 6. In vivo efficacy - Ischemic heart failure

[0092] Male Wistar rats underwent surgery under general anesthesia by inhalation of 4% isoflurane mixed with medical oxygen (1 L / min) in an isoflurane vaporizer, then the animals were maintained under 2% isoflurane inhalation anesthesia until the end of surgery. The animals' body temperature was maintained at 37°C with an adjustable heating pad. When the animals stopped to respond to mechanical stimuli, the neck and chest region was depilated and both jugular veins were exposed, followed by tracheal intubation and mechanical ventilation (VentStar, RWD). The heart was exposed via thoracotomy and a 6-0 suture (Braun Silkam C0760021) was placed around the left anterior descending coronary artery (LAD) ~2 mm from the origin for 30 min. Successful myocardial infarction by LAD occlusion was associated with pale myocardial tissue at risk and ST- segment elevation in the ECG signal. Reperfusion was initiated by removing the suture after 30 min of LAD occlusion. During the reperfusion phase, the animals were placed under a 2-photon microscope to measure the capillary diameter in the area affected by the LAD occlusion. Before visualization, 25 mg / ml fluorescein isothiocyanate (FITC) dissolved in PBS solution was injected intravenously via the right jugular vein. Animals received a bolus treatment of 0 or 10 mg / kg compound 2501 in 30% HPBCD in DPBS, followed by a 1-hour infusion treatment (2 mg / ml compound 2501_with 1 ml / hour flow rate) or vehicle only via the left jugular vein. Changes in capillary diameter were monitored during and for 1 hour after infusion. At the end of 2 hours of reperfusion, animals were sacrificed, hearts were excised and placed at -20°C. In a separate set of experiments, animals received a bolus treatment of 0, 2, 5, 10, or 20 mg / kg compound 2501, with no subsequent infusion.

[0093] When the hearts were semi-frozen, they were sliced into four equal thickness slices. The slices were then incubated in 1% 2,3,5-triphenyltetrazolium chloride(TTC) solution for 15 min at 37°C and fixed in 10% formaldehyde for 1 hour. Viable myocardium was stained red, while the infarcted area appeared pale white. After formalin fixation, the heart slices were carefully flattened so that they could be photographed to measure the infarct area in each slice.

[0094] Image analysis of TTC-stained hearts was performed using Fiji image analysis software. The background of the images was removed together with the TTC negative pericardial layer. To obtain the highest contrast difference between the TTC positive and TTC negative areas of the heart sections for further study, data were extracted from the green channel of the images. The pixels of the images were grouped into three intensity fractions: low, medium and high intensity. The areas of the high intensity pixel fraction, corresponding to TTC negative necrotic areas were quantified. The relative ischemic areas were determined by dividing the areas of the TTC negative area by the total area of the slice. TTC negative area in the context of the whole ventricular part was calculated from the weighted averages of the slices.

[0095] Results: Effect of LADO and subsequent administration of compound 2501 on heart function (ECG, Fig 5 A), heart capillary diameter (Fig 5B), area of ischemic tissue (Fig 5C, E) and survival time of the animals (Fig 5D) is presented.Example 7. In vivo efficacy - effect on LAD blood flow

[0096] Left anterior ascending artery (LAD) blood flow was measured with coronary flow probes in open-heart landrace pig. After stable LAD blood flow baseline rate was achieved, catheter-guided intracoronary bolus injection (20 ml in 10 seconds) of 30% hydroxypropyl-P-cyclodextrin (HPBCD) dissolved in DPBS (vehicle control), was followed by 20 ml bolus injection of 5 mM, 12.5 mM, and 25 mM MPH-2501 in 30% HPBCD, corresponding to 0.6 mg / kg, 1.5 mg / kg and 2.9 mg / kg dose levels. Similarly, 3.54 pg / kg nitroglycerine was also administered for comparison. LAD blood flow as well as arterial blood pressure was continuously monitored, and each bolus injection was introduced after a stable baseline level of LAD blood flow was restored.

[0097] Results: Effect of intracoronary bolus administration of different doses of compound 2501, 3.54 pg / kg nitroglycerine, or 30% HPBCD on LAD blood flow and arterial pressure (Figs 6, 7, and 8) is presented. Compound 2501 dose-dependently increased LAD blood flow, while preserving autoregulation, as opposed to nitroglycerin.Example 8. Smooth muscle myosin binding

[0098] Azidoblebbistatin is a photoactivatable probe, which generates highly reactive products that can covalently modify its binding partners (DOI:10.1073 / pnas.1202786109). 30 pl pure DMSO (control sample) or DMSO with 16.74 mM (+) azidoblebbistatin (treated sample) was added to 824 pl 22.1 pM SmA (human smooth muscle myosin) and 2.9 mM ATP in buffer (5 mM MOPS, 2mM MgCh, 40 mM NaCl, pH 7.0) and the samples were irradiated using a Hg-Xe lamp (Hamamatsu L2482) at 312 nm under argon atmosphere for 15 min. The addition of 30 pl DMSO with or without 16.74 mM (+) azidoblebbistatin followed by 15 min irradiation was repeated two more times. Absorbance spectrum of 40 ul samples was checked at each stage. After the third photocrosslinking cycle, 200 ul acetonitrile was added to each sample to precipitate the proteins. The solution was centrifuged and the supernatant was removed. The pellet was washed 5 more times with 200 ul acetonitrile, dried and stored at -80 °C. The color of the treated pellet was yellow, while the control was white.

[0099] The dried samples were reconstituted in 260 pl water, lOOpl 4x LDS buffer and 40pl of reducing agent. Samples were heated at 70° for 10 min and then cooled and ran on a 10% NuPAGE Bis-Tris gel with PAGE Prestained Protein Markers. The gel was run on a ThermoFisher Scientific Mini Gel apparatus for 25 min with MES running buffer at a constant voltage of 200V. Gels were then fixed for 10 min with 50% methanol, 40% water and 10% acetic acid. The gel was then stained with colloidal coomassie stain for three hours, then destained with water for 2 hours. The region of the gel that corresponds to the molecular weight of myosin 11 (90-120 kDa) was excised. The excised gel segment was placed in a 1.5 ml Eppendorf tube and the protein was reduced with 45 mM DTT for 30 min at 55 °C and the cysteinyl residues were carbamidom ethylated for 30 min with 100 mM iodoacetamide at room temperature in the dark. The gels were destained and digested overnight with trypsin or GluC at 37 °C. Peptides were extracted with 60% acetonitrile, 0.1% trifluoroacetic acid, the extracts were evaporated under vacuum and then reconstituted in 0.1% formic acid for LC-MS / MS analysis.

[0100] Peptides were resolved on an Aurora Cl 8 analytical column (75 pm x 15 cm, 1.7 pm particle size) (lonOpticks) coupled to a Dionex Ultimate 3000 nanoLC and autosampler. The mobile phase solvents consisted of 0.1% formic acid, 99.9% water (solvent A) and 0.1% formic acid, and 99.9% acetonitrile (solvent B). The peptides weregradient-eluted at a flow rate of 300 nl min1using a 70 min gradient. The Exploris 480 Orbitrap mass spectrometer (Thermo Scientific) equipped with a nano-electrospray ionization source, was used to analyze the eluted peptides. MS / MS spectra were collected using a data-dependent method, with MS 1 spectra acquired at an MS AGC target value of 300%, followed by MS / MS scans of the most abundant ions detected in the preceding MS scan. A maximum MS / MS ion time of 22 ms was used with a MS2 AGC target of 100%. Dynamic exclusion was set to 45 s and HCD collision energy was set to 28%.

[0101] Identification of peptide sequences from MS / MS spectra was done by database search using Thermo Proteome Discoverer 2.5.400. The data was searched against a Homo sapiens database created from the UniprotKB protein database. Data for the peptides was compared on the basis of spectral counts for each peptide-spectrum match to the MYH11 protein.

[0102] A single residue (586Y) was found to be labelled by (+) azidoblebbistatin, which resides in the relay-SHl drug binding cleft, shown on Fig 9.Example 9. Enantiomer ID

[0103] Structure of compound 2501 crystallized in cyclohexane and trace amount of THF was determined from single crystal X-ray diffraction pattern. Crystal structure confirms stereochemistry (Fig 10).Example 10. Synthesis of (+)-(4S)-7,7-dimethoxy-l 1,1 l-dimethyl-5-oxa-3X6-thia-4- azatetracyclo[6.2.1.01,6.©4,6]undecane-3,3-dione

[0104] Step a) Synthesis of (15)-{7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-l-yl}- methanesulfonamide (Compound 10a).600 g (2.58 mol, 1.0 eq.) (15)-{7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-l-yl}- methanesulfonic acid was suspended in 3.8 L chloroform (3.8 L), heated to reflux and 226 ml (3.1 mol, 1.2 eq.) of freshly distilled thionyl chloride was added dropwise over 1 hour. Heating was continued until gas evolution (sulfur dioxide and hydrogen chloride) had ceased (approximately 9-10 hr.). The resultant solution was added dropwise over a period of 1 hour at 0-10 °C to 8.3 L 25% ammonium hydroxide solution (pre-chilled to 0 °C in an ice bath), the reaction mixture was warmed to room temperature, stirred for 4 hours, the organic layer separated, and the aqueous layer extracted with 3X 1.3 L of DCM. The combined organic layers were washed with 1.5 L of water and brine, dried over Na2SO4, then the solvent was evaporated under vacuum. The whole procedure was performed in 3 repeats total. Average yield: 550 g (2.38 mol, 92%) white solid (Compound 10a).'H-NMR 5 (400 MHz CDCh T = 300K): 3.19 (d, J = 12.0 Hz, 1H), 2.99 (d J = 12.0 Hz, 1H), 2.78 (dqa, J = 20.0 Hz and 3.5 Hz, 1H), 2.39 (d, J = 20.0 Hz, 1H), 2.27 (br m, 1H), 2.12-2.05 (overlapping m’s, 2H), 1.77 (broad m, 1H), 1.48 (broad m, 1H), 1.09 (s, 3H), 0.87 (s, 3H).

[0105] Step b) Synthesis of (15)- 10,10-dimethyl-3X6-thia-4-azatricyclo[5.2.1.01,5]- dec-4-ene-3,3-dione (Compound 10b).825 g (3.57 mol, 1.0 eq.) crude Compound 10a and 100 g Amberlyst 15 ion exchange resin were suspended in 10 L toluene. The reaction mixture was heated at reflux for 4h, distilling the water formed during the reaction (water distillation stopped after 3 hours). The still warm solution was filtered, the filter funnel washed with 1 L dichloromethane, and the solvent evaporated. The crude product was purified by re-crystallization from 32 L absolute EtOH. The whole procedure was performed 2 times total. Average yield: 605 g (2.84 mol, 80%) white crystals (Compound 10b). ESI-MS (M+H+) = 213.9.

[0106] Step c) Synthesis of (15)-10,10-dimethyl-3X6-thia-4-azatricyclo[5.2.1.01,5]- dec-4-ene-3, 3, 6-trione (Compound 10c).1210 g (5.68 mol, 1.0 eq.) Compound 10b was dissolved in 4.3 L acetic acid, 950 g (8.56 mol, 1.5 eq.) SeCh was added, and the mixture stirred at reflux for 8 hours. The reaction mixture was poured onto 21.5 L ice, the resultant precipitate was filtered, washed with water and dissolved in 5 L dichloromethane. The solution was washed with water, dried on Na2SO4 and the solvent was evaporated. Yield: 865 g (3.81 mol, 67%) yellow solid (Compound 10c). ESI-MS (M+H+) = 228.1.

[0107] Step d) Synthesis of (l)-6,6-dimethoxy-10,10-dimethyl-3X6-thia-4- azatricyclo[5.2. l.O’ dec-d-ene-S, 3-dione (Compound lOd).865 g (3.81 mol, 1.0 eq.) Compound 10c was dissolved in a mixture of 6.3 L trimethyl orthoformate, 2.1 L methanol and 210 ml (3.91 mol, 1.05 eq.) concentrated sulfuric acid, then stirred at reflux for 5 hours. The reaction mixture was cooled to room temperature, and the resultant precipitate was filtered and washed with cool methanol. Yield: 990 g (3.62 mol, 95%) white solid (Compound lOd). ESI-MS (M+H+) = 273.9.

[0108] Step e) Synthesis of (45)-7,7-dimethoxy-l l,l l-dimethyl-5-oxa-3X6-thia-4- azatetracyclofd^. l.O1,6.©4,6]undecane-3,3-dione (Compound lOe).990 g (3.62 mol, 1.0 eq.) Compound lOd was dissolved in 7.8 L dichloromethane with 110 ml aliquot 336. Then 1650 ml (21 mol, 5.8 eq.) of a 30% solution of H2O2 and 1650 g (11.9 mol, 3.3 eq.) of K2CO3 were added in three portions alternately. The mixture was warmed to 40 °C and stirred vigorously for 8 hours, then cooled to room temperature and the phases separated. To the organic layer 55 ml aliquot 336 was added, then again 1650 ml of 30% H2O2 solution and 1650 g K2CO3 were added in three portions, alternately. Again, this reaction mixture was warmed to 40 °C then stirred vigorously for 8 hours. It was cooled down to room temperature and the phases separated. Again, 55 ml aliquot 336 was added to the organic layer, followed by 1650 ml 30% H2O2 solution and 1650 g K2CO3 added in three portions, alternately. The solution was warmed to 40 °C and stirred vigorously for 8 hours. The mixture was cooled down to room temperature, the phases separated and the organic phase washed with 2 L saturated Na2SO3 solution and 2 L water, then dried on Na2SO4 and the solvent evaporated. The crude product was crystallized from MeOH. Yield: 1000 g (3.46 mol, 96%) white solid (Compound lOe, R-Davis reagent).'H-NMR 5 (400 MHz CDCI3 T = 300K): 3.27 (s, 3H), 3.33 and 3.32 [overlapping d (J = 12.0 Hz), 1H and s, 3H], 3.07 (d, J = 12.0 Hz, 1H), 2.27 (m, 1H), 1.85-1.50 (overlapping m’s, 4H), 1.32 (s, 3H), 1.05 (s, 3H).Example 11 / 1. Synthesis of (9A)-12-[4-(dimethylamino)phenyl]-9-hydroxy-5-methyl-4- thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (compound 2501)

[0109] Step a) Preparation of methyl 2-amino-5-methylthiophene-3-carboxylate (Compound I la).169 ml (1.91 mol, 1.0 eq.) methyl cyanoacetate was dissolved in a mixture of 144 ml tetraethylammonium and 240 ml dimethylformamide, then 61.4 g (1.91 mol, 1.0 eq.) octasulfur was added. The reaction mixture was warmed to 60 °C, then 138 ml (1.91 mol, 1.0 eq.) propionaldehyde was added dropwise through the course of 1 hour. The reaction mixture was allowed to cool down to room temperature, then it was stirred for 12 hours. The reaction mixture was poured into 1.5 L water, and the formed precipitate filtered and washed with water and methanol. The crude product was purified by recrystallization from a 1 : 1 mixture of toluene and isopropanol. Yield: 155 g (0.905 mol, 47.3%) yellow solid (Compound I la). ESI-MS (M+H+) = 171.82. 'H-NMR 5 (500 MHz DMSO-de T = 300K): 2.13 (p, J = 7.4 Hz, 2H), 2.57 (t, J = 8.0 Hz, 2H), 2.92 (s, 6H), 3.80 (t, J = 7.0 Hz, 2H), 6.74 (d, J = 9.1 Hz, 2H), 7.41 (d, J = 9.1 Hz, 2H).13C DEPTq NMR 5 (126 MHz DMSO- de T = 300K): 18.1, 32.4, 40.9, 49.4, 113.0, 121.9, 129.4, 148.1, 173.8.

[0110] Step b) Preparation of 4-chloro-N-[4-(dimethylamino)phenyl]butanamide (Compound 1 lb).30.0 g (220 mmol, 1.0 eq.) N,N-dimethyl-p-phenylenediamine was dissolved in 200 ml dichloromethane and 33.77 ml (242 mmol, 1.1 eq.) tetraethylammonium, cooled to 5 °C, then 27.11 ml (242 mmol, 1.1 eq.) 4-chlorobutyryl chloride was added dropwise and the reaction mixture was stirred for 72 hours at rt. The solution was washed with water, dried on Na2SO4 and the solvent was evaporated. The crude product was redissolved intodichloromethane, filtered through a silica pad and the dichloromethane evaporated. The pure product was obtained by crystallization from methyl tert-butyl ether. Yield: 33.75 g (140 mmol, 63.7%), white solid (Compound 11b).

[0111] Step c) Preparation of l-[4-(dimethylamino)phenyl]pyrrolidin-2-one (Compound 11c).33.75 g (140 mmol, 1.0 eq.) Compound 1 lb was dissolved in 300 ml tetrahydrofuran. The solution was cooled to 0 °C and 6.17 g (168 mmol, 1.2 eq.) NaH (60% in mineral oil) was added, then the reaction mixture was stirred for 12 hours at room temperature. The excess of NaH was neutralized by addition of MeOH, then the solvents were evaporated. The residue was dissolved in dichloromethane, washed with water and brine, dried on Na2SO4 and the solvent evaporated. The crude product was crystallized with toluene. Yield: 15.69 g (76.8 mmol, 54.8%), beige solid (Compound 11c). ESI-MS (M+H+) = 204.9.

[0112] Step d) Preparation of methyl 2-{[(2E)-l-[4-(dimethylamino)phenyl]- pyrrolidin-2-ylidene]amino}-5-methylthiophene-3-carboxylate (Compound l id).14.82 g (72.5 mmol, 1.0 eq.) Compound 11c was dissolved in 250 ml chloroform, then 7.10 ml (76.1 mmol, 1.05 eq.) POCh was added dropwise and the solution stirred for 2 hours at rt. 12.42 g (72.5 mmol, 1.0 eq.) Compound I la was added, and the reaction mixture stirred for 12 hours at reflux, cooled to room temperature and neutralized with solutions of Na2COs and NaHCCh. The solution was extracted, washed with water and brine, dried on Na2SO4 and the solvent evaporated. The crude product was filtered through a silica pad. Yield: 18.75 g (52.5 mmol, 72.4%) red oil (Compound l id). ESI-MS (M+H+) = 358.3.

[0113] Step e) Preparation of 12-[4-(dimethylamino)phenyl]-5-methyl-4-thia-2, 12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound l ie).18.73 g (52.4 mmol, 1.0 eq.) Compound l id was dissolved in 200 ml tetrahydrofuran, then 11.76 g (105 mmol, 2.0 eq.) potassium tert-butoxide was added and the reactionmixture stirred for 5 hours at reflux and 12 hours at room temperature. The solvent was evaporated, the residue redissolved into water and acidified with AcOH, then neutralized with solutions of Na2COs and NaHCCh. The resulting suspension was extracted with di chloromethane, then washed with water and brine, dried on Na2SO4 and the solvent evaporated. The crude product was crystallized with isopropanol and methyl tert-butyl ether. Yield: 6.07 g (18.6 mmol, 35.5%) ochre solid (Compound l ie).

[0114] Step f) Preparation of (9A)-12-[4-(dimethylamino)phenyl]-9-hydroxy-5- methyl-4-thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 2501).4.0 g (12.3 mmol, 1.0 eq.) Compound l ie was dissolved in 245 ml tetrahydrofuran, then the solution cooled to -20 °C. 14.7 ml (14.7 mmol, 1.2 eq.) 1 mol / L LiHMDS solution was added. The solution was incubated on room temperature, then cooled to -78 °C. Compound lOe (4.3 g, 14.7 mmol, 1.2 eq.) was added and the solution stirred at room temperature overnight. The solution was washed with a solution of NH4CI, extracted with EtOAc, dried on Na2SO4, filtered, washed with EtOAc and the solvent evaporated. The crude product was recrystallized twice by redissolving in warm ACN, filtering then crystallizing at rt. Yield: 0.34 g (1.0 mmol, 8.1%) orange solid (Compound 2501). ESI-MS (M+H+) = 342.20.Example 11 / H. Scaled-up synthesis of (9A)-12-[4-(dimethylamino)phenyl]-9-hydroxy-5- methyl-4-thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 2501)

[0115] Step a) Preparation of l-[4-(dimethylamino)phenyl]pyrrolidin-2-one (Compound 11c).A 30 L Biichi midiplot glass reactor was charged under inert atmosphere (N2) with 948.8 g (4.7 mol, l.Oeq.) 4- Bromo-N,N-dimethylaniline, 3834.2 g (45 mol, 9.5eq.) 2- pyrrolidinone, 180.63 g (0.95 mol, 0.2eq.) copper(I) iodide, 97.78 g (0.95 mol, 0.2eq.) N,N- dimethylglycine, and 1310.81 g (9.56 mol, 2.0eq.) potassium carbonate. Reaction mixture was heated to 125 °C (internal temperature) and stirred for 18 hours (Temperature of the jacket = 130°C). The reaction mixture was cooled to 70 °C, poured into 13 L ice water, and the resulting mixture was stirred for 1 hour. The formed precipitate was filtered, dissolved in 16 L di chloromethane, and washed with 10% ammonium hydroxide (2x 6.2 L, lx 3.2 L). The light brown organic layer was then washed with brine (2 L), dried over sodium sulfate (150g), filtered, and concentrated under vacuum. The crude product (beige solid) was triturated with ethyl acetate (1.25 L), stirred for Ihour at 50 °C, filtered, and dried. Yield: 853.0 g (4.2 mol, 89%) light beige solid (Compound 11c). GC- MS = 204. 'H-NMR 5 (500 MHz CDCh T = 300K): 7.42 (A part of an AA’XX’ spin system JAX = 9.2 Hz, 2H), 6.74 (X part of an AA’XX’ spin system JAX = 9.2 Hz, 2H), 3.81 (t, J =7.5 Hz, 2H), 2.92 (s, 6H), 2.57 (t, J =7.5 Hz, 2H), 2.13 (qi, J =7.5 Hz, 2H).

[0116] Step b) Preparation of methyl 2-{[(2E)-l-[4-(dimethylamino)phenyl]- pyrrolidin-2-ylidene]amino}-5-methylthiophene-3-carboxylate (Compound l id).A 30 L Biichi midiplot glass reactor was charged under inert atmosphere (N2) with 853 g (4.2 mol, 1.0 eq.) Compound 11c and 17 L / l L 1,4-Dioxane / di chloromethane at 20 °C, and 704 g (4.6 mol, 1.1 eq.) phosphorus oxychloride was added during 15 min. The resulting mixture was stirred at 20 °C for 3 hours, during which the beige slurry turned to a reddish brown, turbid solution. Then the mixture was heated to 80 °C (internal temperature, temperature of the jacket = 85 °C) and 1201 g (7 mol, 1.7 eq.) methyl 2- amino-5-methylthiophene-3-carboxylate was added in 4X 300 g portions over 1.5 hours. The mixture was incubated under reflux for 1 hour, then the heating was switched off and the mixture was stirred overnight slowly cooling down to 22 °C. A yellow precipitate was formed overnight. It was filtered and washed 2 times with ethyl acetate (2.4 L then 2 L, respectively). The solid was transferred into a 30 L Biichi midiplot glass reactor, dissolved in 8.5 L dichloromethane, and carefully quenched with 8.5 L sat. sodium carbonate solution. The aqueous phase was extracted with 2X 1.5 L di chloromethane. The pooled dark red organic phase was washed with brine (3.4 L), dried over sodium sulfate, filtered and concentrated under vacuum, yielding 1410 g crude product. The isolated red resin was then triturated with 1.9 L ethyl acetate, sonicated for 1 hour at 25°C, filtered, then washed with 1.7 L water and twice with ethanol (1.2 L and 0.6 L, respectively), then dried under vacuum. Yield: 514.7 g (1.44 mol, 34%) yellow solid (Compound l id). 'H-NMR 5 (500 MHz CDCh T = 300K): 7.59 (A part of an AA’XX’ spin system JAX = 9.2 Hz, 2H), 7.04 (s, 1H), 6.75 (X part of an AA’XX’ spin system JAX = 9.2 Hz, 2H), 3.86 (t, J =7.5 Hz, 2H), 3.77 (s, 3H), 2.92 (s, 6H), 3.77 (s, 3H), 2.74 (t, J =7.5 Hz, 2H), 2.11 (s, 3H), 2.09 (qi, J =7.5 Hz, 2H).

[0117] Step c) Preparation of 12-[4-(dimethylamino)phenyl]-5-methyl-4-thia-2, 12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound l ie).A 3-neck 6 L round bottom flask was charged with 250 g (0.7 mol, 1.0 eq.) Compound l id, 625 ml toluene, 2425 ml dry tetrahydrofuran, and 100 g activated 4 A molecular sieves. Separately, 156.95 g (5.6 mol, 2.0 eq.) potassium tert-butoxide was dissolved in 625 ml dry tetrahydrofuran and transferred into a dropping funnel. The reaction mixture was preheated to 90°C and the potassium tert-butoxide solution was added dropwise over the course of 1 hour. The resulting red-brown mixture was stirred at 90 °C for 4.5 hours, then the heating was switched off and the mixture was stirred overnight slowly cooling down to 22°C.1 L water was added, the turbid mixture was filtered in order to separate molecular sieves, then quenched with 240 ml (4.2 eq.) glacial acetic acid. The mixture was immediately concentrated under reduced pressure till p=140mbar. The pH of the resulting crude beige slurry was adjusted to pH = 10.0 by adding 2 L 20% sodium carbonate solution. The formed beige precipitate was filtered, washed with 2X 400 ml water, 2X 500 ml ethanol, and 500 ml ethyl acetate, then and dried under vacuum. Yield: 160g 80% pure Compound l ie (containing 20 m / m% acid of Compound l id). This material was dissolved in a mixture of 930 ml dichloromethane and 930 ml methanol, the di chloromethane was evaporated, and the precipitated Compound l ie was filtered then dried under vacuum. Traces of methanol were eliminated by corotation with 2X 250 ml tetrahydrofuran. Yield: 83.35 g (215 mmol, 31%, with 16 m / m% tetrahydrofuran) beige solid (Compound l ie).'H-NMR 5 (500 MHz DMSO-de T = 300K): 10.28 (s, 1H), 7.60 (d J = 9.2 Hz, 2H), 6.98 (s, 1H), 6.78 (d, J= 9.2 Hz, 2H), 3.97 (t, J = 8.5 Hz, 2H), 3.05 (t, J = 8.5 Hz, 2H), 2.84 (s, 6H), 2.44 (s, 3H).

[0118] Step d) Preparation of (9R)-12-[4-(dimethylamino)phenyl]-9-hydroxy-5- methyl-4-thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 2501).A 3-neck 2L round bottom flask was charged under inert atmosphere (N2) with 47.62 g Compound l ie (84% pure, 40g = 123 mmol, 1.0 eq.) and 900 ml dry tetrahydrofuran. The suspension was cooled to -20 °C, then 147 ml (147 mmol, 1.2 eq.) IM LiHMDS solution in THF was added dropwise over 60 minutes. The resulting dark brown solution was stirred at -10°C for 1 hour, then 44.4 g (154 mmol, 1.25 eq.) compound lOe was added in a single portion. The reaction was stirred at -10°C for 2 hours, then at ambient temperature overnight. The resulting orange-red suspension was diluted with 200 ml dichloromethane, then poured into mixture of 600 ml dichloromethane and 600 ml saturated ammonium chloride solution. The phases were separated and the aqueous phase was extracted with 3X 100 ml di chloromethane. The pooled organic phase was washed 2X 400 ml water, dried over sodium sulfate, filtered, and concentrated under vacuum. The red residue was purified by washing with 300 mL hot ethyl acetate and 200 ml acetonitrile, respectively, then dried under vacuum at 40 °C for 12 hours. Yield: 25.16 g (74 mmol, 60%) red solid (Compound 2501). ESI-MS (M+H+) = 342.1H-NMR 5 (500 MHz DMSO-de T = 300K): 7.69 (d, J = 9.5 Hz, 2H), 7.60 (d J = 9.2 Hz, 2H), 6.98 (s, 1H), 6.79 and 6.75 [partly overlapping s and d (J = 9.5 Hz) 1H and 2H], 6.73 (s, 1H), 4.11 (m, 1H), 3.96 (m, 1H), 2.91 (s, 6H), 2.31 (s, 3H), 2.19 (m, 2H).Example 12. Synthesis of (97?)-9-hydroxy-5-methyl-12-[4-(pyrrolidin-l-yl)-phenyl]-4- thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound R2507).

[0119] Step a) Preparation of l-(4-iodophenyl)pyrrolidine (Compound 12a).8.76 g (40 mmol, 1.0 eq.) 4-iodoaniline was dissolved in 20.0 ml toluol, then 14.0 ml (80 mmol, 2.0 eq.) ethyldiisopropylamine was added. A mixture of 4.8 ml (40 mmol, 1.0 eq) 1,4-dibromobutan and 10 ml toluol was added dropwise at room temperature. The reaction was stirred at reflux for 4 hours, then allowed to cool to room temperature and further stirred overnight. The mixture was chilled then stirred for 1 hour at ice, filtered and washed with 100 ml water. The crude product was recrystallized in 60 ml isopropanol, stirred on ice, filtered and dried at room temperature on atmospheric pressure. Yield: 6.7 g (24.5 mmol, 61%) beige crystalline solid (Compound 12a).

[0120] Step b) Preparation of 4-chloro-N-[(4-methoxyphenyl)methyl]butanamide (Compound 12b).786 g (554 mmol, 1.0 eq.) 4-methoxybenzylamine was dissolved in 370 ml dry dichloromethane. 93 ml (665 mmol, 1.2 eq.) tetraethylammonium and 39 g (28 mmol, 0.05 eq.) N,N-dimethylaminopyridine were added in an inert flask under argon atmosphere, and the reaction mixture cooled to 5 °C. Then a mixture of 68.5 ml (610 mmol, 1.1 eq.) 4- chlorobutyryl chloride and 40 ml dry dichloromethane was added dropwise at 5-15 °C. The mixture was warmed to, then stirred for 24 hours at room temperature, followed bywashing with 400 ml water, 2X 400 ml 5% citric acid solution and 100 ml saturated NaHCCh solution. The mixture was separated, the organic layer dried on Na2SO4, filtered and the solvent evaporated. The resulting crystals were stirred at room temperature with 200 ml heptane overnight, the suspension filtered through a glass filter, washed with cold heptane, then dried at 40 °C under vacuum. Yield: 107.27 g (443 mol, 80%) white solid (Compound 12b).

[0121] Step c) Preparation of l-[(4-methoxyphenyl)methyl]pyrrolidin-2-one (Compound 12c).105 g (434 mmol, 1.0 eq.) Compound 12b was dissolved in 330 ml dry tetrahydrofuran in an inert flask under argon atmosphere, then 58.39 g (521 mmol, 1.2 eq.) potassium tertbutylate was added dropwise as its tetrahydrofuran solution (20.1 w / w%, 321 ml) in a controlled way, so the reaction mixture remained in mild reflux. The dropping funnel was washed with a further 40 ml tetrahydrofuran into the flask. The mixture was stirred at reflux for 2 hours, then cooled down and 15 ml (262 mmol, 0.6 eq.) acetic acid was added (exothermic reaction). 60 ml water was added and the solution decanted from the solid residue. The solid was washed with 50 ml methyl tert-butyl ether and the combined organic phases evaporated. The residue was dissolved in 200 ml methyl tert-butyl ether, extracted with 100 ml saturated NaHCCh, and the pH of the aqueous phase was adjusted to 8. The organic phase was separated, the aqueous phase further extracted with 100 ml of ethyl acetate, the combined organic layers dried first over 100 ml NaCl, then on Na2SO4, and the solvent evaporated under vacuum. The crude product was further purified by vacuum distillation at 151-158 °C, 0.64 mbar. Yield: 77.03 g (375 mmol, 86%) Compound 12c.

[0122] Step d) Preparation of methyl 2-{[(2E)-l-[(4-methoxyphenyl)methyl]- pyrrolidin-2-ylidene]amino}-5-methylthiophene-3-carboxylate (Compound 12d).24.95 g (121.54 mmol, 1.0 eq.) Compound 12c was dissolved in 100 ml dry dichloromethane in an inert flask under argon atmosphere. The solution was cooled to 5- 10 °C, then a mixture of 20.45 ml trifluoromethanesulfonic anhydride (121.54 mmol, 1.0 eq.) and 20 ml dry dichloromethane was added dropwise over 20 minutes. The reaction mixture was allowed to warm to room temperature then stirred for 1 hour. Then it was cooled to 5-15 °C and a solution of 22.89 g (133.7 mmol, 1.1 eq.) of Compound I la in 160 ml dry dichloromethane was added dropwise in 15 min. The mixture was stirred at reflux for 24 hours, cooled down, then 140 ml of saturated Na2COs solution was added slowly and stirred for 10-15 min. The phases were separated, the organic layer washed with 2X 100 ml water then evaporated. The residue was dissolved in 100 ml isopropyl-acetate and extracted with 10X 60 ml of 10% citric acid. The aqueous extract was pooled and washed with 60 ml isopropyl-acetate. pH of the aqueous phase was adjusted to 9 by adding 100 ml saturated Na2COs and 50 ml 50% NaOH, followed by extraction with 3X 100 ml isopropyl-acetate. The organic layer was dried over Na2SO4, filtered and concentrated. Yield: 20.1 g (56.07 mmol, 46%) yellow oil (Compound 12d).

[0123] Step e) Preparation of 12-[(4-methoxyphenyl)methyl]-5-methyl-4-thia- 2,12-diazatricyclo[7.3.0.03,7]dodeca-l(9),3(7),5-trien-8-one (Compound 12e).30.28 g (84.47 mmol) of Compound 12d was dissolved in 750 ml toluene, of which 500 ml was distilled away using a water separator and the residual solvent evaporated under vacuum. The residue was dissolved in 340 ml dry tetrahydrofuran in an inert flask under argon atmosphere, then 94.31g of 20.1 m / m % solution of potassium-tert-butylate in tetrahydrofuran (containing 18.96 g KOtBu, 168.9 mmol, 2 eq.) was added. The reaction mixture was stirred at reflux, while the reaction was followed using thin layer chromatography (hexane:ethyl acetate 7:3). After depletion of the starting material the mixture was cooled down and 38 ml (664.4 mmol, 7.8 eq.) of acetic acid was added followed by concentration under vacuum. The residue was suspended in 200 ml toluene, the toluene was evaporated and the residue was suspended in 300 ml water. pH of the mixture was adjusted to 8 by adding 600 ml saturated NaHCCh solution, then it was stirred for at least 30 min, filtered on a glass filter, washed with 3X 150 ml water, then with 3X 150 ml methyl-tert-butyl ether. The filtered material was dried under vacuum at room temperature. Yield: 18.83 g (57.69 mmol, 68.29%) Compound 12e.

[0124] Step f) Preparation of 5-methyl-4-thia-2,12-diazatricyclo[7.3.0.03,7]- dodeca-l(9),3(7),5-trien-8-one (Compound 12f).18.82 g (57.66 mmol) Compound 12e was mixed with 62 ml (570.46 mmol, 9.89 eq.) anisole in an inert flask under argon atmosphere. 35.5 ml (461.26 mmol, 8 eq.) trifluoroacetic acid was added dropwise to the mixture at room temperature, then the solution was stirred for 19 hours at 70 °C. After cooling the reaction mixture to 0-5 °C,340 ml cc. hydrochloric acid was added resulting in precipitation of the hydrochloride salt of the product. The suspension was stirred for 10-15 min, then filtered through a glass filter, washed with 50 ml cold acetonitrile and 50 ml cold diethyl ether, and dried under vacuum at 30 °C. The resulting HC1 salt was suspended in 90 ml saturated NHCO3 solution, stirred for 30 min, filtered, washed on a glass filter with 2X 40 ml water and 20 ml methyl-tert-butyl ether, then dried under vacuum. The filtrate of the first filtration step was also processed by setting its pH to 9, followed by filtering and washing with aqueous and cold acetonitrile, yielding a further 4.65 g product. Total yield: 11.33 g (54.93 mmol, 95.29%) white solid (Compound 12f).

[0125] Step g) Preparation of (97?)-9-hydroxy-5-methyl-4-thia-2,12-diazatricyclo-[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 12g).5.15g (25 mmol) Compound 12f was dissolved in 40.0 ml dry tetrahydrofuran. 10.9 g (37.5 mmol, 1.5 eq.) Compound lOe was added, the resulting suspension was cooled to -5- 0 °C, then 50 ml (50 mmol, 2.0 eq.) of an 1 mol / 1 solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran was added dropwise, while maintaining the temperature at a maximum of 0 °C. The solution was allowed to warm to room temperature, then stirred overnight. It was washed with 100 ml NH4Q and extracted with 150 ml 2-methyltetrahydrofuran. The aqueous phase was further extracted with 4 X 50 ml 2-methyltetrahydrofuran. The combined organic phase was washed with 50 ml water, dried on Na2SO4, filtered, washed and the solvent evaporated under vacuum at 40 °C. The crude product was dissolved in 50 ml acetonitrile, heated to 70 °C, stirred at room temperature for 1 hour, chilled on ice, stirred on ice for 3 hours, filtered and washed with 5.0 ml acetonitrile, then dried under vacuum at room temperature. Yield: 5.23 g (23.5 mmol, 94%) brown solid (Compound 12g). ESI-MS mlz)'. 223 (M+H)+.

[0126] Step h) Preparation of (97?)-9-hydroxy-5-methyl-12-[4-(pyrrolidin-l- yl)phenyl]-4-thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound R2507).56.0 mg (0.25 mmol) Compound 12g was dissolved in 3.0 ml dimethylformamide, then 57.0 mg (0.3 mmol, 1.2 eq.) Cui, 43.0 mg (0.3 mmol, 1.2 eq.) Nondimethyl cyclohexane- 1,2-diamine, 244.0 mg (0.75 mmol, 3.0 eq.) CS2CO3 and 103.0 mg (0.37 mmol, 1.5 eq.) Compound 12a was added. The reaction mixture was heated to 60 °C and stirred overnight under argon atmosphere. The crude product was filtered, washed with methanol then the solvent evaporated under vacuum. The residue was suspended in 20 ml saturated NaCl solution and 20 ml 2-methyltetrahydrofuran, and the aqueous phase further extracted with 2X 50 ml 2-methyltetrahydrofuran. The pooled organic phase was dried on Na2SO4, filtered, washed then the solvent evaporated under vacuum at 40 °C. Yield: 0.7 g brown oil (Compound R2507).Example 13. Synthesis of ethyl l-[12-(4-iodophenyl)-5-methyl-8-oxo-4-thia-2,12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-9-yl]-4,5-dihydro-lH-l,2,3-triazole-4- carboxylate (Compounds R3003 and S3003).

[0127] Step a) Preparation of 4-chloro-N-(4-iodophenyl)butanamide (Compound 13a).120.2 g (549 mmol, 1.0 eq.) 4-iodoaniline was dissolved in 480 ml dichloromethane, then91.6 ml (657 mmol, 1.2 eq.) triethylamine was added. The solution was chilled on ice to 0-5 °C, then 67.4 ml (601 mmol, 1.1 eq.) 4-chlorobutyryl chloride was added dropwise overthe course of 20 min, while maintaining the temperature below 5 °C. The reaction mixture was allowed to warm to 20-25 °C with constant cooling on ice, and stirred for 4 days. 100 ml water was added to the mixture, and the formed precipitate was recovered by filtering. The organic phase of the filtrate was washed with 100 ml 20% solution of HC1 and 100 ml water, and the solvent evaporated under vacuum at 40 °C. The residue was united with the residue of the filtration, suspended in 400 ml toluol and evaporated under vacuum at 45 °C. Yield: 171.7 g (531 mmol, 96.7%) peach solid (Compound 13a).

[0128] Step b) Preparation of l-(4-iodophenyl)pyrrolidin-2-one (Compound 13b).120 g (371 mmol, 1.0 eq.) of Compound 13a was dissolved in 600 ml tetrahydrofuran, chilled on ice to 0-5 °C, then 9.8 g (408 mmol, 1.1 eq.) NaH was added slowly over 1.5 hours, while maintaining the temperature below 10 °C. The reaction mixture was stirred overnight at room temperature, then 20.0 ml MeOH was added to neutralize excess NaH and the solvent was evaporated under vacuum at 45 °C. The residue was suspended in 500 ml water and 300 ml EtOAc, and the undissolved solid recovered with filtering. The wet residue was dried under vacuum at 50 °C. Yield: 103.2 g (359 mmol, 96.8%) creamy white crystalline solid (Compound 13b).

[0129] Step c) Preparation of methyl 2-{[(2E)-l-(4-iodophenyl)pyrrolidin-2- ylidene]amino}-5-methylthiophene-3-carboxylate (Compound 13c).50.0 g (0.174 mol, 1.0 eq.) Compound 13b was dissolved in 250.0 ml 1,4-dioxane, 19.6 ml (0.2 mol, 1.2 eq.) POCh was added dropwise over 5 minutes, and the reaction mixture (tan suspension) was stirred overnight at room temperature. The suspension was warmed to 50- 60 °C and stirred until it turned into an opaque yellow solution. 30.0 g (0.174 mol, 1.0 eq.) of Compound I la was added, and the solution stirred at reflux for 3 hours, during which it first turned more thick, suspension-like, then cleared out again. The mixture was allowed to cool to room temperature and stirred overnight. The reaction mixture was slowly poured into 300 ml of a pre-chilled (10 °C) saturated solution of Na2COs, then extracted with 200 ml dichloromethane. Solid matter remained at the boundary of the two phase, which was removed by filtering and dissolved in 300 ml di chloromethane. The pooled organic phase was dried over Na2SO4, filtered, washed with dichloromethane and the solvent evaporated under vacuum at 45 °C. The residue was recrystallized in methyl tert-butyl ether. Yield: 54.25 g (0.123 mol, 70,7%) of tan powder (Compound 13c).

[0130] Step d) Preparation of 12-(4-iodophenyl)-5-methyl-4-thia-2, 12- diazatricyclo[7.3.0.03,7]dodeca-l(9),3(7),5-trien-8-one (Compound 13d).53.90 g (0.122 mol, 1.0 eq.) of Compound 13c was dissolved in 320.0 ml tetrahydrofuran, 24.72 g (0.220 mol, 1.8 eq.) potassium tert-butylate was added and the mixture was stirred at reflux for 3 hours. The solvent was evaporated, the residue suspended in 300 ml water, filtered, washed with 4X 100 ml MeOH, filtered again and evaporated under vacuum at 50 °C. Yield: 41.97 g (0.103 mol, 84.4%) tan powder (Compound 13d).

[0131] Step e) Preparation of 9-chloro-12-(4-iodophenyl)-5-methyl-4-thia-2,12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 13e).40.83 g (100 mmol, 1.0 eq.) Compound 13d was dissolved in 100 ml tetrahydrofuran and 100 ml water. The suspension was cooled to 10 °C on ice, 14.08 g (55 mmol, 0.55 eq.) of sodium dichloroisocyanurate was added in 3 portions over the course of 3 minutes, and the reaction mixture stirred for 2h at room temperature. Finally, the mixture was filtered, washed with water and dichloromethane, then dried. Yield: 38.07 g (86 mmol, 86%) dark red powder (Compound 13e).

[0132] Step f) Preparation of 9-azido-12-(4-iodophenyl)-5-methyl-4-thia-2, 12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compound 13f).110.0 mg (0.25 mmol, 1.0 eq.) of Compound 13e was dissolved in 10.0 ml of acetonitrile and the solution chilled on ice to 0-5 °C. 49.0 mg (0.75 mmol, 3.0 eq.) of sodium azide was added, then the mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated to 1 / 10 volume by evaporation under vacuum at 30 °C. The residue was dissolved in 10.0 ml water and 10.0 ml EtOAc, and the aqueous phase was extracted again with a further 20.0 ml EtOAc. The pooled organic phase was dried on Na2SO4, filtered, washed with EtOAc, and evaporated under vacuum at 30 °C. Yield: 140 mg orange amorphous solid (crude Compound 13f).

[0133] Step g) Preparation of ethyl l-[12-(4-iodophenyl)-5-methyl-8-oxo-4-thia- 2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-9-yl]-4,5-dihydro-lH-l,2,3-triazole-4- carboxylate (Compounds R3003 and S3003).50.8 mg (0.113 mmol, 1.0 eq.) of Compound 13f and 11.0 mg (0.113 mmol, 1.0 eq.) of Cui were dissolved in 1 ml DMSO, then 58.0 pL (0.565 mmol, 5.0 eq.) of ethyl propionate was added and the mixture stirred overnight at room temperature. The reaction mixture was washed with 30 ml water and extracted with 30 ml EtOAc. 10 ml of saturated NaCl solution was also added to aid separation. The aqueous phase was further extracted with 2X 30 ml EtOAc. The pooled organic phase was dried on Na2SO4, filtered, and evaporated under vacuum at 40 °C. The resulting orange amorphous solid was dissolved in 20.0 ml acetonitrile and purified using HPLC. The resulting pure product was a racemic mixture of compounds R3003 and S3003, which were separated on a cellulose HPLC column.Example 14. Synthesis of 9-amino-12-[4-(dimethylamino)phenyl]-5-methyl-4-thia-2,12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compounds S3403 and R3403)

[0134] Step a) Preparation of 9-amino-12-(4-iodophenyl)-5-methyl-4-thia-2, 12- diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compounds S3400 and R3400)10.0 g (22.6 mmol, 1.0 eq.) of Compound 13e was suspended in 200 ml tetrahydrofuran in a 250 ml Schott glass bottle. With constant cooling on ice, ammonia gas was flowed through the mixture applying a weak flow over 15 minutes. The reaction was incubated at 40 °C for 48 hours. The mixture was concentrated under vacuum, then separated between water (pH 8-9) and dichloromethane. The organic phase was concentrated, then 50 ml dichloromethane and 20 ml 5N HC1 was added, resulting in a dark red suspension. The precipitate was filtered, then redissolved in a mixture of 130 ml concentrated Na2COs and 180 ml acetic acid, followed by the addition of 10 ml 5N HC1. The precipitate was filtered, redissolved in a mixture of 70 ml concentrated Na2COs and 150 ml acetic acid, the phases were separated, the organic phase was dried on Na2SO4, then the solvent was evaporated under vacuum. Yield: 4.05 g (9.6 mmol, 42%) solid (racemic mixture of Compounds S3400 and R3400).

[0135] Step b) Preparation of 9-amino-12-[4-(dimethylamino)phenyl]-5-methyl-4- thia-2,12-diazatricyclo[7.3.0.03,7]dodeca-l,3(7),5-trien-8-one (Compounds S3403 and R3403)1.2 g (2.61 mmol, 1.0 eq.) Compound 3400 (racemic mixture of Compounds S3400 and R3400) was dissolved in 8 ml DMSO. 149.1 mg (0.78 mmol, 0.3 eq.) Cui, 180.3 mg (1.56 mmol, 0.6 eq.) L-proline, and 901.7 mg (6.52 mmol, 2.5 eq.) was added, followed by 10.5 ml (20.88 mmol, 8.0 eq.) of 2 mol / 1 dimethylamine dissolved in tetrahydrofuran. The reaction mixture was stirred overnight at room temperature, resulting in a dark brown suspension. The mixture was concentrated under vacuum, then 100 ml acetic acid, 100 ml water , and 20 ml 20% NaOH solution was added. Aqueous phase was extracted with 3X 100 ml acetic acid. The organic phase was dried on Na2SO4, washed with acetic acid, then the solvent was evaporated at 40 °C under vacuum, resulting in a brown solid. The crude product was resuspended in 20 ml MTBE, and incubated at 10 °C overnight. The suspension was filtered, wased with 2X 10 ml MTBE, then the solvent evaporated. Yield: 690 mg (2.03 mmol, 78%) reddish brown powder (racemic mixture of S3403 and R3403). The two enantiomers were separated on a cellulose HPLC column.Example 15. Synthesis of (3aR)-3a-hydroxy-l-{imidazo[l,2-a]pyridin-2-yl}-6-methyl- lH,2H,3H,3aH,4H-pyrrolo[2,3-b]quinolin-4-one (Compound R165).

[0136] Step a) Preparation of methyl 2-{[(2E)-l-[(4- methoxyphenyl)methyl]pyrrolidin-2-ylidene]amino}-5-methylbenzoate (Compound 15a)39.92 g (195 mmol, 1.0 eq.) of Compound 12c was dissolved in 15 ml of dry dichloromethane under argon atmosphere. The solution was cooled to 5-10 °C, then a solution of 54.86 g (195 mmol, 1.0 eq.) trifluoromethanesulfonic anhydride in 30 ml dry dichloromethane was added through the course of 20 minutes, with constant cooling to maintain the temperature between 5-15 °C. The solution was stirred for 1 hour at room temperature, then cooled to 5-10 °C. A solution of 35.42 g (214 mmol, 1.1 eq.) methyl 2- amino-5-methylbenzoate in 160 ml of dry di chloromethane was added dropwise between 5-15 °C over 15 minutes. The mixture was stirred at reflux for 24 hours, then cooled to room temperature. 220 ml of saturated sodium carbonate solution was added slowly to the mixture, then the reaction was stirred for 15 minutes. The organic phase was separated, washed twice with 160 ml water, then the solvent was evaporated. The residue was dissolved in 160 ml of isopropyl acetate and extracted with 1 OX 100 ml 10% citric acid solution. The pooled aqueous phase was washed with 100 ml isopropyl acetate, its pH was set to pH 9 by adding a 50% solution of sodium hydroxide, then it was extracted with 3X 160 ml isopropyl acetate. The pooled organic phase was dried over sodium sulfate, filtered, and the solvent was evaporated under vacuum. Yield: 32.2 g (90 mmol, 46.1%), yellow oil (Compound 15a).

[0137] Step b) Preparation of l-[(4-methoxyphenyl)methyl]-6-methyl- lH,2H,3H,4H,9H-pyrrolo[2,3-b]quinolin-4-one (Compound 15b)30.28 g (86 mmol, 1.0 eq.) Compound 15a was dissolved in 750 ml of toluene and refluxed in a Dean-Stark apparatus until no more water distilled out. The incubation was continued until 500 ml toluene was distilled, then the remaining toluene was evaporated under vacuum. The residue was dissolved in 340 ml dry tetrahydrofuran and transferred to a 3-necked flask fitted with reflux condenser and dropping funnel. Then 18.96 g (169 mmol, 2 eq.) KO / Bu as its 20.1% w / w solution in tetrahydrofuran was added slowly, and the reaction incubated at reflux for 5 hours. The mixture was cooled to room temperature, and 12 ml (12.6 g, 210 mmol, 2.5 eq.) acetic acid was added dropwise, then the solvent was evaporated under vacuum. The residue was suspended in 200 ml toluene, the solvent evaporated under vacuum, then the residue was suspended again in 300 ml water. The pH of the suspension was set to 8 by adding a saturated solution of sodium bicarbonate, then it was stirred for 30 minutes at room temperature. The suspension was filtered, washed with 3X 150 ml water, then with 3X 150 ml methyl-tert-butyl ether, and dried under vacuum. Yield: 18.83 g (58 mmol, 68.3%) Compound 15b.

[0138] Step c) Preparation of 6-Methyl-lH,2H,3H,4H,9H-pyrrolo[2,3-b]quinolin- 4-one (Compound 15 c)In a round-bottom flask, fitted with a reflux condenser and a dropping funnel, 18.82 g (58 mmol, 1 eq.) of Compound 15b was suspended in 57 ml (56.45 g, 522 mmol, 9 eq.) anisole. The solid was dissolved by slowly adding 35.5 ml (52.56 g, 461 mmol, 8 eq.)trifluoroacetic acid to the suspension, then the solution was stirred for 24 hours at 70 °C under argon atmosphere. The solution was cooled to 0-5 °C, followed by the addition 340 ml concentrated hydrochloric acid, then the reaction was stirred for 15 minutes. The resulting hydrochloride salt was filtered, the pH of the filtrate was set to pH 9, then the precipitate was filtered, washed with water, acetonitrile, and diethyl ether, and dried under vacuum at 30 °C, yielding 4.65 g of Compound 15c. The hydrochloride salt (7.76 g) was suspended in 90 ml saturated sodium bicarbonate solution, stirred at room temperature for 30 minutes, then filtered. The solid was washed with 2X 40 ml water, then with 20 ml of methyl tert-butyl ether. Yield: 6.16 g (31 mmol, 53.0%) Compound 15c.'HNMR (500 MHz, CD3OD): 5 = 2.41 (s, 3H), 3.07 (t, J= 8.5 Hz, 2H), 3.75 (t, J = 8.5 Hz, 2 H), 7.23 (d, J= 8.4 Hz, 1H), 7.34 (dd, J= 1.5, 8.4 Hz, 1H), 7.90 (br, 1H).

[0139] Step d) Preparation of (3aR)-3a-hydroxy-6-methyl-lH,2H,3H,3aH,4H- pyrrolo[2,3-b]quinolin-4-one (Compound 15d)3.00 g (14.5 mmol, 1 eq.) Compound 15c and 6.31 g (21.8 mmol, 1.5 eq.) Compound lOe were dissolved in 30 ml dry tetrahydrofuran under argon atmosphere, then cooled to 0-5 °C. 26.5 ml (4.87 g, 29.1 mmol, 2 eq.) of a 20.7% w / w LiHMDS solution in tetrahydrofuran was added drop-wise, while keeping the temperature between 0-5 °C. The reaction was stirred at room temperature for 24 hours, the 50 ml saturated ammonium chloride solution, 150 ml 2-methyltetrahydrofuran, and 50 ml water were added, respectively. The organic phase was separated, filtered, and the residue was washed with 2-methyltetrahydrofuran yielding 0.34 g yellow solid product. The filtrate was dried over sodium sulfate, filtered, and concentrated to 100 ml. The precipitate was filtered and washed with 2-methyltetrahydrofuran, yielding 0.87 g product. The remaining filtrate was further concentrated to 30 ml and the precipitate was filtered and washed with 2- methyltetrahydrofuran, yielding 1.27 g product. Combined yield: 2.47 g (11.1 mmol, 77%) yellow solid (Compound 15d). 'HNMR (500 MHz, DMSO-t / e): 5 = 2.23 (s, 3H), 2.08 (m, 2H), 3.60 (m, 2 H), 6.90 (d, J = 8.3 Hz, 1H), 7.27 (dd, J= 2.1, 8.3 Hz, 1H), 7.44(d, J= 2.1Hz, 1H). DEPTq (500 MHz, DMSO- e): 8 = 20.0, 32.0, 48.6, 76.7, 119.0, 119.1, 129.2, 136.3, 145.7, 165.9, 193.6.

[0140] Step e) Preparation of (3aR)-3a-hydroxy-l-{imidazo[l,2-a]pyridin-2-yl}-6- methyl-lH,2H,3H,3aH,4H-pyrrolo[2,3-b]quinolin-4-one (Compound R165)Compound R165 was prepared by coupling 1.3 eq. 2-iodoimidazo[l,2-a]pyridine to 1.0 eq. Compound 15d in an Ullmann-type arylation, in the presence of 1.2 eq. Cui, 1.2 eq. N,N' -Dimethyl- 1,2-cy cl ohexanediamine, and 3.0 eq. K2CO3, using DMF as solvent. The reaction mixture was stirred for 12 hours at 60 °C, then it was diluted with EtOAc, washed with water and brine, dried on Na2SO4 and the solvent was evaporated.

[0141] Synthesis products were purified via HPLC. Enantiomers were separated on a cellulose HPLC column.

[0142] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

[0143] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0144] The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0145] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0146] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0147] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0148] Other embodiments are set forth in the following claims.

Claims

1. WHAT IS CLAIMED IS:

1. A compound represented by Formula (IR) or (IIR), or a pharmaceutically acceptable salt or tautomer thereof,(|R) (HR)wherein:Q1, Q2, Q3, and Q4are each independently CR20;Q5, Q6, and Q7are each independently S, O, N, NR30or CR31, provided that the aromatic nature of the ring is retained, wherein at least one of Q5, Q6, and Q7is S, O, N, or NR30;R20is H, or C1-C6 alkyl;R30is H, or C1-C6 alkyl;R31is H, or C1-C6 alkyl;R2, R3, R4, and R5are each independently H, F, Cl, Br, I, or C1-C6 alkyl;R1is H or a group of formula:wherein: n = 0, 1, 2, 3, 4, 5, or 6; and“D ring” is a saturated, partially saturated, unsaturated or aromatic, 5- or 6- membered cyclic, or 9- or 10-membered fused bicyclic group, which is optionally deuterated or tritiated, which optionally contains one or more N, O, or S atoms, and which is optionally substituted by one or more groups selected from the group consisting of: F; Cl; Br; I;C1-C6 straight chain alkyl;C1-C6 haloalkyl;-OH;C1-C6 hydroxyalkyl;C1-C6 hydroxyalkynyl;C1-C6 alkoxy, or Cl -6 alkoxyalkyl, wherein the alkyl group is optionally substituted by any number of F, Cl, Br, or I atoms;C1-C6 carboxylic acid; azido;-NH2;C1-C6 alkylamino, or Cl -6 dialkylamino, wherein the alkyl groups are optionally deuterated or tritiated, and are optionally substituted by any number of F, Cl, Br, or I atoms; sulfonylamino; a 4-7 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, or CO group; and a C4-C12 fused bicycle, which is optionally substituted by C1-C6 alkyl, or C1-C6 carboxylic acid; andR6is selected from the group consisting of:H;F; Cl; Br; I;-OH;C1-C6 alkoxy;-CN;-NH2;C1-C6 alkylamino;C1-C6 dialkylamino; azido; amido; and a 5 or 6 membered saturated, partially saturated, unsaturated, or aromatic heterocyclic group containing one or more N, O, or S atoms, which is optionally substituted by one or more groups selected from the group consisting of:C1-C6 (straight chain) alkyl;-OH;C1-C6 hydroxyalkyl;C1-C6 carboxylic acid; andC1-C6 ester of a carboxylic acid and a saturated alcohol, unsaturated alcohol, or aryl alcohol.

3. A compound that is:or a pharmaceutically acceptable salt or tautomer thereof.

4. The compoundor a pharmaceutically acceptable salt or tautomer thereof.

5. The compound of any one of claims 1-4 for targeting relay-SHl drug binding cleft on smooth muscle myosin 2.

6. The compound of any one of claims 1-4 for inhibiting smooth muscle myosin 2.

7. The compound of any one of claims 1-4 for treating mooth muscle myosin 2 related diseases.The compound of any one of claims 1-4 for treating conditions involving no-reflow.

9. The compound of any one of claims 1-4 for treating myocardial infarction, acute kidney injury, stroke, hypertension, or gastrointestinal motility disorder.

10. A method of inhibiting smooth muscle myosin 2, the method comprising contacting a compound of any one of claims 1-4 with smooth muscle myosin 2.

11. A method of treating a disease mediated by smooth muscle myosin 2, the method comprising administering the compound of any one of claims 1-4 to an individual in need thereof.

12. The method of claim 11, wherein the disease mediated by smooth muscle myosin 2 comprises conditions involving no-reflow.

13. A method of treating myocardial infarction, acute kidney injury, stroke, hypertension, or gastrointestinal motility disorder in an individual, the method comprising administering the compound of any one of claims 1-4, or a formulation comprising the compound of any one of claims 1-4 to the individual.

14. A method of preparing a compound of formula, the method comprising: adding lithium bis(trimethylsilyl)amide to a solution of a compound of formulaadding aqueous ammonium chloride; and isolating the compound of formula5. The method of claim 14 further comprising preparing the compound of formulaadding a solution of a base in an ether solvent to a solution of a compound of formulafollowed by quenching with acetic acid; isolating the compound of formula6. The method of claim 15 further comprising preparing the compound of formulaadding phosphorus oxychloride to a solutionmixture ot1,4-di oxane and di chloromethane; adding methyl 2-amino-5-methylthiophene-3-carboxylate and heating at reflux; collecting the compound of formula

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