Methods and materials for treating ectopic tissue mineralization

Abstract

This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, methods and materials for using soluble guanylate cyclase (sGC) agonists of Formula I, Group I, and / or Group II to reduce ectopic tissue mineralization formation within a mammal and / or to slow ectopic tissue mineralization progression within a mammal are provided. sGC agonists of Formula I, Group I, and / or Group II and compositions containing sGC agonists of Formula I, Group I, and / or Group II also are provided.

Description

[0001] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0002] METHODS AND MATERIALS FOR TREATING ECTOPIC TISSUE MINERALIZATION TECHNICAL FIELD

[0003] This document relates to methods and materials involved in treating ectopic tissue mineralization. For example, this document provides methods and materials for using soluble guanylate cyclase (sGC) agonists (e.g., oxidized or non-oxidized sGC agonists) of Formula I. Group I, and / or Group II to reduce formation and / or to slow progression of ectopic tissue mineralization.

[0004] BACKGROUND

[0005] Ectopic tissue mineralization refers to the deposition of mineralized complexes in tissues other than those in which it normally occurs. Ectopic tissue mineralization can occur in various tissues such as organs, muscles, tendons, and other soft tissues and may impair normal tissue function.

[0006] SUMMARY

[0007] This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, this document provides soluble guanylate cyclase (sGC) agonists of Formula I, Group I. and / or Group II, compositions containing one or more sGC agonists of Formula I, Group I, and / or Group II, and methods for using sGC agonists of Formula I, Group I, and / or Group II and / or compositions containing one or more sGC agonists of Formula I, Group I, and / or Group II to treat mammals having, or likely to develop, ectopic tissue mineralization.

[0008] As demonstrated herein, loss of sGC signaling (e g., through genetic inactivation of enzymes in pathways that regulate nitric oxide signaling) resulted in significant increases in ectopic tissue calcification. These results demonstrate that sGC agonists of Formula I, Group I, and / or Group II can be used to treat ectopic tissue mineralization and / or to slow progression of ectopic tissue mineralization formation. Having the ability to reduce formation and / or slow progression of ectopic tissue mineralization as described herein can allow clinicians to treat patients who have ectopic tissue mineralization, allowing patients to live longer and happier lives.

[0009] In general, one aspect of this document features a method for reducing ectopic tissue mineralization formation. The method can include (or consist essentially of, orAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0010] consist of) administering a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II to a mammal having ectopic tissue mineralization or likely to develop ectopic tissue mineralization, wherein formation of the ectopic tissue mineralization within the mammal is reduced. The mammal can be a human. Formation of ectopic tissue mineralization within the mammal can be delayed.

[0011] Formation of ectopic tissue mineralization within the mammal can be delayed for at least 6 months. The mammal can be a mammal having ectopic tissue mineralization. The mammal can be a mammal likely to develop ectopic tissue mineralization.

[0012] In another aspect, this document features a method for slowing ectopic tissue mineralization progression. The method can include (or consist essentially of, or consist of) administering a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II to a mammal having ectopic tissue mineralization, wherein progression of the ectopic tissue mineralization within the mammal is slowed. The mammal can be a human. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 10 percent. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 25 percent. Progression of ectopic tissue mineralization within the mammal can be slowed by at least 50 percent.

[0013] In another aspect, this document features a compound of Formula I:

[0014]

[0015] or a pharmaceutically acceptable salt thereof, wherein:

[0016] R1is selected from NH2, morpholinyl, piperidinyl, pyrrolidinyl, and 2-oxa-6-azaspiro[3.3]heptanyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, OH, C1-3 alkoxy, C1-3 haloalkyl, C1-3 haloalkoxy, COOH, and CH2COOH;

[0017] X1is a 5-membereed heteroaryl, optionally substituted with halo, OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy;Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0018] R2is selected fromH, C(=O)Ci-3 alkyl, C(=O)Ci-3 haloalkyl, and S(=O)2R4; R4is selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy;

[0019] n is 1, 2, or 3; and

[0020] each R3is independently selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy. X1can be a 5-membereed heteroaryl. X1can be selected from 1,2,3-triazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-oxadiazolyl, and 1,3,4-thiadiazolyl. The compound of Formula I can have formula:

[0021]

[0022] or a pharmaceutically acceptable salt thereof. The compound of Formula I can have formula:

[0023]

[0024] or a pharmaceutically acceptable salt thereof. R2can be H. R2can be selected from C(=O)Ci-3 alkyl and C(=O)Ci-3 haloalkyl. The compound can have formula:

[0025]

[0026] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0027] or a pharmaceutically acceptable salt thereof. R4can be selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, and C1-3 alkoxy. The compound can have formula:

[0028]

[0029] or a pharmaceutically acceptable salt thereof. N can be 1 or 2. R3can be selected from halo, C1-3 alkyl, and C1-3 alkoxy. The compound can have formula:

[0030]

[0031] or a pharmaceutically acceptable salt thereof. The compound can have formula:

[0032]

[0033] or a pharmaceutically acceptable salt thereof. The compound can have formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0034]

[0035] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0036]

[0037] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0038]

[0039] or a pharmaceutically acceptable salt thereof.

[0040] In another aspect, this document features a compound selected from:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0041]

[0042] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0043]

[0044] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0045]

[0046] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0047]

[0048] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0049]

[0050] , andAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0051]

[0052] or a pharmaceutically acceptable salt thereof.

[0053] In another aspect, this document features a pharmaceutical composition containing (or consisting essentially of, or consisting of) a compound provided herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety'. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0055] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below'. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0056] DESCRIPTION OF DRAWINGS

[0057] Figure 1 is a graph plotting expression of DDAH1, DDAH2, PRMT1, and SLC7A2 in aortic valve tissue from humans with end-stage aortic stenosis undergoing valve replacement surgery. Expression of DDAH1 and SLC7A2 was significantly increased whereas expression of PRMT1 was reduced in calcified valve tissue suchAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0058] that: 1) DDAH1 and DDAH2, the enzy mes that degrade the endogenous inhibitor of nitric oxide synthase ADMA, are both upregulated in diseased tissue.

[0059] Figures 2A-2B contain results demonstrating changes in aortic valve calcification (Figure 2A) following 6 months of Western diet feeding, and changes in aortic valve function (Figure 2B) in LA-D+ / +and LA-D+ / " mice. Reductions in cusp separation distance are indicative of poorer valve function. Valvular calcification and valvular dysfunction were significantly increased in LA-D+‘ mice compared to LA-D+ / +littermates.

[0060] Figures 3A-3C contain results demonstrating changes in canonical BMP signaling (pSMADl / 5 / 8, Figure 3A), Osterix protein levels (Figure 3B), and SMAD target genes (Idl. Id2, and Hey, Figure 3C; note that Osterix (Osx) transcript levels were not detectable) in aortic valve from LA-D+ +and LA-D+ / ‘ mice. Increased valvular calcification and valvular dysfunction observed in LA-D+ / " mice (Figures 2A-2B) was associated with increases in pSMAD 1 / 5 / 8 protein levels, osteogenic transcription factor proteins, and target genes of canonical BMP signaling.

[0061] Figures 4A-4B contain results demonstrating changes USF-1 protein levels (Figure 4A) and the expression levels of USF-1 target genes osteopontin and alkaline phosphatase (Figure 4B) in aortic valve tissue from LA-D+ / +and LA-D+ / " mice. USF-1 protein levels and SPP 1 expression were increased in LA-D mice, suggesting that there was increased USF-1 signaling in LA-D ’ mice.

[0062] Figures 5A-5C contain results demonstrating changes in protein levels of osteopontin (Figure 5 A) and protein (Figure 5B) and mRNA (Figure 5C) levels of tartrate-resistant alkaline phosphatase (TRAP) in aortic valve tissue from LA-D+ / +and LA-D ' mice, suggesting that post-translational regulation of SPP1 (e.g., protein phosphorylation) may limit the anti-calcific effects of SPP1 in valve tissue.

[0063] Figures 6A-6B contain results demonstrating changes in expression of genes related to ADMA degradation, synthesis, and transport (Figure 6A), USF-1 protein levels (Figure 6B), and USF-1 target gene expression (Figure 6B) in aortic valve interstitial cells following 24 hours of treatment with vehicle, ADMA, or PD404182. Treatment with exogenous ADMA did not significantly alter gene expression or protein levels; however inhibition of DDAH1 with PD404182 significantly increased both SPP1 expression and USF-1 protein levels.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0064] Figure 7 is a graph plotting expression of DDAH1, DDAH2, PRMT1, and SLC7A2 in aortic punch biopsies from humans with advanced coronary artery disease undergoing coronary artery' bypass surgery. Expression of DDAH1, DDAH2, and SLC7A2 did not differ between normal and diseased aortic vessel punch tissue; however, PRMT1 expression was significantly reduced in the aorta of diseased samples.

[0065] Figures 8A-8B contain results demonstrating changes in aortic calcification following 6 months of Western diet feeding (Figure 8A) and changes in endothelium-dependent relaxation to acetylcholine in aortic rings (Figure 8B) from LA-D / +and LA-D " mice. While mice of both genoty pes developed advanced atherosclerotic plaques, LA-D / _mice demonstrated increased intimal plaque calcification compared to LA-D+ +mice.

[0066] Figure 9 contains results demonstrating changes in vasomotor function in thoracic aorta from LA-D+ +and LA-D ' mice following 6 months of Western diet feeding. There were no significant changes in vascular relaxation to sodium nitroprusside or vascular constriction to prostaglandin F2αor serotonin (5-HT).

[0067] Figures 10A-10C contain results demonstrating changes in canonical BMP signaling (pSMADl / 5 / , Figure 10A), osterix protein levels (Figure 10B), and SMAD target genes (Idl, Id2, Heyl, Osx, Figure 10C) in aorta from LA-D+ / +and LA-D " mice. Vascular calcification was associated with increases in canonical BMP signaling, but not increased expression of markers of mature osteoblast like cells or pSMAD target genes, suggesting a functional disconnect between upstream BMP signaling and induction of dow nstream ectopic osteogenic gene programs.

[0068] Figures 11A-11B contain results demonstrating changes in USF-1 protein levels (Figure 11 A) and the USF-1 target genes osteopontin and alkaline phosphatase (Figure 1 IB) in aorta from LA-D+ +and LA-D " mice. No increases in USF-1 or its target genes were observed in aorta from LA-D+‘ mice.

[0069] Figures 12A-12C contain results demonstrating changes in protein levels of osteopontin (Figure 12A) and protein (Figure 12B) and mRNA (Figure 12C) levels of tartrate-resistant alkaline phosphatase (TRAP) in aorta from LA-D+ +and LA-D " mice. There was no significant difference in SPP1 protein levels and TRAP protein and mRNA levels were variable, suggesting a possible dysregulation of mechanisms that protect against calcification in aorta.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0070] Figures 13A-13B contain results demonstrating changes in expression of genes related to ADMA degradation, synthesis, and transport (Figure 13A). USF-1 protein levels, and USF-1 target gene expression (Figure 13B) in aortic smooth muscle cells follow ing 24 hours of treatment with vehicle, ADMA, or PD404182. Treatment of cells with exogenous ADMA did not alter expression of DDAH1, DDAH2, ADMA transporters, USF-1 protein levels, or USF-1 molecular targets. Inhibition of DDAH1 with PD404182 failed to drive changes in expression or protein levels of DDAH isoforms or USF-1 protein or targets.

[0071] DETAILED DESCRIPTION

[0072] This document provides methods and materials for reducing formation and / or slowing progression of ectopic tissue mineralization. For example, this document provides soluble guanylate cyclase (sGC) agonists of Formula I, Group I, and / or Group II, compositions containing one or more sGC agonists of Formula I, Group I, and / or Group II, and methods for using sGC agonists of Fonnula I, Group I, and / or Group II and / or compositions containing one or more sGC agonists of Formula 1, Group I, and / or Group II to treat mammals having, or likely to develop, ectopic tissue mineralization.

[0073] As described herein, one or more sGC agonists of Formula I, Group I, and / or Group II can be administered to a mammal (e.g., a human) to reduce ectopic tissue mineralization formation and / or to slow ectopic tissue mineralization progression within the mammal. For example, one or more sGC agonists of Formula I, Group I, and / or Group II can be administered to a mammal (e.g., a human) having ectopic tissue mineralization (or likely to develop ectopic tissue mineralization) to reduce ectopic tissue mineralization and / or to slow ectopic tissue mineralization progression.

[0074] Any type of mammal having or likely to develop ectopic tissue mineralization can be treated as described herein. Examples of mammals that can have or be likely to develop ectopic tissue mineralization and can be treated as described herein include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. In some cases, a human having ectopic tissue mineralization can be treated as described herein. In some cases, a human likely to develop ectopic tissue mineralization can be treated as described herein.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0075] When treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can be associated with a disease or condition. Examples of diseases and conditions that can be associated with ectopic tissue mineralization and treated as described herein to reduce ectopic tissue mineralization and / or to slow ectopic tissue mineralization progression include, without limitation, age-associated tendinous degeneration and calcification, chronic kidney disease, atherosclerosis, aortic valve stenosis, osteoarthritis, calcific tendinitis, diabetes-associated calcific tendinitis, hypothyroidism-associated calcific tendinitis, ligamentous calcification, myositis ossificans, pseudoxanthoma elasticum, Keutel syndrome, heterotopic ossification, heterotopic calcification, x-linked hypophosphatemia, fibrodysplasia ossificans progressiva, ankylosing spondylitis, diffuse idiopathic skeletal hyperostosis, hypertrophic osteoarthritis, Paget’s disease, diabetes, post-traumatic arthritis, osteonecrosis, rheumatoid arthritis, vitamin and mineral imbalances, accelerated aging conditions, Hutchinson-Gilford syndrome. Werner syndrome, and radiation-induced soft tissue calcification of tendons and other connective tissues. Ectopic tissue mineralization, particularly ectopic ossification, may be associated with previous tissue trauma or injury, for example, following surgery or traumatic blast injuries commonly seen in warfare.

[0076] When treating a mammal (e.g.. a human) having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can be present in any appropriate tissue within the mammal. Examples of tissues that can undergo ectopic tissue mineralization and that can be treated as described herein include, without limitation, skin, cartilage, muscle (e.g., skeletal muscle such as quadricep, gastrocnemius, bicep, or hamstring), tendons, joints, valves, vessels, arteries, brain and nervous system tissue, ligaments, calcific bursitis, and other soft tissues. In some cases, when treating a mammal having or likely to develop ectopic tissue mineralization as described herein, the ectopic tissue mineralization can be present in an organ within the mammal. Examples of organs that can undergo ectopic tissue mineralization can include, without limitation, heart, kidney, lung, breast, liver, and brain.

[0077] Any appropriate method can be used to identify a mammal (e.g., a human) as being a mammal having ectopic tissue mineralization. For example, urinalysis, bloodAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0078] tests, x-ray, computed tomography scanning, fluoroscopic x-ray imaging, ultrasound imaging, physical palpation, self-reported symptoms consistent with mineralization (e.g., pain with movement), and / or other clinical assessments can be used to identify a mammal (e.g., a human) as being a mammal having ectopic tissue mineralization. Any appropriate method can be used to identify a mammal (e.g., a human) as being a mammal likely to develop ectopic tissue mineralization. For example, patient questioning or review of a patient’s medical history, recent tissue injury, chronic joint inflammation, systemic inflammatory conditions, and / or other clinical risk factors associated with soft tissue mineralization can be used to identify a mammal (e.g., a human) as being a mammal likely to develop ectopic tissue mineralization.

[0079] In some cases, one or more (e.g.. one, two. three, four, or more) sGC agonists of Formula I, Group I, and / or Group II can be administered to a mammal having ectopic tissue mineralization (or likely to develop ectopic tissue mineralization) to reduce ectopic tissue mineralization fonnation by delaying or preventing the formation of ectopic tissue mineralization within the mammal. For example, one or more sGC agonists of Formula 1. Group I, and / or Group II can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or likely to develop ectopic tissue mineralization) to reduce the formation of ectopic tissue mineralization within the mammal. In some cases, the methods and materials described herein can be effective to delay the formation of ectopic tissue mineralization within a mammal by, for example, at least 6 months (e.g., at least about 7 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 2.5 years, at least about 3 years, at least about 4 years, at least about 5 years, 10 to 20 years, or for the remaining lifetime of the mammal being treated). In some cases, the formation of ectopic tissue mineralization within a mammal being treated as described herein (e.g., by administration of one or more sGC agonists of Formula I, Group I, and / or Group II) can be delayed for as long as the mammal is administered one or more sGC agonists of Formula I, Group I, and / or Group II.

[0080] In some cases, one or more (e g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II can be administered to a mammal having ectopic tissue mineralization to slow progression of the ectopic tissue mineralization. For example, one or more sGC agonists of Formula I, Group I, and / or Group II can beAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0081] administered to a mammal (e.g., a human) in need thereof (e.g., a human having ectopic tissue mineralization) to slow progression (e.g., to slow further growth) of that ectopic tissue mineralization within the mammal. In some cases, the methods and materials described herein can be effective to slow progression of ectopic tissue mineralization within a mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the progression of existing ectopic tissue mineralization within a mammal being treated as described herein (e.g.. by administration of one or more sGC agonists of Formula I, Group I, and / or Group II) can be reduced to no observable growth for as long as the mammal is administered one or more sGC agonists of Formula I, Group I, and / or Group II.

[0082] Any appropriate sGC agonist of Formula I, Group I. and / or Group II can be administered to a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein.

[0083] In some cases, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be administered or instructed to self-administer one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II. For example, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be administered a composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I. Group I, and / or Group II to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein. In some cases, a mammal (e.g., a human) having (or likely to develop) ectopic tissue mineralization can be instructed to self-administer a composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein.

[0084] In some cases, when a composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I. Group I, and / or Group II is administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization, the composition can be designed for oral or parenteral (including, without limitation, subcutaneous, intramuscular, intravenous, intradermal, intracerebral, intrathecal, intraabdominal, and intraperitoneal injections) administration toAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0085] the mammal. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and nonaqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the fonnulation isotonic with the blood of the intended recipient. In some cases, a pharmaceutically acceptable composition including one or more sGC agonists of Formula I. Group I, and / or Group II can be administered locally or systemically. For example, a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II can be administered systemically by injection to a mammal (e.g., a human). In some cases, each sGC agonist of Formula I. Group I, and / or Group II to be administered when two or more sGC agonists of Formula I, Group I, and / or Group II are to be administered can be administered by the same or different routes. For example, one sGC agonist of Formula I, Group I, and / or Group II can be administered orally, and a second sGC agonist of Formula I. Group I, and / or Group II can be administered by injection.

[0086] In some cases, a composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II can be in the form of a sterile injectable suspension (e.g., a sterile injectable aqueous or oleaginous suspension). This suspension may be fonnulated using, for example, suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Examples of acceptable vehicles and solvents that can be used include, without limitation, saline, mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In some cases, sterile, fixed oils can be used as a solvent or suspending medium. In some cases, a bland fixed oil can be used such as synthetic mono- or di-glycerides.

[0087] When one or more (e.g., one. two, three, four, or more) sGC agonists of Fonnula I, Group I, and / or Group II are formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e g., a human) having or likely to develop ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein, the composition can deliver the sGC agonist(s) of Formula I.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0088] Group I, and / or Group II in any manner. In some cases, a composition including one or more sGC agonists of Formula I, Group I, and / or Group II can be an immediate release composition (e.g., an immediate release oral dosage form). In some cases, a composition including one or more sGC agonists of Formula I, Group I, and / or Group II can be a controlled (e.g., delayed and / or sustained) release compositions (e.g., a controlled release oral dosage form). For example, a controlled release composition including one or more sGC agonists of Formula I. Group I, and / or Group II can be designed to release the one or more sGC agonists of Formula I, Group I, and / or Group II over 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0089] When designing a composition (e.g., a pharmacally acceptable composition) containing one or more sGC agonists of Formula I, Group I, and / or Group II for administration to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization to reduce ectopic tissue mineralization formation and / or slow ectopic tissue mineralization progression as described herein, the composition (e.g., pharmaceutically acceptable composition) can include any appropriate effective amount of the one or more sGC agonists of Formula 1, Group I, and / or Group II. For example, a therapeutically effective amount of one or more sGC agonists of Formula I, Group I, and / or Group II can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents. Examples of pharmacally acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, cyclodextrins (e.g., betacyclodextrins such as KLEPTOSE®), dimethylsulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil. serumAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0090] proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g.. saline such as phosphate buffered saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and com oil. If required, the solubility and bioavailability of the one or more sGC agonists of Formula I, Group I, and / or Group II in a pharmaceutically acceptable composition can be enhanced using lipid excipients and / or block copolymers of ethylene oxide and propylene oxide. See, e g., U. S. Patent No. 7,014,866 and U. S. Patent Application Publication Nos. 2006 / 0094744 and 2006 / 0079502.

[0091] In some cases, a composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II can be designed to be in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.

[0092] One or more sGC agonists of Formula I, Group I, and / or Group II (e.g., a composition containing one or more sGC agonists of Formula 1, Group I, and / or Group II) can be administered to a mammal in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to reduce formation and / or to slow progression of ectopic tissue mineralization).

[0093] An effective amount of a sGC agonist of Formula I, Group I, and / or Group II that can be administered to a mammal as described herein can be any amount that reduces formation of ectopic tissue mineralization within the mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. For example, an effective amount of a sGC agonist of Formula I, Group I, and / or Group II can be from about 0.0001 mg of sGC agonist / kg of body weight to about 8000 mg of sGC agonist / kg of body weight (e.g., from about 0.0001 mg / kg to about 5000 mg / kg, from about 0.0001 mg / kg to about 1000 mg / kg, from about 0.0001 mg / kg to about 500 mg / kg, from about 0.0001 mg / kg to about 100 mg / kg, from about 0.0001 mg / kg to about 50 mg / kg. from about 0.0001 mg / kg to about 20 mg / kg, from about 0.0001 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.1 mg / kg, from about 0.0001 mg / kg to about 0.01 mg / kg, from about 0.0001 mg / kg to about 0.001 mg / kg, from about 0.001 mg / kg to about 8000 mg / kg. from about 0.01 mg / kg to aboutAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0094] 8000 mg / kg, from about 0.1 mg / kg to about 8000 mg / kg, from about 1 mg / kg to about 8000 mg / kg, from about 10 mg / kg to about 8000 mg / kg, from about 20 mg / kg to about 8000 mg / kg, from about 50 mg / kg to about 8000 mg / kg, from about 100 mg / kg to about 8000 mg / kg, from about 500 mg / kg to about 8000 mg / kg, from about 1000 mg / kg to about 8000 mg / kg, from about 5000 mg / kg to about 8000 mg / kg, from about 0.001 mg / kg to about 5000 mg / kg, from about 0.01 mg / kg to about 1000 mg / kg, from about 0.1 mg / kg to about 500 mg / kg, from about 1 mg / kg to about 100 mg / kg, from about 10 mg / kg to about 50 mg / kg, from about 0.0001 mg / kg to about 0.01 mg / kg, from about 0.001 mg / kg to about 0.1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 20 mg / kg, from about 10 mg / kg to about 50 mg / kg, from about 20 mg / kg to about 100 mg / kg. from about 50 mg / kg to about 500 mg / kg, from about 100 mg / kg to about 1000 mg / kg, from about 500 mg / kg to about 5000 mg / kg, or from about 1000 mg / kg to about 8000 mg / kg). In some cases, between about 0.1 mg and 200 mg (e.g., between about 0.1 mg and 150 mg, between about 0.1 mg and 100 mg. between about 0.1 mg and 50 mg, between about 0.1 mg and 20 mg, between about 0.1 mg and 10 mg, between about 0.1 mg and 5 mg, between about 0.1 mg and 1 mg, between about 1 mg and 200 mg, between about 5 mg and 200 mg, between about 10 mg and 200 mg, between about 20 mg and 200 mg, between about 50 mg and 200 mg, between about 100 mg and 200 mg, between about 150 mg and 200 mg, between about 1 mg and 150 mg, between about 5 mg and 100 mg, between about 10 mg and 50 mg, between about 0.1 mg and 5 mg, betw een about 1 mg and 10 mg, betw een about 5 mg and 20 mg, between about 10 mg and 50 mg, betw een about 20 mg and 100 mg, between about 50 mg and 150 mg, or between about 100 mg and 200 mg) of a sGC agonist of Formula I, Group I, and / or Group II can be administered to an average sized human (e.g., about 70 kg human) daily for about 20 weeks. If a particular mammal fails to respond to a particular amount, then the amount of sGC agonist of Formula I, Group I, and / or Group II can be increased by, for example, two-fold. After receiving this higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. Reciprocally, if a mammal experiences side effects at an initial dose (e.g., blood pressure lowering), the amount of sGC agonist of Fonmila I, Group I, and / or Group II can be decreased while still monitoring for efficacy with regards to ectopic tissue mineralization. TheAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0095] effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., ectopic tissue mineralization formation and / or progression) can require an increase or decrease in the actual effective amount administered.

[0096] A composition containing one or more (e g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II can be administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization in any appropriate frequency. The frequency of administration can be any frequency that reduces formation of ectopic tissue mineralization within the mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about three times a day. or from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II can include rest periods. For example, a composition containing one or more sGC agonists of Formula I. Group I, and / or Group II can be administered daily over a two week period followed by a two week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., ectopic tissue mineralization formation and / or progression) can require an increase or decrease in administration frequency.

[0097] A composition containing one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I. and / or Group II can be administered to a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization for any appropriate duration. An effective duration for administering a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II can be any duration that reduces fonnation of ectopic tissue mineralization within theAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0098] mammal and / or slows progression of ectopic tissue mineralization within the mammal without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several weeks, months, or years. In general, the effective duration for the treatment of ectopic tissue mineralization as described herein can range in duration from several months to several years. In some cases, an effective duration can be for as long as an individual mammal is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e.g., ectopic tissue mineralization formation and / or progression) being treated.

[0099] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein (e.g., by administering one or more sGC agonists of Formula I, Group I, and / or Group II) can include administering to the mammal the one or more (e.g., one, two, three, four, or more) sGC agonists of Formula 1, Group I, and / or Group II as the sole active ingredient(s) to treat ectopic tissue mineralization formation and / or progression within the mammal. For example, a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II can include the one or more sGC agonists of Formula I, Group I, and / or Group II as the sole active ingredient in the composition that is effective to reduce formation of ectopic tissue mineralization within the mammal and / or to slow progression of ectopic tissue mineralization within the mammal.

[0100] In some cases, methods for treating a mammal (e.g., a human) having or likely to develop ectopic tissue mineralization as described herein (e.g., by administering one or more sGC agonists of Formula I, Group I, and / or Group II) can include administering to the mammal the one or more (e.g., one, two, three, four, or more) sGC agonists of Formula I, Group I, and / or Group II in combination with one or more cGMP-specific phosphodiesterase 5A (PDE5A) inhibitors. In some cases, the one or more sGC agonists of Formula I. Group I, and / or Group II can be administered together as a single composition with the one or more PDE5A inhibitors. In some cases, the one or more sGC agonists of Formula I, Group I, and / or Group II and the one more PDE5A inhibitors can be administered to the mammal being treated at the same time as separate compositions. In some cases, the one or more sGC agonists ofAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0101] Formula I, Group I, and / or Group II can be administered to the mammal first followed by the one more PDE5A inhibitors. In some cases, the one more PDE5A inhibitors can be administered to the mammal first followed by the one or more sGC agonists of Formula I, Group I, and / or Group II. Examples of PDE5A inhibitors include, without limitation, sildenafil, vardenafil, tadalafil, EMD 360527, DA 8159, UK-343-664 (Walker et al., Xenobiotica, 31:651-664 (2001)), UK-427-387, UK-357903 ([1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-pyrazolo[4.3-d]pyri mi din-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulphonyl}piperazine]) (Gardiner et al., J.

[0102] Pharmacol. Exp. Ther., 312:265-271 (2005)), UK-371800 (Pfizer), UK-313794 (Pfizer), UK-343664 (Abel et al., Xenobiotica, 31:665-76 (2001)), TA-1790 (Tanabe Seiyaku). CP-248 (Osi Pharmaceuticals), CP-461 (Osi Pharmacals), exisulind (Deguchi et al., Mol. Cancer Thcr.. 803-809 (2002); (Osi Pharmaceuticals)), pyrazolinone, EMD82639 (Merck KgaA, Darmstadt, DE; (4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hydro-pyrazol-l-yl)-benzoic acid; Senzaki et a., FASEB J.. 15:1718-1726 (2001), and Scutt et al.. BMC Pharmacol., 4:10 (2004)). EMD360527 (Merck KgaA. Darmstadt. DE; [7-(3-Chloro-4-methoxy-benzylamino)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-5-ylmethoxy]-acetic acid; Scutt et al., BMC Pharmacol., 4: 10 (2004)), EMD221829 (Merck KgaA, Dannstadt, DE; 4-[4-(3-Chloro-4-methoxy-benzylamino)-benzo[4,5]thieno[2,3-d]-pyrimidin-2-yl]-cyclohexanecarboxylic acid, ethanolamin salt; Scutt et al., BMC Pharmacol., 4:10 (2004)), EMD1 71827 (Merck KgaA, Darmstadt, DE; 5-[4-(3-Chloro-4-methoxy-benzylamino)-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimi din-2 -yl] -pentanoic acid; Scutt et al., BMC Pharmacol., 4:10 (2004)), DA-8259 (3-(l-Methyl-7-oxo-3-propyl-6,7-dihydro-lH-pyrazolo-[4,3-d]pyrimidin-5-yl)-N-|2-(l-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide), E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med., 160:858-865 (1999)), pentoxifylline, and FR22934 (Fujisawa). Additional examples of PDE5A inhibitors can be set forth in U. S. PatentNos: 6.916,927, 6,911.542, 6,903,099, 6,878,711, 6,872,721, 6.858,620, 6,825,197, 6.774,128, 6,723.719, 6,699,870. 6,670,366, 5,859,006, and 5.250,534 and International Patent Application Publication No. WO 03 / 063875, WO 03 / 1012761, WO 2004 / 037183, and WO 98 / 38168.

[0103] In some cases, a course of treatment and the severity of ectopic tissue mineralization formation and / or progression can be monitored. Any appropriateAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0104] method can be used to detennine whether or not treatment with one or more sGC agonists of Formula I, Group I, and / or Group II as described herein is effective to reduce ectopic tissue mineralization formation and / or to slow ectopic tissue mineralization progression within the mammal being treated. For example, ectopic tissue mineralization progression during a course of treatment described herein can be assessed using imaging techniques at different time points.

[0105] This document also provides sGC agonists of Formula I, Group I. and / or Group II and compositions containing one or more sGC agonists of Formula I, Group I, and / or Group II. The sGC agonists of Group I are GPHR312770, GPHR312771, GPHR312772, GPHR312773, GPHR312853, GPHR312854, GPHR312855, GPHR312856, GPHR312858, Compound Al. Compound A2, Compound A3, Compound A4, Compound A5, and Compound A6. The sGC agonists of Group II are GPHR312768, GPHR312769, GPHR312844, GPHR312845, GPHR312846, GPHR312847, GPHR312848, GPHR312849, GPHR312850, GPHR312857, GPHR312859, Compound Bl, Compound B2, Compound B3, Compound B4, Compound B5, and Compound B6. In some cases, a composition containing one or more sGC agonists of Formula I, Group I, and / or Group II can include one or more PDE5A inhibitors.

[0106] Compounds of Formula (I)

[0107] This document also provides compounds of Formula (I).

[0108]

[0109] or a pharmaceutically acceptable salt thereof, wherein:

[0110] R1is selected from NH2, morpholinyl, piperidinyl, pyrrolidinyL and 2-oxa-6-azaspiro[3.3]heptanyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, OH, C1-3 alkoxy, C1-3 haloalkyl, C1-3 haloalkoxy, COOH, and CH2COOH;Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0111] X1is a 5-membereed heteroaryl, optionally substituted with halo, OH, C1-3 alkyl. C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy;

[0112] R2is selected from H, C(=O)Ci-3 alkyl, C(=O)Ci-3 haloalkyl, and S(=O)2R4; R4is selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl. and C1-3 haloalkoxy;

[0113] n is 1, 2, or 3; and

[0114] each R3is independently selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy.

[0115] In some embodiments, R1is NH2.

[0116] In some embodiments, R1is selected from morpholinyl, piperidinyl, pyrrolidinyl, and 2-oxa-6-azaspiro[3.3]heptanyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, OH, C1-3 alkoxy, C1-3 haloalkyl, C1-3 haloalkoxy, COOH, and CH2COOH.

[0117] In some embodiments, R1is morpholinyl. In some embodiments, R1is piperidinyl, optionally substituted with COOH or CH2COOH. In some embodiments, R1is pyrrolidinyl, optionally substituted with OH. In some embodiments, R1is 2-oxa-6-azaspiro[3.3]heptanyl.

[0118] In some embodiments, the compound of Formula I has formula:

[0119]

[0120] or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, X1is a 5-membereed heteroaryl. In some embodiments, X1is selected from 1,2,3-triazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-oxadiazolyl, and 1,3,4-thiadiazolyl,

[0122] In some embodiments, the compound of Formula I has formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0123]

[0124] or a pharmaceutically acceptable salt thereof.

[0125] In some embodiments, the compound of Formula I has formula:

[0126]

[0127] or a pharmaceutically acceptable salt thereof.

[0128] In some embodiments, R2is H. In some embodiments, R2is selected from C(=O)Ci-3 alkyl and C(=O)Ci-3 haloalky 1. In some embodiments, R2is C(=O)Ci-3 alkyl. In some embodiments, R2is C(=O)Ci-3 haloalkyl.

[0129] In some embodiments, R2is S(=O)2R4.

[0130] In some embodiments, the compound of Formula I has formula:

[0131]

[0132] or a pharmaceutically acceptable salt thereof.

[0133] In some embodiments, R4is selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, and C1.3 alkoxy. In some embodiments. R4is selected from thiophenyl and phenyl, each of which is optionally substituted with halo, CN, or C1-3 alkoxy.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0134] In some embodiments, R4is thiophenyl, optionally substituted with halo. In some embodiments, R4is phenyl, optionally substituted with halo, CN, or C1.3 alkoxy.

[0135] In some embodiments, the compound of Formula I has formula:

[0136]

[0137] or a pharmaceutically acceptable salt thereof.

[0138] In some embodiments, n is 1. In some embodiments, n is 1 or 2. In some embodiments, n is 2. In some embodiments, n is 2 or 3. In some embodiments, n is 3.

[0139] In some embodiments, R3is selected from halo, C1-3 alkyl, and C1-3 alkoxy. In some embodiments, R3is selected from halo and C1-3 alkoxy.

[0140] In some embodiments, R3is halo. In some embodiments, R3is C1-3 alkoxy. In some embodiments, n is 1 and R3is halo. In some embodiments, n is 2 and each R3is halo. In some embodiments, n is 2, one R3is halo, and the other R3is C1-3 alkoxy. In some embodiments, n is 2 and each R3is C1-3 alkoxy.

[0141] In some embodiments, the compound of Formula I has fonnula:

[0142]

[0143] or a pharmaceutically acceptable salt thereof.

[0144] In some embodiments, the compound of Formula I has formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0145]

[0146] or a pharmaceutically acceptable salt thereof.

[0147] In some embodiments, the compound of Formula I is selected from any one of the following compounds:

[0148]

[0149] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0150]

[0151] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0152]

[0153]

[0154] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0155]

[0156] (Compound A6), or a pharmaceutically acceptable salt thereof.

[0157] In some embodiments, this document provides a compound of formula:

[0158]

[0159] or a pharmaceutically acceptable salt thereof.

[0160] In some embodiments, this document provides a compound of formula:

[0161]

[0162] or a pharmaceutically acceptable salt thereof.

[0163] In some embodiments, this document provides a compound of formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0164]

[0165] or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, this document provides a compound of formula:

[0167]

[0168] or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments, this document provides a compound of formula:

[0170]

[0171] or a pharmaceutically acceptable salt thereof.

[0172] In some embodiments, this document provides a compound of formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0173]

[0174] or a pharmaceutically acceptable salt thereof.

[0175] In some embodiments, this document provides a compound of formula:

[0176]

[0177] or a pharmaceutically acceptable salt thereof.

[0178] In some embodiments, this document provides a compound of formula:

[0179]

[0180] (GPHR312849), or a pharmaceutically acceptable salt thereof.

[0181] In some embodiments, this document provides a compound of formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0182]

[0183] or a pharmaceutically acceptable salt thereof.

[0184] In some embodiments, this document provides a compound of formula:

[0185]

[0186] or a pharmaceutically acceptable salt thereof.

[0187] In some embodiments, this document provides a compound of formula:

[0188]

[0189] or a pharmaceutically acceptable salt thereof.

[0190] In some embodiments, this document provides a compound of formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0191]

[0192] or a pharmaceutically acceptable salt thereof.

[0193] In some embodiments, this document provides a compound of formula:

[0194]

[0195] or a pharmaceutically acceptable salt thereof.

[0196] In some embodiments, this document provides a compound of formula:

[0197]

[0198] or a pharmaceutically acceptable salt thereof.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0199] In some embodiments, this document provides a compound of formula:

[0200]

[0201] or a pharmaceutically acceptable salt thereof.

[0202] In some embodiments, this document provides a compound of formula:

[0203]

[0204] or a pharmaceutically acceptable salt thereof.

[0205] In some embodiments, this document provides a compound of formula:

[0206]

[0207] or a pharmaceutically acceptable salt thereof.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0208] In some embodiments, a salt of a compound of Formula I, Group I, and / or Group II can be formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group, for use as described herein. In some cases, a sGC agonist of Formula I, Group I, and / or Group II can be a pharmaceutically acceptable acid addition salt of a sGC agonist of Formula I, Group I, and / or Group II.

[0209] In some cases, acids commonly used to form pharmaceutically acceptable salts of a sGC agonist of Formula I, Group I, and / or Group II can include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as paratoluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, capry late, acry late, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4-dioate, hexyne-1.6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, pheny lbutyrate, citrate, lactate, -hydroxybutyrate, gly colate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthal ene-1 -sulfonate, naphthalene-2- sulfonate, mandelate and other salts. In some cases, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

[0210] In some cases, bases commonly used to form pharmaceutically acceptable salts of a sGC agonist of Formula I, Group I, and / or Group II can include hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0211] substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(Cl-C6)-alkylamine), such as N, N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. In some cases, a compound of Formula I, Group I, and / or Group II, or a pharmaceutically acceptable salt thereof, can be substantially isolated.

[0212] A sGC agonist of Formula I, Group I, and / or Group II, including salts thereof, can be prepared using organic synthesis techniques and can be synthesized according to any of numerous possible sy nthetic routes. For example, appropriate synthetic protocols can be selected and implemented to synthesize the sGC agonists of Formula I, Group I, and / or Group II described herein.

[0213] Suitable synthetic methods of starting materials, intermediates and products can be identified by reference to the literature, including reference sources such as; Advances in Heterocyclic Chemistry. Vol. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vol. 1-49 (J. Heterocyclic Chem., 1964-2012); Carreira et al. (Ed.) Science of Synthesis, Vol. 1-48 (2001-2010) and Knowledge Updates KU2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012); Katritzky et al. (Ed.) Comprehensive Organic Functional Group Transfomiations, (Pergamon Press, 1996); Katritzky et al. (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, 2nd Edition, 2004); Katritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984); Katritzky et al., Comprehensive Heterocyclic Chemistry' II. (Pergamon Press, 1996); Smith et al., March’s Advanced Organic Chemistry; Reactions. Mechanisms, and Structure, 6th Ed. (Wiley, 2007); and Trost et al. (Ed.). Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0214] The reactions for preparing the compounds of Formula I, Group I, and / or Group II provided herein can be carried out in suitable solvents that can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e g., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature. A given reaction can be carried out in one solvent or a mixture of moreAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0215] than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

[0216] Preparation of the compounds of Formula I, Group I, and / or Group II provided herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed Wiley & Sons, Inc., New York (2006).

[0217] Selected definitions

[0218] As used herein, the term "‘about” means “approximately” (e.g., plus or minus approximately 10% of the indicated value).

[0219] At various places in this document, substituents of compounds of Formula I, Group I, and / or Group II are disclosed in groups or in ranges. It is specifically intended that the subject matter include each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and Ce alkyl.

[0220] As used herein, the phrase “optionally substituted” means unsubstituted or substituted. The substituents are independently selected, and substitution may be at any chemically accessible position. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms. It is to be understood that substitution at a given atom is limited by valency.

[0221] Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C1.4, Ci-6, and the like.

[0222] As used herein, the term “Cn-m alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, w-propyl, isopropyl, / 7-butyl. tert-butyl, isobutyl, sec-butyl: higher homologs such as 2-methyl-l -butyl, n-pentyl, 3-pentyl, «-hexyl, 1,2,2-trimethylpropyl. and the like. In some embodiments, the alkylAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0223] group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0224] As used herein, the term “Cn-mhaloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+ 1 halogen atoms which may be the same or different, where “s’" is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0225] As used herein, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula -O-alkyl, wherein the alky l group has n to m carbons. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., / 7-propox and isopropoxy), butoxy (e.g., / 7-buto.xy and tertbutoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0226] As used herein, “Cn-mhaloalkoxy” refers to a group of formula -O-haloalkyl having n to m carbon atoms. An example haloalkoxy group is OCF3. In some embodiments, the haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0227] As used herein, “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl. or Br. As used herein, the term “oxo” refers to an oxygen atom as a divalent substituent, forming a carbonyl group when attached to a carbon (e.g., C=O), or attached to a heteroatom forming a sulfoxide or sulfone group.

[0228] The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0229] The compounds of Formula I, Group I, and / or Group II described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of Formula I, Group I, and / or Group II that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. ManyAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0230] geometric isomers of olefins, C=N double bonds, N=N double bonds, and the like can also be present in the compounds of Formula I, Group I, and / or Group II described herein, and all such stable isomers are contemplated. Cis and trans geometric isomers of the compounds of Formula I, Group I, and / or Group II provided herein are described and may be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound of Formula I, Group I, and / or Group II has the / / / / -configuration. In some embodiments, the compound of Formula I, Group I. and / or Group II has the / S / -con figuration.

[0231] Compounds of Formula I, Group I, and / or Group II provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam -lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole.

[0232] Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0233] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0234] EXAMPLES

[0235] Example 1 - Increases in DP AHI are adaptive in aortic valve disease This example identifies increases in DDAH1, a modulator of nitric oxide signaling, per se as adaptive in aortic disease and protective against fundamental molecular processes that drive osteogenesis, valvular calcification, and stenosis.

[0236] Human tissue samples

[0237] Non-calcified, non-stenotic human aortic valve tissue was acquired from explanted hearts at the time of transplant or from hearts not suitable for transplant, as described previously (Hagler et al., Cardiovasc. Res., 99:175-184 (2013)). CalcifiedAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0238] valve tissue was acquired from patients with severe aortic valve stenosis undergoing aortic valve replacement surgery.

[0239] Non-atheroscl erotic aortic tissue samples were obtained from explanted hearts that were not suitable for transplant. Aortic tissue samples from patients with severe atherosclerotic heart disease were obtained from patients undergoing coronary7artery7bypass surgery (i.e., tissue punches removed for initiation of anastomosis on ascending aorta).

[0240] Animals

[0241] To experimentally alter DDAH1 levels, we used low density7lipoprotein receptor- deficient, apolipoprotein BlOO-only (LA) mice that were either wild-type (D+ +) or heterozygous (D ) for DDAH1. Mice were studied following six months of Western diet feeding (TD88137, Harlan Teklad). Prior work from our group suggests that this model faithfully recaptures multiple phenoty pic and molecular changes observed in tissues from aged humans with end-stage disease (Miller et al., Arterioscler. Thromb. Vase. Biol., 33:459-465 (2013); and Weiss et al.. Circulation, 114:2065-2069 (2006)). Littermate-matched pairs were studied at each time point whenever possible. Animals were euthanized using intraperitoneal injection of pentobarbital sodium (>100 mg / kg).

[0242] Cell culture

[0243] Aortic valves interstitial cells from C57BL / 6 mice (pooled in groups of three) were harvested. All three valve leaflets were used for cell culture. As described in previous study (Hagler et al., supra), aortic valves were pooled in keeping with standard culture techniques to ensure the growth of a large and sufficient number of cells for quantitative assays. Valvular cells were grown in complete media using standard outgrowth techniques (40% Dulbecco’s modified Eagles medium, 40% Ham's F-10, 20% fetal bovine serum, 100 U / mL penicillin and 100 pg / mL streptomycin) between passages 6 and 8, and harvested at 90-100% confluence. Cells were treated separately for different concentration of ADMA (50 pmol / L) and PD404182 (50 pmol / L) or normal saline as a control. Tw enty-four hour treatments were used to represent a standard duration at which transcriptional changes can be observed.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0244] Quantitative Real-Time RT-PCR

[0245] Quantitative real-time PCR was performed on a StepOne Plus RT-PCR machine (Applied Biosystems). Gene expression for DDAH isoforms, ADMA transporters (SLC7A2), osteogenic pathways, and USF-1 related genes were measured using TaqMan Gene Expression Assay primers (Life Technologies). Gene expression levels were normalized by GADPH and hypoxanthine phosphoribosyltransferase 1 (HPRT1), both commonly used housekeeper genes and expressed using the AACt method.

[0246] Table 1. Primer sets used for gene expression assays. All primer sets were purchased from Life Technologies.

[0247]

[0248] Circulating glucose and ADMA levels

[0249] Mice were fasted for 12 hours prior to euthanasia. Whole blood glucose was measured at the time of sacrifice using a point-of-care device (AccuChek). Plasma samples were collected at the time of sacrifice and stored at -80°C. Plasma ADMA concentration was measured using ELISA / EIA kits (Immundiagnostik AG).

[0250]

[0251] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0252] Cardiac and aortic valve function (in vivo)

[0253] Left ventricular ejection fraction, aortic valve cusp separation distance, and peak transvalvular velocity were assessed under light isoflurane anesthesia using echocardiography (GE Vivid 7 ultrasound system, 13 MHz linear probe) as described previously (Casaclang-Verzosa et al., J Vis Exp 120:54110 (2017)).

[0254] In brief, echocardiography was performed on all animals using 2-D. M-mode, pulse- wave and tissue Doppler imaging techniques. Each mouse was sedated using 1.5% isoflurane delivered via a nose cone, while keeping heart rate above 450 bpm. Left ventricular ejection fraction, fractional shortening and LV mass were calculated from the maximal diastolic and systolic dimensions obtained using M-mode imaging.

[0255] Aortic valve cusp separation distance was measured using M-mode imaging. Peak aortic velocity was obtained from Doppler flow velocity spectral display. The mitral, tricuspid and pulmonic valves were assessed using color flow imaging.

[0256] Blood pressure (in vivo)

[0257] Systolic blood pressure was measured non-invasively at six month time point using a tail -cuff method (CODA system, Kent Scientific). More specifically, blood pressure was measured for 5 consecutive days, with the first 3 days considered to be “acclimatization” to the stress of gentle restraint, and the mean of systolic blood pressure measurements during the last two days being used for data analysis.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0258] RNA Extraction and cDNA Synthesis

[0259] Thoracic aortic arch tissue was carefully taken from test animal with perivascular adipose tissue removed, homogenized and stored in TRIzol regent (Invitrogen). Aortic valve tissue were carefully dissected and stored in TRIzol reagent. All aortic and aortic valve tissue was stored at -80°C prior to RNA extraction. RNA samples were isolated using spin columns and chloroform extractions (Ambion), as described previously (Hagler et al., supra).

[0260] Vasomotor function

[0261] Rings from descending thoracic aorta were used for studies of vasomotor function, as described previously (Roos et al., J. Physiol. Heart Circ. Physiol., 305: H1428-1439 (2013)). Responses to serotonin, acetylcholine, sodium nitroprusside, and prostaglandin F2alpha (PGF-2a) were evaluated in all mice.

[0262] Immunohistochemistry

[0263] Murine descending thoracic aorta (1-2 mm length), aortic valve, and kidney were embedded in tissue freezing medium (Triangle Biomedical Sciences). Crosssections of the frozen embedded tissue were subsequently cryosectioned (10 pm thickness), and used for histological and immunohistochemical analyses. Osterix (1:500 dilution), USF-1 (1:200 dilution), and pSMADl / 5 / 8 (1:100 dilution. Cell Signaling Technology) primary antibodies were used on individual slides and costained with species matched Alexa Fluor 647 secondary antibodies. Primary antibody incubation duration was 24 hours for USF-1 and pSMADl / 5 / 8 experiments (4° C) and one hour incubation for osterix (room temperature), and secondary antibody incubation duration was 1 hour (room temperature). Sections were mounted in ProLong Gold DAPI solution, and then imaged on a Zeiss 780 LSM confocal microscope at 20x magnification. Subsequently, images were quantified and analyzed using the software ImageJ (NIH).Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0264] Table 2. Antibodies used for immunohistochemistry / immunofluorescence experiments. All antibody sets were purchased from Life Technologies.

[0265]

[0266] *CST, Cell Signaling Technology; LT, Life Technologies

[0267] Western blotting

[0268] Protein was obtained from lysed valvular and aortic cells. Gel electrophoresis was carried out using Bis-Tris 4-12% gels under reducing conditions, and subsequently transferred to nitrocellulose membranes. Membrane incubation with anti- USF-1, and anti-HPRTl was performed at dilutions of 1: 1000 and for 18 h at 4°C. Thereafter, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody for 1.5 hours at room temperature (1:7500). Following the addition of chemiluminescent substrate, bands w ere detected by densitometric analysis performed using digital imaging techniques (Fluorochem M) and quantified using Cell Biosystems software. The density of pSMAD2 and pSMADl / 5 / 8 protein bands was normalized to housekeeper, hypoxanthine phosphoribosyltransferase 1 (HPRT1). Quantitation w as carried out using AlphaView software (ProteinSimple™).

[0269] Table 3. Antibodies used for western blotting experiments. All antibody sets were purchased from Life Technologies.

[0270]

[0271] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0272] Statistical analyses

[0273] Between-group differences were detected using unpaired, Bonferrom-corrected t-tests in GraphPad Prism Software. All data are expressed as mean ± SE.

[0274] Results

[0275] To determine whether ADMA synthesizing or hydrolyzing enzymes were altered in diseased tissue from aged humans with end-stage calcific aortic valve stenosis, RT-qPCR was performed on samples acquired from patients undergoing valve replacement surgery. Compared to normal valves, only expression of DDAH1 was significantly increased (Figure 1A) in valve tissue from patients with CAVS. whereas expression of the ADMA-synthesizing enzyme PRMT1 was reduced in calcified valve tissue (Figure 1 A). In the context of the working hypothesis, these changes would be expected to be adaptive and protect against increases in intracellular ADMA (Morales et al., J. Biol. Chem., 290:14915-14926 (2015); and Pope et al., Pharmacol. Res., 60:461-465 (2009)). In contrast, expression of SLC7A2 — a key ADMA transporter was increased in calcified valve tissue compared to non-calcified control samples (Kawahara et al., Brain Res. Mol. Brain Res., 90: 165-173 (2001); and Yeramian et al., J. Immunol., 176:5918-5924 (2006)) (Figure 1), which we propose would be maladaptive and result in deleterious increases in cytosolic ADMA (Strobel et al, Amino Acids, 45:989-1002 (2013)).

[0276] To determine the mechanistic contribution of DDAH1 in the evolution of calcific aortic valve disease, low density lipoprotein receptor-deficient / apolipoprotein BlOO-only mice (a previously validated model of valvular stenosis) were generated that were deficient in one copy of DDAH1 (LA-D ). Consistent with previous reports (Hu et al., Circulation, 120:2222-2229 (2009)), these mice had significantly elevated plasma ADMA levels, increased blood pressure, and preserved left ventricular function.

[0277] In line with the working hypothesis, valvular calcification (Figure 2A) and valvular dysfunction (Figure 2B) were significantly increased in LA-D+' mice compared to their LA-D+ / +littermates. These increases in valvular calcification and valvular dysfunction were associated with increases in pSMADl / 5 / 8 protein levels (Figure 3A), osteogenic transcription factor proteins such as Osterix (Figure 3B). andAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0278] target genes of canonical BMP signaling (Figure 3C) (Bostrom et al., J. Clin. Invest., 91:1800-1809 (1993); Miller et al., Circ. Res.. 108:1392-1412 (2011); New & Aikawa, Circ. Res., 108: 1381-1391 (2011); Seya et al., J. Pharmacol. Sci., 115:8-14 (2011); and Yangawa et al., J. Thorac. Cardiovasc. Surg., 144:256-262 (2012)). Based on a recent report associating increased nuclear methylarginine levels with osteogenic differentiation (Cheng et al., Circ. Res., 117:142-156 (2015)), upstream stimulatory factor- 1 (USF-1) protein levels (Figure 4A) were measured and expression of the USF-1 target genes osteopontin (SPP1) and alkaline phosphatase (ALPL, both depicted in Figure 4B) and strong evidence of increased USF-1 signaling in LA-D+ / " mice was found. Importantly, protein levels of SPP1 were modestly but not significantly elevated in valve tissue from LA-D+mice (Figure 5A), suggesting that post-translational regulation of SPP1 (e.g., protein phosphorylation) may limit the anti-calcific effects of SPP1 in valve tissue (Cheng et al., supra,' Jono et al., J. Biol. Chem., 275:20197-20203 (2000)). Consistent with this hypothesis, significant increases in tartrate-resistant acid phosphatase (TRAP) protein levels - an enzyme that cleaves and inactivates phosphorylated osteopontin (Hailing et al., Calcif. Tissue Int., 101:92-101 (2017); and Wolak, Atherosclerosis, 236:327-337 (2014)) - were found in aortic valves from LA-D+ / " mice compared to their LA-D+ / + littermates (Figures 5B-5C).

[0279] To determine the relative contributions of ADMA and DDAH1 to valve calcification, an in vitro model system of cultured murine aortic valve interstitial cells (VICs) was used. Interestingly, treatment of VICs with exogenous ADMA did not significantly alter expression of DDAH1, DDAH2, or ADMA transporters in vitro (Figure 6A), nor did it alter USF-1 protein levels or osteopontin (SPP1) expression (Figure 6B). In contrast, inhibition of DDAH1 with PD404182 significantly increased both SPP1 expression and USF-1 protein levels (Figure 6B). Importantly, treatment of cells with the combination of ADMA or PD404182 did not exhibit an additive effect compared to PD404182 alone.

[0280] Collectively, these data suggest increases in DDAH1 per se are adaptive in aortic valve disease and protect against fundamental molecular processes that drive osteogenesis, valvular calcification, and stenosis, including suppression of canonical BMP signaling, USF-1 activity, and their nuclear targets.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0281] Example 2 - Preservation of DDAH1 levels is an adaptive response and protects against valvular calcification

[0282] This Example identifies the preservation DDAH1 levels in diseased blood vessels as an adaptive response, protective against vascular calcification in conduit blood vessels independent of changes in osteogenic signaling or differentiation.

[0283] Results

[0284] Aortic tissue from patients undergoing coronary bypass surgery were acquired to determine changes in enzymes related to ADMA synthesis or metabolism. As a positive control, increased expression of inflammatory and osteogenic genes in diseased aortic vascular tissue was confirmed. Unlike observations from patients with end-stage aortic valve disease, expression of DDAH1 or DDAH2 did not differ between normal and diseased aortic vessel punch tissue, nor did expression of the ADMA transporter SLC7A2 (Figure 7). Changes in expression of arginine methyltransferases paralleled those observed in CAVS, however, with PRMT1 being significantly reduced in aorta from CABG patients compared to disease-free control subjects (Figure 7). While these data depict an overall adaptive strategy7to minimize local ADMA synthesis in advanced atherosclerosis in conduit vessels, these data also depict fundamental differences in expression of DDAH1 and SLC7A2 between diseased aortic valve and aortic tissue.

[0285] To determine whether reducing DDAH1 accelerates intimal calcification, aortic tissue from the same LA-D+ +and LA-D+ / ‘ mice, which develop advanced atherosclerotic plaques and calcified intimal lesions following 6 months of Western diet feeding, was examined. Consistent with previous studies, mice with either genotype developed advanced atherosclerotic plaques, and there were no significant differences in plaque size between groups. Much like observations in aortic valve, however, loss of one copy of DDAH1 increased intimal plaque calcification in LA-D+' mice compared to their LA-D+ / +littermates (Figure 8A). When taken in the context of prior reports that overexpression of DDAH1 can reduce atherosclerotic burden in hyperlipidemic mice (Jacobi et al., Am. J. Pathol., 176:2559-2570 (2010)), this data suggested that losses in DDAH1 are likely to have a predominant impact on atherosclerotic plaque composition. Consistent with previous observations (Roos et al., supra), experimentally reducing DDAH1 did not, however, further impairAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0286] vascular relaxation to acetylcholine (Figure 8B) or other indices of vasomotor function (Figure 9).

[0287] When combined with the observation that haploinsufficiency of DDAH1 does not further increase atherosclerotic plaque size, these data suggested there may be a “floor effect’' where reductions in DDAH1 activity7may not be sufficient to drive overt changes in nitric oxide bioavailability or vascular function in the context of severe atherosclerosis.

[0288] Interestingly, while increases in vascular calcification were associated with increases in canonical BMP signaling (Figure 10A), no increased expression of markers of mature osteoblast like cells (Figure 10B) or pSMADl / 5 / 8 target genes (Figure 10C) were observed, suggesting a functional disconnect between upstream BMP signaling and induction of downstream ectopic osteogenic gene programs. Furthermore — and in contrast to findings in aortic valve — no increases in USF-1 (Figure 11 A) or its target genes osteopontin and alkaline phosphatase (ALPL) in aorta from LA-D+' mice were observed (Figure 1 IB). Similar to observations in aortic valve — there were no significant increases in SPP1 protein levels in aortic tissue (Figure 12A) in LA-D mice compared to LA-D mice. Also, in contrast to findings in aortic valve, changes in TRAP protein and mRNA levels were variable and failed to reach statistical significance between genotypes in aortic tissue (Figure 12B-C). Altogether, the failure to increase SPP 1 expression and / or protein levels may represent the dysregulation of mechanisms that protect against calcification in aorta (Ge et al., Sci. Rep., 7:40253 (2017); and Lee-Kirsch et al., Semin. Immunopathol., 37:349-357 (2015)).

[0289] To determine the relative contributions of ADMA and DDAH1 to vascular calcification, an in vitro model system of cultured murine vascular smooth muscle cells (VSMCs) was used. Similar to aortic valve cells, treatment of VSMCs with exogenous ADMA did not alter expression of DDAH1, DDAH2, ADMA transporters (Figure 13A), or USF-1 protein levels or molecular targets (Figure 13B). Unlike aortic valve interstitial cells, however, inhibition of DDAH1 with PD404182 failed to drive changes in expression or protein levels of DDAH isoforms or USF-1 protein or targets (Figure 13B).

[0290] Collectively, these data suggest that preservation of DDAH1 levels is an adaptive response in diseased blood vessels, and protects against vascular calcification in conduit blood vessels independent of changes in osteogenic signalingAttorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0291] or differentiation. These results also demonstrate that sGS agonists of Formula I, Group I, and / or Group II can be used to reduce formation of ectopic tissue mineralization within mammals and / or to slow progression of ectopic tissue mineralization within mammals, in some cases, by restoring impaired nitric oxide signaling.

[0292] Example 3 - Compound Synthesis

[0293] Compounds 1 and 2 were prepared by methylation of 5-chloro-2-((5-chl orothiophene)-2-sulfonamido)-7V-(4-(piperi din-1 -ylsulfonyl)phenyl)benzamide (see, e.g., International Patent Application Publication No. W02000 / 002851). See, Scheme 1.

[0294] Scheme 1:

[0295]

[0296] Compound 3 was prepared from previously reported carboxylic acid (International Patent Application Publication No. W02000 / 002851) and commercially available N-methyl-4-(morpholinosulfonyl)aniline by using HATU as an amide coupling agent. See, Scheme 2.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0297] Scheme 2:

[0298]

[0299] 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)-N-methyl-N-(4-(morpholinosulfonyl)phenyl)benzamide (GPHR312768)

[0300] Results: m / z: ESI / APCI LM+H]+: 590.1

[0301]

[0302] 5-chloro-2-((5-chloro-N-methylthiophene)-2-sulfonamido)-N-methyl-N-(4-(morpholinosulfonyl)phenyl)benzamide (GPHR312845)

[0303] Results: m / z: ESI / APCI [M+H]+: 604.3

[0304]

[0305] 5-chloro-2-((5-chloro-N-methylthiophene)-2-sulfonamido)-N-(4- (morpholinosulfonyl)phenyl)benzamide (GPHR312844)

[0306] Results: m / z: ESI / APCI [M+H]+: 590.1

[0307]

[0308] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0309] Compounds 4-9 were prepared by following the standard procedures (International Patent Application Publication No. W02000 / 002851) by employing commercially available reagents.

[0310] 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)-N-(4- sulfamoylphenyl)benzamide (GPHR312850)

[0311] Results: m / z: ESI / APCI [M+H]+: 505.9

[0312]

[0313] (R)-5-chloro-2-((5-chlorothiophene)-2-sulfonamido)-N-(4-((3-hydroxypyrrolidin- l-yl)sulfonyl)phenyl)benzamide (5, GPHR312847)

[0314] Results: m / z: ESI / APCI [M+H]1: 576.1

[0315]

[0316] (S)-5-chloro-2-((5-chlorothiophene)-2-sulfonamido)-N-(4-((3-hydroxypyrrolidin- l-yl)sulfonyl)phenyl)benzamide (6)

[0317] Results: m / z: ESI / APCI [M+H]+: 576.0

[0318]

[0319] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0320] 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)-N-(4-(pyrrolidin-l- ylsulfonyl)phenyl)benzamide (7, GPHR312849)

[0321] Results: m / z: ESI / APCI [M+H]+: 561.0

[0322]

[0323] 5-chloro-2-((4-fluorophenyl)sulfonamido)-N-(4- (morpholinosulfonyl)phenyl)benzamide (8, GPHR312859)

[0324] Results: m / z: ESI / APCI [M+H]+: 554.1

[0325]

[0326] Compounds 10 was prepared by the cycloaddition reaction of commercially available acetylene derivative and azide obtained according to a previously reported procedure (Sanphanyaet al., Bioorg. Med. Chem. Lett., 23(10):2962-7 (2013)).

[0327] Compound 11 was derived from compound 10 by reacting with 5-chloro-2- methylsulfonyl chloride (Scheme 3). Acylation of compound 10 in pyridine / DMF

[0328] provided compound 12.

[0329]

[0330] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0331] 4-chloro-2-(l-(4-(morpholinosulfonyl)phenyl)-lH-l,2,3-triazol-4-yl)aniline (10, GPHR312854)

[0332] Results: m / z: ESI / APCI [M+H]+: 420.3

[0333]

[0334] 5-chloro-N-(4-chloro-2-(l-(4-(morpholinosulfonyl)phenyl)-lH-l,2,3-triazol-4-yl)phenyl)thiophene-2-sulfonamide (11, GPHR312770)

[0335] Results: m / z: ESI / APCI [M+H]+: 600.1

[0336]

[0337] Compound 12 was prepared by the cycloaddition reaction of commercially available acetylene derivative and 2-azido-4-chloroaniline (obtained by Cu-catalyzed coupling of azide with commercially available 2-iodo-4-chloroaniline). Compound 14

[0338] was derived from compound 13 by reacting with 5-chloro-2-methylsulfonyl chloride (Scheme 4).

[0339] Scheme 4:

[0340]

[0341] Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0342] 4-chloro-2-(4-(4-(morpholinosulfonyl)phenyl)-lH-l,2,3-triazol-l-yl)aniline (12, GPHR312855)

[0343] Results: m / z: ESI / APCI [M+H]+: 420.1

[0344]

[0345] 5-chloro-N-(4-chloro-2-(4-(4-(morpholinosulfonyl)phenyl)- 1H- 1,2,3-triazol- 1-yl)phenyl)thiophene-2-sulfonamide (13, GPHR312771)

[0346] Results: m / z: ESI / APCI [M+H]+: 600.2

[0347]

[0348] A-(4-chloro-2-(4-(4-(morpholinosulfonyl)phenyl)-lH-l,2,3-triazol-l-yl)phenyl)acetamide (GPHR312858)

[0349] Results: m / z: ESI / APCI [M+H]+: 462.1

[0350]

[0351] 4-(morpholinosulfonyl)benzonitrile was treated with hydroxylamine hydrochloride and sodium carbonate in EtOH to obtain N-hydroxy-4-(morpholinosulfonyl)benzimidamide that upon reacting with the activated 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)benzoic acid under microwave conditions provided compound 15 (Scheme 5).Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0352] Scheme 5:

[0353]

[0354] 5-chloro-N-(4-chloro-2-(3-(4-(morpholinosulfonyl)phenyl)-l,2,4-oxadiazol-5-yl)phenyl)thiophene-2-sulfonamide (15, GPHR312853)

[0355] Results: m / z: ESI / APCI [M+H]+: 601.0

[0356]

[0357] Compound 16, a hydrazide, was obtained from 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)benzoic acid and 4-(4-morphoUnylsulfonyl)benzoyl chloride using a previously known procedure Compound 17 was obtained by treatment of compound 16 with POCh under reflux in toluene Treatment of compound 16 with Lawesson’s reagent under reflux resulted in compound 18, a 1,3,4" thiadiazole Scheme 6.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0358] Scheme 6:

[0359]

[0360] yl)sulfonyl)benzoyl)hydrazine-l-carbonyl)phenyl)thiophene-2-sulfonamide (16, GPHR312857)

[0361] Results: m / z: ESI / APCI [M+H]+: 619.1

[0362]

[0363] 5-chloro-N-(4-chloro-2-(5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)phenyl)thiophene-2-sulfonamide (17, GPHR312772)

[0364] Results: m / z: ESI / APCI [M+H]+: 601.3

[0365]

[0366] 5-chloro-N-(4-chloro-2-(5-(4-(morpholinosulfonyl)phenyl)-l,3,4-thiadiazol-2-yl)phenyl)thiophene-2-sulfonamide (18, GPHR312856)

[0367] Results: m / z: ESI / APCI [M+H]+: 616.9Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0368]

[0369] 5-chloro-2-((5-chlorothiophene)-2-sulfonamido)benzoic acid was treated with oxalyl chloride followed by hydrazine to obtain hydrazinamide intermediate that upon heating with 4-(4-morpholinylsulfonyl)benzonitrile in wBuOH at 150°C provided compound 19. Scheme 7.

[0370] Scheme 7:

[0371]

[0372] Cl

[0373]

[0374] 5-chloro-N-(4-chloro-2-(5-(4-(morpholinosulfonyl)phenyl)-4H-l,2,4-triazol-3-yl)phenyl)thiophene-2-sulfonamide (GPHR312773)

[0375] Results: m / z: ESI / APCI [M+H]+: 600.2

[0376]

[0377] Example 4 - Treating Ectopic Tissue Mineralization Formation A human identified as having ectopic tissue mineralization formation is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of an sGC agonist of Formula I, Group I, and / or Group II daily.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257

[0378] After administering the sGC agonist of Formula I, Group I, and / or Group II daily for 30 days, ectopic tissue mineralization formation within the human is reduced.

[0379] Example 5 - Preventing Development of Ectopic Tissue Mineralization Formation A human identified as being susceptible to ectopic tissue mineralization formation is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of an sGC agonist of Formula I. Group I, and / or Group II daily. While receiving daily administration of the sGC agonist of Formula I, Group I, and / or Group II daily, the human does not develop any new ectopic tissue mineralization formation.

[0380] Example 6 - Treating Vascular and / or Cardiovascular Tissue Mineralization Formation

[0381] A human identified as having calcified cardiac valves is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of an sGC agonist of Formula 1, Group I, and / or Group 11 daily. After administering the sGC agonist of Formula I, Group I, and / or Group II daily for 30 days, the calcification of the cardiac valves within the human is reduced.

[0382] Example 7 - Preventing Vascular and / or Cardiovascular Tissue Mineralization Formation

[0383] A human identified as being susceptible to forming calcified cardiac valves is administered a composition containing 0.01 mg to 400 mg (e.g., 1 mg to 300 mg, or 10 mg to 250 mg) of an sGC agonist of Formula I, Group I, and / or Group II daily. While receiving daily administration of the sGC agonist of Formula I, Group I, and / or Group II daily, the human does not develop any new cardiac valve calcification.

[0384] OTHER EMBODIMENTS

[0385] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257WHAT IS CLAIMED IS:

1. A method for reducing ectopic tissue mineralization formation, wherein said method comprises administering a composition comprising one or more sGC agonists of Formula I, Group I, and / or Group II to a mammal having ectopic tissue mineralization or likely to develop ectopic tissue mineralization, wherein formation of said ectopic tissue mineralization within said mammal is reduced.

2. The method of claim 1, wherein said mammal is a human.

3. The method of any one of claims 1-2, wherein formation of ectopic tissue mineralization within said mammal is delayed.

4. The method of any one of claims 1-2, wherein formation of ectopic tissue mineralization within said mammal is delayed for at least 6 months.

5. The method of any one of claims 1-4, wherein said mammal is a mammal having ectopic tissue mineralization.

6. The method of any one of claims 1-5, wherein said mammal is a mammal likely to develop ectopic tissue mineralization.

7. A method for slowing ectopic tissue mineralization progression, wherein said method comprises administering a composition comprising one or more sGC agonists of Formula I, Group I, and / or Group II to a mammal having ectopic tissue mineralization, wherein progression of said ectopic tissue mineralization within said mammal is slowed.

8. The method of claim 7, wherein said mammal is a human.

9. The method of any one of claims 7-8, wherein progression of ectopic tissue mineralization within said mammal is slowed by at least 10 percent.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-25710. The method of any one of claims 7-8, wherein progression of ectopic tissue mineralization within said mammal is slowed by at least 25 percent.

11. The method of any one of claims 7-8, wherein progression of ectopic tissue mineralization within said mammal is slowed by at least 50 percent.

12. A compound of Formula I:or a pharmaceutically acceptable salt thereof, wherein:R1is selected from NH2, morpholinyl, piperidiny 1. pyrrolidinyl, and 2-oxa-6-azaspiro[3.3]heptanyl, each of which is optionally substituted with 1 or 2 substituents independently selected from C1-3 alkyl, OH, C1-3 alkoxy, C1-3 haloalkyl, C1-3 haloalkoxy, COOH, and CH2COOH;X1is a 5-membereed heteroaryl, optionally substituted with halo, OH, C1-3 alkyl. C1-3 alkoxy, C1-3 haloalkyl, and C1-3 haloalkoxy;R2is selected from H, C(=O)Ci-s alkyl, C(=O)Ci-3 haloalkyl, and S(=O)2R4: R4is selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl. and C1-3 haloalkoxy;n is 1, 2, or 3; andeach R3is independently selected from halo, C1-3 alkyd, C1-3 alkoxy, C1-3 haloalky l, and C1-3 haloalkoxy.

13. The compound of claim 12, wherein X1is a 5-membereed heteroaryl.

14. The compound of claim 12, wherein X1is selected from 1,2,3-triazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-oxadiazolyl, and 1,3,4-thiadiazoly 1.Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-25715. The compound of claim 12, the compound of Formula I has formula:or a pharmaceutically acceptable salt thereof.

16. The compound of claim 12, the compound of Formula I has formula:or a pharmaceutically acceptable salt thereof.

17. The compound of any one of claims 12-16, wherein R2is H.

18. The compound of any one of claims 12-16, wherein R2is selected from C(=O)Ci-3 alkyl and C(=O)Ci-3haloalkyl.

19. The compound of any one of claims 12-16, wherein the compound has formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257or a pharmaceutically acceptable salt thereof.

20. The compound of any one of claims 12-19, wherein R4is selected from thiophenyl and phenyl, each of which is optionally substituted with 1 or 2 substituents independently selected from halo, CN, and C1-3 alkoxy.

21. The compound of claim 20, wherein the compound has formula:or a pharmaceutically acceptable salt thereof.

22. The compound of any one of claims 12-21, wherein n is 1 or 2.

23. The compound of any one of claims 12-22, wherein R3is selected from halo, C1-3 alkyl, and C1-3 alkoxy.

24. The compound of any one of claims 12-19, wherein the compound has formula:or a pharmaceutically acceptable salt thereof.

25. The compound of claim 24, wherein the compound has formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257or a pharmaceutically acceptable salt thereof.

26. The compound of claim 12, wherein the compound has formula:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257(Compound A6), or a pharmaceutically acceptable salt thereof.

27. A compound selected from:Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257Attorney Docket No.: 07039-2394WO1 / 2018-223 / 2024-257or a pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising a compound of any one of claims 12-27, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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