ИНГИБИТОРЫ PDE1

Abstract

В настоящем изобретении предложены соединение формулы (I), причем каждый из R1, R2, R3, R4 и R5 определены, как указано в настоящем документе, или его фармацевтически приемлемая соль, для применения в качестве ингибитора человеческого PDE1.

Description

[0001] PDE1 INHIBITORS

[0002] The present invention relates to certain human PDE1 inhibitors, to pharmaceutical compositions comprising the compounds, to methods of using the compounds to treat physiological disorders, and to intermediates and processes useful in the synthesis of the compounds.

[0003] Phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of cAMP and cGMP by controlling the rate at which these cyclic nucleotides are hydrolyzed. PDE1, a calcium and calmodulin-dependent PDE, is one of at least 11 known PDE families. PDE1 is expressed in many tissues, including the brain, heart, lung, kidney, and smooth muscle. In addition, PDE1 is comprised of a family of three known isoforms, PDE1A, PDE1B, and PDE1C.

[0004] Patients suffering from diabetes often develop a form of chronic kidney disease referred to as diabetic kidney disease (or diabetic nephropathy). It has been estimated that diabetic kidney disease may affect as many as 40 percent of diabetic patients. Treatment options for diabetic kidney disease is limited and includes use of medications that lower blood pressure, management of blood glucose levels, diet, and weight, and implementation of regular physical activity. Thus, there is a need for additional treatment choices for patients suffering from chronic kidney disease, particularly diabetic kidney disease.

[0005] United States Patent No.8, 299, 080 discloses certain quinoxaline derivatives for treating various disorders such as dysuria and hypertension. In addition, European Patent No. 0 040 401 discloses certain substituted triazol oquinoxalin-4-ones possessing antihypertensive activity. Certain other PDE1 inhibitor compounds are disclosed in WO 2018 / 039051, WO 2017 / 139186, WO 2019 / 032383, WO 2019 / 156861 and WO 2019 / 027783.

[0006] There remains a need for PDE1 inhibitor therapy with a compound that exhibits desirable potency, minimizes the induction of cytochrome P450 (cyp) enzymes in patients, and that minimizes toxicity in patients, e.g., with respect to undesired cardiac effects. The present invention provides certain novel compounds that are inhibitors of human PDE1. In addition, the present invention provides certain novel compounds that are selective inhibitors of human PDE1A, PDE1B, and PDE1C relative to other human PDEs, such as PDE3A, PDE4D, and PDE6AB. Furthermore, the present invention provides certain novel compounds that may have antihypertensive effects and may also improve renal blood flow. In addition, certain compounds of the present invention may reduce renal fibrosis.

[0007] Accordingly, the present invention provides a compound of Formula I:

[0008] Formula I wherein R1, R2, and R3are each independently H, halogen, or C1-C3 alkyl optionally substituted with one or more halogen;

[0009] R4is H, halogen, C1-C4 alkyl optionally substituted with one or more halogen, cyclopropyl, or CH=CH-CH3; and

[0010] R5is C1-C3 alkyl optionally substituted with one or more halogen or cyclopropyl; or a pharmaceutically acceptable salt thereof.

[0011] The PDE1 pathway is associated with conditions and disease such as kidney disease, cardiovascular disease, hypertension, pulmonary hypertension, Parkinson’s Disease, artherosclerotic, neurocognitive disorders, chizophrenia, cognitive deficits associated with Alzheimer’s disease, movement disorder, and attention deficit and hyperactivity disorders (Samidurai A, et al., Pharmacol Ther. 2021 October; 226: 107858. doi: 10.1016 / j.pharmthera.2021.107858).

[0012] Accordingly, the present invention also provides a method of treating chronic kidney disease, diabetic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artherosclerotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated with Alzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

[0013] In addition, the invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in therapy. The invention further provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in for the treatment of diabetic kidney disease, chronic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artherosclerotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated with Alzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders.

[0014] Furthermore, the invention provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of diabetic kidney disease, chronic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artherosclerotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated with Alzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders.

[0015] The invention further provides a pharmaceutical composition, comprising a compound of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents, or excipients. The invention further provides a process for preparing a pharmaceutical composition, comprising admixing a compound of Formula I or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable carriers, diluents, or excipients. This invention also encompasses novel intermediates and processes for the synthesis of compounds of Formula I.

[0016] As used herein, the terms “treating”, “treatment”, or “to treat” includes prohibiting, restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

[0017] As used herein, the term "patient" refers to a mammal, such as a dog or a human, with a human being preferred.

[0018] As used herein, the term “effective amount” refers to the amount or dose of compound of the invention, or a pharmaceutically acceptable salt thereof which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment. As used herein, “C1-C3 alkyl” refers to methyl, ethyl, n-propyl, and isopropyl;

[0019] “C1-C4 alkyl” refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, and isobutyl.

[0020] As used herein, “halogen” refers to F, Cl, Br, and I. Examples of alkyl substituted with halogen include trifluoromethyl, chloromethyl, and the like.

[0021] An effective amount can be readily determined by one skilled in the art using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered by one skilled in the art, including, but not limited to: the patient’s size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0022] The compounds of the present invention are formulated as pharmaceutical compositions administered by any route which makes the compound bioavailable, including oral and parenteral routes. Most preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing same are well known in the art (See, e.g., Remington: The Science and Practice of Pharmacy, L. V. Allen, Editor, 22ndEdition, Pharmaceutical Press, 2012).

[0023] The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

[0024] The compounds of Formula I are particularly useful in the treatment methods of the invention, but certain groups, substituents, and compounds are preferred. The following paragraphs describe such preferred groups, substituents, and compounds. It will be understood that these preferences are applicable both to the treatment methods and to the new compounds of the invention.

[0025] It is preferred that R1, R2, and R3are H.

[0026] It is preferred that R4is C1-C4 alkyl. It is preferred that R5is C 1 -C3 alkyl .

[0027] It is further preferred that R4is ethyl.

[0028] It is further preferred that R5is methyl.

[0029] It is further preferred that the compounds of Formula I are in the free base form.

[0030] It is especially preferred that when R1, R2, and R3are H, R4is ethyl. It is especially preferred that when R1, R2, and R3are H, R5is methyl.

[0031] It is especially preferred that when R4is ethyl, R5is methyl.

[0032] Examples of the compounds disclosed herein include: pharmaceutically acceptable salts thereof.

[0033] The compound of the formula: and the pharmaceutically acceptable salts thereof are most preferred with the free base of the compound directly above being most especially preferred.

[0034] A pharmaceutically acceptable salt of the compound of the invention may be formed, for example, by reaction of an appropriate free base of the compound of the invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P.L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R. J., et al. “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4: 427-435 (2000); and Berge, S.M., et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1- 19, (1977).

[0035] Individual isomers, enantiomers, and diastereomers may be separated or resolved by one of ordinary skill in the art at any convenient point in the synthesis of compounds of the invention, by methods such as selective crystallization techniques or chiral chromatography (See for example, J. Jacques, et al., "Enantiomers, Racemates, and Resolutions" , John Wiley and Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen,” Stereochemistry of Organic Compounds", Wiley-Interscience, 1994). The designations “isomer 1” and “isomer 2” refer to the compounds that elute from chiral chromatography under specified conditions, first and second, respectively.

[0036] Certain abbreviations are defined as follows: “ACN” refers to acetonitrile; “AcOH” refers to glacial acetic acid; “DBU” refers to l,8-diazabicyclo[5.4.0]undec-7- ene; “DCM” refers to dichloromethane or methylene chloride; “DIPEA” refers to N,N- diisopropylethylamine; “DMF” refers to N,N-dimethylformamide; “DMSO” refers to dimethylsulfoxide; “EDCI” refers to l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; “ES / MS” refers to Electrospray Mass Spectrometry; “EtOAc” refers to ethyl acetate; “Et2O” refers to diethyl ether; “EtOH” refers to ethanol; “HMDS” refers to hezamethyldisilazane; “HOBT” refers to hydroxybenzotriazole; “IP A” refers to isopropanol; “TFA” refers to trifluoroacetic acid; “HATU” refers to l- [bis(dimethylamino)methylene]-lZ / -l,2,3-triazolo[4,5-Z>]pyridinium 3-oxid hexafluorophosphate; “LCMS” refers to Liquid Chromatography Mass Spectrometry; “RBF” refers to round bottomed flask; “TR” refers to retention time; “hr” refers to hour or hours; “IC50” refers to the concentration of an agent that produces 50% of the maximal inhibitory response possible for that agent; “pmol” refers to micromole or micromoles; “min” refers to minute or minutes; “MeOH” refers to methanol or methyl alcohol;

[0037] “MTBE” refers to methyl -tert-butyl ether; “NiNTA” refers to chromatography with an agarose stationary phase functionalized with nitrilotriacetic acid as chelator; “POCI3” refers to phosphorus oxychloride; “RT” refers to room temperature; “SNAr” refers to nucleophilic aromatic substitution; “TEA” refers to triethylamine; “THF” refers to tetrahydrofuran; “Tris” refers to 2-Amino-2-hydroxymethyl-propane- 1,3 -diol; “U / ml” refers to units per milliliter; “wt” refers to weight; and “Pd(OAc)2” refers to Palladium(II) acetate.

[0038] The compounds of the present invention may be prepared by a variety of procedures known to one of ordinary skill in the art, some of which are illustrated in the schemes, preparations, and examples below. One of ordinary skill in the art recognizes that the specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the invention. The products of each step below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. Without limiting the scope of the invention, the following representative schemes, preparations, and examples are provided to further illustrate the invention.

[0039] Scheme 1

[0040] Preparation 1 Synthesis of 2-fluoro-l-nitro-3-vinylbenzene

[0041] Scheme 1, Step A: l-Bromo-2-fluoro-3 -nitro-benzene (9.500 g, 43.182 mmol;), 4,4,5,5-Tetramethyl-2-vinyl-l,3,2-dioxaborolane (9.976 g, 64.773 mmol), S-phos (1.772 g, 4.318 mmol), and potassium phosphate, tribasic, N-hydrate (18.332 g, 86.364 mmol) were combined in a mixture of 1,4-Dioxane (100 mL, 1.171 mol) and Water (10 mL, 555.084 mmol). The mixture was purged with nitrogen for 30 minutes. Pd(OAc)2 (969.4805 mg, 4.318 mmol) was added, and it was then heated to 80 °C over the weekend. The mixture was cooled to ambient temperature and poured through filter paper. The filtrate was then partitioned between ethyl acetate and brine. The aqueous was removed and the organic was washed IX with brine, dried over sodium sulfate, and then reduced to a brown oil. The material was purified on silica gel using 5% ethyl acetate / hexanes as solvent to give an orange oil as the title compound (7.2 g, quantitative yield).

[0042] Preparation 2

[0043] Synthesis of N-(4-methoxybenzyl)-2-nitro-6-vinylaniline

[0044] Scheme 1, Step B: 2-fluoro-l-nitro-3-vinylbenzene (7.22 g, 43.197 mmol) and 4- methoxy-benzenemethanamine (11.851 g, 86.395 mmol) were combined in ethanol (150 mL, 2.576 mol) and the mixture was then heated to reflux overnight. LCMS indicated the reaction was complete. The mixture was cooled to ambient temperature and the solvent was removed under vacuum. The residue was partitioned between ethyl acetate and water. The aqueous was removed and the organic was washed IX with water, IX with brine, dried over sodium sulfate, and then reduced to an oil. The material was purified on silica gel using 5% ethyl acetate / hexanes as solvent to give an orange oil as the title compound (11.73 g, 96% yield).

[0045] Preparation 3

[0046] Synthesis of 6-ethyl-N1-(4-methoxybenzyl)benzene-l,2-diamine

[0047] Scheme 1, Step C: To a 2250 ml Parr shaker bottle was added N-(4- methoxybenzyl)-2-nitro-6-vinylaniline (11.73 g, 41.257 mmol), 5% Pt / C (2.65 g, 11.670 mmol) and ethyl acetate (1000 mL). The reaction vessel was placed on the shaker for 4 hours at 60 psig under room temperature. LCMS indicated the formation of the product. The reaction was filtered and rinsed with ethyl acetate. The solvent was removed under vacuum to give a yellow oil as the title compound (9.94 g, 89% yield). MS (m / z): 257.1 (M+l).

[0048] Preparation 4

[0049] Synthesis of 8-ethyl-l-(4-methoxybenzyl)-l,4-dihydroquinoxaline-2,3-dione

[0050] Scheme 1, Step D: 6-ethyl-N1-(4-methoxybenzyl)benzene-l,2-diamine (9.94 g, 36.875 mmol) was dissolved in ethanol (270 mL, 4.638 mol). Sodium ethoxide (25.093 g, 77.438 mmol) and diethyl oxalate (10.778 g, 73.751 mmol) were then added. The reaction was stirred at ambient temperature overnight. LCMS indicated the reaction was complete. The solvent was then removed under vacuum to give a tan foam. The foam was sonicated in water and then stirred at ambient temperature for 2 hours. The resulting solid was isolated by vacuum filtration, washed with water, dried for 30 minutes, and then placed in a vacuum oven at 50°C overnight to give a yellow solid after drying as the title compound (10.91 g, 95% yield). MS (m / z): 309.0 (M-l).

[0051] Preparation 5

[0052] Synthesis of 3-chloro-8-ethyl-l-(4-methoxybenzyl)quinoxalin-2(lH)-one

[0053] Scheme L Step E: 8-ethyl- 1 -(4-m ethoxybenzyl)- 1 ,4-dihydroquinoxaline-2,3 - dione (6.00 g, 19.333 mmol) was combined with thionyl chloride (12 mL, 164.713 mmol) and dimethylformamide (3 mL, 38.798 mmol) in toluene (150 mL, 1.418 mol). The mixture was heated at 115 °C for 45 minutes. LCMS indicated the starting material was consumed. The toluene was then removed under vacuum. The resulting residue was taken up in toluene and it was again removed under vacuum to give a brown oil as the title compound (6.36 g, quantitative yield). The material was carried forward without further purification. MS (m / z): 329.0 (M+l).

[0054] Preparation 6

[0055] Synthesis of 8-ethyl-3-hydrazineyl-l -(4-methoxybenzyl)quinoxalin-2(lH)-one

[0056] Scheme 1, Step F: 3-chloro-8-ethyl-l-(4-methoxybenzyl)quinoxalin-2(lH)-one (6.355 g, 19.33 mmol) was suspended in ethanol (80 mL, 1.374 mol) and hydrazine (3.097 mL, 96.651 mmol) was added. The mixture was heated at reflux for 3 hours and then cooled to ambient temperature. The reaction was stirred over the weekend. The resulting precipitate was isolated by vacuum filtration. The cake was washed with ethanol and dried for 2 hours to give a yellow solid as the title compound (4.23 g, 67% yield). MS (m / z): 325.2 (M+l).

[0057] Preparation 7

[0058] Synthesis of N'-(5-ethyl-4-(4-methoxybenzyl)-3-oxo-3,4-dihydroquinoxalin-2-yl)-l- methylcyclopropane- 1 -carbohydrazide

[0059] Scheme L Step G: 8-ethyl-3-hydrazineyl-l-(4-methoxybenzyl)quinoxalin-2(lH)- one (4.23 g, 13.04 mmol), 1 -methylcyclopropane- 1 -carboxylic acid (22.219 g, 22.168 mmol), HATU (8.429 g, 22.168 mmol), and diisopropylethylamine (7.960 mL, 45.640 mmol) were combined in dimethylformamide (80 mL, 1.035 mol) and stirred at ambient temperature overnight. The mixture was partitioned between ethyl acetate and water. The aqueous was removed and the organic was washed 2X with saturated sodium bicarbonate, washed 2X with brine, dried over sodium sulfate, and then reduced to residue. The residue was purified on silica gel using 50% ethyl acetate / hexanes as solvent to give a yellow foam as the title compound (3.0 g, 56% yield). MS (m / z): 407.2

[0060] (M+l).

[0061] Example 1

[0062] Synthesis of 6-ethyl-l-(l-methylcyclopropyl)-[l,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one

[0063] Scheme E Step H: N'-(5-ethyl-4-(4-methoxybenzyl)-3-oxo-3,4- dihydroquinoxalin-2-yl)-l -methylcyclopropane- 1 -carbohydrazide (3.00 g, 7.380 mmol) was dissolved in acetic acid (30 mL, 523.541 mmol) and heated to 115 °C overnight. LCMS indicated the starting material was consumed. The reaction was cooled to ambient temperature and allowed to stir overnight. The acetic acid was removed under vacuum. The resulting yellow semi-solid was taken up in TFA (8 ml) and heated in a microwave at 90 °C for 1 hour. The mixture was allowed to stand at ambient temperature overnight. The TFA was removed under vacuum and the residue was sonicated in diethyl ether. The resulting solid was isolated by vacuum filtration. The cake was washed with ether and dried for 4 hours to give an off-white solid. The material was purified on silica gel using 70% ethyl acetate / hexanes to 100% ethyl acetate to give an off-white solid as the title compound (1.22 g, 62% yield). MS (m / z): 269.0 (M+H), molecular weight: 268.

[0064] Generation of PDE proteins

[0065] The nucleotide sequences encoding full-length human PDE1 A

[0066] (NP_001003683.1), PDE1C (NP_005011.1), PDE5 A (NP_001074.2), PDE7B

[0067] (NP 061818.1) and PDE9A (NP 002597.1) are inserted into pFastBacl (Invitrogen) vector with an N-terminal HIS tag. The nucleotide sequences encoding full-length human PDE4D (NP 006194.2) and catalytic domain (residue 641-1141) ofPDE3A

[0068] (NP 000912.3) are inserted into pFastBacl (Invitrogen) vector with a C-terminal HIS tag. The nucleotide sequences encoding full-length human PDE8A (NP 002596.1) and PDE11A (AAI12394.1) are inserted into pFastBacl (Invitrogen) vector with an N- terminal Flag tag. The nucleotide sequences encoding full-length human PDE10A (AAD32595.1) are inserted into pFastBacl (Invitrogen) vector with a C-terminal Flag- His tag. The nucleotide sequences encoding full-length human PDE6A (NP 000431.2) and PDE6B (AAH00249.1) are inserted into pFastBacDual (Invitrogen) vector with an N- terminal HIS tag and N-terminal Flag tag, respectively, for production of PDE6A / 6B dimer. Baculovirus generation and protein expression in Sf9 cells are carried out according to the protocol of Bac-to-Bac Baculovirus Expression system (Invitrogen). The nucleotide sequences encoding full-length human PDE1B (NP 000915.1) and PDE2A (NP_002590.1) are inserted into p!EX4 (Novagen) with a C-terminal HIS tag, and both protein productions in Sf9 cells are carried out according to the vendor’s protocol (Novagen). The His tagged PDE proteins are purified using Ni-NTA agarose (Qiagen) followed by size exclusion chromatography on a SUPERDEX® 200 column (GE Healthcare) in storage buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% Glycerol). The Flag tagged PDE proteins including PDE6A / 6B are purified using anti-Flag M2- agarose (Sigma), after purification through NiNTA column chromatography and eluted in storage buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% Glycerol, 0.1 mg / ml Flag peptide). All purified proteins are stored at -80°C in small aliquots.

[0069] Phosphodiesterase enzyme assays

[0070] All 3’, 5’ cyclic nucleotide phosphodiesterase (PDE) enzyme activities are measured with a radiometric enzyme assay based on SPA detection system (scintillation proximity assay). Compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using ten point concentration response curves. Maximal compound concentration in the reaction mixture is either 10 or 100 pM. Compounds at the appropriate concentration are preincubated with either of the PDE enzymes for 30 minutes before the reaction is started by the addition of substrate. Reactions are allowed to proceed for 60 minutes at room temperature. Next, reactions are stopped by addition of SPA beads. Samples are read 12 hours later in a MICROBETA™ TRILUX® Counter. “IC50” refers to the concentration of the compound that produces 50% of the maximal inhibitory response possible for that compound. IC50 values are calculated by plotting the normalized data vs. log [compound] and fitting the data using a four parameter logistic equation.

[0071] Ca2+- calmodulin dependent PDE enzyme assays

[0072] PDE1B, PDE1 A, and PDE1C are cloned and purified following standard protein generation procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 50 mM MgCh, 4 mM CaCh, 0.1% Bovine serum albumin and 6 U / ml Calmodulin in water, at pH 7.5. The final enzyme concentration is 0.25, 0.074 and 0.0012 nM, for PDE1 A, PDE1B and PDE1C respectively. The reactions are started by addition of the substrate, [3H]cAMP, to give a final concentration of 47 nM.

[0073] Table 1: In vitro potency of Example 1 against human PDE1A, PDE1B, and PDE1C.

[0074] The data in Table 1 demonstrate that the compound of Example 1 inhibits human PDE1A, PDE1B, and PDE1C enzyme activity in vitro.

[0075] PDE enzyme assays using [3H]cAMP as substrate

[0076] The following phosphodiesterase activities are measured using [3H]cAMP as reaction substrate: human PDE3 A (catalytic domain), human PDE4D, human PDE7B and human PDE8A. All these enzymes are cloned and purified following standard procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 8.3 mM MgCh, 1.7 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% Bovine serum albumin at pH 7.5. Final enzyme concentrations are 0.008, 0.021, 0.5 and 0.06 nM for PDE3A, PDE4D, PDE7B and PDE8A respectively. Reactions are started by addition of the substrate, [3H]cAMP, to give a final concentration of 47 nM.

[0077] Table 2: In vitro potency of Example 1 against human PDE3A (catalytic domain), PDE4D, PDE7B and PDE8A,

[0078] PDE enzyme assays using cGMP as substrate

[0079] The following phosphodiesterase activities are measured using [3H]cGMP as reaction substrate: human PDE2A, human PDE5A, human PDE6A / 6B, human PDE9A, human PDE10A and human PDE11 A. The catalytic active form of human PDE6 is a dimer composed of a a (human PDE6A) and 0 subunits (human PDE6B). The dimer of human PDE6A / 6B is produced by the expression and purification strategy, using two purification steps, z.e., NiNTA and anti -FLAG Sepharose chromatography. The rest of the enzymes are cloned and purified in house following standard procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 8.3 mM MgCh, 1.7 mM EDTA and 0.1% Bovine serum albumin at pH 7.5. Final enzyme concentrations are 0.2, 0.002, 5, 1, 0.03 and 0.03 nM for human PDE2A, human PDE5A, human PDE6AB, human PDE9A, human PDE10A and human PDE11 A, respectively. The reactions are started by addition of the substrate, [3H]cGMP, to give a final concentration of 80 nM in the case of human PDE2A, human PDE10A, human PDE5A, human PDE6AB and human PDE11 A assays, whereas for human PDE9A 20 nM of [3H]cGMP is used.

[0080] Table 3: In vitro potency of Example 1 against PDE2A. PDE5A, PDE6AB, PDE9A. PDElOA and PDE11A,

[0081] The data in Tables 1, 2, and 3 demonstrate that the compound of Example 1 is a selective inhibitor of human PDE1A, PDE1B, and PDE1C relative to human PDE2A, PDE3A, PDE4D, PDE5A, PDE6AB, PDE7B, PDE8A, PDE9A, PDE10A, and PDE11 A in vitro.

[0082] Example 2

[0083] Other compounds were prepared in a manner analogous to the method of Example 1, as shown below.

[0084]

Claims

WE CLAIM:

1. A compound of the formula:wherein R1, R2, and R3are each independently H, halogen, or C1-C3 alkyl optionally substituted with one or more halogen;R4is H, halogen, C1-C4 alkyl optionally substituted with one or more halogen, cyclopropyl, or CH=CH-CH3; andR5is C1-C3 alkyl optionally substituted with one or more halogen or cyclopropyl; or a pharmaceutically acceptable salt thereof.

2. The compound or salt according to claim 1 wherein R1, R2, R3are H.

3. The compound or salt according to either claim 1 or claim 2, wherein R4is C1-C4 alkyl.

4. The compound or salt according to any one of claims 1 to 3, wherein R4is ethyl.

5. The compound or salt according to any one of claims 1 to 4, wherein R5is C1-C3 alkyl.

6. The compound or salt according to any one of claims 1 to 5, wherein R5is methyl.

7. The compound or salt according to any one of claims 1 to 6, wherein the compound is in the free base form.

8. The compound or salt according to claim 1 wherein the compound isor a pharmaceutically acceptable salt thereof.

9. The compound or salt according to claim 8, wherein the compound is10. The compound or salt according to claim 8, which is a pharmaceutically acceptable salt of11. A method of treating diabetic kidney disease, chronic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artherosclerotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated withAlzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders in a patient, comprising administering to a patient in need thereof an effective amount of a compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof.

12. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10 for use in therapy.

13. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10 for use in the treatment of diabetic kidney disease, chronic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artheroscl erotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated with Alzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders.

14. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of diabetic kidney disease, chronic kidney disease, acute kidney injury, hypertension, resistant hypertension, Parkinson’s Disease, artherosclerotic cardiovascular disease, angina, myocardial infarction, heart failure, pulmonary hypertension, ischemic stroke, cognitive deficits associated with schizophrenia, cognitive deficits associated with Alzherimer’s disease, movement disorder, or attention deficit and hyperactivity disorders.

15. A pharmaceutical composition, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

16. A process for preparing a pharmaceutical composition, comprising admixing a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

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