WO2025091010A1

WO2025091010A1 - Organic compounds

Info

Publication number: WO2025091010A1
Application number: PCT/US2024/053216
Authority: WO
Inventors: Peng Li; Hailin Zheng; Robert E. Davis
Original assignee: Intra Cellular Therapies Inc
Current assignee: Intra Cellular Therapies Inc
Priority date: 2023-10-26
Filing date: 2024-10-28
Publication date: 2025-05-01
Anticipated expiration: 2026-04-26

Abstract

Provided are PDE1 inhibitors of Formula I, processes for their production, their use as pharmaceuticals, and pharmaceutical compositions comprising them.

Description

ORGANIC COMPOUNDS

TECHNICAL FIELD

[001] The present invention relates to novel PDE1 inhibitory compounds of Formula I as described below, processes for their production, and their use as pharmaceuticals and pharmaceutical compositions comprising them.

BACKGROUND OF THE INVENTION

[002] From a therapeutic standpoint, PDEs are of particular therapeutic interest, as their structure is amenable to specific and potent small molecule inhibitors, and cell-specific expression provides selective organ targeting. Eleven families of phosphodiesterases (PDEs) have been identified totaling over 100 isoforms, but only PDEs in Family I, the Ca²⁺- calmodulin-dependent phosphodiesterases (CaM-PDEs), have been shown to mediate both the calcium and cyclic nucleotide (e.g., cAMP and cGMP) signaling pathways. These PDEs are therefore active in stimulated conditions when intra-cellular calcium levels rise, leading to increased hydrolysis of cyclic nucleotides. The three known CaM-PDE genes, PDE1 A, PDE1B, and PDE1C, are all expressed in human central nervous system tissue. PDE1 A is expressed in the brain with high levels in the CAI to CA3 layers of the hippocampus and cerebellum and at a low level in the striatum. PDE1 A is also expressed in the heart. PDE1B is predominately expressed in the striatum, dentate gyrus, olfactory tract and in the prefrontal cortex colocalized with the dopamine DI receptor. Its expression generally correlates with brain regions having high levels of dopaminergic innervation. Although PDE1B is primarily expressed in the central nervous system, it is present in neutrophils. PDE1C is more ubiquitously expressed in the brain and is expressed in the heart and vascular smooth muscle.

[003] PDE1 inhibitors have been found to be useful for the treatment or prophylaxis of many disorders, including those characterized by low levels of cAMP and/or cGMP in cells expressing PDE1; and/or reduced dopamine DI receptor signaling activity (e.g., Parkinson’s disease, Tourette’s Syndrome, Autism, fragile X syndrome, ADHD, restless leg syndrome, depression, cognitive impairment of schizophrenia, narcolepsy); and/or ophthalmic disorders (e.g., glaucoma, elevated intraocular pressure, etc.); and/or any disease or condition that may be ameliorated by the enhancement of progesterone signaling; and/or any disease or condition characterized by adenosine A2 dysfunction or which would benefit from adenosine A2

1

SUBSTITUTE SHEET (RULE 26) stimulation (e.g., cardiovascular diseases and disorders, muscular dystrophy (e.g., Duchenne muscular dystrophy), amyotrophic lateral sclerosis, etc.); and/or diseases or disorders characterized by inflammation; and/or cancers or tumors which over-express PDE1 (e.g., melanoma, neuroblastoma, renal cell and colon carcinomas, osteosarcoma, glioblastoma multiforme (GBM), etc.). This list of disorders is exemplary and not intended to be exhaustive.

[004] There is thus a continued need for novel compounds that selectively inhibit PDE1 activity.

SUMMARY OF THE INVENTION

[005] In an aspect, the present disclosure provides a compound of Formula I:

Formula I wherein

(i) Ri is H or C1-4 alkyl (e.g., methyl or ethyl);

(ii) R2 and R3 are independently H or C1-6 alkyl (e.g., methyl or ethyl);

(iii) R4 is H or C1-4 alkyl (e.g., methyl or ethyl);

(iv) R5 is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated heterocycloalkyl comprising one or more oxygens or nitrogens (e.g., saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or nitrogens, e.g., tetrahydrofuryl, dihydrofuryl, or aziridyl) and (b) -C(=O)-X, wherein X is saturated or unsaturated heterocycloalkyl comprising one or more oxygens or nitrogens (e.g., saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or nitrogens, e.g., tetrahydrofuryl, dihydrofuryl, or aziridyl);

2

SUBSTITUTE SHEET (RULE 26) (v) Re and R7 are independently H or aryl (e.g., phenyl) optionally substituted with one or more groups independently selected from Cue alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), for example unsubstituted phenyl or phenyl substituted with one or more halogen (e.g., F) or phenyl substituted with one or more C1-6 alkyl and one or more halogen or phenyl substituted with one C1-6 alkyl and one halogen, for example 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro- 3 -methylphenyl; and

(vi) n is 1, 2, 3, or 4, in free, salt, or prodrug form, e.g., pharmaceutically acceptable salt form, including enantiomers, diastereomers and racemates, thereof.

[006] In some embodiments, the present disclosure provides a compound of Formula I, wherein

(i) Ri is methyl;

(ii) R2 and R3 are both methyl;

(iii) R4 is H;

(v) R7 is H and Re is 4-fluorophenyl; and

(vi) n is 1, in free, salt, or prodrug form, e.g., pharmaceutically acceptable salt form, including enantiomers, diastereomers and racemates, thereof.

[007] In some embodiments, the compound of Formula I is selected from:

3

SUBSTITUTE SHEET (RULE 26)

in free, salt, or prodrug form, e.g., pharmaceutically acceptable salt form, including enantiomers, diastereomers and racemates, thereof.

[008] In another aspect, the present disclosure provides a pharmaceutical composition comprising a Compound of the Invention, in free, pharmaceutically acceptable salt, or prodrug form, in admixture with a pharmaceutically acceptable diluent or carrier.

[009] In another aspect, the present disclosure provides a method for the prophylaxis or treatment of a disease or disorder selected from neurodegenerative diseases (e.g., Parkinson’s disease, restless leg, tremors, dyskinesias, Huntington’s disease, Alzheimer’s disease, and drug-induced movement disorders); neuroinflammation and/or diseases or disorders associated with neuroinflammation and/or microglial function; mental disorders (e.g., depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorders, e.g., narcolepsy, cognitive impairment, e.g., cognitive impairment of schizophrenia, Tourette’s syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and drug addiction); chemobrain (cognitive impairment caused by cancer treatment (e.g., chemotherapy) or cancer itself); disorders associated with dementia; circulatory and cardiovascular disorders (e.g., cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, and sexual dysfunction); respiratory

SUBSTITUTE SHEET (RULE 26) and inflammatory disorders (e.g., asthma, chronic obstructive pulmonary disease, and allergic rhinitis); diseases which may be alleviated by the enhancement of progesterone signaling (e.g., female sexual dysfunction); psychosis; ophthalmic disorders (e.g., glaucoma and elevated intraocular pressure); traumatic brain injury; cancers or tumors (e.g., glioma, colon cancer, and breast cancer); renal disorders (e.g., kidney fibrosis, chronic kidney disease, renal failure, glomerulosclerosis, nephritis); cardiotoxicity consequent to administration of a chemotherapeutic agent and/or radiation therapy; any disease or condition characterized by low levels of cAMP and/or cGMP in cells expressing PDEl(e.g., angina, stroke, renal failure, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension, congestive heart failure, an inflammatory disease or disorder, fibrosis, cardiac hypertrophy, vascular remodeling, a connective tissue disease or disorder, chronic heart failure, myocardial inflammation, myocardial ischemia, myocardial hypoxia, reperfusion injury, left ventricular dysfunctions, vascular leakage, acute vascular inflammation, and amyotrophic lateral sclerosis); bacterial infections; viral infections; obesity; and any disease or condition characterized by reduced dopamine DI receptor signaling activity (e.g., attention deficit, cognition, PTSD, memory, and/or inhibitory processing), wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of the compound of the invention, e.g., compounds of Formula I, e.g., any compounds described in this disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0010] In an aspect, the invention provides a compound of Formula I (Compound 1.0):

wherein

(i) Ri is H or C1-4 alkyl (e.g., methyl or ethyl);

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SUBSTITUTE SHEET (RULE 26) (ii) R2 and R3 are independently H or C1-6 alkyl (e.g., methyl or ethyl);

(iii) R4 is H or C1-4 alkyl (e.g., methyl or ethyl);

(vii) R5 is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated heterocycloalkyl comprising one or more oxygens or nitrogens (e.g., saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or nitrogens, e.g., tetrahydrofuryl, dihydrofuryl, or aziridyl) and (b) -C(=O)-X, wherein X is saturated or unsaturated heterocycloalkyl comprising one or more oxygens or nitrogens (e.g., saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or nitrogens, e.g., tetrahydrofuryl, dihydrofuryl, or aziridyl);

(iv) Re and R7 are independently H or aryl (e.g., phenyl) optionally substituted with one or more groups independently selected from Cue alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), for example unsubstituted phenyl or phenyl substituted with one or more halogen (e.g., F) or phenyl substituted with one or more C1-6 alkyl and one or more halogen or phenyl substituted with one C1-6 alkyl and one halogen, for example 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro- 3 -methylphenyl; and

(v) n is 1, 2, 3, or 4, in free, salt, or prodrug form, e.g., pharmaceutically acceptable salt form, including enantiomers, diastereomers and racemates, thereof.

[0011] The invention further provides compounds of Formula I as follows:

1.1 Compound 1.0, wherein n is 1, 2, or 3;

1.2 Compound 1.0, wherein n is 1 or 2;

1.3 Compound 1.0, wherein n is 1;

1.4 Any of Compounds 1.0-1.3, wherein Ri is H or C1-3 alkyl (e.g., methyl);

1.5 Any of Compounds 1.0-1.3, wherein Ri is H;

1.6 Any of Compounds 1.0-1.3, wherein Ri is C1-4 alkyl;

1.7 Any of Compounds 1.0-1.3, wherein Ri is methyl;

1.8 Any of Compounds 1.0-1.7, wherein R2 and R3 are independently H or C1-5 alkyl (e.g., methyl or ethyl);

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SUBSTITUTE SHEET (RULE 26) Any of Compounds 1.0-1.7, wherein R2 and R3 are independently H or C1-4 alkyl (e.g., methyl); Any of Compounds 1.0-1.7, wherein R2 and R3 are both C1-6 alkyl (e.g., C1-4 alkyl, e.g., methyl); Any of Compounds 1.0-1.7, wherein R2 and R3 are both C1-4 alkyl (e.g., methyl); Any of Compounds 1.0-1.7, wherein R2 and R3 are both methyl; Any of Compounds 1.0-1.12, wherein R4 is H or C1-3 alkyl (e.g., methyl or ethyl); Any of Compounds 1.0-1.12, wherein R4 is H; Any of Compounds 1.0-1.14, wherein R5 is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens and (b) - C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens. Any of Compounds 1.0-1.14, wherein R5 is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens. Any of Compounds 1.0-1.14, wherein R5 is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 5 membered heterocycloalkyl comprising one or more oxygens or (b) -C(=O)-X, wherein X is saturated or unsaturated 5 membered heterocycloalkyl comprising one or more oxygens. Any of Compounds 1.0-1.14, wherein R5 is phenyl substituted with tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl). Any of Compounds 1.0-1.14, wherein R5 is phenyl substituted with tetrahydrofuryl. Any of Compounds 1.0-1.14, wherein R5 is phenyl substituted with dihydrofuryl (e.g., 2,3- dihydrofuryl). Any of Compounds 1.0-1.14, wherein R5 is phenyl substituted with -C(=O)-X, wherein X is tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl). Any of Compounds 1.0-1.14, wherein R5 is phenyl substituted with -C(=O)-X, wherein X is tetrahydrofuryl.

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SUBSTITUTE SHEET (RULE 26) Any of Compounds 1.0-1.14, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is dihydrofuryl (e.g., 2,3- dihydrofuryl). Any of Compounds 1.0-1.14, wherein Rs is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens and (b) - C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens. Any of Compounds 1.0-1.14, wherein Rs is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens. Any of Compounds 1.0-1.14, wherein Rs is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3 membered heterocycloalkyl comprising one or more nitrogens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3 membered heterocycloalkyl comprising one or more nitrogens. Any of Compounds 1.0-1.14, wherein Rs is phenyl substituted with aziridyl. Any of Compounds 1.0-1.14, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is aziridyl. Any of Compounds 1.0-1.28, wherein Re and R? are independently H or aryl (e.g., phenyl) substituted with one or more groups independently selected from Ci-4 alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), for example phenyl substituted with one or more (e.g., two) halogen (e.g., F) or phenyl substituted with one or more Ci-4 alkyl (e.g., methyl) and one or more halogen (e.g., F) or phenyl substituted with one Ci-4 alkyl (e.g., methyl) and one halogen (e.g., F), for example 4-fluorophenyl or 3,4-difluorophenyl or 4- fluoro-3 -methylphenyl . Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one or more groups independently selected from Cue alkyl (e.g., Ci-4 alkyl, e.g., methyl) and halogen (e.g., F or Cl), for example Re is phenyl substituted with one or more (e.g., two) halogen (e.g., F) or phenyl substituted with one Ci-6 alkyl (e.g., Ci-4 alkyl, e.g., methyl) and one halogen (e.g., F), for example wherein Re is 4-fluorophenyl or 3,4-difluorophenyl or 4- fluoro-3 -methylphenyl .

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SUBSTITUTE SHEET (RULE 26) Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one or more groups independently selected from Ci-4 alkyl (e.g., methyl) and halogen (e.g., F), for example Re is phenyl substituted with one or more (e.g., two) halogen (e.g., F) or phenyl substituted with one Ci-4 alkyl (e.g., methyl) and one halogen (e.g., F), for example wherein Re is 4- fluorophenyl or 3,4-difluorophenyl or 4-fluoro-3 -methylphenyl. Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one or more halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with two halogens (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with two F. Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one F. Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one or more Ci-6 alkyl (e.g., Ci-4 alkyl, e.g., methyl) and one or more halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one or more Ci-4 alkyl (e.g., methyl) and one or more halogen (e g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one Ci-6 alkyl (e.g., Ci-4 alkyl, e.g., methyl) and one halogen (e g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one Ci-4 alkyl (e.g., methyl) and one halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is aryl (e.g., phenyl) substituted with one methyl and one F. Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one or more halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with two halogens (e.g., F).

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SUBSTITUTE SHEET (RULE 26) Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one halogen (e.g., F). Any of Compounds 1.0-1.28, wherein Re is phenyl substituted with two F; Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one F. Any of Compounds 1.0-1.28, wherein R? is H and Re is 3,4-difluorophenyl. Any of Compounds 1.0-1.28, wherein R? is H and Re is 4 -fluorophenyl. Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one or more Cue alkyl (e.g., Ci-4 alkyl, e.g., methyl) and one or more halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one or more Ci-4 alkyl (e.g., methyl) and one or more halogen (e.g., F); Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one Cue alkyl (e.g., Ci-4 alkyl, e.g., methyl) and one halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one Ci-4 alkyl (e.g., methyl) and one halogen (e.g., F). Any of Compounds 1.0-1.28, wherein R? is H and Re is phenyl substituted with one methyl and one F. Any of Compounds 1.0-1.28, wherein R? is H and Re is 4-fluoro-3- methylphenyl. Compound 1.0, wherein

(i) Ri is methyl;

(ii) R2 and R3 are both methyl;

(iii) R4 is H;

(iv) R7 is H and Re is 4 -fluorophenyl; and

(v) n is 1. Compound 1.55, wherein R5 is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens and (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens. Compound 1.55, wherein R5 is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more

10

SUBSTITUTE SHEET (RULE 26) oxygens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens. Compound 1.55, wherein Rs is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 5 membered heterocycloalkyl comprising one or more oxygens or (b) -C(=O)-X, wherein X is saturated or unsaturated 5 membered heterocycloalkyl comprising one or more oxygens. Compound 1.55, wherein Rs is phenyl substituted with tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl). Compound 1.55, wherein Rs is phenyl substituted with tetrahydrofuryl. Compound 1.55, wherein Rs is phenyl substituted with dihydrofuryl (e.g., 2,3- dihydrofuryl). Compound 1.55, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl). Compound 1.55, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is tetrahydrofuryl. Compound 1.55, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is dihydrofuryl (e.g., 2,3- dihydrofuryl). Compound 1.55, wherein Rs is aryl (e.g., phenyl) substituted with one or more groups independently selected from (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens and (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens. Compound 1.55, wherein Rs is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens. Compound 1.55, wherein Rs is aryl (e.g., phenyl) substituted with (a) saturated or unsaturated 3 membered heterocycloalkyl comprising one or more nitrogens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3 membered heterocycloalkyl comprising one or more nitrogens. Compound 1.55, wherein Rs is phenyl substituted with aziridyl. Compound 1.55, wherein Rs is phenyl substituted with -C(=O)-X, wherein X is aziridyl.

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SUBSTITUTE SHEET (RULE 26) Compound 1.0, wherein the compound is selected from:

Compound 1.0, wherein the compound is selected from:

in free or salt form. Compound 1.0, wherein the compound is selected from:

SUBSTITUTE SHEET (RULE 26)

in free or salt form. Compound 1.0, wherein the compound is

in free or salt form. Compound 1.0, wherein the compound is

in free or salt form. Compound 1.0, wherein the compound is

SUBSTITUTE SHEET (RULE 26)

in free or salt form.

1.76 Compound 1.0, wherein the compound is

in free or salt form.

1.77 Any of the preceding compounds, wherein the compound inhibits phosphodiesterase-mediated (e.g., PDE1 -mediated) hydrolysis of cGMP, e.g., with an IC50 of less than 1 pM, preferably less than 500 nM, more preferably less than 50 nM, still more preferably less than 10 nM, most preferably less than or equal to 5 nM in an immobilized-metal affinity particle reagent PDE assay, for example, as described in Example 5.

[0012] If not otherwise specified or clear from context, the following terms herein have the following meanings:

(a) “Alkyl” as used herein is a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having one to six carbon atoms, preferably having one to four carbon atoms, which may be linear or branched, and may be optionally mono-, di- or tri- substituted, e.g., with halogen (e.g., Cl or F) or carboxy.

(b) “Hydroxyalkyl” as used herein is a saturated hydrocarbon moiety, preferably having one to six carbon atoms, preferably having one to four carbon atoms,

14

SUBSTITUTE SHEET (RULE 26) which may be linear or branched, and is mono-, di- or tri- substituted with hydroxy.

(c) “Haloalkyl” as used herein is a saturated hydrocarbon moiety, preferably having one to six carbon atoms, preferably having one to four carbon atoms, which may be linear or branched, and is mono-, di- or tri- substituted with halogen. For di- or tri- substituted haloalkyl, the halogens may be the same (e.g., dichloromethyl) or different (e.g., chlorofluoromethyl).

(d) “Aryl” as used herein is a mono or bicyclic aromatic hydrocarbon, preferably having 5-10 carbon atoms, preferably phenyl, which may be optionally substituted, e.g., optionally substituted with one or more groups independently selected from Ci-6 alkyl (e.g., methyl), halogen (e.g., Cl or F), Ci-6-haloalkyl (e.g., trifluoromethyl), hydroxy, and carboxy. In some embodiments, aryl, in addition to being substituted with the groups disclosed herein, is further substituted with an aryl or a heteroaryl to form, e.g., biphenyl or pyridylphenyl.

(e) “Heteroaryl” as used herein is an aromatic moiety preferably having 5-10 atoms wherein one or more of the atoms making up the aromatic ring is sulfur or nitrogen rather than carbon, e.g., pyridyl or thiadiazolyl, which may be optionally substituted, e.g., optionally substituted with one or more groups independently selected from Ci-6 alkyl (e.g., methyl), halogen (e.g., Cl or F), Ci-6-haloalkyl (e.g., trifluoromethyl), hydroxy, and carboxy.

(f) “Heterocycloalkyl” as used herein is a non-aromatic moiety preferably comprising three to six atoms, wherein one or more of the atoms making up the non-aromatic ring is oxygen or nitrogen rather than carbon, e.g., tetrahydrofuryl, dihydrofuryl, or aziridyl.

(g) “Hydroxy” as used herein is -OH.

(h) “Carboxy” as used herein is -COOH.

(i) “Halogen” as used herein is F, Cl, Br, or I.

[0013] Compounds of the Invention, e.g., compounds of Formula I, e.g., any of Compounds 1.0-1.77, may exist in free or salt form, e.g., as acid addition salts. In this specification unless otherwise indicated, language such as “Compounds of the Invention” is to be understood as embracing the compounds in any form, for example free or acid addition salt form, or where the compounds contain acidic substituents, in base addition salt form.

15

SUBSTITUTE SHEET (RULE 26) The Compounds of the Invention are intended for use as pharmaceuticals, therefore pharmaceutically acceptable salts are preferred. Salts which are unsuitable for pharmaceutical uses may be useful, for example, for the isolation or purification of free Compounds of the Invention or their pharmaceutically acceptable salts, are therefore also included.

[0014] Compounds of the Invention may in some cases also exist in prodrug form. A prodrug form is compound which converts in the body to a Compound of the Invention. For example, when the Compounds of the Invention contain hydroxy or carboxy substituents, these substituents may form physiologically hydrolysable and acceptable esters. As used herein, “physiologically hydrolysable and acceptable ester” means esters of Compounds of the Invention which are hydrolysable under physiological conditions to yield acids (in the case of Compounds of the Invention which have hydroxy substituents) or alcohols (in the case of Compounds of the Invention which have carboxy substituents) which are themselves physiologically tolerable at doses to be administered. Therefore, wherein the Compound of the Invention contains a hydroxy group, for example, Compound-OH, the acyl ester prodrug of such compound, i.e., Compound-O-C(O)-Ci-4alkyl, can hydrolyze in the body to form physiologically hydrolysable alcohol (Compound-OH) on the one hand and acid on the other (e.g., HOC(O)-Ci-4alkyl). Alternatively, wherein the Compound of the Invention contains a carboxylic acid, for example, Compound-C(O)OH, the acid ester prodrug of such compound, i.e., Compound-C(O)O-Ci-4alkyl, can hydrolyze to form Compound-C(O)OH and HO-Ci- 4alkyl. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free, pharmaceutically acceptable salt, or prodrug form, in admixture with a pharmaceutically acceptable diluent or carrier. The Compound of the Invention may be provided in the form of a pharmaceutical composition (e.g., a dosage form), for example for oral administration, e.g., in the form of pills (tablets or capsules), or for parenteral administration. In some embodiments, the Compound of the Invention is provided in the form of a long-acting depot for administration by injection to provide sustained release (e.g., intramuscular or subcutaneous injection). In some embodiments, the solid drug for oral administration or as a depot may be embedded, dissolved, dispersed, or suspended, in a suitable polymer matrix to provide delayed release of

16

SUBSTITUTE SHEET (RULE 26) the active compound, for example, in polymeric microspheres or in a solvent-based carrier. In some embodiments, the Compound of the Invention is provided in the form of nasal spray or inhaler.

[0016] In some embodiments, the pharmaceutical composition is in the form of an oral dosage form (such as a tablet or capsule), an intranasal dosage form (e.g., a nasal spray or mister), a pulmonary dosage form (e.g., an inhaler), or an injectable dosage form (e.g., an intravenous, intramuscular or subcutaneous injection, such as a long-acting depot injection). In some embodiments, the composition is in the form of a tablet or capsule. In some embodiments, the composition is in the form of a nasal spray, mister, or inhaler. In some embodiments, the composition is in the form of an injectable dosage form (e.g., an intravenous, intramuscular or subcutaneous injection, such as a long-acting depot injection).

[0017] In another aspect, the present disclosure provides a method for the prophylaxis or treatment of a patient, e.g., a human, suffering from a disorder selected from the following disorders: i. Neurodegenerative diseases, including Parkinson’s disease, restless leg, tremors, dyskinesias, Huntington’s disease, Alzheimer’s disease, and drug-induced movement disorders; ii. Mental disorders, including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorders, e.g., narcolepsy, cognitive impairment, e.g., cognitive impairment of schizophrenia, dementia, Tourette’s syndrome, autism, fragile X syndrome, psychostimulant withdrawal, chemobrain (cognitive impairment caused by cancer treatment (e.g., chemotherapy and/or radiotherapy) or cancer itself), and drug addiction; iii. Circulatory and cardiovascular disorders, including cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, e.g., pulmonary arterial hypertension, and sexual dysfunction, including cardiovascular diseases and related disorders as described in International Application No. PCT/US2014/16741, the contents of which are incorporated herein by reference; iv. Respiratory and inflammatory disorders, including asthma, chronic obstructive pulmonary disease, and allergic rhinitis, as well as autoimmune and inflammatory diseases;

17

SUBSTITUTE SHEET (RULE 26) v. Diseases that may be alleviated by the enhancement of progesterone- signaling such as female sexual dysfunction; vi. A disease or disorder such as psychosis, glaucoma, or elevated intraocular pressure; vii. Traumatic brain injury; viii. Cancers or tumors, e.g., brain tumors, a glioma (e.g., ependymoma, astrocytoma, oligodendrogliomas, brain stem glioma, optic nerve glioma, or mixed gliomas, e.g., oligoastrocytomas), an astrocytoma (e.g., glioblastoma multiforme), osteosarcoma, melanoma, leukemia, neuroblastoma or leukemia; ix. Renal disorders, e.g., kidney fibrosis, chronic kidney disease, renal failure, glomerulosclerosis and nephritis; x. Any disease or condition characterized by low levels of cAMP and/or cGMP (or inhibition of cAMP and/or cGMP signaling pathways) in cells expressing PDE1; and/or xi. Any disease or condition characterized by reduced dopamine DI receptor signaling activity, comprising administering to a patient in need thereof a therapeutically effective of compound of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77.

Methods of Making Compounds of the Invention

[0018] The Compounds of the Invention and their pharmaceutically acceptable salts may be made using the methods as described and exemplified herein and by methods similar thereto and by methods known in the chemical art. Such methods include, but are not limited to, those described below. If not commercially available, starting materials for these processes may be made by procedures, which are selected from the chemical art using techniques which are similar or analogous to the synthesis of known compounds. Various starting materials, intermediates and/or Compounds of the Invention may be prepared using methods described or similarly described in WO 2006/133261, WO 2009/075784, WO 2010/065148, WO 2010/065149, WO 2010/065151, and/or WO2014/151409. All references cited herein are hereby incorporated by reference in their entirety.

18

SUBSTITUTE SHEET (RULE 26) [0019] The Compounds of the Invention include their enantiomers, diastereoisomers and racemates, as well as their polymorphs, hydrates, solvates and complexes. Some individual compounds within the scope of this invention may contain double bonds. Representations of double bonds in this invention are meant to include both the E and the Z isomer of the double bond. In addition, some compounds within the scope of this invention may contain one or more asymmetric centers. This invention includes the use of any of the optically pure stereoisomers as well as any combination of stereoisomers.

[0020] It is also intended that the Compounds of the Invention encompass their stable and unstable isotopes. Stable isotopes are nonradioactive isotopes which contain one additional neutron compared to the abundant nuclides of the same species (i.e., element). It is expected that the activity of compounds comprising such isotopes would be retained, and such compound would also have utility for measuring pharmacokinetics of the non-isotopic analogs. For example, the hydrogen atom at a certain position on the Compounds of the Invention may be replaced with deuterium (a stable isotope which is non-radioactive). Examples of known stable isotopes include, but not limited to, deuterium, ¹³ C, ¹⁵ N, ¹⁸ O. Alternatively, unstable isotopes, which are radioactive isotopes which contain additional neutrons compared to the abundant nuclides of the same species (i.e., element), e.g., ¹²³1, ¹³¹I, ¹²⁵I, ⁿC, ¹⁸F, may replace the corresponding abundant species of I, C, and F. Another example of useful isotope of the compound of the invention is the ⁿC isotope. These radio isotopes are useful for radio-imaging and/or pharmacokinetic studies of the compounds of the invention. Methods of making isotopes of PDE1 inhibitors disclosed in WO 2011/043816, the contents of which are incorporated by reference in their entirety, may be used for making the isotopes of the compounds of the current invention.

[0021] Melting points are uncorrected and (dec) indicates decomposition. Temperatures are given in degrees Celsius (°C); unless otherwise stated, operations are carried out at room or ambient temperature, that is, at a temperature in the range of 18-25 °C. Chromatography means flash chromatography on silica gel; thin layer chromatography (TLC) is carried out on silica gel plates. NMR data is in the delta values of major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Conventional abbreviations for signal shape are used. Coupling constants (J) are given in Hz. For mass spectra (MS), the lowest mass major ion is reported for molecules where isotope splitting results in multiple mass spectral peaks. Solvent mixture compositions

19

SUBSTITUTE SHEET (RULE 26) are given as volume percentages or volume ratios. In cases where the NMR spectra are complex, only diagnostic signals are reported.

[0022] Terms and abbreviations:

BOP = benzotri azole- l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate

BOC = tert-butyloxycarbonyl,

CAN = ammonium cerium (IV) nitrate,

DBU = l,8-diazabicyclo[5.4.0]undec-7-ene

DIPEA = diisopropylethylamine,

DMF = N,N-dimethylformamide,

DMSO = dimethyl sulfoxide,

Et2O = diethyl ether, EtOAc = ethyl acetate, equiv. = equivalent(s), h = hour(s),

HPLC =high performance liquid chromatography,

LDA = lithium diisopropylamide,

LiHMDS = lithium bis(trimethylsilyl)amide, MeOH = methanol,

NBS = N-bromosuccinimide,

NCS = N-chlorosuccinimide,

NMP = N-methyl-2-pyrrolidone,

NaHCOs = sodium bicarbonate,

NH4OH = ammonium hydroxide,

Pd2(dba)s = tris[dibenzylideneacetone]dipalladium(0)

PMB = p-methoxybenzyl,

POCI3 = phosphorous oxychloride,

SOCh = thionyl chloride,

TFA = tri fluoroacetic acid,

TFMSA = trifluoromethanesulfonic acid, and

THF = tetrahydrofuran.

[0023] The synthetic methods in this invention are illustrated below. The significances for the R groups are as set forth above for Formula I unless otherwise indicated.

20

SUBSTITUTE SHEET (RULE 26) [0024] In an aspect of the invention, intermediate compounds of formula lib can be synthesized by reacting a compound of formula Ila with malonic acid and acetic anhydride in acetic acid with heating, e.g., to about 90°C for about 3 hours, and then cooled:

(Ila) (^,,b> wherein Ri is H or C1-4 alkyl, e.g., methyl.

[0025] Intermediate lie can be prepared by for example reacting intermediate lib with for example a chlorinating compound such as POCI3, sometimes with small amounts of water and heat, e.g., heating to about 80°C for about 4 hours, and then cooled:

(lib) (lie)

[0026] Intermediate lid may be formed by reacting intermediate lie with for example P^J-L in a solvent such as DMF and a base such as K2CO3, sodium bicarbonate, cesium carbonate, sodium hydroxide, triethylamine, diisopropylethylamine or the like at room temperature or with heating:

wherein P¹ is a protective group [e.g., -methoxybenzyl group (PMB) or

BOC]; L is a leaving group such as a halogen, mesylate, or tosylate. Preferably, P¹ is PMB and the base is potassium carbonate.

[0027] Intermediate He may be prepared by reacting intermediate Hd with hydrazine or hydrazine hydrate in a solvent such as methanol and with heating, e.g. refluxed for about 4 hours, and then cooled:

21

SUBSTITUTE SHEET (RULE 26)

(lid) (He)

[0028] Intermediate IVa may be formed by for example reacting intermediate He with POCI3 and DMF :

(lie) (IVa) wherein Ri is as defined previously for Formula I, e.g., such as a methyl group.

[0029] Intermediate IVb may be formed by reacting intermediate IVa with for example F'-X in a solvent such as DMF with a base such as K2CO3 at room temperature

(Reaction 1):

wherein F¹ is for example benzyl substituted with a halogen such as 4-bromobenzyl and X is a halogen (e.g., Br).

[0030] Intermediate IVc may be synthesized from intermediate IVb by removing the protective group P¹ with an appropriate method. For example, if P¹ is a PMB group, then it can be removed with CAN or TFA/TFMSA at room temperature (Reaction 2):

wherein if P¹ is BOC, the compound may be deprotected by using acid such as hydrochloric acid or TFA.

22

SUBSTITUTE SHEET (RULE 26) [0031] Intermediate IVd can be prepared by reacting intermediate IVc with for example a chlorinating compound such as POCI3 and optionally with heating, e.g., reflux for about 2 days or more, or heated at 150~200°C for about 5-10 minutes in a sealed vial with a microwave instrument and then cooled (Reaction 3):

Vc)

[0032] Intermediate IVe can be formed by reacting intermediate IVd with an amino alcohol under basic condition in a solvent such as DMF or NMP and heated then cooled

(Reaction 4A):

wherein Ri, R2, R3, and R4 are as defined previously for Formula I.

[0033] Alternatively, intermediate IVe can be synthesized directly from intermediate IVc by reacting with an amino alcohol and a coupling reagent such as BOP in the presence of a base such as DBU (Reaction 4B):

wherein Ri, R2, R3, and R4 are as defined previously for Formula I.

[0034] Intermediate IVf may be formed by reacting a compound of IVe with, for example, a dehydrating/halogenating agent such as SOCI2 in a solvent such as CH2Q2 at room temperature or heated at 35 °C for several hours, and then cooled (Reaction 5):

23

SUBSTITUTE SHEET (RULE 26)

[0035] Intermediate IVg may be formed by reacting intermediate IVf with, for example, catalysts such as a copper salt and 2,2,6,6-tetramethylheptane-3,5-dione and a base such as cesium carbonate in a solvent such as NMP with heat for several hours (Reaction 6):

wherein, F² is a diaryl ether.

[0036] Intermediate IVh may be formed by reacting intermediate IVg with, for example, TFA and TFMSA in a solvent such as CH2CI2 at room temperature (Reaction 7):

[0037] Intermediate IVi may be formed by reacting intermediate IVh with

R5- (CH2)_n- L in the presence of a base, for example K2CO3, in a solvent such as DMF at room temperature (Reaction 8):

SUBSTITUTE SHEET (RULE 26) wherein Rs and n are as defined previously for Formula I and L is a leaving group such as a halogen (e.g., Br).

[0038] Intermediate IVj wherein X is halogen (e.g., Cl) may be formed by reacting intermediate IVi with, for example, a halogenating agent such as hexachloroethane, NCS, NBS, I2 and a base such as LiHMDS in a solvent such as THF at low temperature (Reaction 9):

[0039] Compounds of the Invention may be formed by reacting intermediate IVj wherein X is halogen (e.g., Cl) with NHReR? and a catalyst with heating (Reaction 10):

wherein Re and R7 are as defined previously for Formula I.

[0040] In another aspect of the invention, intermediate Ilf may be prepared by reacting intermediate lie with hydrazine or hydrazine hydrate in a solvent such as methoxy ethanol and refluxed for about 30 minutes and then cooled:

(lie) (Ilf)

[0041] Intermediate Va can be synthesized by reacting a compound of formula He with for example an aryl isothiocyanate or isocyanate in a solvent such as DMF and heated at 110° C for about 2 days and then cooled:

25

SUBSTITUTE SHEET (RULE 26)

(lie) (Va) wherein Re is as defined previously for Formula I.

[0042] Intermediate Vb may be formed by removing the protective group P¹ with an appropriate method. For example, if P¹ is a PMB group, then it can be removed with AICI3 or

TFA/TFMSA at room temperature. Intermediate Vb may also be prepared directly from a compound of Ilf using the similar methods, but the yields are relatively low.

[0043] Intermediate Vc can be prepared by for example reacting intermediate Vb with for example a chlorinating compound such as POCI3. The reaction may be carried out at atmospheric pressure and refluxed for about 2 days or heated at 150~200°C for about 10 minutes in a sealed vial with a microwave instrument and then cooled (Reaction 11):

(Vb) (Vc)

[0044] Intermediate Vd can be prepared by reacting intermediate Vc with an amino alcohol under basic condition in a solvent such as DMF. The reaction may be heated overnight and then cooled (Reaction 12):

26

SUBSTITUTE SHEET (RULE 26)

wherein Ri, R2, R3, R4, and Re are as defined previously for Formula I.

[0045] Intermediate Ve can be formed by reacting intermediate Vd with for example a dehydrating agent such as SOCI2 in a solvent such as CH2CI2 at room temperature overnight or heated at 35° C for about 4 hours, and then cooled (Reaction 13):

[0046] Compounds of the Invention may be formed by reacting intermediate Ve with for example R5-(CH2)_n~L in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating (Reaction 14):

Compounds of Formuta I wherein R5 is as defined previously for Formula I and L is a leaving group such as a halogen, mesylate, or tosylate.

[0047] In some embodiments, R5 is phenyl substituted with dihydrofuryl. The compound VIb may be formed by reacting a compound of formula Via with 2,3- dihydrofuran in the presence of K2CO3, Pd(OAc)2, and PPI13 with heating.

27

SUBSTITUTE SHEET (RULE 26)

[0048] In some embodiments, Rs is phenyl substituted with tetrahydrofuryl. The compound Vic may be formed by reacting Compound VIb with hydrogen gas (H2) in the presence of Pd/C in methanol at room temperature.

[0049] In some embodiments, Rs is phenyl substituted with aziridyl. The Compound

VIf may be prepared by reactions shown below.

28

SUBSTITUTE SHEET (RULE 26)

[0050] Intermediate Vid may be formed by reacting a compound of formula Via with 2-aminoethanol, CuCl and KOH with heating. Intermediate Vie may be formed by reacting Vid with a chlorinating agent such as thionyl chloride in dichloromethane (DCM) at room temperature. The compound VIf may be prepared by reacting Vie with K2CO3 and Nal in dimethylformamide (DMF) with heating.

[0051] In some embodiments, R5 is phenyl substituted with -C(=O)-aziridyl. The compound Vlld may be formed by reactions shown below.

29

SUBSTITUTE SHEET (RULE 26)

[0052] Intermediate Vllb may be formed by reacting a compound of formula Vila with 2-aminoethanol and tert-butyl hydroperoxide at room temperature. Intermediate Vile may be formed by reacting Vllb with a chlorinating agent such as thionyl chloride in dichloromethane (DCM) at room temperature. The compound VHd may be prepared by reacting Vile with K2CO3 and Nal in dimethylformamide (DMF) with heating.

Methods of using Compounds of the Invention

[0053] The Compounds of the Invention are useful in the prophylaxis or treatment of a variety of diseases including any one or more of the following conditions:

(i) Neurodegenerative diseases, including Parkinson’s disease, restless leg, tremors, dyskinesias, Huntington’s disease, Alzheimer’s disease, and drug- induced movement disorders, and related disorders and diseases as described in International Application No. PCT/US2014/030412 and PCT/US2015/050814, the contents of which are incorporated herein by reference;

(ii) Neuroinflammation and/or diseases or disorders associated with neuroinflammation and/or microglial function, such as neuroinflammation associated with neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and demyelinating

30

SUBSTITUTE SHEET (RULE 26) conditions, e.g., multiple sclerosis and prion diseases; neuroinflammation associated with damage sue to stroke, cardiac arrest, hypoxia, intracerebral hemorrhage or traumatic brain injury; neuroinflammation associated with chronic CNS infections, e.g., Lyme disease, syphilis, or CNS infection consequent to an immunosuppressive condition, e.g., HIV dementia; and neuroinflammation consequent to chemotherapy; and related disorders as described in International Application No. PCT/US2017/051220, the contents of which are incorporated herein by reference;

(iii) Mental disorders, including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorders, e.g., narcolepsy, cognitive impairment, e.g., cognitive impairment of schizophrenia, Tourette’s syndrome, autism, fragile X syndrome, psychostimulant withdrawal, chemobrain (cognitive impairment caused by cancer treatment (e.g., chemotherapy) or cancer itself), and drug addiction, and related disorders as described in International Application No. PCT/US2014/025666, the contents of which are incorporated herein by reference;

(iv) Disorders associated with dementia, including disorders associated with Huntington's disease, Parkinson's disease, Multiple sclerosis, Amyotrophic lateral sclerosis, Down syndrome, Elderly depression, Wernicke- Korsakoff s syndrome, corticobasal degenerations, and prion disease; disorders associated with mild cognition impairment and dementing illnesses including senile dementia, Alzheimer’s disease, Pick’s disease, frontotemporal dementia, parasupranculear palsy, dementia with Lewy bodies, and vascular dementia; and behavioral or mood disorders such as agitation/irritation, aggressive/assaultive behavior, anger, physical or emotional outbursts, psychosis, depression and sleep disorders in patients suffering from dementia, particularly Alzheimer’s disease (e.g., sleep maintenance insomnia, advanced sleep-phase syndrome, delayed sleepphase syndrome, frequent awakenings, and waking up feeling unrefreshed); and related disorders as described in International Application No. PCT/US2017/024575, the contents of which are incorporated herein by reference;

31

SUBSTITUTE SHEET (RULE 26) (v) Circulatory and cardiovascular disorders, including cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, e.g., pulmonary arterial hypertension, and sexual dysfunction, including cardiovascular diseases and related disorders as described in International Application No. PCT/US2014/016741, the contents of which are incorporated herein by reference;

(vi) Respiratory and inflammatory disorders, including asthma, chronic obstructive pulmonary disease, and allergic rhinitis, as well as autoimmune and inflammatory diseases, including the inflammatory disorders as described in International Application No. PCT7US2019/053032, the contents of which are incorporated herein by reference;

(vii) Diseases that may be alleviated by the enhancement of progesterone- signaling such as female sexual dysfunction, including the diseases as described in International Application No. PCT7US2007/024866;

(viii) Ophthalmic disorders, such as glaucoma, or elevated intraocular pressure, and other disorders as described in International Application No. PCT/US2010/000534, the contents of which are incorporated herein by reference;

(ix) Cancers or tumors, including acoustic neuroma/vestibular schwannoma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (e.g., glioblastoma multiforme, Brain stem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal (PNET), schwannoma, adenomas (e.g., basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibroids (fibrous histiocytoma), fibromas, hemangiomas, lipomas (e.g., angiolipoma, myelolipoma, fibrolipoma, spindle cell lipoma, hibernoma, atypical lipoma), myxoma, osteoma, leukemias, preleukemias, rhadomyoma, papilloma, seborrheic keratosis, skin adnexal tumors, hepatic adenomas, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, hydatidiform moles, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C cell hyperplasia, pheochromocytoma, insulinoma,

32

SUBSTITUTE SHEET (RULE 26) gastrinoma, carcinoids, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (e.g., metaplasia of the lung), leukoplakia, hemangioma, lymphangioma, carcinoma (e.g., squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, embryonal cell carcinoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, oat cell carcinoma, islet cell carcinoma, malignant carcinoid,), sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), blastoma (e.g., medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, a tumor in the retina of the eye, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant skin adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant shwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, mercell cell neoplasm, cystosarcoma phylloides, Wilms tumor, colorectal cancer, breast cancer, and related disorders as described in International Application No. PCT/US2019/033941, PCT/US2020/012578, PCT/US2020/049248, and PCT7US2023/064202, the contents of which are incorporated herein by reference;

(x) Any disease or condition characterized by low levels of cAMP/PKA and/or cGMP/PKG (or inhibition of cAMP and/or cGMP signaling pathways) in cells expressing PDE1, such as angina, stroke, renal failure, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension, congestive heart failure, an inflammatory disease or disorder, fibrosis, cardiac hypertrophy, vascular remodeling, a connective tissue disease or disorder (e.g., Marfan Syndrome), chronic heart failure (e.g., chronic systolic heart failure), myocardial inflammation, myocardial ischemia, myocardial hypoxia, reperfusion injury, left ventricular dysfunctions (e.g.,

33

SUBSTITUTE SHEET (RULE 26) myocardial infarction, ventricular expansion), vascular leakage (i.e., consequent to hypoxia), acute vascular inflammation (i.e., consequent to vascular injury), cardiac hypertrophy muscular dystrophy (e.g., Duchenne muscular dystrophy), or amyotrophic lateral sclerosis, and related disorders as described in U.S. Pub. No. 2020/0289519, the contents of which are incorporated herein by reference;

(xi) Cardiotoxicity consequent to administration of a chemotherapeutic agent and/or radiation therapy, as described in U.S. Pub. No. 2020/0289519, the contents of which are incorporated herein by reference;

(xii) Renal disorders, including kidney fibrosis, chronic kidney disease, renal failure, glomerulosclerosis, nephritis, and related disorders as described in International Application No. PCT/US2020/047451, the contents of which are incorporated herein by reference; and/or

(xiii) Any disease or condition characterized by reduced dopamine DI receptor signaling activity, including but not limited to attention deficit, cognition, PTSD, memory, and/or inhibitory processing, as discussed in International Application No. PCT/US2019/057260 and International Application No. PCT/US2020/062400, the contents of each of which are incorporated herein by reference.

[0054] In an embodiment, the invention provides methods of treatment or prophylaxis for narcolepsy. In this embodiment, PDE1 Inhibitors may be used as a sole therapeutic agent, but may also be used in combination or for co-administration with other active agents. Thus, the invention further comprises a method of treating narcolepsy comprising administering simultaneously, sequentially, or contemporaneously therapeutically effective amounts of

(i) a PDE1 Inhibitor, e.g., a compound according to Formula I, e.g., any of Compounds

1.0 to 1.77, and

(ii) a compound to promote wakefulness or regulate sleep, e.g., selected from (a) central nervous system stimulants-amphetamines and amphetamine like compounds, e.g., methylphenidate, dextroamphetamine, methamphetamine, and pemoline; (b) modafinil, (c) antidepressants, e.g., tricyclics (including imipramine, desipramine, clomipramine, and protriptyline) and selective serotonin reuptake inhibitors (including fluoxetine and sertraline); and/or (d) gamma hydroxybutyrate (GHB),

34

SUBSTITUTE SHEET (RULE 26) in free or pharmaceutically acceptable salt or prodrug form, to a human or animal patient in need thereof.

[0055] In another embodiment, the invention further provides methods of treatment or prophylaxis of a condition which may be alleviated by the enhancement of the progesterone signaling comprising administering an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, to a human or animal patient in need thereof. Diseases or conditions that may be ameliorated by enhancement of progesterone signaling include, but are not limited to, female sexual dysfunction, secondary amenorrhea (e.g., exercise amenorrhoea, anovulation, menopause, menopausal symptoms, hypothyroidism), pre-menstrual syndrome, premature labor, infertility, for example infertility due to repeated miscarriage, irregular menstrual cycles, abnormal uterine bleeding, osteoporosis, autoimmune disease, multiple sclerosis, prostate enlargement, prostate cancer, and hypothyroidism. For example, by enhancing progesterone signaling, the PDE1 inhibitors may be used to encourage egg implantation through effects on the lining of uterus, and to help maintain pregnancy in women who are prone to miscarriage due to immune response to pregnancy or low progesterone function. The novel PDE1 inhibitors, e.g., as described herein, may also be useful to enhance the effectiveness of hormone replacement therapy, e.g., administered in combination with estrogen/estradiol/estriol and/or progesterone/progestins in postmenopausal women, and estrogen-induced endometrial hyperplasia and carcinoma. The methods of the invention are also useful for animal breeding, for example to induce sexual receptivity and/or estrus in a nonhuman female mammal to be bred.

[0056] In this embodiment, PDE1 Inhibitors may be used in the foregoing methods of treatment or prophylaxis as a sole therapeutic agent, but may also be used in combination or for co-administration with other active agents, for example in conjunction with hormone replacement therapy. Thus, the invention further comprises a method of treating disorders that may be ameliorated by enhancement of progesterone signaling comprising administering simultaneously, sequentially, or contemporaneously therapeutically effective amounts of

1.0 to 1.77, and

(ii) a hormone, e.g., selected from estrogen and estrogen analogues (e.g., estradiol, estriol, estradiol esters) and progesterone and progesterone analogues (e.g., progestins)

35

[0057] In yet another embodiment, the invention provides a method of treating neuroinflammation and/or diseases or disorders associated with neuroinflammation and/or microglial function comprising administering a therapeutically acceptable amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form to a patient in need thereof, i.e., a patient with increased levels of one or more pro-inflammatory cytokines (e.g., ILip, TNF-a, and Ccl2, or combinations thereof). In this embodiment, a therapeutic amount of a Compound of the Invention is an amount effective to (i) reduce or inhibit activation of Ml microglia, and/or (ii) an amount effective to reduce levels of one or more pro-inflammatory cytokines (e.g., ILip, TNF- a, and Ccl2, or combination thereof); to a patient in need thereof. Neuroinflammation and/or diseases or disorders associated with neuroinflammation and/or microglial function include, but are not limited to, neuroinflammation associated with neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and demyelinating conditions, e.g., multiple sclerosis and prion diseases; neuroinflammation associated with damage sue to stroke, cardiac arrest, hypoxia, intracerebral hemorrhage or traumatic brain injury; neuroinflammation associated with chronic CNS infections, e.g., Lyme disease, syphilis, or CNS infection consequent to an immunosuppressive condition, e.g., HIV dementia; and neuroinflammation consequent to chemotherapy.

[0058] The invention further provides a method of promoting resolution of inflammation for the treatment or prophylaxis of inflammation and/or diseases related to inflammation and/or microglial function, the method comprising administering a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, to a patient in need thereof. In this context, it is believed that an effective amount of a Compound of the Invention is an amount effective to promote macrophage activation from the Ml activation state to the M2 activation state. Diseases or disorders associated with inflammation and/or microglial function, e.g., including bacterial infections (e.g., Salmonella typhi, Salmonella typhimurium, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium avium infections),' viral infections (e.g., African Swine Fever Virus, Classical Swine Fever Virus, Dengue Virus, Foot and Mouth Disease Virus, Human Immunodeficiency

36

SUBSTITUTE SHEET (RULE 26) Virus (HIV) (e.g., HIV1), Influenza A Virus, Porcine Circovirus-2, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Pseudorabies Virus, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome Coronavirus, West Nile Virus, Viral Hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C), and coronavirus infections (e.g., a Severe Acute Respiratory Syndrome Coronavirus (e.g., SARS-CoV, SARS-CoV-2, COVID-19), a Middle East Respiratory Syndrome coronavirus (MERS), 229E coronavirus, NL63 coronavirus, OC43 coronavirus, HKU1 coronavirus)) (see U.S. Publication No. 2021/0369715, the contents of which are incorporated herein by reference); parasitic infestations (e.g., Taenia crassiceps. Toxoplasma gondii, Leishmania infantum, Schistosoma mansoni infestations), ' atopic dermatitis; pneumonia; cardiovascular diseases, such as atherosclerosis; obesity and insulin resistance; asthma; pulmonary fibrosis; cardiac obstructive pulmonary disease (COPD); neuropathic pain; stroke; diabetes; sepsis; nonalcoholic steatoheptatitis (NASH); autoimmune hepatitis; systemic lupus erythematosus (SLE); wound healing; pleurisy; peritonitis; and cystic fibrosis.

[0059] In another embodiment, the invention provides a method for treating a cancer or tumor characterized by an increased expression of PDE1 (i.e., PDE1C) relative to normal cells of the same tissue type as the cancerous or tumorous cells comprising administering a pharmaceutically acceptable amount of a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, either alone or in combination with an antitumor agent, chemotherapeutic, gene therapeutic, immunologic treatment, corticosteroid, and/or an antihistamine. By increased expression of PDE1 it is means an increased PDE1 RNA expression, DNA copy number, PDE1 binding (e.g., PET or radio-isotope retention of PDE1 inhibitor molecules), or PDE1 enzymatic activity (e.g., as measured in an enzymatic assay or as reflected in low levels of cAMP in the cancer cells or subcellular domain, e.g. microtubule domains, of the cancer cells) relative to normal cells of the same tissue type as the cancer or tumor cells. Such cancers or tumors include, but are not limited to, acoustic neuroma/vestibular schwannoma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, gliomas (e.g., Brain stem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectodermal (PNET), schwannoma, adenomas (e.g., basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, islet adenoma, fibroadenoma), fibroids (fibrous histiocytoma), fibromas, hemangiomas, lipomas (e.g., angiolipoma,

37

SUBSTITUTE SHEET (RULE 26) myelolipoma, fibrolipoma, spindle cell lipoma, hibernoma, atypical lipoma), myxoma, osteoma, preleukemias, rhadomyoma, papilloma, seborrheic keratosis, skin adnexal tumors, hepatic adenomas, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, hydatidiform moles, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoids, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (e.g., metaplasia of the lung), leukoplakia, hemangioma, lymphangioma, carcinoma (e.g., squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, embryonal cell carcinoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, oat cell carcinoma, islet cell carcinoma, malignant carcinoid,), sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), blastoma (e.g., medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, a tumor in the retina of the eye, osteoblastoma, bone tumors, neuroblastoma), germ cell tumor, mesothelioma, malignant skin adnexal tumors, hypernephroma, seminoma, glioma, malignant meningioma, malignant shwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, mercell cell neoplasm, cystosarcoma phylloides, or Wilms tumor.

[0060] In another embodiment, the present application provides a method of inhibiting cytokine release syndrome, comprising administering an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, to a patient in need thereof, e.g. wherein the patient is suffering from cancer and is receiving one or more of chemotherapeutic treatment, immunologic treatment, gene therapy and/or antibody therapy (including antibodies directed to cancer antigens and/or antibodies to immune checkpoint targets), and wherein the method optionally further comprises administration of corticosteroids and/ antihistamines to the patient.

[0061] The invention also provides a method for the prophylaxis or treatment of disorders associated with dementia comprising administering to the patient a combination of (i) a therapeutically effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form; and (ii) a therapeutically effective amount of a 5-HT2A or 5-

38

SUBSTITUTE SHEET (RULE 26) HT2A/D2 receptor ligand, for example a substituted heterocycle fused gamma-carbolines as described herein, in free, pharmaceutically acceptable salt or prodrug form. Also encompassed by the present invention are compositions comprising (i) a therapeutically effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form; and (ii) a therapeutically effective amount of a 5-HT2A or 5-HT2A/D2 receptor ligand. Similar methods and compositions are set forth in International Application No. PCT/US2017/024575, the contents of which are incorporated herein by reference.

[0062] In this embodiment, the disorders associated with dementia include disorders associated with Huntington's disease, Parkinson's disease, Multiple sclerosis, Amyotrophic lateral sclerosis, Down syndrome, Elderly depression, Wernicke-Korsakoff s syndrome, corticobasal degenerations, and prion disease; disorders associated with mild cognition impairment and dementing illnesses including senile dementia, Alzheimer’s disease, Pick’s disease, frontotemporal dementia, parasupranculear palsy, dementia with Lewy bodies, and vascular dementia; and behavioral or mood disorders such as agitation/irritation, aggressive/assaultive behavior, anger, physical or emotional outbursts, psychosis, depression and sleep disorders in patients suffering from dementia, particularly Alzheimer’s disease (e.g., sleep maintenance insomnia, advanced sleep-phase syndrome, delayed sleep-phase syndrome, frequent awakenings, and waking up feeling unrefreshed).

[0063] The invention also provides a method for treating diseases characterized by disruption of or damage to cGMP/PKG and/or cAMP/PKA signaling mediated pathways, e.g., as a result of increased expression of PDE1 or decreased expression of cGMP/PKG or cAMP/PKA activity due to inhibition or reduced levels of inducers of cyclic nucleotide synthesis, such as dopamine and nitric oxide (NO). It is believed that by inhibiting PDE1, for example, that this action could reverse or prevent the attenuation of cGMP/PKG or cAMP/PKA signaling (e.g., enhance cGMP or cAMP, respectively). Therefore, administration or use of a preferred PDE1 inhibitor as described herein, e.g., a PDE1 inhibitor as hereinbefore described could provide a potential means to provide a treatment for various cardiovascular diseases and disorders.

[0064] In this embodiment, the present invention provides for a method of enhancing the effect of an adenosine A2 receptor agonist in the treatment, mitigation or prophylaxis of a disease or condition characterized by inotropic and/or lusitropic dysfunction comprising administration of an effective amount of a Compound of the Invention, e.g., a compound

39

SUBSTITUTE SHEET (RULE 26) according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, to a patient in need thereof. Diseases and conditions characterized by inotropic and/or lusitropic dysfunction include, but are not limited to, angina, stroke, renal failure, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension, congestive heart failure, an inflammatory disease or disorder, fibrosis, cardiac hypertrophy, vascular remodeling, a connective tissue disease or disorder (e.g., Marfan Syndrome), chronic heart failure, myocardial inflammation, myocardial ischemia, myocardial hypoxia, reperfusion injury, left ventricular dysfunctions (e.g., myocardial infarction, ventricular expansion), vascular leakage (i.e., consequent to hypoxia), acute vascular inflammation (i.e., consequent to vascular injury), cardiac hypertrophy muscular dystrophy (e.g., Duchenne muscular dystrophy), or amyotrophic lateral sclerosis. In such methods, the Compound of the Invention may be administered in conjunction with an additional therapeutic agent, such as an adenosine A2 agonist, a beta-adrenergic receptor antagonist (i.e., a beta-blocker); ACE inhibitor, a calcium channel blocker; angiotensin receptor blockers (ARBs); neprilysin inhibitors or combinations thereof.

[0065] In a related embodiment, the invention provides for a method of treating, mitigating or preventing cardiotoxicity consequent to administration of a chemotherapeutic agent and/or radiation therapy, comprising administration of an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form to a patient in need thereof. Chemotherapeutic agents known to cause cardiotoxicity include, but are not limited to, cyclophosphamide, ifosfamide, cisplatin, carmustine, busulfan, chlormethine, mitomycin, paclitaxel, etoposide, teniposide, the vinca alkaloids, fluorouracil, cytarabine, amsacrine, cladribine, asparaginase, tretinoin, pentostatin, and antagonists of human epidermal growth factor receptor 2 (HER2)/neu, e.g., trastuzumab.

[0066] In yet another related embodiment, the invention provides for a method of treating diseases or disorders mediated by cyclic nucleotides (cAMP or cGMP) and/or epoxygenated fatty acids, comprising administering an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form to a patient in need thereof, optionally in combination with a soluble epoxide hydrolase (sEH) inhibitor. Such diseases

40

SUBSTITUTE SHEET (RULE 26) and disorders include, but are not limited to, pain, neurodegenerative disorders, mental disorders, circulatory and cardiovascular disorders, respiratory disorders, inflammatory disorders, and other disorders set forth in International Application No.

PCT/US2020/066138, the contents of which are incorporated herein by reference in their entirety.

[0067] Methods of treating a renal disorder, including but not limited to kidney fibrosis, chronic kidney disease, renal failure, glomerulosclerosis and nephritis, by administering to a patient in need thereof an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, are also contemplated. The renal disorder being treated may be consequent to diabetes, an injury to a kidney, high blood pressure, a cancerous growth (e.g., polycystic kidney disease), or a cardiovascular disorder (e.g. angina, stroke, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension, congestive heart failure, myocardial, angina, and stroke, hypertension, an inflammatory disease or disorder, fibrosis, cardiac hypertrophy, vascular remodeling, and an connective tissue disease or disorder, e.g., Marfan Syndrome).

[0068] The present disclosure also provides a method for enhancing or potentiating dopamine DI intracellular signaling activity in a cell or tissue comprising contacting said cell or tissue with an amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, sufficient to inhibit PDE1 activity.

[0069] The present disclosure also provides a method for treating a PDEl-related disorder, a dopamine DI receptor intracellular signaling pathway disorder, or disorders that may be alleviated by the enhancement of the progesterone signaling pathway in a patient in need thereof comprising administering to the patient an effective amount of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, that inhibits PDE1, wherein PDE1 activity modulates phosphorylation of DARPP-32 and/or the GluRl AMPA receptor.

[0070] The present disclosure further provides a method for enhancing the positive effects, and mitigating side effects, of dopamine replacement therapy in a patient suffering from a disease or disorder associated with the dopamine DI receptor intracellular pathway, comprising administering a therapeutically effective amount of a Compound of the Invention,

41

SUBSTITUTE SHEET (RULE 26) e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt or prodrug form, to a patient in need thereof (i.e., a patient suffering from Parkinson’s Disease and receiving dopamine replacement therapy). In some aspects of this embodiment, the side effects encompass dyskinesias and motor impairment. Methods for enhancing the positive effects, and mitigating side effects, of dopamine replacement therapy in a patient suffering from a disease or disorder associated with the dopamine DI receptor intracellular pathway are discussed in International Application No. PCT/US2019/57260, the contents of which are incorporated herein by reference.

[0071] In another aspect, the present disclosure also provides a method for the treatment for glaucoma or elevated intraocular pressure comprising topical administration of a therapeutically effective amount of a PDE1 Inhibitor of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt form, in an ophthalmically compatible carrier to the eye of a patient in need thereof. However, treatment may alternatively include a systemic therapy. Systemic therapy includes treatment that can directly reach the bloodstream, or oral methods of administration, for example.

[0072] The present disclosure further provides a pharmaceutical composition for topical ophthalmic use comprising a PDE1 inhibitor; for example an ophthalmic solution, suspension, cream or ointment comprising a PDE1 Inhibitor of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or ophthalmologically acceptable salt form, in combination or association with an ophthalmologically acceptable diluent or carrier, optionally administered sequentially or simultaneously with a second drug useful for treatment of glaucoma or elevated intraocular pressure.

[0073] In addition to the above-mentioned methods, the PDE1 Inhibitor of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, in free or pharmaceutically acceptable salt form is useful to treat psychosis, for example, any conditions characterized by psychotic symptoms such as hallucinations, paranoid or bizarre delusions, or disorganized speech and thinking, e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, and mania, such as in acute manic episodes and bipolar disorder.

[0074] PDE 1 Inhibitors may be used in the foregoing methods of treatment or prophylaxis as a sole therapeutic agent, but may also be used in combination or for co-

42

SUBSTITUTE SHEET (RULE 26) administration with other active agents known to be effective for the treatment or prophylaxis of a particular disease or disorder.

[0075] The present disclosure also provides

(i) a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or pharmaceutically acceptable salt form for example for use in any method or in the treatment of any disease or condition as hereinbefore set forth,

(ii) the use of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or pharmaceutically acceptable salt form, (in the manufacture of a medicament) for treating any disease or condition as hereinbefore set forth,

(iii) a pharmaceutical composition comprising a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or pharmaceutically acceptable salt form, in combination or association with a pharmaceutically acceptable diluent or carrier, and

(iv) a pharmaceutical composition comprising a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or pharmaceutically acceptable salt form, in combination or association with a pharmaceutically acceptable diluent or carrier for use in the treatment of any disease or condition as hereinbefore set forth.

[0076] Therefore, the present disclosure provides use of a Compound of the Invention, e.g., a compound according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or pharmaceutically acceptable salt form, or a Compound of the Invention in a pharmaceutical composition form (in the manufacture of a medicament) for the treatment or prophylactic treatment of any one or more of the diseases or conditions as hereinbefore set forth.

[0077] The phrase “Compounds of the Invention” or “PDE1 Inhibitor of the Invention” encompasses any and all of the compounds disclosed herewith, e.g., compounds according to Formula I, e.g., any of Compounds 1.0 to 1.77, as hereinbefore described, in free or salt form.

43

SUBSTITUTE SHEET (RULE 26) [0078] The words “treatment” and “treating” are to be understood accordingly as embracing prophylaxis and treatment or amelioration of symptoms of disease as well as treatment of the cause of the disease. In one embodiment, the invention provides a method for the treatment of the disease or disorder disclosed herein. In another embodiment, the invention provides a method for the prophylaxis of a disease or disorder as disclosed herein.

[0079] For methods of treatment, the word “effective amount” is intended to encompass a therapeutically effective amount to treat a specific disease or disorder.

[0080] The term “patient” includes human or non-human (i.e., animal) patient. In one embodiment, the invention encompasses both human and nonhuman. In another embodiment, the invention encompasses nonhuman. In other embodiment, the term encompasses human.

[0081] The term “comprising” as used in this disclosure is intended to be open-ended and does not exclude additional, unrecited elements or method steps.

[0082] Compounds of the Invention, in free or pharmaceutically acceptable salt form, may be used as a sole therapeutic agent, but may also be used in combination or for coadministration with other active agents. For example, as Compounds of the Invention potentiate the activity of DI agonists, such as dopamine, they may be simultaneously, sequentially, or contemporaneously administered with conventional dopaminergic medications, such as levodopa and levodopa adjuncts (carbidopa, COMT inhibitors, MAO-B inhibitors), dopamine agonists, and anticholinergics, e.g., in the treatment of a patient having Parkinson’s disease. In addition, the novel PDE1 inhibitors, e.g., as described herein, may also be administered in combination with estrogen/estradiol/estriol and/or progesterone/progestins to enhance the effectiveness of hormone replacement therapy or treatment of estrogen-induced endometrial hyperplasia or carcinoma. The novel PDE1 inhibitors as described herein may also be administered in combination with an antitumor agent, chemotherapeutic, gene therapeutic, immunologic treatment, corticosteroid, and/or an antihistamine to treat a cancer or tumor characterized by an increased expression of PDE1. The Compounds of the Invention may also be administered in combination with a therapeutically effective amount of a 5-HT2A or 5-HT2A/D2 receptor ligand for the prophylaxis or treatment of disorders associated with dementia. In addition, the novel PDE1 inhibitors of the Invention may be administered in combination with an adenosine A2 agonist for the treatment, mitigation or prophylaxis of a disease or condition characterized by inotropic and/or lusitropic dysfunction. Furthermore, the Compounds of the Invention may be

44

SUBSTITUTE SHEET (RULE 26) administered in combination with a soluble epoxide hydrolase (she) inhibitor in a method of treating diseases or disorders mediated by cAMP or cGMP and/or epoxygenated fatty acids.

[0083] Dosages employed in practicing the present invention will of course vary depending, e.g., on the particular disease or condition to be treated, the particular Compound of the Invention used, the mode of administration, and the therapy desired. Compounds of the Invention may be administered by any suitable route, including orally, parenterally, transdermally, or by inhalation, but are preferably administered orally. In general, satisfactory results, e.g., for the treatment of diseases as hereinbefore set forth are indicated to be obtained on oral administration at dosages of the order from about 0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicated daily dosage for oral administration will accordingly be in the range of from about 0.75 to 150 mg, conveniently administered once, or in divided doses 2 to 4 times, daily or in sustained release form. Unit dosage forms for administration (e.g., oral administration) thus for example may comprise from about 0.2 to 150 or 300 mg, e.g., from about 0.2 or 2.0 to 10, 25, 50, 75, 100, 150, or 200 mg, from about 1 to 100 mg, from about 1 to 50 mg, from about 1 to 10 mg, from about 5 to 10 mg, from about 10 to 50 mg, 1 mg, 3 mg, 5 mg, 10 mg, 30 mg, or 90 mg, of a Compound of the Invention, together with a pharmaceutically acceptable diluent or carrier therefor.

[0084] Pharmaceutical compositions comprising Compounds of the Invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus, oral dosage forms may include tablets, capsules, solutions, suspensions and the like. In some embodiments, the composition is in the form of an oral dosage form (such as a tablet or capsule), an intranasal dosage form (e.g., a nasal spray or mister), a pulmonary dosage form (e.g., an inhaler), or an injectable dosage form (e.g., an intravenous, intramuscular or subcutaneous injection, such as a long-acting depot injection).

EXAMPLES

[0085] The synthetic methods for various Compounds of the Invention are illustrated below. The intermediates of Compounds of the Invention as well as other Compounds of the Invention and their salts may be made using the methods as similarly described below and/or by methods similar to those generally described in the detailed description and by methods known in the chemical art.

45

SUBSTITUTE SHEET (RULE 26) Example 1: Synthesis of 2-(4-(4,5-dihydrofuran-2-yl)benzyl)-3-((4-fluorophenyl)amino)- 5,7,7-trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 1)

Compound 1

[0086] A mixture of K2CO3 (410 mg, 3.0 mmol), PPI13 (53 mg, 0.2 mmol), Pd(OAc)2 (22 mg, 0.1 mmol), and (2-(4-bromobenzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8- dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (497 mg, 1.0 mmol) in 2,3- dihydrofuran (3 mL) is bubbled with argon at room temperature for 2 mins. The resulting mixture is heated at 110 °C in a microwave reactor for 2 h. The reaction solution is diluted with DCM (20 mL) and washed with water (20 mL). The obtained DMC phase is evaporated to dryness. The residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 : 1 : 0.1 volume). The product (Compound 1) is given as a beige solid (243 mg, 50% yield). MS (ESI) m/z 487.24 [M + H]⁺. ¹ H NMR (500 MHz, DMSO) 5 8.65 (s, 1H), 7.20 (d, J= 8.0 Hz, 2H), 7.09 (d, J= 8.1 Hz, 2H), 7.03 - 6.94 (m, 2H), 6.82 - 6.74 (m, 2H), 6.09 (dq, J= 5.8, 1.8 Hz, 1H), 5.91 (dq, J= 6.2, 2.2 Hz, 1H), 5.68 (ddt, J= 5.9, 3.8, 1.9 Hz, 1H), 5.14 (s, 2H), 4.73 (ddt, J= 13.0, 6.1, 2.0 Hz, 1H), 4.66 - 4.58 (m, 1H), 3.57 (s, 2H), 3.08 (s, 3H), 1.25 (s, 6H). ¹³C NMR (126 MHz, DMSO) 5 157.57, 157.01, 155.70, 150.88, 148.48, 141.71, 141.58, 139.32, 135.86, 129.82, 127.20, 126.73, 126.23, 117.44, 117.38, 115.23, 115.05, 89.70, 86.57, 75.19, 64.28, 57.34, 50.39, 29.40, 28.33.

Example 2: Synthesis of 3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(4- (tetrahydrofuran-2-yl)benzyl)-7,8-dihydro-2H-imidazo [1,2-a] pyrazolo [4,3-e] pyrimidin- 4(5H)-one (Compound 2)

46

SUBSTITUTE SHEET (RULE 26)

Compound 2

[0087] To a solution of Compound 1 (150 mg, 0.308 mmol) in methanol (6 mL) is added 10% Pd/C (50 mg). The mixture is degassed and backfilled with Ar three times, then stirred under H2 (1 atm) for 1 h. The reaction mixture was filtered through a pad of celite and the pad is washed with methanol (10 mL). The filtrate is combined and concentrated to dryness. The residue is purified by silica-gel column chromatography using a gradient of 0- 100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 : 1 : 0.1 volume). The product (Compound 2) is given as an off-white solid (126 mg, 83% yield). MS (ESI) m/z 489.27 [M + H]⁺. ¹ H NMR (300 MHz, CDCI3) 8 7.25 - 7.16 (m, 2H), 7.00 - 6.83 (m, 6H), 6.70 (s, 1H), 4.83 (d, J= 4.0 Hz, 3H), 4.07 (dt, J= 8.3, 6.8 Hz, 1H), 3.91 (dt, J= 8.3, 6.9 Hz, 1H), 3.68 (s, 2H), 3.33 (s, 3H), 2.38 - 2.21 (m, 1H), 2.07 - 1.91 (m, 2H), 1.83 - 1.65 (m, 1H), 1.39 (s, 6H). ¹³C NMR (126 MHz, DMSO) 5 157.56, 157.02, 155.68, 150.88, 148.47, 142.83, 141.55, 139.33, 135.27, 127.01, 125.69, 117.41, 117.35, 115.20, 115.03, 89.73, 79.44, 67.78, 64.29, 57.35, 50.42, 34.24, 29.40, 28.32, 25.49.

Example 3: Synthesis of 2-(4-(aziridin-l-yl)benzyl)-3-((4-fluorophenyl)amino)-5,7,7- trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 3)

SUBSTITUTE SHEET (RULE 26)

Compound 3

(a) 3-((4-fluorophenyl)amino)-2-(4-((2-hydroxyethyl)amino)benzyl)-5,7,7-trimethyl-7,8- dihydro-2H-imidazo [1,2-a] pyrazolo [4,3- e] pyrimidin-4(5H)-one.

[0088] A mixture of 2-(4-bromobenzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl- 7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (497 mg, 1.0 mmol), CuCl (12 mg, 0.1 mmol), and KOH (112 Mg, 2.0 mmol) in anhydrous 2-aminoethanol (2 mL) is stirred at 90 °C for 12 h. To the reaction mixture is added water (10 mL) and the resulting mixture is extracted with di chloromethane (DCM) (15 mL). The extract is evaporated to dryness and the residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 : 1 : 0.1 volume). The product (3-((4- fhiorophenyl)amino)-2-(4-((2-hydroxyethyl)amino)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H- imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one) is given as a beige solid (198 mg, 41% yield). MS (ESI) m/z 478.22 [M + H]⁺. 'H NMR (500 MHz, CDCI3) 8 7.00 - 6.93 (m, 3H), 6.88 (dd, J= 8.9, 4.6 Hz, 3H), 6.84 (dd, J= 8.8, 2.9 Hz, 2H), 6.66 (s, 1H), 6.55 - 6.49 (m, 2H), 4.75 (s, 2H), 3.82 (t, J= 5.2 Hz, 2H), 3.68 (s, 2H), 3.31 (s, 3H), 3.28 (t, J= 5.3 Hz, 2H), 1.38 (s, 6H).

(b) 2-(4-((2-chloroethyl)amino)benzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8- dihydro-2H-imidazo [1,2-a] pyrazolo [4,3-e] pyrimidin-4(5H)-one

[0089] To a solution of 3-((4-fhiorophenyl)amino)-2-(4-((2- hydroxyethyl)amino)benzyl)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3- e]pyrimidin-4(5H)-one) (195 mg, 0.408 mmol) in DCM (1.5 mL) at room temperature under argon is added thionyl chloride (1.5 mL). The resulting solution is stirred at room temperature for 24 h, and the solvents is removed under reduced pressure. To the residue is added sat.

48

SUBSTITUTE SHEET (RULE 26) NaHCCh (8 mL) and the obtained mixture is extracted with DCM. The extract is evaporated to dryness and the residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NHs in methanol, 10 :1: 0.1 volume). The product (2-(4-((2- chloroethyl)amino)benzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8-dihydro-2H- imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one) is given as a beige solid (60 mg, 30% yield). MS (ESI) mlz 496.22 [M + H]⁺.

(c) 2-(4-(aziridin-l-yl)benzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8-dihydro- 2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 3)

[0090] A suspension of 2-(4-((2-chloroethyl)amino)benzyl)-3-((4- fluorophenyl)amino)-5,7,7-trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3- e]pyrimidin-4(5H)-one (59 mg, 0.119 mmol), K2CO3 (49 mg, 0.357 mmol), and Nal (36 mg, 0.238 mmol) in DMF (1 mL) is stirred at 100 °C for 3 h. The reaction mixture is evaporated to dryness and the residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 : 1 : 0.1 volume). The final product (Compound 3) is given as an off-white solid (35 mg, 64% yield). MS (ESI) mlz 460.25 [M + H]⁺. 'H NMR (500 MHz, CDCI3) 8 6.98 - 6.92 (m, 2H), 6.87 (q, J= 4.0 Hz, 3H), 6.84 (td, J= 6.6, 2.1 Hz, 2H), 6.70 (s, 1H), 4.77 (s, 2H), 3.69 (s, 2H), 3.33 (s, 3H), 2.07 (s, 4H), 1.39 (s, 6H). ¹³C NMR (126 MHz, DMSO) 5 157.51, 157.02, 155.64, 154.59, 150.88, 148.40, 141.33, 139.41, 129.90, 127.95, 120.76, 117.32, 117.26, 115.20, 115.03, 89.79, 64.29, 57.36, 50.27, 29.40, 28.32, 27.09.

Example 4: Synthesis of 2-(4-(aziridine-l-carbonyl)benzyl)-3-((4-fluorophenyl)amino)- 5,7,7-trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 4)

SUBSTITUTE SHEET (RULE 26)

Compound 4

(a) 4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H- imidazo [1,2-a] pyrazolo [4,3-e] pyrimidin-2-yl)methyl)-N-(2-hydroxyethyl)benzamide

[0091] To a suspension of 2-(4-acetylbenzyl)-3-((4-fluorophenyl)amino)-5,7,7- trimethyl-7,8-dihydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (360 mg, 0.781 mmol) and 2-aminoethanol (286 mg, 4.69 mmol) in hexane (6 mL) at room temperature under argon is added tert-butyl hydroperoxide (70% in water, 0.7 mL, 8.467 mmol) . The resulting mixture is stirred at room temperature for 4 h, and solid Na2S20s (200 mg) is added to quench the reaction. The reaction mixture is evaporated and the residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 :1: 0.1 volume). The product (4-((3-((4-fhiorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8- tetrahydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)-N-(2- hydroxyethyl)benzamide) is given as a beige solid (198 mg, 48% yield). MS (ESI) mlz 506.24 [M + H]⁺.

(b) N-(2-chloroethyl)-4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8- tetrahydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)benzamide

[0092] To a solution of 4-((3-((4-fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8- tetrahydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)-N-(2- hydroxyethyl)benzamide (193 mg, 0.382 mmol) in DCM (1.5 mL) at room temperature under argon is added thionyl chloride (1.5 mL). The resulting solution is stirred at room temperature for 24 h, and the solvents is removed under reduced pressure. The residue is further dried under high vacuum for 24 h to yield the product (N-(2-chloroethyl)-4-((3-((4- fluorophenyl)amino)-5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[l,2- a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)benzamide) as a beige solid (200 mg, crude). This

50

SUBSTITUTE SHEET (RULE 26) crude product is directly used for the next reaction without further purification. MS (ESI) m/z 524.19 [M + H]⁺.

(c) 2-(4-(aziridine-l-carbonyl)benzyl)-3-((4-fluorophenyl)amino)-5,7,7-trimethyl-7,8- dihydro-2H-imidazo [1,2-a] pyrazolo [4,3- e] pyrimidin-4(5H)-one (Compound 4)

[0093] A suspension ofN-(2-chloroethyl)-4-((3-((4-fluorophenyl)amino)-5,7,7- trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-2- yl)methyl)benzamide (200 mg, 0.382 mmol), K2CO3 (207 mg, 1.50 mmol), and Nal (111 mg, 0.740 mmol) in DMF (2 mL) is stirred at 100 °C for 1 h. The reaction mixture is evaporated to dryness and the residue is dissolved in DCM (15 mL). The obtained mixture is washed with water (10 mL) and evaporated to dryness. The residue is purified by silica-gel column chromatography using a gradient of 0-100% solvent B in solvent A as eluent (solvent A: DCM; solvent B: a mixture of DCM : methanol : 7 N NH3 in methanol, 10 :1: 0.1 volume). The final product (Compound 4) is given as an off-white solid (96 mg, 53% yield over 2- step). MS (ESI) m/z 488.25 [M + H]⁺. 'H NMR (500 MHz, DMSO) 5 8.66 (s, 1H), 7.81 - 7.75 (m, 2H), 7.21 - 7.16 (m, 2H), 7.02 - 6.94 (m, 2H), 6.82 - 6.74 (m, 2H), 5.23 (s, 2H), 4.37 (t, J= 9.4 Hz, 2H), 3.93 (t, J= 9.5 Hz, 2H), 3.58 (s, 2H), 3.09 (s, 3H), 1.25 (s, 6H). ¹³C NMR (126 MHz, DMSO) 5 162.57, 157.63, 156.99, 155.76, 150.87, 148.59, 141.88, 140.03, 139.15, 127.86, 127.13, 126.63, 117.63, 117.57, 115.20, 115.02, 89.65, 67.31, 64.32, 57.36, 54.89, 54.37, 50.40, 29.41, 28.34.

Example 5

Measurement of PDE1 inhibition in vitro using IMAP Phosphodiesterase Assay Kit

[0094] Phosphodiesterase 1 (PDE1) is a calcium/calmodulin dependent phosphodiesterase enzyme that converts cyclic guanosine monophosphate (cGMP) to 5'- guanosine monophosphate (5'-GMP). PDE1 can also convert a modified cGMP substrate, such as the fluorescent molecule cGMP-fluorescein, to the corresponding GMP -fluorescein. The generation of GMP-fluorescein from cGMP -fluorescein can be quantitated, using, for example, the IMAP (Molecular Devices, Sunnyvale, CA) immobilized-metal affinity particle reagent.

[0095] Briefly, the IMAP reagent binds with high affinity to the free 5 ’-phosphate that is found in GMP-fluorescein and not in cGMP-fluorescein. The resulting GMP- fluorescein - IMAP complex is large relative to cGMP-fluorescein. Small fluorophores that are bound up in a large, slowly tumbling, complex can be distinguished from unbound

51

SUBSTITUTE SHEET (RULE 26) fluorophores, because the photons emitted as they fluoresce retain the same polarity as the photons used to excite the fluorescence.

[0096] In the phosphodiesterase assay, cGMP -fluorescein, which cannot be bound to IMAP, and therefore retains little fluorescence polarization, is converted to GMP-fluorescein, which, when bound to IMAP, yields a large increase in fluorescence polarization (Amp). Inhibition of phosphodiesterase, therefore, is detected as a decrease in Amp.

[0097] Enzyme assay

Materials: All chemicals are available from Sigma-Aldrich (St. Louis, MO) except for IMAP reagents (reaction buffer, binding buffer, FL-GMP and IMAP beads), which are available from Molecular Devices (Sunnyvale, CA).

Assay: The following phosphodiesterase enzymes may be used: 3’,5’-cyclic-nucleotide- specific bovine brain phosphodiesterase (Sigma, St. Louis, MO) and recombinant full length human PDE1 A and PDE1B (r-hPDEl A and r-hPDElB, respectively) which may be produced e.g., in HEK or SF9 cells by one skilled in the art. The PDE1 enzyme is reconstituted with 50% glycerol to 2.5 U/ml. One unit of enzyme will hydrolyze 1.0 pmole of 3’,5’-cAMP to 5’-AMP per min at pH 7.5 at 30°C. One part enzyme is added to 1999 parts reaction buffer (30 pM CaCh, 10 U/ml of calmodulin (Sigma P2277), lOmM Tris-HCl pH 7.2, lOmM MgCh, 0.1% BSA, 0.05% NaNs) to yield a final concentration of 1.25mU/ml. 99 pl of diluted enzyme solution is added into each well in a flat bottom 96-well polystyrene plate to which 1 pl of test compound dissolved in 100% DMSO is added. The test compounds are mixed and pre-incubated with the enzyme for 10 min at room temperature.

[0098] The FL-GMP conversion reaction is initiated by combining 4 parts enzyme and inhibitor mix with 1 part substrate solution (0.225 pM) in a 384-well microtiter plate. The reaction is incubated in dark at room temperature for 15 min. The reaction is halted by addition of 60 pl of binding reagent (1 :400 dilution of IMAP beads in binding buffer supplemented with 1 : 1800 dilution of antifoam) to each well of the 384-well plate. The plate is incubated at room temperature for 1 hour to allow IMAP binding to proceed to completion, and then placed in an Envision multimode microplate reader (PerkinElmer, Shelton, CT) to measure the fluorescence polarization (Amp).

[0099] A decrease in GMP concentration, measured as decreased Amp, is indicative of inhibition of PDE activity. IC50 values are determined by measuring enzyme activity in the presence of 8 to 16 concentrations of compound ranging from 0.0037 nM to 80,000 nM

52

SUBSTITUTE SHEET (RULE 26) and then plotting drug concentration versus AmP, which allows IC50 values to be estimated using nonlinear regression software (XLFit; IDBS, Cambridge, MA).

[00100] The Compounds of the Invention were tested for PDE1 inhibitory activity.

Exemplified Compounds of the Invention (e.g., Compounds 1-4) have IC50 values of less than or equal to 10 nm. IC50 values for Exemplified Compounds of the Invention (Compounds 1- 4) are as shown in Table 1 below.

Table 1

SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS What is claimed is:

1. A compound of Formula I:

wherein

(i) Ri is H or C1-4 alkyl (e.g., methyl or ethyl);

(ii) R2 and R3 are independently H or C1-6 alkyl (e.g., methyl or ethyl);

(iii) R4 is H or C1-4 alkyl (e.g., methyl or ethyl);

(v) Re and R7 are independently H or aryl (e.g., phenyl) optionally substituted with one or more groups independently selected from Cue alkyl (e.g., methyl or ethyl) and halogen (e.g., F or Cl), for example unsubstituted phenyl or phenyl substituted with one or more halogen (e.g., F) or phenyl substituted with one or more C1-6 alkyl and one or more halogen or phenyl substituted with one C1-6 alkyl and one

54

SUBSTITUTE SHEET (RULE 26) halogen, for example 4-fluorophenyl or 3,4-difluorophenyl or 4-fluoro- 3 -methylphenyl; and

(vi) n is 1, 2, 3, or 4, in free, salt, or prodrug form, e.g., pharmaceutically acceptable salt form, including enantiomers, diastereomers, and racemates, thereof.

2. The compound according to claim 1, wherein

(i) Ri is methyl;

(ii) R2 and R3 are both methyl;

(iii) R4 is H;

(iv) R7 is H and Re is 4-fluorophenyl; and

(v) n is 1.

3. The compound according to claim 1 or 2, wherein R5 is phenyl substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens or (b) -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more oxygens.

4. The compound according to claim 3, wherein R5 is phenyl substituted with tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl).

5. The compound according to claim 3, wherein R5 is phenyl substituted with -C(=O)-X, wherein X is tetrahydrofuryl or dihydrofuryl (e.g., 2,3- dihydrofuryl).

6. The compound according to claim 1 or 2, wherein R5 is phenyl substituted with (a) saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens or -C(=O)-X, wherein X is saturated or unsaturated 3-5 membered heterocycloalkyl comprising one or more nitrogens.

7. The compound according to claim 6, wherein R5 is phenyl substituted with aziridyl.

8. The compound according to claim 6, wherein R5 is phenyl substituted with -C(=O)-X, wherein X is aziridyl.

9. The compound according to claim 1, wherein the compound is selected from:

55

SUBSTITUTE SHEET (RULE 26)

in free or salt form.

10. The compound according to claim 1, wherein the compound is selected from:

in free or salt form.

11. The compound according to claim 1, wherein the compound is selected from:

SUBSTITUTE SHEET (RULE 26)

in free or salt form.

12. A pharmaceutical composition comprising a compound according to any of claims 1-11, in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier.

13. The composition of claim 12, wherein the composition is in the form of an oral dosage form (such as a tablet or capsule), an intranasal dosage form (e.g., a nasal spray or mister), a pulmonary dosage form (e.g., an inhaler), or an injectable dosage form (e.g., an intravenous, intramuscular or subcutaneous injection, such as a long-acting depot injection).

14. A method of preventing or treating a disease or disorder selected from neurodegenerative diseases (e.g., Parkinson’s disease, restless leg, tremors, dyskinesias, Huntington’s disease, Alzheimer’s disease, and drug-induced movement disorders); neuroinflammation and/or diseases or disorders associated with neuroinflammation and/or microglial function; mental disorders (e.g., depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorders, e.g., narcolepsy, cognitive impairment, e.g., cognitive impairment of schizophrenia, Tourette’s syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and drug addiction); chemobrain (cognitive impairment caused by cancer treatment (e.g., chemotherapy) or cancer itself); disorders associated with dementia; circulatory and cardiovascular disorders (e.g., cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, and sexual dysfunction); respiratory and inflammatory disorders (e.g., asthma, chronic obstructive pulmonary disease, and allergic rhinitis); diseases which may be alleviated by the enhancement of progesterone signaling (e.g., female sexual dysfunction); psychosis; ophthalmic disorders (e.g., glaucoma and elevated intraocular pressure); traumatic brain injury; cancers or tumors (e.g., glioma, colon cancer, and breast

57

SUBSTITUTE SHEET (RULE 26) cancer); renal disorders (e.g., kidney fibrosis, chronic kidney disease, renal failure, glomerulosclerosis, nephritis); cardiotoxicity consequent to administration of a chemotherapeutic agent and/or radiation therapy; any disease or condition characterized by low levels of cAMP and/or cGMP in cells expressing PDEl(e.g., angina, stroke, renal failure, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension associated with diabetes, hypertension associated with atherosclerosis, renovascular hypertension, congestive heart failure, an inflammatory disease or disorder, fibrosis, cardiac hypertrophy, vascular remodeling, a connective tissue disease or disorder, chronic heart failure, myocardial inflammation, myocardial ischemia, myocardial hypoxia, reperfusion injury, left ventricular dysfunctions, vascular leakage, acute vascular inflammation, and amyotrophic lateral sclerosis); bacterial infections; viral infections; obesity; and any disease or condition characterized by reduced dopamine DI receptor signaling activity (e.g., attention deficit, cognition, PTSD, memory, and/or inhibitory processing), wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of the compound according to any of claims 1-11 or a therapeutically effective amount of the pharmaceutical composition according to claim 12 or 13.

58

SUBSTITUTE SHEET (RULE 26)