# Patent

## Title
Pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione derivatives useful as inhibitors of phosphodiesterase 1

## Abstract

Optionally substituted (5- or 7-amino)-3,4-dihydro-(optionally 4-oxo, 4-thioxo or 4-imino)-1H-pyrrolo[3,4-d]pyrimidin-2(6H)-ones, Compounds of Formula I, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a United States Application under 35 U.S.C. 371 claiming benefit of PCT Application No. PCT/US2009/006443, filed on Dec. 7, 2009, which claims the benefit of U.S. Provisional Application Nos. 61/120,444, filed on Dec. 6, 2008, and 61/235,888 filed on Aug. 21, 2009, respectively, the contents of each of which are incorporated herein by reference.


TECHNICAL FIELD
The present invention relates to a compound of Formula I as described below, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them. Of particular interest are novel compounds useful as inhibitors of phosphodiesterase 1 (PDE1), e.g., in the treatment of diseases involving disorders of the dopamine D1 receptor intracellular pathway, such as Parkinson's disease, depression, narcolepsy, damage to cognitive function, e.g., in schizophrenia, or disorders that may be ameliorated through enhanced progesterone-signaling pathway, e.g., female sexual dysfunction.
BACKGROUND OF THE INVENTION
Eleven families of phosphodiesterases (PDEs) have been identified 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. The three known CaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in central nervous system tissue. PDE is expressed throughout the brain with higher levels of expression in the CA1 to CA3 layers of the hippocampus and cerebellum and at a low level in the striatum. PDE1A is also expressed in the lung and heart. PDE1B is predominately expressed in the striatum, dentate gyrus, olfactory tract and cerebellum, and its expression correlates with brain regions having high levels of dopaminergic innervation. Although PDE1B is primarily expressed in the central nervous system, it may be detected in the heart. PDE1C is primarily expressed in olfactory epithelium, cerebellar granule cells, and striatum. PDE1C is also expressed in the heart and vascular smooth muscle.
Cyclic nucleotide phosphodiesterases decrease intracellular cAMP and cGMP signaling by hydrolyzing these cyclic nucleotides to their respective inactive 5′-monophosphates (5′AMP and 5′GMP). CaM-PDEs play a critical role in mediating signal transduction in brain cells, particularly within an area of the brain known as the basal ganglia or striatum. For example, NMDA-type glutamate receptor activation and/or dopamine D2 receptor activation result in increased intracellular calcium concentrations, leading to activation of effectors such as calmodulin-dependent kinase II (CaMKII) and calcineurin and to activation of CaM-PDEs, resulting in reduced cAMP and cGMP. Dopamine D1 receptor activation, on the other hand, leads to activation of nucleotide cyclases, resulting in increased cAMP and cGMP. These cyclic nucleotides in turn activate protein kinase A (PKA; cAMP-dependent protein kinase) and/or protein kinase G (PKG; cGMP-dependent protein kinase) that phosphorylate downstream signal transduction pathway elements such as DARPP-32 (dopamine and cAMP-regulated phosphoprotein) and cAMP responsive element binding protein (CREB). Phosphorylated DARPP-32 in turn inhibits the activity of protein phosphates-1 (PP-1), thereby increasing the state of phosphorylation of substrate proteins such as progesterone receptor (PR), leading to induction of physiologic responses. Studies in rodents have suggested that inducing cAMP and cGMP synthesis through activation of dopamine D1 or progesterone receptor enhances progesterone signaling associated with various physiological responses, including the lordosis response associated with receptivity to mating in some rodents. See Mani, et al., Science (2000) 287: 1053, the contents of which are incorporated herein by reference.
CaM-PDEs can therefore affect dopamine-regulated and other intracellular signaling pathways in the basal ganglia (striatum), including but not limited to nitric oxide, noradrenergic, neurotensin, CCK, VIP, serotonin, glutamate (e.g., NMDA receptor, AMPA receptor), GABA, acetylcholine, adenosine (e.g., A2A receptor), cannabinoid receptor, natriuretic peptide (e.g., ANP, BNP, CNP), DARPP-32, and endorphin intracellular signaling pathways.
Phosphodiesterase (PDE) activity, in particular, phosphodiesterase 1 (PDE1) activity, functions in brain tissue as a regulator of locomotor activity and learning and memory. PDE1 is a therapeutic target for regulation of intracellular signaling pathways, preferably in the nervous system, including but not limited to a dopamine D1 receptor, dopamine D2 receptor, nitric oxide, noradrenergic, neurotensin, CCK, VIP, serotonin, glutamate (e.g., NMDA receptor, AMPA receptor), GABA, acetylcholine, adenosine (e.g., A2A receptor), cannabinoid receptor, natriuretic peptide (e.g., ANP, BNP, CNP), endorphin intracellular signaling pathway and progesterone signaling pathway. For example, inhibition of PDE should act to potentiate the effect of a dopamine D1 agonist by protecting cGMP and cAMP from degradation, and should similarly inhibit dopamine D2 receptor signaling pathways, by inhibiting PDE1 activity. Chronic elevation in intracellular calcium levels is linked to cell death in numerous disorders, particularly in neurodegerative diseases such as Alzheimer's, Parkinson's and Huntington's Diseases and in disorders of the circulatory system leading to stroke and myocardial infarction. PDE1 inhibitors are therefore potentially useful in diseases characterized by reduced dopamine D1 receptor signaling activity, such as Parkinson's disease, restless leg syndrome, depression, narcolepsy and cognitive impairment. PDE1 inhibitors are also useful in diseases that may be alleviated by the enhancement of progesterone-signaling such as female sexual dysfunction.
There is thus a need for compounds that selectively inhibit PDE1 activity, especially PDE1A and/or PDE activity.
SUMMARY OF THE INVENTION
In the first embodiment, the invention provides a compound of Formula II




wherein

    
    
        (i) L is S, SO or SO2;
        (ii) R1 is H or C1-6alkyl (e.g., methyl or ethyl);
        (iii) R2 is
        
            H,
            C1-6alkyl (e.g., isopropyl, isobutyl, neopentyl, 2-methylbutyl, 2,2-dimethylpropyl) wherein said alkyl group is optionally substituted with halo (e.g., fluoro) or hydroxy (e.g., 1-hydroxypropan-2-yl, 3-hydroxy-2-methylpropyl),
            —C0-4-alkyl-C3-8cycloalkyl (e.g., cyclopentyl, cyclohexyl) optionally substituted with one or more amino (e.g., —NH2), for example, 2-aminocyclopentyl or 2-aminocyclohexyl), wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl (e.g., 1-methyl-pyrrolindin-2-yl, 1-methyl-pyrrolindin-3-yl, 1-methyl-pyrrolindin-2-yl-methyl or 1-methyl-pyrrolindin-3-yl-methyl),
            C3-8heterocycloalkyl (e.g., pyrrolidinyl, for example, pyrrolidin-3-yl) optionally substituted with C1-6alkyl (e.g., methyl), for example, 1-methylpyrrolidin-3-yl,
            C3-8cycloalkyl-C1-6alkyl (e.g., cyclopropylmethyl),
            haloC1-6alkyl (e.g., trifluoromethyl, 2,2,2-trifluoroethyl),
            —N(R14)(R15)—C1-6alkyl (e.g., 2-(dimethylamino)ethyl,2-aminopropyl),
            hydroxyC1-6alkyl (e.g., (e.g., 3-hydroxy-2-methylpropyl, 1-hydroxyprop-2-yl),
            arylC0-6alkyl (e.g., benzyl),
            heteroarylC1-6alkyl (e.g., pyridinylmethyl),
            C1-6alkoxyarylC1-6alkyl (e.g., 4-methoxybenzyl);
            -G-J wherein:
            
                G is a single bond or, alkylene (e.g., methylene);
                J is cycloalkyl or heterocycloalkyl (e.g., oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl) optionally substituted with C1-6alkyl (e.g., (1-methylpyrrolidin-2-yl));
            
            
        
        
        (iv) R3 is attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A
    
    






    
    
        
        
            wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F);
            and R10 is
            
                halogen,
                C1-6alkyl,
                C3-8cycloalkyl,
                heteroC3-8cycloalkyl (e.g., pyrrolidinyl or piperidinyl)
                haloC1-6alkyl (e.g., trifluoromethyl),
                aryl (e.g., phenyl),
                heteroaryl (e.g., pyridyl, (for example, pyrid-2-yl) or e.g., thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), diazolyl, triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl),
                alkyl sulfonyl (e.g., methyl sulfonyl),
                arylcarbonyl (e.g., benzoyl), or
                heteroarylcarbonyl,
                alkoxycarbonyl, (e.g., methoxycarbonyl),
                aminocarbonyl;
                preferably phenyl, pyridyl, e.g., 2-pyridyl, piperidinyl, or pyrrolidinyl;
                wherein the aryl, heteroaryl cycloalkyl or heterocycloalkyl is optionally substituted with one or more halo (e.g., F or Cl), C1-4alkyl, C1-4alkoxy, C1-4haloalkyl (e.g., trifluoromethyl), and/or —SH,
                provided that when X, Y or X is nitrogen, R8, R9 or R10, respectively, is not present;
            
            
        
        
        (v) R4 is
        
            H,
            C1-6alkyl (e.g., methyl, isopropyl),
            C3-8cycloalkyl (e.g., cyclopentyl),
            C3-8heterocycloalkyl (e.g., pyrrolidin-3-yl),
            aryl (e.g., phenyl) or heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) wherein said aryl or heteroaryl is optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkyl, C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        
        
        (vi) R14 and R15 are independently H or C1-6alkyl,

in free or salt form.

    
    


In still another embodiment, the invention provides a Compound of Formula II as described above, wherein

    
    
        R4 is:
        
            C1-6alkyl (e.g., methyl, isopropyl),
            C3-8cycloalkyl (e.g., cyclopentyl),
            C3-8heterocycloalkyl (e.g., pyrrolidin-3-yl),
            aryl (e.g., phenyl) or heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) wherein said aryl or heteroaryl is optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkyl, C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        
        
        and
        R3 is attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A
    
    






    
    
        
        
            wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F);
            and R10 is
            C3-8cycloalkyl
            
                heteroC3-8cycloalkyl (e.g., pyrrolidinyl or piperidinyl)
                aryl (e.g., phenyl), or
                heteroaryl (e.g., pyridyl, (for example, pyrid-2-yl) or e.g., thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), diazolyl, triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl),
                wherein the aryl or heteroaryl is optionally substituted with one or more halo (e.g., F or Cl), C1-4alkyl, C1-4alkoxy, C1-4haloalkyl (e.g., trifluoromethyl), and/or —SH,
                provided that when X, Y or X is nitrogen, R8, R9 or R10, respectively, is not present;

in free or salt form.

            
            
        
        
    
    


The invention also provides a compound of formula I




wherein

    
    
        (i) L is S, SO or SO2;
        (ii) R1 is H or C1-6alkyl (e.g., methyl or ethyl);
        (iii) R2 is
        
            H,
            C1-6alkyl (e.g., isopropyl, isobutyl, neopentyl, 2-methylbutyl, 2,2-dimethylpropyl) wherein said alkyl group is optionally substituted with halo (e.g., fluoro) or hydroxy (e.g., 1-hydroxypropan-2-yl, 3-hydroxy-2-methylpropyl),
            —C0-4-alkyl-C3-8cycloalkyl (e.g., cyclopentyl, cyclohexyl) optionally substituted with one or more amino (e.g., —NH2), for example, 2-aminocyclopentyl or 2-aminocyclohexyl), wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl (e.g., 1-methyl-pyrrolindin-2-yl, 1-methyl-pyrrolindin-3-yl, 1-methyl-pyrrolindin-2-yl-methyl or 1-methyl-pyrrolindin-3-yl-methyl),
            C3-8heterocycloalkyl (e.g., pyrrolidinyl, for example, pyrrolidin-3-yl) optionally substituted with C1-6alkyl (e.g., methyl), for example, 1-methylpyrrolidin-3-yl,
            C3-8cycloalkyl-C1-6alkyl (e.g., cyclopropylmethyl), haloC1-6alkyl (e.g., trifluoromethyl, 2,2,2-trifluoroethyl),
            —N(R14)(R15)—C1-6alkyl (e.g., 2-(dimethylamino)ethyl,2-aminopropyl),
            hydroxyC1-6alkyl (e.g., (e.g., 3-hydroxy-2-methylpropyl, 1-hydroxyprop-2-yl),
            arylC0-6alkyl (e.g., benzyl),
            heteroarylC1-6alkyl (e.g., pyridinylmethyl),
            C1-6alkoxyarylC1-6alkyl (e.g., 4-methoxybenzyl);
            -G-J wherein:
            
                G is a single bond or, alkylene (e.g., methylene);
                J is cycloalkyl or heterocycloalkyl (e.g., oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl) optionally substituted with C1-6alkyl (e.g., (1-methylpyrrolidin-2-yl));
            
            
        
        
        (iv) R3 is
        
            1) -D-E-F wherein:
            
                D is a single bond, C1-6alkylene (e.g., methylene), or arylalkylene (e.g., pbenzylene or —CH2C6H4—);
                E is
                
                    a single bond,
                    C1-4alkylene (e.g., methylene, ethynylene, prop-2-yn-1-ylene),
                    —C0-4alkylarylene (e.g., phenylene or —C6H4—, -benzylene- or —CH2C6H4—), wherein the arylene group is optionally substituted with halo (e.g., Cl or F),
                    heteroarylene (e.g., pyridinylene or pyrimidinylene),
                    aminoC1-6alkylene (e.g., —CH2N(H)—),
                    amino (e.g., —N(H)—);
                    C3-8cycloalkylene optionally containing one or more heteroatom selected from N or O (e.g., piperidinylene),
                
                
                F is
                
                    H,
                    halo (e.g., F, Br, Cl),
                    C1-6alkyl (e.g., isopropyl or isobutyl),
                    haloC1-6alkyl (e.g., trifluoromethyl),
                    aryl (e.g., phenyl),
                    C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O (e.g., cyclopentyl, cyclohexyl, piperidinyl, pyrrolidinyl, tetrahydro-2H-pyran-4-yl, or morpholinyl), and optionally substituted with C1-6alkyl (e.g., methyl or isopropyl), for example, 1-methylpyrrolidin-2-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, piperidin-2-yl, 1-methylpiperidin-2-yl, 1-ethylpiperidin-2-yl,
                    heteroaryl optionally substituted with C1-6alkyl, (e.g., pyridyl, (for example, pyrid-2-yl), pyrimidinyl (for example, pyrimidin-2-yl, pyrimidin-4-yl), thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), oxazolyl (e.g., oxazol-2-yl), diazolyl (e.g., pyrazolyl (for example, pyrazol-1-yl) or imidazolyl (for example, imidazol-1-yl, 4-methylimidazolyl, 1-methylimidazol-2-yl,), triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), wherein said heteroaryl is optionally substituted with halo (e.g., fluoro) or haloC1-6alkyl;
                    amino (e.g., —NH2),
                    C1-6alkoxy,
                    —O-haloC1-6alkyl (e.g., —O—CF3),
                    C1-6alkylsulfonyl (for example, methylsulfonyl or —S(O)2CH3),
                    —C(O)—R13,
                    —N(R14)(R15); or
                
                
            
            
            2) a substituted heteroarylaklyl, e.g., substituted with haloalkyl; or
            3) attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A
        
        
    
    






    
    
        
        
            
            
                wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F); and R10 is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, (for example, pyrid-2-yl) or e.g., thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), diazolyl, triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl), alkyl sulfonyl (e.g., methyl sulfonyl), arylcarbonyl (e.g., benzoyl), or heteroarylcarbonyl, alkoxycarbonyl, (e.g., methoxycarbonyl), aminocarbonyl; preferably phenyl or pyridyl, e.g., 2-pyridyl; provided that when X, Y or X is nitrogen, R8, R9 or R10, respectively, is not present;
            
            
        
        
        (v) R4 is selected from:
        
            H,
            C1-6alkyl (e.g., methyl, isopropyl),
            C3-8cycloalkyl (e.g., cyclopentyl),
            C3-8heterocycloalkyl (e.g., pyrrolidin-3-yl),
            aryl (e.g., phenyl) or heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) wherein said aryl or heteroaryl is optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkyl, C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        
        
        (vi) R13 is —N(R14)(R15), C1-6alkyl (e.g., methyl), —OC1-6alkyl (e.g., —OCH3), haloC1-6alkyl(trifluoromethyl), aryl (e.g., phenyl), or heteroaryl; and
        (vii) R14 and R15 are independently H or C1-6alkyl, in free or salt form.
    
    


The invention further provides compounds of Formula I as follows:

    
    
        1.1 Formula I, wherein R3 is -D-E-F;
        1.2 Formula 1.1, D is a single bond, C1-6alkylene (e.g., methylene), or arylalkylene (e.g., benzylene or —CH2C6H4—);
        1.3 Formula 1.1, wherein D is a single bond;
        1.4 Formula 1.1, wherein D is C1-6alkylene (e.g., methylene);
        1.5 Formula 1.1, wherein D is methylene;
        1.6 Formula 1.1, wherein D is arylalkylene (e.g., benzylene or —CH2C6H4—);
        1.7 Formula 1.1, wherein D is benzylene or —CH2C6H4—;
        1.8 Any of formulae 1.1-1.7, wherein E is a single bond, C2-4alkylene (e.g., methylene, ethynylene, prop-2-yn-1-ylene), —C0-4alkylarylene (e.g., phenylene or —C6H4—, -benzylene- or —CH2C6H4—), wherein the arylene group is optionally substituted with halo (e.g., Cl or F), heteroarylene (e.g., pyridinylene or pyrimidinylene), aminoC1-6alkylene (e.g., —CH2N(H)—), amino (e.g., —N(H)—); C3-8cycloalkylene optionally containing one or more heteroatom selected from N or O (e.g., piperidinylene);
        1.9 Any of formulae 1.1-1.7, wherein E is a single bond;
        1.10 Any of formulae 1.1-1.7, wherein E is C2-4alkylene (e.g., methylene, ethynylene, prop-2-yn-1-ylene);
        1.11 Any of formulae 1.1-1.7, wherein E is methylene;
        1.12 Any of formulae 1.1-1.7, wherein E is ethynylene;
        1.13 Any of formulae 1.1-1.7, wherein E is prop-2-yn-1-ylene;
        1.14 Any of formulae 1.1-1.7, wherein E is —C0-4alkylarylene (e.g., phenylene or —C6-H4—, -benzylene- or —CH2C6H4—), wherein the arylene group is optionally substituted with halo (e.g., Cl or F);
        1.15 Any of formulae 1.1-1.7, wherein E is phenylene or —C6H4—;
        1.16 Any of formulae 1.1-1.7, wherein E is heteroarylene (e.g., pyridinylene or pyrimidinylene);
        1.17 Any of formulae 1.1-1.7, wherein E is pyridinylene;
        1.18 Any of formulae 1.1-1.7, wherein E is pyrimidinylene;
        1.19 Any of formulae 1.1-1.7, wherein E is aminoC1-6alkylene (e.g., —CH2N(H)—);
        1.20 Any of formulae 1.1-1.7, wherein E is amino (e.g., —N(H)—);
        1.21 Any of formulae 1.1-1.7, wherein E is C3-8cycloalkylene optionally containing one or more heteroatom selected from N or O (e.g., piperidinylene);
        1.22 Any of formulae 1.1-1.21, wherein F is H, halo (e.g., F, Br, Cl), C1-6alkyl (e.g., isopropyl or isobutyl), haloC1-6alkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O (e.g., cyclopentyl, cyclohexyl, piperidinyl, pyrrolidinyl, tetrahydro-2H-pyran-4-yl, or morpholinyl), and optionally substituted with C1-6alkyl (e.g., methyl or isopropyl), for example, 1-methylpyrrolidin-2-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, piperidin-2-yl, 1-methylpiperidin-2-yl, 1-ethylpiperidin-2-yl; heteroaryl optionally substituted with C1-6alkyl (e.g., pyridyl, (for example, pyrid-2-yl), pyrimidinyl (for example, pyrimidin-2-yl, pyrimidin-4-yl), thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), oxazolyl (e.g., oxazol-2-yl), diazolyl (e.g., pyrazolyl (for example, pyrazol-1-yl) or imidazolyl (for example, imidazol-1-yl, 4-methylimidazolyl, 1-methylimidazol-2-yl,), triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl), amino (e.g., —NH2), C1-6alkoxy, —O-haloC1-6alkyl (e.g., —O—CF3), C1-6alkylsulfonyl (for example, methylsulfonyl or —S(O)2CH3), C(O)—R13 or —N(R14)(R15);
        1.23 Any of formulae 1.1-1.22, wherein F is H,
        1.24 Any of formulae 1.1-1.22, wherein F is halo (e.g., F, Br, Cl);
        1.25 Any of formulae 1.1-1.22, wherein F is fluoro;
        1.26 Any of formulae 1.1-1.22, wherein F is C1-6alkyl (e.g., isopropyl or isobutyl);
        1.27 Any of formulae 1.1-1.22, wherein F is isopropyl;
        1.28 Any of formulae 1.1-1.22, wherein F is isobutyl;
        1.29 Any of formulae 1.1-1.22, wherein F is haloC1-6alkyl (e.g., trifluoromethyl);
        1.30 Any of formulae 1.1-1.22, wherein F is trifluoromethyl;
        1.31 Any of formulae 1.1-1.22, wherein F is aryl (e.g., phenyl);
        1.32 Any of formulae 1.1-1.22, wherein F is phenyl;
        1.33 Any of formulae 1.1-1.22, wherein F is C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O (e.g., cyclopentyl, cyclohexyl, piperidinyl, pyrrolidinyl tetrahydro-2H-pyran-4-yl, morpholinyl); and optionally substituted with C1-6alkyl (e.g., methyl or isopropyl), for example, 1-methylpyrrolidin-2-yl,), for example, 1-methylpyrrolidin-2-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, piperidin-2-yl, 1-methylpiperidin-2-yl, 1-ethylpiperidin-2-yl;
        1.34 Any of formulae 1.1-1.22, wherein F is cyclopentyl or cyclohexyl;
        1.35 Any of formulae 1.1-1.22, wherein F is 1-methylpyrrolidin-2-yl;
        1.36 Any of formulae 1.1-1.22, wherein F is heteroaryl optionally substituted with C1-6alkyl (e.g., pyridyl, (for example, pyrid-2-yl), pyrimidinyl (for example, pyrimidin-2-yl, pyrimidin-4-yl), thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), oxazolyl (e.g., oxazol-2-yl), diazolyl (e.g., pyrazolyl (for example, pyrazol-1-yl) or imidazolyl (for example, imidazol-1-yl, 4-methylimidazolyl, 1-methylimidazol-2-yl,), triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl), wherein said heteroaryl is optionally substituted with halo (e.g., fluoro) or haloC1-6alkyl;
        1.37 Any of formulae 1.1-1.22, wherein F is pyrid-2-yl optionally substituted with halo (e.g., fluoro);
        1.38 Any of formulae 1.1-1.22, wherein F is 6-fluoro-pyrid-2-yl;
        1.39 Any of formulae 1.1-1.22, wherein F is pyrimidinyl (for example, pyrimidin-2-yl, pyrimidin-4-yl);
        1.40 Any of formulae 1.1-1.22, wherein F is oxazolyl (e.g., oxazol-2-yl),
        1.41 Any of formulae 1.1-1.22, wherein F is triazolyl (e.g., 1,2,4-triazol-1-yl);
        1.42 Any of formulae 1.1-1.22, wherein F is diazolyl (e.g., pyrazolyl (for example, pyrazol-1-yl) or imidazolyl (for example, imidazol-1-yl, 4-methylimidazolyl, 1-methylimidazol-2-yl,);
        1.43 Any of formulae 1.1-1.22, wherein F is C1-6alkyl-oxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazolyl);
        1.44 Any of formulae 1.1-1.22, wherein F is amino (e.g., —NH2);
        1.45 Any of formulae 1.1-1.22, wherein F is C1-6alkoxy;
        1.46 Any of formulae 1.1-1.22, wherein F is —O-haloC1-6alkyl (e.g., —O—CF3);
        1.47 Any of formulae 1.1-1.22, wherein F is —C(O)—R13;
        1.48 Any of formulae 1.1-1.22, wherein F is —N(R14)(R15);
        1.49 Any of formulae 1.1-1.22, wherein F is C1-6alkylsulfonyl;
        1.50 Any of formulae 1.1-1.22, wherein F is methylsulfonyl or —S(O)2CH3;
        1.51 Formula I or any of 1.1-1.21, wherein R3 is a substituted heteroarylaklyl, e.g., substituted with haloalkyl;
        1.52 Formula I or any of 1.1-1.21, wherein R3 is attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A
    
    






    
    
        
        
            wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F); and R10 is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, (for example, pyrid-2-yl) or e.g., thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), diazolyl, triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl), alkyl sulfonyl (e.g., methyl sulfonyl), arylcarbonyl (e.g., benzoyl), or heteroarylcarbonyl, alkoxycarbonyl, (e.g., methoxycarbonyl), aminocarbonyl; preferably phenyl or pyridyl, e.g., 2-pyridyl; provided that when X, Y or X is nitrogen, R8, R9 or R10, respectively, is not present;
        
        
        1.53 Formula 1.52, wherein R3 is a moiety of Formula A, R8, R9, R11 and R12 are each H and R10 is phenyl;
        1.54 Formula 1.52, wherein R3 is a moiety of Formula A, R8, R9, R11 and R12 are each H and R10 is pyridyl or thiadiazolyl;
        1.55 Formula 1.52, wherein R3 is a moiety of Formula A, R8, R9, R11 and R12 are each H and R10 is pyrid-2-yl optionally substituted with halo (e.g., fluoro);
        1.56 Formula 1.52, wherein R3 is a moiety of Formula A and X, Y and Z are all C,
        1.57 Formula 1.52, wherein R10 is pyrimidinyl;
        1.58 Formula 1.52, wherein R10 is 5-fluoropyrmidinyl;
        1.59 Formula 1.52, wherein R10 is pyrazol-1-yl;
        1.60 Formula 1.52, wherein R10 is 1,2,4-triazol-1-yl;
        1.61 Formula 1.52, wherein R10 is aminocarbonyl;
        1.62 Formula 1.52, wherein R10 is methylsulfonyl;
        1.63 Formula 1.52, wherein R10 is 5-methyl-1,2,4-oxadiazol-3-yl;
        1.64 Formula 1.52, wherein R10 is 5-fluoropyrimidin-2-yl;
        1.65 Formula 1.52, wherein R10 is trifluoromethyl;
        1.66 Formula 1.52, wherein R3 is a moiety of Formula A, X and Z are C, and Y is N,
        1.67 Formula I or any of 1.1-1.66, wherein R2 is H; C1-6alkyl (e.g., isopropyl, isobutyl, neopentyl, 2-methylbutyl, 2,2-dimethylpropyl) wherein said alkyl group is optionally substituted with halo (e.g., fluoro) or hydroxy (e.g., 1-hydroxypropan-2-yl, 3-hydroxy-2-methylpropyl); —C0-4alkyl-C3-8cycloalkyl (e.g., cyclopentyl, cyclohexyl) optionally substituted with one or more amino (e.g., —NH2), for example, 2-aminocyclopentyl or 2-aminocyclohexyl), wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl (e.g., 1-methyl-pyrrolindin-2-yl, 1-methyl-pyrrolindin-3-yl, 1-methyl-pyrrolindin-2-yl-methyl or 1-methyl-pyrrolindin-3-yl-methyl); C3-8heterocycloalkyl (e.g., pyrrolidinyl, for example, pyrrolidin-3-yl) optionally substituted with C1-6alkyl (e.g., methyl), for example, 1-methylpyrrolidin-3-yl; C3-8cycloalkyl-C1-6alkyl (e.g., cyclopropylmethyl); haloC1-6alkyl (e.g., trifluoromethyl, 2,2,2-trifluoroethyl); —N(R14)(R15)—C1-6alkyl (e.g., 2-(dimethylamino)ethyl,2-aminopropyl); hydroxyC1-6alkyl (e.g., (e.g., 3-hydroxy-2-methylpropyl, 1-hydroxyprop-2-yl); arylC0-6alkyl (e.g., benzyl); heteroarylC1-6alkyl (e.g., pyridinylmethyl); C1-6alkoxyarylC1-6alkyl (e.g., 4-methoxybenzyl); -G-J wherein: G is a single bond or, alkylene (e.g., methylene) and J is cycloalkyl or heterocycloalkyl (e.g., oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl) optionally substituted with C1-6alkyl (e.g., (1-methylpyrrolidin-2-yl));
        1.68 Formula 1.66, wherein R2 is H,
        1.69 Formula 1.66, wherein R2 is C1-6alkyl (e.g., isopropyl, isobutyl, neopentyl, 2-methylbutyl, 2,2-dimethylpropyl) wherein said alkyl group is optionally substituted with halo (e.g., trifluoroethyl) or hydroxy (e.g., 1-hydroxypropan-2-yl, 3-hydroxy-2-methylpropyl);
        1.70 Formula 1.66, wherein R2 is isobutyl;
        1.71 Formula 1.66, wherein R2 is 3-hydroxy-2-methylpropyl;
        1.72 Formula 1.66, wherein R2 is 1-hydroxypropan-2-yl;
        1.73 Formula 1.66, wherein R2 is —C0-4alkyl-C3-8cycloalkyl (e.g., cyclopentyl, cyclohexyl) optionally substituted with one or more amino (e.g., —NH2), for example, 2-aminocyclopentyl or 2-aminocyclohexyl), wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl (e.g., 1-methyl-pyrrolindin-2-yl, 1-methyl-pyrrolindin-3-yl, 1-methyl-pyrrolindin-2-yl-methyl or 1-methyl-pyrrolindin-3-yl-methyl);
        1.74 Formula 1.66, wherein R2 is 1-methyl-pyrrolindin-2-yl, 1-methyl-pyrrolindin-3-yl, 1-methyl-pyrrolindin-2-yl-methyl or 1-methyl-pyrrolindin-3-yl-methyl;
        1.75 Formula 1.66, wherein R2 is C3-8heterocycloalkyl (e.g., pyrrolidinyl, for example, pyrrolidin-3-yl) optionally substituted with C1-6alkyl (e.g., methyl), for example, 1-methylpyrrolidin-3-yl;
        1.76 Formula 1.66, wherein R2 is 1-methylpyrrolidin-3-yl;
        1.77 Formula 1.66, wherein R2 is C3-8cycloalkyl-C1-6alkyl (e.g., cyclopropylmethyl);
        1.78 Formula 1.66, wherein R2 is —N(R14)(R15)—C1-6alkyl (e.g., 2-(dimethylamino)ethyl, 2-aminopropyl);
        1.79 Formula 1.66, wherein R2 is heteroarylC1-6alkyl (e.g., pyridinylmethyl),
        1.80 Formula 1.66, wherein R2 is C1-6alkoxyarylC1-6alkyl (e.g., 4-methoxybenzyl;
        1.81 Formula 1.66, wherein R2 is arylC0-6alkyl (e.g., benzyl);
        1.82 Formula 1.66, wherein R2 is cyclopentyl or cyclohexyl;
        1.83 Formula I or any of 1.1-1.66, wherein R2 is -G-J; G is a single bond or, alkylene (e.g., methylene); and J is cycloalkyl or heterocycloalkyl (e.g., oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl) optionally substituted with C1-6alkyl (e.g., (1-methylpyrrolidin-2-yl));
        1.84 Formula 1.83, wherein G is alkylene (e.g., methylene);
        1.85 Formula 1.83, wherein G is methylene;
        1.86 Formula 1.83, wherein J is cycloalkyl or heterocycloalkyl (e.g., oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl) optionally substituted with alkyl (e.g., 1-methylpyrrolidin-2-yl);
        1.87 Formula 1.83, wherein J is oxetan-2-yl, pyrolyin-3-yl, pyrolyin-2-yl;
        1.88 Formula 1.83, wherein J is (1-methylpyrrolidin-2-yl);
        1.89 Any of the preceding formulae wherein R4 is selected from H, C1-6alkyl (e.g., methyl, isopropyl), C3-8cycloalkyl (e.g., cyclopentyl), C3-8heterocycloalkyl (e.g., pyrrolidin-3-yl), or aryl (e.g., phenyl) or heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) wherein said aryl or heteroaryl is optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkoxy C1-6alkyl, or C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        1.90 Formula 1.89, wherein either R4 is H,
        1.91 Formula 1.89, wherein either R4 or R5 is C1-6alkyl (e.g., methyl, isopropyl);
        1.92 Formula 1.89, wherein either R4 is isopropyl;
        1.93 Formula 1.89, wherein either R4 or R5 is C3-8cycloalkyl (e.g., cyclopentyl);
        1.94 Formula 1.89, wherein either R4 or R5 is C3-8heterocycloalkyl (e.g., pyrrolidin-3-yl);
        1.95 Formula 1.89, wherein either R4 or R5 is aryl (e.g., phenyl) optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkyl, C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        1.96 Formula 1.89, wherein either R4 or R5 is 4-hydroxyphenyl;
        1.97 Formula 1.89, wherein either R4 or R5 is 4-fluorophenyl;
        1.98 Formula 1.89, wherein either R4 or R5 is heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) optionally substituted with halo (e.g., 4-fluorophenyl), hydroxy (e.g., 4-hydroxyphenyl), C1-6alkyl, C1-6alkoxy or another aryl group (e.g., biphenyl-4-ylmethyl);
        1.99 Formula 1.89, wherein either R4 or R5 is phenyl;
        1.100 Any of the foregoing formulae, wherein R13 is —N(R14)(R15), C1-6alkyl (e.g., methyl), —OC1-6alkyl (e.g., —OCH3), haloC1-6alkyl, aryl (for example phenyl), or heteroaryl;
        1.101 Formula 1.100, wherein R13 is —N(R14)(R15);
        1.102 Formula 1.100, wherein R13 is —NH2;
        1.103 Formula 1.100, wherein R13 is C1-6alkyl (e.g., methyl);
        1.104 Formula 1.100, wherein R13 is —OC1-6alkyl (e.g., —OCH3),
        1.105 Formula 1.100, wherein R13 is —OCH3;
        1.106 Formula 1.100, wherein R13 is haloC1-6alkyl (e.g., trifluoromethyl);
        1.107 Formula 1.100, wherein R13 is trifluoromethyl;
        1.108 Formula 1.100, wherein R13 is aryl (e.g., phenyl);
        1.109 Formula 1.100, wherein R13 is heteroaryl (e.g., pyridyl);
        1.110 Any of the preceding formulae, wherein R14 and R15 are independently H or C1-6alkyl (e.g., methyl);
        1.111 Formula I or any of 1.1-1.110, wherein either R14 or R15 is independently H,
        1.112 Formula I or any of 1.1-1.110, wherein either R14 or R15 is C1-6alkyl (e.g., methyl);
        1.113 Formula I or any of 1.1-1.110, wherein either R14 or R15 is methyl;
        1.114 Formula I or any of 1.1-1.110, wherein R14 and R15 are methyl;
        1.115 any of the preceding formulae wherein R3 is selected from a group consisting of 4-(pyrimidin-2-yl)benzyl, 4-(1,2,4-triazol-1-yl)benzyl, 4-(1-methylpyrrolidin-2-yl)benzyl, 4-(1-methylpiperid-2-yl)benzyl, 4-(pyrid-2-yl)benzyl, 4-(pyrazol-1-yl)benzyl, 4-(pyrrolidin-3-yl)benzyl, (6-chloropyridin-3-yl)methyl, (6-fluoropyridin-3-yl)methyl, 4-(imidazol-1-yl)benzyl, 4-(pyrimidin-4-yl)benzyl), 4-(oxazol-2-yl)benzyl, 4-(dimethylamino)benzyl, 4-(methylsulfonyl)benzyl, 4-(pyrrolidin-3-yl)benzyl, (1-isopropylpyiperidin-4-yl)methyl, and —CH2—C2H4—C(O)—NH2;
        1.116 formula 1.115 wherein R3 is 4-(pyrimidin-2-yl)benzyl;
        1.117 formula 1.115 wherein R3 is 4-(1,2,4-triazol-1-yl)benzyl;
        1.118 formula 1.115 wherein R3 is 4-(1-methylpyrrolidin-2-yl)benzyl;
        1.119 formula 1.115 wherein R3 is 4-(1-methylpiperid-2-yl)benzyl;
        1.120 formula 1.115 wherein R3 is 4-(pyrid-2-yl)benzyl;
        1.121 formula 1.115 wherein R3 is 4-(pyrazol-1-yl)benzyl;
        1.122 formula 1.115 wherein R3 is 4-(pyrrolidin-3-yl)benzyl;
        1.123 formula 1.115 wherein R3 is (6-chloropyridin-3-yl)methyl or (6-fluoropyridin-3-yl)methyl;
        1.124 formula 1.115 wherein R3 is 4-(imidazol-1-yl)benzyl;
        1.125 formula 1.115 wherein R3 is 4-(pyrimidin-4-yl)benzyl);
        1.126 formula 1.115 wherein R3 is 4-(oxazol-2-yl)benzyl;
        1.127 formula 1.115 wherein R3 is 4-(dimethylamino)benzyl;
        1.128 formula 1.115 wherein R3 is 4-(methylsulfonyl)benzyl;
        1.129 formula 1.115 wherein R3 is 4-(pyrrolidin-3-yl)benzyl;
        1.130 formula 1.115 wherein R3 is (1-isopropylpyiperidin-4-yl)methyl;
        1.131 formula 1.115 wherein R3 is —CH2—C2H4—C(O)—NH2;
        1.132 any of the preceding formulae wherein compound of formula I is
    
    






    
    
        1.133 any of the preceding formulae wherein compound of formula I is
    
    






    
    
        1.134 any of the preceding formulae wherein compound of formula I is selected from a group consisting of:
    
    















    
    
        1.135 any of the preceding formulae wherein the compounds inhibit phosphodiesterase-mediated (e.g., PDE1-mediated, especially PDE1B-mediated) hydrolysis of cGMP, e.g., with an IC50 of less than 10 μM, preferably less than 1 μM, preferably less than 500 nM, preferably less than 200 nM in an immobilized-metal affinity particle reagent PDE assay, for example, as described in Example 16, in free or salt form.
    
    


In a further embodiment, the Compound of the Invention is a Compound of Formula I, wherein R3 is C5-6heteroarylC1-alkyl optionally substituted with halo, C1-6alkyl (e.g., (6-fluoropyrid-3-yl)methyl, (6-chloropyrid-3-yl)methyl).
In another embodiment, the Compound of the Invention is a Compound of Formula I, wherein R3 is pyridylC1-6alkyl (e.g., pyridylmethyl) optionally substituted with halo, haloalkyl alkyl sulfonyl, —C(O)N(C0-6alkyl)(C0-6alkyl), heteroaryl (e.g., pyridyl, pyrimidinyl, oxazole, imidazole, triazolyl, pyrazolyl), amino, C1-6alkylamino, and heterocycloalkyl (e.g., piperidinyl, pyrrolidinyl) which heterocycloalkyl, heteroaryl are further optionally substituted with C1-6alkyl or halo.
In still another embodiment, the Compound of the Invention is a Compound of Formula I, wherein R3 is benzyl substituted with aryl, heteroaryl (e.g., pyridyl, pyrimidinyl, oxazole, imidazole, triazolyl, pyrazolyl), C3-7cycloalkyl, heteroC3-7cycloalkyl (e.g., piperidinyl, pyrrolidinyl), alkyl sulfonyl, —C(O)N(C0-6alkyl)(C0-6 alkyl), amino, C1-6alkylamino, which aryl, heterocycloalkyl, heteroaryl are further optionally substituted with C1-6alkyl or halo.
In a another embodiment, the Compound of the Invention is a Compound of Formula I wherein

    
    
        (ii) R1 is H or alkyl (e.g., methyl);
        (iii) R2 is H, alkyl (e.g., isobutyl, 2-methylbutyl, 2,2-dimethyl propyl), cycloalkyl (e.g., cyclopentyl, cyclohexyl), haloalkyl (e.g., trifluoromethyl, 2,2,2-trifluoroethyl), alkylaminoalkyl (e.g., 2-(dimethylamino)ethyl), hydroxyalkyl (e.g., 3-hydroxy-2-methyl propyl), arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), or alkoxyarylalkyl (e.g., 4-methoxybenzyl);
        (iv) R3 is a substituted heteroarylaklyl, e.g., substituted with haloalkyl
        or
        R3 is attached to one of the nitrogens on the pyrazolo portion of Formula I and is
        a moiety of Formula A
    
    






wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F); and R10 is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, (for example, pyrid-2-yl) or e.g., thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), diazolyl, triazolyl (e.g., 1,2,4-triazol-1-yl), tetrazolyl (e.g., tetrazol-5-yl), alkoxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol), pyrazolyl (e.g., pyrazol-1-yl), alkyl sulfonyl (e.g., methyl sulfonyl), arylcarbonyl (e.g., benzoyl), or heteroarylcarbonyl, alkoxycarbonyl, (e.g., methoxycarbonyl), aminocarbonyl; preferably phenyl or pyridyl, e.g., 2-pyridyl; provided that when X, Y or X is nitrogen, R8, R9 or R10, respectively, is not present;

    
    
        (v) R4 is aryl (e.g., phenyl) or heteroaryl;
        (vi) R5 is H, alkyl, cycloalkyl (e.g., cyclopentyl), heteroaryl, aryl, p-benzylaryl (e.g., biphenyl-4-ylmethyl);
        (vii) R6 is H, C1-6alkyl (e.g., methyl) or C3-8cycloalkyl;
        (viii) R13 is —N(R14)(R15), C1-6alkyl (e.g., methyl), —OC1-6alkyl (e.g., —OCH3), haloC1-6alkyl(trifluoromethyl), aryl (e.g., phenyl), or heteroaryl; and
        (vii) R14 and R15 are independently H or C1-6alkyl,

in free or salt form (hereinafter, Compound of Formula I(i)).

    
    


In still another embodiment, the Compound of the Invention is a Compound of Formula I wherein

    
    
        (i) R1 is H or alkyl (e.g., methyl);
        (ii) R2 is H, alkyl (e.g., isopropyl, isobutyl, 2-methylbutyl, 2,2-dimethyl propyl), cycloalkyl (e.g., cyclopentyl, cyclohexyl), haloalkyl (e.g., trifluoromethyl, 2,2,2-trifluoroethyl), alkylaminoalkyl (e.g., 2-(dimethylamino)ethyl), hydroxyalkyl (e.g., 3-hydroxy-2-methyl propyl), arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), or alkoxyarylalkyl (e.g., 4-methoxybenzyl);
        (iii) R3 is D-E-F wherein
        
            1. D is single bond, alkylene (e.g., methylene), or arylalkylene (e.g., benzylene or —CH2C6H4—);
            2. E is a alkylene (e.g., methylene, ethynylene, prop-2-yn-1-ylene), arylene (e.g., phenylene or —C6H4—), alkylarylene (e.g., -benzylene- or —CH2C6H4—), aminoalkylene (e.g., —CH2N(H)—) or amino (e.g., —N(H)—); and
            3. F is alkyl (e.g., isobutyl), aryl (e.g., phenyl), heteroaryl (e.g., pyrid-2-yl, 1,2,4-triazolyl), heteroC3-8cycloalkyl (e.g., pyrrolidin-1-yl), amino (e.g., —NH2), C1-6alkoxy, or —O-haloalkyl (e.g., —O—CF3);
        
        
        (iv) R4 is aryl (e.g., phenyl), heteroaryl (e.g., pyrid-4-yl, pyrid-2-yl or pyrazol-3-yl) or heterocycloalkyl (e.g., pyrrolidin-3-yl); and
        (v) R5 is H, alkyl, cycloalkyl (e.g., cyclopentyl), heteroaryl, aryl, p-benzylaryl (e.g., biphenyl-4-ylmethyl);
        (vi) R6 is H, C1-6alkyl (e.g., methyl) or C3-8cycloalkyl;
        (vii) R13 is —N(R14)(R15), C1-6alkyl (e.g., methyl), —OC1-6alkyl (e.g., —OCH3), haloC1-6alkyl(trifluoromethyl), aryl (e.g., phenyl), or heteroaryl; and
        (viii) R14 and R15 are independently H or alkyl,

wherein “alk”, “alkyl”, “haloalkyl” or “alkoxy” refers to C1-6 alkyl and “cycloalkyl” refers to C3-8 cycloalkyl unless specifically specified;

in free or salt form (hereinafter, Compound of Formula I(ii)).

    
    


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, which may be linear or branched, and may be optionally mono-, di- or tri-substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy.
        (b) “Cycloalkyl” as used herein is a saturated or unsaturated nonaromatic hydrocarbon moiety, preferably saturated, preferably comprising three to eight carbon atoms, at least some of which form a nonaromatic mono- or bicyclic, or bridged cyclic structure, and which may be optionally substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy. Wherein the cycloalkyl optionally contains one or more atoms selected from N and O and/or S, said cycloalkyl may also be a heterocycloalkyl.
        (c) “Heterocycloalkyl” is, unless otherwise indicated, saturated or unsaturated nonaromatic hydrocarbon moiety, preferably saturated, preferably comprising three to nine carbon atoms, at least some of which form a nonaromatic mono- or bicyclic, or bridged cyclic structure, wherein at least one carbon atom is replaced with N, O or S, which heterocycloalkyl may be optionally substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or carboxy.
        (d) “Aryl” as used herein is a mono or bicyclic aromatic hydrocarbon, preferably phenyl, optionally substituted, e.g., with alkyl (e.g., methyl), halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoromethyl), hydroxy, carboxy, or an additional aryl or heteroaryl (e.g., biphenyl or pyridylphenyl).
        (e) “Heteroaryl” as used herein is an aromatic moiety 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., with alkyl, halogen, haloalkyl, hydroxy or carboxy.
        (1) Wherein E is phenylene, the numbering is as follows:
    
    






    
    
        (g) It is intended that wherein the substituents end in “ene”, for example, alkylene, phenylene or arylalkylene, said substitutents are intended to bridge or be connected to two other substituents. Therefore, methylene is intended to be —CH2— and phenylene intended to be —C6H4— and arylalkylene is intended to be —C6H4—CH2— or —CH2—C6H4—.
    
    


Compounds of the Invention 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 of Formula I, or any of 1.1-1.135, a Compound of Formula I(i) or I(ii), in any form, for example free or acid addition salt form, or where the compounds contain acidic substituents, in base addition salt form. 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.
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)—C1-4alkyl, 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)—C1-4alkyl). Alternatively, wherein the Compound of the Invention contains a carboxylic acid, for example, Compound —C(O)OH, the acid ester prodrug of such compound, Compound —C(O)O—C1-4alkyl can hydrolyze to form Compound —C(O)OH and HO—C1-4alkyl. As will be appreciated, the term thus embraces conventional pharmaceutical prodrug forms.
The invention also provides methods of making the Compounds of the Invention and methods of using the Compounds of the Invention for treatment of diseases and disorders as set forth below (especially treatment of diseases characterized by reduced dopamine D1 receptor signaling activity, such as Parkinson's disease, Tourette's Syndrome, Autism, fragile X syndrome, ADHD, restless leg syndrome, depression, cognitive impairment of schizophrenia, narcolepsy and diseases that may be alleviated by the enhancement of progesterone-signaling such as female sexual dysfunction), or a disease or disorder such as psychosis or glaucoma). This list is not intended to be exhaustive and may include other diseases and disorders as set forth below.
In another embodiment, the invention further provides a pharmaceutical composition comprising a Compound of the Invention, e.g., a Compound of Formula I, or any of 1.1-1.135, or a Compound of Formula I(i) or I(ii), or any described in this specification, in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable carrier.


DETAILED DESCRIPTION OF THE INVENTION
Methods of Making Compounds of the Invention
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 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. In particular, the intermediates and starting materials for the Compounds of the Invention may be prepared by methods and processes as described in PCT/US2007/070551. All references cited herein are hereby incorporated by reference in their entirety.
The Compounds of the Invention include their enantiomers, diastereoisomers, tautomers 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.
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, 13C, 15N, 18O. 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., 123I, 131I, 125I, 11C, 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 11C isotope. These radio isotopes are useful for radio-imaging and/or pharmacokinetic studies of the compounds of the invention.
Melting points are uncorrected and (dec) indicates decomposition. Temperature 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 are given as volume percentages or volume ratios. In cases where the NMR spectra are complex, only diagnostic signals are reported.
Terms and Abbreviations:
BuLi=n-butyllithium
ButOH=tert-butyl alcohol,
CAN=ammonium cerium (IV) nitrate,
DIPEA=diisopropylethylamine,
DMF=N,N-dimethylforamide,
DMSO=dimethyl sulfoxide,
Et2O=diethyl ether,
EtOAc=ethyl acetate,
equiv.=equivalent(s),
h=hour(s),
HPLC=high performance liquid chromatography,
LDA=lithium diisopropylamide
MeOH=methanol,
NBS=N-bromosuccinimide
NCS=N-chlorosuccinimide
NaHCO3=sodium bicarbonate,
NH4OH=ammonium hydroxide,
Pd2(dba)3=tris[dibenzylideneacetone]dipalladium(0)
PMB=p-methoxybenzyl,
POCl3=phosphorous oxychloride,
SOCl2=thionyl chloride,
TFA=trifluoroacetic acid,
THF=tetrahedrofuran.
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.
In an aspect of the invention, intermediate compounds of formula IIb can be synthesized by reacting a compound of formula IIa with a dicarboxylic acid, acetic anhydride and acetic acid mixing with heat for about 3 hours and then cooled:




wherein R1 is H or C1-4alkyl [e.g., methyl].
Intermediate IIc can be prepared by for example reacting a compound of IIb with for example a chlorinating compound such as POCl3, sometimes with small amounts of water and heated for about 4 hours and then cooled:




Intermediate IId may be formed by reacting a compound of IIc with for example a P1—X in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:




wherein P1 is a protective group [e.g., p-methoxybenzyl group (PMB)]; X is a leaving group such as a halogen, mesylate, or tosylate.

Intermediate IIe may be prepared by reacting a compound of IId with hydrazine or hydrazine hydrate in a solvent such as methanol and refluxed for about 4 hours and then cooled:




Alternatively, Intermediate IIIa may be formed by reacting a compound of IIc with for example a R2—X wherein X is a leaving group such as a halogen, mesylate, or tosylate, in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:




Intermediate IIIb may be prepared by reacting a compound of IIIa with hydrazine or hydrazine hydrate in a solvent such as methanol and heated for several hours and then cooled:




Intermediate IVa may be formed by for example reacting a compound of IIIb with POCl3 and DMF.




Intermediate IVb may be formed by reacting a compound of IVa with for example a R3—X wherein X here is a leaving group, e.g., halogen, mesylate, or tosylate, in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating.




The thio Compounds of the Invention, e.g., Formula I wherein L is S or Compound (I)-E may be prepared by reacting a compound (IVb) with a disulfide R4-L-L-R4 or a thiol R4-LH in the presence of a strong base, such as a lithium reagent (e.g. LiHMDS) in a solvent such as THF.




The sulfinyl or sulfonyl derivative, e.g. Formula I wherein L is SO or SO2, may be formed by reacting a 3-thio compounds (I)-E with an oxidizer such as a peroxide (e.g. oxone or hydrogen peroxide) at room temperature in a solvent such as acetonitrile and methanol.
Alternatively, a compound of Formula (I)-E can be prepared by reacting, for example, Compounds 1-A with, for example, R3—X, in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:




wherein all the substituents are as defined previously; X is a leaving group such as a halogen, mesylate, or tosylate.

Methods of Using Compounds of the Invention

The Compounds of the Invention are useful in the treatment of diseases characterized by disruption of or damage to cAMP and cGMP mediated pathways, e.g., as a result of increased expression of PDE1 or decreased expression of cAMP and cGMP due to inhibition or reduced levels of inducers of cyclic nucleotide synthesis, such as dopamine and nitric oxide (NO). By preventing the degradation of cAMP and cGMP by PDE1B, thereby increasing intracellular levels of cAMP and cGMP, the Compounds of the Invention potentiate the activity of cyclic nucleotide synthesis inducers.
The invention provides methods of treatment of 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;
        (ii) Mental disorders, including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorders, e.g., narcolepsy, cognitive impairment, dementia, Tourette's syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and drug addiction;
        (iii) Circulatory and cardiovascular disorders, including cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, and sexual dysfunction;
        (iv) Respiratory and inflammatory disorders, including asthma, chronic obstructive pulmonary disease, and allergic rhinitis, as well as autoimmune and inflammatory diseases;
        (v) 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
        (vi) Any disease or condition characterized by reduced dopamine D1 receptor signaling activity,

comprising administering an effective amount of a Compound of the Invention, e.g., a compound according to any of Formula I or 1.1-1.135, or a composition comprising a Compound of the Invention, e.g., a compound according to any of Formula I or 1.1-1.135, or any described in this specification, in free or pharmaceutically acceptable salt form, to a human or animal patient in need thereof. In another aspect, the invention provides a method of treatment of the conditions disclosed above comprising administering a therapeutically effective amount of a Compound of Formula II as hereinbefore described, in free or pharmaceutically acceptable salt form, to a human or animal patient in need thereof.

    
    


In an especially preferred embodiment, the invention provides methods of treatment or prophylaxis for narcolepsy. In this embodiment, PDE 1 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 administering therapeutically effective amounts of

    
    
        (i) a PDE 1 Inhibitor of the Invention, e.g., a compound according to any of Formula I or any of 1.1-1.135, or I(i) or I(ii), or any described in this specification; 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),

in free or pharmaceutically acceptable salt form, to a human or animal patient in need thereof. In another embodiment, the invention provides methods of treatment or prophylaxis for narcolepsy as herein before described, wherein the PDE1 inhibitor is in a form of a pharmaceutical composition. In still another embodiment, the methods of treatment or prophylaxis for narcolepsy as hereinbefore described, comprises administering a therapeutically effective amount of a Compound of Formula II as hereinbefore described, in free or pharmaceutically acceptable salt form, as a sole therapeutic agent or use in combination for co-administered with another active agent.

    
    


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 any of Formula 1.1-1.135 or Formula I, I(i) or I(ii), or any described in this specification, in free or pharmaceutically acceptable salt form, to a human or animal patient in need thereof. The invention also provides methods of treatment as disclosed here, comprising administering a therapeutically effective amount of a Compound of Formula II, in free or pharmaceutically acceptable salt form. Disease or condition 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 PDE 1 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 PDE 1 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.
In this embodiment, 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-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 administering therapeutically effective amounts of

    
    
        (i) a PDE 1 Inhibitor, e.g., a compound according to any of Formula I, any of 1.1-1.135 or I(i) or I(ii), or any described in this specification 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)

in free or pharmaceutically acceptable salt form, to a human or animal patient in need thereof. In another embodiment, the invention provides the method described above wherein the PDE 1 inhibitor is a Compound of Formula II, in free or pharmaceutically acceptable salt form.

    
    


The invention also provides a method for enhancing or potentiating dopamine D1 intracellular signaling activity in a cell or tissue comprising contacting said cell or tissue with an amount of a Compound of the Invention sufficient to inhibit PDE1 B activity.
The invention also provides a method for enhancing or potentiating progesterone signaling activity in a cell or tissue comprising contacting said cell or tissue with an amount of a Compound of the Invention sufficient to inhibit PDE1B activity.
The invention also provides a method for treating a PDE1-related, especially PDE1B-related disorder, a dopamine D1 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 that inhibits PDE1B, wherein PDE1B activity modulates phosphorylation of DARPP-32 and/or the GluR1 AMPA receptor.
In another aspect, the invention also provides a method for the treatment for glaucoma or elevated intraocular pressure comprising topical administration of a therapeutically effective amount of a phosphodiesterase type I (PDE1) Inhibitor of the Invention, in free or pharmaceutically acceptable salt form, in an opthalmically 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.
The invention 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, in free or ophthamalogically acceptable salt form, in combination or association with an ophthamologically acceptable diluent or carrier.
Optionally, the PDE1 inhibitor may be administered sequentially or simultaneously with a second drug useful for treatment of glaucoma or elevated intraocular pressure. Where two active agents are administered, the therapeutically effective amount of each agent may be below the amount needed for activity as monotherapy. Accordingly, a subthreshold amount (i.e., an amount below the level necessary for efficacy as monotherapy) may be considered therapeutically effective and also may also be referred alternatively as an effective amount. Indeed, an advantage of administering different agents with different mechanisms of action and different side effect profiles may be to reduce the dosage and side effects of either or both agents, as well as to enhance or potentiate their activity as monotherapy.
The invention thus provides the method of treatment of a condition selected from glaucoma and elevated intraocular pressure comprising administering to a patient in need thereof an effective amount, e.g., a subthreshold amount, of an agent known to lower intraocular pressure concomitantly, simultaneously or sequentially with an effective amount, e.g., a subthreshold amount, of a PDE1 Inhibitor of the Invention, in free or pharmaceutically acceptable salt form, such that amount of the agent known to lower intraocular pressure and the amount of the PDE 1 inhibitor in combination are effective to treat the condition.
In one embodiment, one or both of the agents are administered topically to the eye. Thus the invention provides a method of reducing the side effects of treatment of glaucoma or elevated intraocular pressure by administering a reduced dose of an agent known to lower intraocular pressure concomitantly, simultaneously or sequentially with an effective amount of a PDE1 inhibitor. However, methods other than topical administration, such as systemic therapeutic administration, may also be utilized.
The optional additional agent or agents for use in combination with a PDE1 inhibitor may, for example, be selected from the existing drugs comprise typically of instillation of a prostaglandin, pilocarpine, epinephrine, or topical beta-blocker treatment, e.g. with timolol, as well as systemically administered inhibitors of carbonic anhydrase, e.g. acetazolamide. Cholinesterase inhibitors such as physostigmine and echothiopate may also be employed and have an effect similar to that of pilocarpine. Drugs currently used to treat glaucoma thus include, e.g.,

    
    
        1. Prostaglandin analogs such as latanoprost (Xalatan), bimatoprost (Lumigan) and travoprost (Travatan), which increase uveoscleral outflow of aqueous humor. Bimatoprost also increases trabecular outflow.
        2. Topical beta-adrenergic receptor antagonists such as timolol, levobunolol (Betagan), and betaxolol, which decrease aqueous humor production by the ciliary body.
        3. Alpha2-adrenergic agonists such as brimonidine (Alphagan), which work by a dual mechanism, decreasing aqueous production and increasing uveo-scleral outflow.
        4. Less-selective sympathomimetics like epinephrine and dipivefrin (Propine) increase outflow of aqueous humor through trabecular meshwork and possibly through uveoscleral outflow pathway, probably by a beta2-agonist action.
        5. Miotic agents (parasympathomimetics) like pilocarpine work by contraction of the ciliary muscle, tightening the trabecular meshwork and allowing increased outflow of the aqueous humour.
        6. Carbonic anhydrase inhibitors like dorzolamide (Trusopt), brinzolamide (Azopt), acetazolamide (Diamox) lower secretion of aqueous humor by inhibiting carbonic anhydrase in the ciliary body.
        7. Physostigmine is also used to treat glaucoma and delayed gastric emptying.
    
    


For example, the invention provides pharmaceutical compositions comprising a PDE1 Inhibitor of the Invention and an agent selected from (i) the prostanoids, unoprostone, latanoprost, travoprost, or bimatoprost; (ii) an alpha adrenergic agonist such as brimonidine, apraclonidine, or dipivefrin and (iii) a muscarinic agonist, such as pilocarpine. For example, the invention provides ophthalmic formulations comprising a PDE-1 Inhibitor of the Invention together with bimatoprost, abrimonidine, brimonidine, timolol, or combinations thereof, in free or ophthamalogically acceptable salt form, in combination or association with an ophthamologically acceptable diluent or carrier. In addition to selecting a combination, however, a person of ordinary skill in the art can select an appropriate selective receptor subtype agonist or antagonist. For example, for alpha adrenergic agonist, one can select an agonist selective for an alpha 1 adrenergic receptor, or an agonist selective for an alpha2 adrenergic receptor such as brimonidine, for example. For a beta-adrenergic receptor antagonist, one can select an antagonist selective for either β1, or β2, or β3, depending on the appropriate therapeutic application. One can also select a muscarinic agonist selective for a particular receptor subtype such as M1-M5.
The PDE 1 inhibitor may be administered in the form of an ophthalmic composition, which includes an ophthalmic solution, cream or ointment. The ophthalmic composition may additionally include an intraocular-pressure lowering agent.
In yet another example, the PDE-1 Inhibitors disclosed may be combined with a subthreshold amount of an intraocular pressure-lowering agent which may be a bimatoprost ophthalmic solution, a brimonidine tartrate ophthalmic solution, or brimonidine tartrate/timolol maleate ophthalmic solution.
In addition to the above-mentioned methods, it has also been surprisingly discovered that PDE1 inhibitors are 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. Without intending to be bound by any theory, it is believed that typical and atypical antipsychotic drugs such as clozapine primarily have their antagonistic activity at the dopamine D2 receptor. PDE1 inhibitors, however, primarily act to enhance signaling at the dopamine D1 receptor. By enhancing D1 receptor signaling, PDE1 inhibitors can increase NMDA receptor function in various brain regions, for example in nucleus accumbens neurons and in the prefrontal cortex. This enhancement of function may be seen for example in NMDA receptors containing the NR2B subunit, and may occur e.g., via activation of the Src and protein kinase A family of kinases.
Therefore, the invention provides a new method for the treatment of psychosis, e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, and mania, such as in acute manic episodes and bipolar disorder, comprising administering a therapeutically effective amount of a phosphodiesterase-1 (PDE1) Inhibitor of the Invention, in free or pharmaceutically acceptable salt form, to a patient in need thereof.
PDE 1 Inhibitors may be used in the foregoing methods of treatment prophylaxis 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 psychosis, e.g., schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, or mania, comprising administering simultaneously, sequentially, or contemporaneously administering therapeutically effective amounts of:

    
    
        (i) a PDE 1 Inhibitor of the invention, in free or pharmaceutically acceptable salt form; and
        (ii) an antipsychotic, e.g.,
        
            Typical antipsychotics, e.g.,
            
                Butyrophenones, e.g. Haloperidol (Haldol, Serenace), Droperidol (Droleptan);
                Phenothiazines, e.g., Chlorpromazine (Thorazine, Largactil), Fluphenazine (Prolixin), Perphenazine (Trilafon), Prochlorperazine (Compazine), Thioridazine (Mellaril, Melleril), Trifluoperazine (Stelazine), Mesoridazine, Periciazine, Promazine, Triflupromazine (Vesprin), Levomepromazine (Nozinan), Promethazine (Phenergan), Pimozide (Orap);
                Thioxanthenes, e.g., Chlorprothixene, Flupenthixol (Depixol, Fluanxol), Thiothixene (Navane), Zuclopenthixol (Clopixol, Acuphase);
            
            
            Atypical antipsychotics, e.g.,
            
                Clozapine (Clozaril), Olanzapine (Zyprexa), Risperidone (Risperdal), Quetiapine (Seroquel), Ziprasidone (Geodon), Amisulpride (Solian), Paliperidone (Invega), Aripiprazole (Abilify), Bifeprunox; norclozapine,
            
            
        
        
        in free or pharmaceutically acceptable salt form, to a patient in need thereof.
    
    


In a particular embodiment, the Compounds of the Invention are particularly useful for the treatment or prophylaxis of schizophrenia.
Compounds of the Invention, in free or pharmaceutically acceptable salt form, are particularly useful for the treatment of Parkinson's disease, schizophrenia, narcolepsy, glaucoma and female sexual dysfunction.
In still another aspect, the invention provides a method of lengthening or enhancing growth of the eyelashes by administering an effective amount of a prostaglandin analogue, e.g., bimatoprost, concomitantly, simultaneously or sequentially with an effective amount of a PDE 1 inhibitor of the Invention, in free or pharmaceutically acceptable salt form, to the eye of a patient in need thereof.
In yet another aspect, the invention provides a method for the treatment or prophylaxis of traumatic brain injury comprising administering a therapeutically effective amount of a PDE1 inhibitor of the invention, in free or pharmaceutically acceptable salt form, to a patient in need thereof. Traumatic brain injury (TBI) encompasses primary injury as well as secondary injury, including both focal and diffuse brain injuries. Secondary injuries are multiple, parallel, interacting and interdependent cascades of biological reactions arising from discrete subcellular processes (e.g., toxicity due to reactive oxygen species, overstimulation of glutamate receptors, excessive influx of calcium and inflammatory upregulation) which are caused or exacerbated by the inflammatory response and progress after the initial (primary) injury. Abnormal calcium homeostasis is believed to be a critical component of the progression of secondary injury in both grey and white matter. For a review of TBI, see Park et al., CMAJ (2008) 178(9):1163-1170, the contents of which are incorporated herein in their entirety. Studies have shown that the cAMP-PKA signaling cascade is downregulated after TBI and treatment of PDE IV inhibitors such as rolipram to raise or restore cAMP level improves histopathological outcome and decreases inflammation after TBI. As Compounds of the present invention is a PDE1 inhibitor, it is believed that these compounds are also useful for the treatment of TBI, e.g., by restoring cAMP level and/or calcium homeostasis after traumatic brain injury.
The present invention also provides

    
    
        (i) a Compound of the Invention for use as a pharmaceutical, 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 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 in combination or association with a pharmaceutically acceptable diluent or carrier, and
        (iv) a pharmaceutical composition comprising a Compound of the Invention 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.
    
    


Therefore, the invention provides use of a Compound of the Invention for the manufacture of a medicament for the treatment or prophylactic treatment of the following diseases: Parkinson's disease, restless leg, tremors, dyskinesias, Huntington's disease, Alzheimer's disease, and drug-induced movement disorders; depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep disorder, narcolepsy, cognitive impairment, dementia, Tourette's syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and/or drug addiction; cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, and/or sexual dysfunction; asthma, chronic obstructive pulmonary disease, and/or allergic rhinitis, as well as autoimmune and inflammatory diseases; and/or female sexual dysfunction, exercise amenorrhoea, anovulation, menopause, menopausal symptoms, hypothyroidism, pre-menstrual syndrome, premature labor, infertility, irregular menstrual cycles, abnormal uterine bleeding, osteoporosis, multiple sclerosis, prostate enlargement, prostate cancer, hypothyroidism, estrogen-induced endometrial hyperplasia or carcinoma; and/or 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 by reduced dopamine D1 receptor signaling activity; and/or any disease or condition that may be ameliorated by the enhancement of progesterone signaling.
The invention also provides use of a Compound of the Invention, in free or pharmaceutically acceptable salt form, for the manufacture of a medicament for the treatment or prophylactic treatment of:

    
    
        a) glaucoma or elevated intraocular pressure,
        b) 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,
        c) traumatic brain injury.
    
    


The phrase “Compounds of the Invention” or “PDE 1 inhibitors of the Invention” encompasses any and all of the compounds disclosed within this specification.
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
For methods of treatment, the word “effective amount” is intended to encompass a therapeutically effective amount to treat a specific disease or disorder.
The term “pulmonary hypertension” is intended to encompass pulmonary arterial hypertension.
The term “patient” include human or non-human (i.e., animal) patient. In particular embodiment, the invention encompasses both human and nonhuman. In another embodiment, the invention encompasses nonhuman. In other embodiment, the term encompasses human.
The term “comprising” as used in this disclosure is intended to be open-ended and does not exclude additional, unrecited elements or method steps.
Compounds of the Invention are in particular useful for the treatment of Parkinson's disease, narcolepsy and female sexual dysfunction.
Compounds of the Invention may be used as a sole therapeutic agent, but may also be used in combination or for co-administration with other active agents.
For example, as Compounds of the Invention potentiate the activity of D1 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 PDE 1 inhibitors of the Invention, e.g., the Compounds of the Invention 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.
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 oral administration thus for example may comprise from about 0.2 to 75 or 150 mg, e.g. from about 0.2 or 2.0 to 50, 75 or 100 mg of a Compound of the Invention, together with a pharmaceutically acceptable diluent or carrier therefor.
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.
EXAMPLES
The synthetic methods for various Compounds of the Present Invention are illustrated below. 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.
Example 1
2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




1) 2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione
5-methyl-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (200 mg, 0.847 mmol), 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole (202 mg, 0.847 mmol) and K2CO3 (117 mg, 0.847 mmol) are suspended in 5 mL of anhydrous DMF. The reaction mixture is stirred at room temperature for 5 h, and then evaporated to dryness under reduced pressure. The residue is treated with water, and then extracted with dichloromethane three times. The combined organic phase is dried with anhydrous sodium sulfate, filtered, and then evaporated to dryness to give 337 mg of crude product, which is used in the next step without further purification. MS (ESI) m/z 394.2 [M+H]+.
2) 2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione
2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (52 mg, 0.132 mmol) and methyl disulfide (12 μL, 0.13 mmol) are dissolved in 2 mL of anhydrous CH2Cl2, and then 1.0 M LiHMDS (190 μL, 0.19 mmol) in THF is added dropwise. The reaction mixture is stirred at room temperature for 30 min, and then quenched with saturated ammonium chloride aqueous solution. After routine workup, the obtained crude product is purified by silica gel column chromatography to give pure product as off-white solids. MS (ESI) m/z 440.2 [M+H]+.
Example 2
5-methyl-3-(methylthio)-7-neopentyl-2-(4-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 1 wherein 1-(bromomethyl)-4-(trifluoromethyl)benzene is used in step 1 instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 441.2 [M+H]+.
Example 3
5-methyl-7-neopentyl-3-(phenylthio)-2-(4-(trifluoromethyl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 1 wherein 1-(bromomethyl)-4-(trifluoromethyl)benzene is used in step 1 instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole, and phenyl disulfide is used in step 2 instead of methyl disulfide. MS (ESI) m/z 503.2 [M+H]+.
Example 4
2-(4-methoxybenzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 1 wherein 4-methoxybenzyl bromide is used in step 1 instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole, and phenyl disulfide is used in step 2 instead of methyl disulfide. MS (ESI) m/z 465.2 [M+H]+.
Example 5
2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




2-(4-methoxybenzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (2.7 g, 5.9 mmol) is dissolved in CH2Cl2 (20 mL) containing TFA (4.2 mL) and TFMSA (1.0 mL). The mixture is stirred at room temperature overnight. Solvents are removed, and the residue is dissolved in ethyl acetate (250 mL), followed by washing with saturate NaHCO3 and water successively. After dried over anhydrous sodium sulfate, the organic phase is evaporated to dryness. The obtained crude product is used in the next step without further purification.
Crude 5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (40 mg, 0.12 mmol), 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole (28 mg, 0.12 mmol) and K2CO3 (16 mg, 0.12 mmol) are suspended in 5 mL of anhydrous DMF. The reaction mixture is stirred at room temperature overnight, and then evaporated to dryness under reduced pressure. The residue is purified by silica gel column chromatography to give pure product as white solids. MS (ESI) m/z 502.2 [M+H]+.
Example 6
2-(4-methoxybenzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 1 wherein 4-methoxybenzyl bromide is used in step 1 instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 403.2 [M+H]+.
Example 7
2-(4-(1H-pyrazol-1-yl)benzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 5 wherein 2-(4-methoxybenzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione is used instead of 2-(4-methoxybenzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione, and 1-(4-(bromomethyl)phenyl)-1H-pyrazole is used instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 439.2 [M+H]+.
Example 8
5-methyl-2-(4-(methylsulfonyl)benzyl)-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 5 wherein 1-(bromomethyl)-4-(methylsulfonyl)benzene is used instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 513.2 [M+H]+.
Example 9
5-methyl-7-neopentyl-3-(phenylthio)-2-(4-(pyridin-2-yl)benzyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 1 wherein 2-(4-(bromomethyl)phenyl)pyridine is used in step 1 instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole, and phenyl disulfide is used in step 2 instead of methyl disulfide. MS (ESI) m/z 512.3 [M+H]+.
Example 10
5-methyl-2-(4-(1-methylpiperidin-2-yl)benzyl)-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 5 wherein 2-(4-(chloromethyl)phenyl)-1-methylpiperidine is used instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 532.3 [M+H]+.
Example 11
5-methyl-2-(4-(methylsulfonyl)benzyl)-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 5 wherein 2-(4-methoxybenzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione is used instead of 2-(4-methoxybenzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione, and 1-(bromomethyl)-4-(methylsulfonyl)benzene is used instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 451.1 [M+H]+.
Example 12
5-methyl-3-(methylsulfinyl)-2-(4-(methylsulfonyl)benzyl)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




5-methyl-2-(4-(methylsulfonyl)benzyl)-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (11.4 mg, 0.022 mmol) is dissolved in CH2Cl2 (200 μL) and CH3CN (100 μL), and then 30% H2O2 aqueous solution (75 μL, 0.66 mmol) is added, followed by acetic acid (6.6 mg, 0.11 mmol). The reaction mixture is stirred at room temperature over a weekend, and then purified by a semi-preparative HPLC to give 6 mg of product as white solids. MS (ESI) m/z 467.1 [M+H]+.
Example 13
2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylsulfinyl)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (16 mg, 0.036 mmol) is dissolved in CH2Cl2 (500 μL) and MeOH (500 μL), and then an aqueous solution of Oxone (22.4 mg, 0.036 mmol) is added. The reaction mixture is stirred at room temperature for 2 days, and then purified by a semi-preparative HPLC to give 8 mg of product as off-white solids. MS (ESI) m/z 456.2 [M+H]+.
Example 14
2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylsulfonyl)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is the same as EXAMPLE 13. 2-(4-(1H-1,2,4-triazol-1-yl)benzyl)-5-methyl-3-(methylsulfonyl)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione is obtained as a minor product of the reaction. MS (ESI) m/z 472.2 [M+H]+.
Example 15
5-methyl-2-(4-(1-methylpiperidin-2-yl)benzyl)-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione




The synthetic procedure of this compound is analogous to EXAMPLE 5 wherein 2-(4-methoxybenzyl)-5-methyl-3-(methylthio)-7-neopentyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione is used instead of 2-(4-methoxybenzyl)-5-methyl-7-neopentyl-3-(phenylthio)-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione, and 2-(4-(chloromethyl)phenyl)-1-methylpiperidine is used instead of 1-(4-(bromomethyl)phenyl)-1H-1,2,4-triazole. MS (ESI) m/z 439.2 [M+H]+.
Example 16
Measurement of PDE1B Inhibition In Vitro Using IMAP Phosphodiesterase Assay Kit
Phosphodiesterase 1B (PDE1B) is a calcium/calmodulin dependent phosphodiesterase enzyme that converts cyclic guanosine monophosphate (cGMP) to 5′-guanosine monophosphate (5′-GMP). PDE1B 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, Calif.) immobilized-metal affinity particle reagent.
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 fluorophores, because the photons emitted as they fluoresce retain the same polarity as the photons used to excite the fluorescence.
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 (Δmp). Inhibition of phosphodiesterase, therefore, is detected as a decrease in Δmp.
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, Calif.).
Assay: 3′,5′-cyclic-nucleotide-specific bovine brain phosphodiesterase (Sigma, St. Louis, Mo.) is reconstituted with 50% glycerol to 2.5 U/ml. One unit of enzyme will hydrolyze 1.0 μmole 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 μM CaCl2, 10 U/ml of calmodulin (Sigma P2277), 10 mM Tris-HCl pH 7.2, 10 mM MgCl2, 0.1% BSA, 0.05% NaN3) to yield a final concentration of 1.25 mU/ml. 99 μl of diluted enzyme solution is added into each well in a flat bottom 96-well polystyrene plate to which 1 μl of test compound dissolved in 100% DMSO is added. Selected Compounds of the Invention are mixed and pre-incubated with the enzyme for 10 min at room temperature.
The FL-GMP conversion reaction is initiated by combining 4 parts enzyme and inhibitor mix with 1 part substrate solution (0.225 μM) 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 μl 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, Conn.) to measure the fluorescence polarization (Δmp).
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 and then plotting drug concentration versus ΔmP, which allows IC50 values to be estimated using nonlinear regression software (XLFit; IDBS, Cambridge, Mass.).
The Compounds of the Invention may be selected and tested in an assay as described or similarly described herein for PDE1 inhibitory activity. The exemplified compounds of the invention generally have IC50 values of less than 5 μM, some less than 1 μM, some less than 250 nM, some with PDE1A activities.
Example 17
PDE1 Inhibitor Effect on Sexual Response in Female Rats
The effect of PDE1 inhibitors on Lordosis Response in female rats is measured as described in Mani, et al., Science (2000) 287: 1053. Ovariectomized and cannulated wild-type rats are primed with 2 μg estrogen followed 24 hours later by intracerebroventricular (icy) injection of progesterone (2 μg), PDE1 inhibitors of the present invention (0.1 mg, 1.0 mg or 2.5 mg) or sesame oil vehicle (control). The rats are tested for lordosis response in the presence of male rats. Lordosis response is quantified by the lordosis quotient (LQ=number of lordosis/10 mounts×100). The LQ for estrogen-primed female rats receiving Compounds of the Invention, at 0.1 mg, will likely be similar to estrogen-primed rats receiving progesterone and higher than for estrogen-primed rats receiving vehicle.

## Claims

1. A compound of formula I:




wherein
(i) L is S or SO2;
(ii) R1 is H or C1-6alkyl;
(iii) R2 is
H,
C1-6alkyl wherein said alkyl group is optionally substituted with halo or hydroxy,
—C0-4alkyl-C3-8cycloalkyl optionally substituted with one or more amino, wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl,
haloC1-6alkyl,
N(R14)(R15)—C1-6alkyl,
hydroxyC1-6alkyl,
arylC0-6alkyl,
heteroarylC1-6alkyl,
C1-6alkoxyarylC1-6alkyl;

(iv) R3 is
1) -D-E-F wherein:
D is a single bond, C1-6alkylene or arylalkylene,
E is
a single bond,
C1-4alkylene,
—C0-4alkylarylene, wherein the arylene group is optionally substituted with halo,
heteroarylene,
aminoC1-6alkylene,
amino,
C3-8cycloalkylene optionally containing one or more heteroatom selected from N or O,

F is
haloC1-6alkyl,
aryl,
C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O, and optionally substituted with C1-6alkyl,
heteroaryl optionally substituted with C1-6alkyl, halo or haloC1-6alkyl,
alkoxadiazoly,
amino,
O-haloC1-6alkyl,
C1-6alkylsulfonyl,
—C(O)—R13,
—N(R14)(R15);



2) a haloalkyl substituted heteroarylaklyl; or
3) attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A





wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is C3-8cycloalkyl, haloC1-6alkyl, aryl, heteroaryl, C1-6alkyl sulfonyl, arylcarbonyl alkoxadiazolyl, heteroarylcarbonyl, alkoxycarbonyl, or aminocarbonyl; provided that when X, Y or Z is nitrogen, R8, R9 or R10, respectively, is not present;

(v) R4 is selected from:
H,
C1-6alkyl,
C3-8cycloalkyl,
C3-8heterocycloalkyl,
aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group;

(vi) R13 is —N(R14)(R15), C1-6alkyl, —OC1-6alkyl, haloC1-6alkyl, aryl, or heteroaryl; and
(vii) R14 and R15 are independently H or C1-6alkyl,

in free or salt form.

2. The compound according to claim 1 selected from:













in free or salt form.

3. The compound according to claim 1, selected from any of the following:










in free or salt form.

4. The compound according to claim 1, which is a compound of formula I:




wherein
(i) L is S or SO2;
(ii) R1 is H or C1-6alkyl;
(iii) R2 is
H,
C1-6alkyl wherein said alkyl group is optionally substituted with halo or hydroxy,
—C0-4alkyl-C3-8cycloalkyl optionally substituted with one or more amino, wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl,
haloC1-6alkyl,
N(R14)(R15)—C1-6alkyl,
hydroxyC1-6alkyl,
arylC0-6alkyl,
heteroarylC1-6alkyl,
C1-6alkoxyarylC1-6alkyl;

(iv) R3 is
1) -D-E-F wherein:
D is a C1-6alkylene,
E is
a
phenylene optionally substituted with halo,

F is
haloC1-6alkyl,
aryl,
C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O, and optionally substituted with C1-6alkyl,
heteroaryl optionally substituted with C1-6alkyl, halo or haloC1-6alkyl,
alkoxadiazolyl,
amino,
—O-haloC1-6alkyl,
C1-6alkylsulfonyl,
—C(O)—R13,
—N(R14)(R15);


2) or attached to one of the nitrogens on the pyrazolo portion of Formula I and is a moiety of Formula A







wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is C1-8cycloalkyl, haloC1-6alkyl, aryl, heteroaryl, C1-6alkyl sulfonyl , arylcarbonyl alkoxadiazolyl, heteroarylcarbonyl, alkoxycarbonyl, or aminocarbonyl;


(v) R4 is selected from:
H,
C1-6alkyl,
C3-8cycloalkyl,
C3-8heterocycloalkyl,
aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group;

(vi) R13 is —N(R14)(R15), C1-6alkyl, —OC1-6alkyl, haloC1-6alkyl, aryl, or heteroaryl; and
(vii) R14 and R15 are independently H or C1-6alkyl,

in free or salt form.

5. The compound according to claim 4, wherein said compound is a compound of Formula II:




wherein
(i) L is S or SO2;
(ii) R1 is H or C1-6alkyl;
(iii) R2 is
H,
C1-6alkyl wherein said alkyl group is optionally substituted with halo or hydroxy,
—C0-4alkyl-C3-8cycloalkyl optionally substituted with one or more amino, wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl,
haloC1-6alkyl,
N(R14)(R15)—C1-6alkyl,
hydroxyC1-6alkyl,
arylC0-6alkyl,
heteroarylC1-6alkyl,
C1-6alkoxyarylC1-6alkyl;

(iv) R3 is attached to one of the nitrogens on the pyrazolo portion of Formula II and is a moiety of Formula A





wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is
C1-8cycloalkyl,
haloC1-6alkyl,
aryl,
heteroaryl,
alkoxadiazolyl,
alkyl sulfonyl,
arylcarbonyl
heteroarylcarbonyl,
alkoxycarbonyl, or
aminocarbonyl;


(v) R4 is
H,
C1-6alkyl,
C3-8cycloalkyl,
C3-8heterocycloalkyl,
aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group;

(vi) R14 and R15 are independently H or C1-6alkyl;

in free or salt form.

6. The compound according to claim 5, wherein:
R4 is:
C1-6alkyl,
C3-8cycloalkyl,
C3-8heterocycloalkyl,
aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group;

and
R3 is attached to one of the nitrogens on the pyrazolo portion of Formula II and is a moiety of Formula A





wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is
C3-8cycloalkyl
aryl, or
heteroaryl, alkoxadiazolyl;



in free or salt form.

7. The compound according to claim 4, wherein the compound of Formula I is:




and R2 is C1-6alkyl wherein said alkyl group is optionally substituted with halo or hydroxy, in free or salt form.

8. The compound according to claim 7, wherein R4 is C1-6alkyl, in free or salt form.

9. The compound according to claim 8, wherein R3 is a moiety of Formula A




wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is 1,2,4-triazol-1-yl, in free or salt form.

10. The compound according to claim 8, wherein R3 is -D-E-F wherein F is heteroaryl optionally substituted with C1-6alkyl, halo or haloC1-6alkyl; or F is C3-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O, and optionally substituted with C1-6alkyl, in free or salt form.

11. The compound according to claim 10, wherein R2 is 2,2-dimethylpropyl, in free or salt form.

12. The compound according to claim 11, wherein said compound is:




in free or salt form.

13. The compound according to claim 7, wherein R4 is aryl optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group, in free or salt form.

14. The compound according to claim 7, wherein R4 is phenyl, in free or salt form.

15. The compound according to claim 14, wherein R3 is a moiety of Formula A




wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is methylsulfonyl, in free or salt form.

16. The compound according to claim 14, wherein R3 is a moiety of Formula A




wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is trifluoromethyl, in free or salt form.

17. The compound according to claim 14, wherein R3 is a moiety of Formula A




wherein X, Y and Z are C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is 1,2,4-triazol-1-yl, in free or salt form.

18. The compound according to claim 14, wherein R3 is -D-E-F wherein F is heteroaryl optionally substituted with C1-6alkyl, halo or haloC1-6alkyl; or F is C1-8cycloalkyl optionally containing at least one atom selected from a group consisting of N or O, and optionally substituted with C1-6alkyl, in free or salt form.

19. The compound according to claim 18, wherein R2 is 2,2-dimethylpropyl, in free or salt form.

20. The compound according to claim 4, wherein said compound is:




in free or salt form.

21. The compound according to claim 1, wherein said compound is a compound of Formula II:




wherein
(i) L is S or SO2;
(ii) R1 is H or C1-6alkyl;
(iii) R2 is
H,
C1-6alkyl wherein said alkyl group is optionally substituted with halo or hydroxy,
—C0-4alkyl-C3-8cycloalkyl optionally substituted with one or more amino, wherein said cycloalkyl optionally contains one or more heteroatom selected from N and O and is optionally substituted with C1-6alkyl,
haloC1-6alkyl,
N(R14)(R15)—C1-6alkyl,
hydroxyC1-6alkyl,
arylC0-6alkyl,
heteroarylC1-6alkyl,
C1-6alkoxyarylC1-6alkyl;

(iv) R3 is attached to one of the nitrogens on the pyrazolo portion of Formula II and is a moiety of Formula A





wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen; and R10 is
C3-8cycloalkyl,
haloC1-6alkyl,
aryl,
heteroaryl,
alkoxadiazolyl,
alkyl sulfonyl,
arylcarbonyl
heteroarylcarbonyl,
alkoxycarbonyl, or
aminocarbonyl,
Provided that when X, Y or Z is nitrogen, R8, R9 or R10, respectively, is not present;


(v) R4 is
H,
C1-6alkyl,
C3-8cycloalkyl,
C3-8heterocycloalkyl,
aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with halo, hydroxy, C1-6alkyl, C1-6alkoxy or another aryl group;

(vi) R14 and R15 are independently H or C1-6alkyl;

in free or salt form.

22. A compound of formula:




in free or salt form.

23. A pharmaceutical composition comprising a compound according to claim 5, in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier.