Products and pharmaceutical compositions

Abstract

The present invention relates to a product comprising a PDE1 inhibitor and a PDE2 inhibitor, in free or salt form, pharmaceutical compositions comprising them and their use as pharmaceuticals for the treatment of cAMP and/or cGMP related disorders.

Claims

1. A product comprising (a) PDE1 inhibitor 6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or salt form, and (b) PDE2 inhibitor 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550), in free or salt form.

2. A pharmaceutical composition comprising PDE1 inhibitor, (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or pharmaceutically acceptable salt form, and PDE2 inhibitor 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550), in free or pharmaceutically acceptable salt form, in admixture with a pharmaceutically acceptable diluent or carrier.

3. The pharmaceutical composition according to claim 2, wherein the composition is a fixed pharmaceutical composition wherein the PDE1 and PDE2 therapeutic agents are in a single dosage form.

4. The pharmaceutical composition according to claim 2, wherein the composition is a free pharmaceutical composition, wherein the PDE1 and PDE2 therapeutic agents are in a separate dosage form.

5. The pharmaceutical composition according to claim 2, wherein the PDE1 inhibitor and PDE2 inhibitor, in free or ophthalmologically acceptable salt form is in combination or association with an ophthalmologically acceptable diluent or carrier.

6. A method of treating any of the following conditions: Parkinson's disease, restless leg tremors, dyskinesias, Huntington's disease, Alzheimer's disease, 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, drug addiction, cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, pulmonary arterial hypertension, sexual dysfunction, asthma, chronic obstructive pulmonary disease, allergic rhinitis, autoimmune and inflammatory diseases; female sexual dysfunction, exercise amenorrhoea, anovulation, menopause, menopausal symptoms, 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, glaucoma or elevated intraocular pressure, psychosis, psychotic symptoms, hallucinations, paranoid or bizarre delusions, or disorganized speech and thinking, schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, mania in acute manic episodes and bipolar disorder, or traumatic brain injury, comprising administering an effective amount of the pharmaceutical composition according to claim 4, to a patient in need of such treatment, wherein treatment excludes prophylaxis.

7. The method of claim 6, wherein the condition is selected from the group consisting of Parkinson's disease, schizophrenia, narcolepsy, glaucoma, female sexual dysfunction, cognitive disorder, anxiety and depression.

8. The pharmaceutical composition according to claim 2 for use in the treatment Parkinson's disease, restless leg tremors, dyskinesias, Huntington's disease, Alzheimer's disease, 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, drug addiction, cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, pulmonary arterial hypertension, sexual dysfunction, asthma, chronic obstructive pulmonary disease, allergic rhinitis, autoimmune and inflammatory diseases; female sexual dysfunction, exercise amenorrhoea, anovulation, menopause, menopausal symptoms, 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, glaucoma or elevated intraocular pressure, psychosis, psychotic symptoms, hallucinations, paranoid or bizarre delusions, or disorganized speech and thinking, schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, mania in acute manic episodes and bipolar disorder, or traumatic brain injury, wherein treatment excludes prophylaxis.

9. A method of treating any of the following conditions: Parkinson's disease, restless leg tremors, dyskinesias, Huntington's disease, Alzheimer's disease, 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, drug addiction, cerebrovascular disease, stroke, congestive heart disease, hypertension, pulmonary hypertension, pulmonary arterial hypertension, sexual dysfunction, asthma, chronic obstructive pulmonary disease, allergic rhinitis, autoimmune and inflammatory diseases; female sexual dysfunction, exercise amenorrhoea, anovulation, menopause, menopausal symptoms, 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, glaucoma or elevated intraocular pressure, psychosis, psychotic symptoms, hallucinations, paranoid or bizarre delusions, or disorganized speech and thinking, schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder, delusional disorder, mania in acute manic episodes and bipolar disorder, traumatic brain injury; comprising administering an effective amount of (a) PDE1 inhibitor, (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or pharmaceutically acceptable salt form, and (b) PDE2 inhibitor 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550), in free or pharmaceutically acceptable salt form, to a patient in need of such treatment, wherein treatment excludes prophylaxis.

10. The method of claim 9, wherein the condition is selected from the group consisting of Parkinson's disease, schizophrenia, narcolepsy, glaucoma, female sexual dysfunction, cognitive disorder, anxiety and depression.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph which illustrates the results of the Enzyme Immunoassay described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

(2) PDE1 and PDE2 inhibitors are known in the art. In a particular embodiment of the invention, a PDE1 inhibitor of the current invention includes those disclosed in WO 2006/133261, WO 2007/143705, WO 2008/063505, WO 2008/070095, WO 2009/075784, WO 2009/073210, WO 2010/065153, WO 2010/065148, WO 2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2010/098839, WO 2010/132127, WO 2011/153129, WO 2011/153135, WO 2011/153136, WO 2011/153138, WO 2012/171016, the contents of each of which are incorporated by reference in their entirety. In another particular embodiment, the PDE1 inhibitor is (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or salt form. In another particular embodiment, the PDE1 inhibitor is (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or salt form.

(3) In another particular embodiment, a PDE2 inhibitor of the current invention includes those disclosed in WO 2010/054260, WO 2010/054253, WO 2012/104293, WO 2013/000924, WO 2013/034755, WO 2013/034758, WO 2013/034761, WO 2006/072615, WO 2006/024640, WO 2006/072612, WO 2012/114222, WO 2012/168817, WO 2005/041957, WO 2005/061497, WO 2011/011312, EP 1749824, EP 1548011, EP 1556055, U.S. Pat. No. 4,766,122, WO 2002/068423, WO 2002/050078, WO 2002/009713, WO 98/32755, U.S. Pat. No. 5,861,396, WO 2004/089953, U.S. Pat. No. 5,861,396 and U.S. Pat. No. 6,573,263. In a particular embodiment, the PDE2 inhibitor of the invention is 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550), in free or salt form.

(4) In this specification, unless otherwise indicated, the PDE1 and PDE2 inhibitors of the invention are to be understood as embracing the compounds in any form, for example free or acid addition salt form, or where the compounds contain acidic substituents, in base addition salt form. The PDE1 and PDE2 inhibitors 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 the free compounds or their pharmaceutically acceptable salts, and are therefore also included.

(5) The PDE1 inhibitor and PDE2 inhibitor of the invention may in some cases also exist in prodrug form. A prodrug form of PDE1 or PDE2 inhibitor may be inactive or less active outside the body and converts in the body to an active PDE1 or PDE2 inhibitory compound, respectively. For example when the PDE1 or PDE2 inhibitor of the invention contains a hydroxy or carboxy substituent, this substituent may form physiologically hydrolysable and acceptable esters. As used herein, “physiologically hydrolysable and acceptable ester” means esters of the PDE1 or PDE2 inhibitor of the invention which are hydrolysable under physiological conditions to yield acids (in the case of PDE1 or PDE2 inhibitor of the invention which have hydroxy substituents) or alcohols (in the case of PDE1 or PDE2 inhibitor of the invention which have carboxy substituents) which are themselves physiologically tolerable at doses to be administered. Therefore, wherein the PDE1 or PDE2 inhibitor of the invention contains a hydroxy group, for example, compound-OH, the acyl ester prodrug of such compound, for example, compound-O—C(O)—C.sub.1-4alkyl, 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)—C.sub.1-4alkyl). Alternatively, wherein the compound of the invention contains a carboxylic acid, for example, compound-C(O)OH, the acid ester prodrug of such compound, for example, compound-C(O)O—C.sub.1-4alkyl can hydrolyze to form compound-C(O)OH and HO—C.sub.1-4alkyl. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.

(6) The PDE1 and PDE2 inhibitors of the invention include their enantiomers, diastereoisomers and racemates, as well as their polymorphs, hydrates, solvates and complexes. Some individual compounds within the scope of this invention may contain double bonds. Representations of double bonds in this invention are meant to include both the E and the Z isomer of the double bond. In addition, some compounds within the scope of this invention may contain one or more asymmetric centers. This invention includes the use of any of the optically pure stereoisomers as well as any combination of stereoisomers.

(7) As will be appreciated by those skilled in the art, the PDE1 and PDE2 inhibitors of the invention may exhibit keto-enol tautomerization. Therefore, the invention as defined in the present invention is to be understood as embracing both the structures as set forth herewith and their tautomeric forms.

(8) It is also intended that the PDE1 and PDE2 inhibitors 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, .sup.13C, .sup.15N, .sup.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., .sup.123I, .sup.131I, .sup.125I, .sup.11C, .sup.18F, may replace the corresponding abundant species, e.g., I, C and F respectively. Another example of useful isotope of the compound of the invention is the .sup.11C isotope. These radio isotopes are useful for radio-imaging and/or pharmacokinetic studies of the compounds of the invention. Methods of making isotopes of PDE1 inhibitors disclosed in WO 2011/043816, the contents of which are incorporated by reference in their entirety, may be used for making the isotopes of the compounds of the current invention.

(9) The phrase “PDE1 inhibitor(s) of the invention” encompasses any and all of the compounds disclosed herewith, in free or (pharmaceutically) salt form. Preferably, the PDE1 inhibitors of the invention inhibit phosphodiesterase-mediated (e.g., PDE1-mediated) hydrolysis of cGMP, e.g., with an IC.sub.50 of less than 100 nM in an immobilized-metal affinity particle reagent PDE assay, for example, as described in Example 3. Preferably, the PDE1 inhibitors of the invention are (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one or (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or salt form.

(10) The phrase “PDE2 inhibitor(s) of the invention” encompasses any and all of the compounds disclosed herewith, in free or (pharmaceutically) salt form. Preferably, the PDE2 inhibitors of the invention inhibit phosphodiesterase-mediated (e.g., PDE2-mediated) hydrolysis of cGMP, e.g., with an IC.sub.50 of less than 100 nM in an immobilized-metal affinity particle reagent PDE assay, for example, as described in Example 5. Preferably, the PDE2 inhibitor of the invention is 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550), in free or salt form.

(11) 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.

(12) For methods of treatment, the word “effective amount” is intended to encompass a therapeutically effective amount to treat a specific disease or disorder.

(13) 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.

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

(15) The product and pharmaceutical composition of the invention are in particular useful for the treatment of Parkinson's disease, schizophrenia, narcolepsy, glaucoma, female sexual dysfunction, cognitive disorder (e.g., learning, memory, recognition memory, social interactions and working memory), anxiety and depression.

(16) 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 PDE1 inhibitor and PDE2 inhibitor used, the mode of administration, and the therapy desired. The PDE1 inhibitor and PDE2 inhibitor 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 3.0 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 1.0 or 2.0 to 50, 75 or 100 mg of the PDE1 or PDE2 inhibitor, together with a pharmaceutically acceptable diluent or carrier therefore. The PDE1 inhibitor(s) and PDE2 inhibitor(s) may be in a single dosage form or separate dosage forms. The PDE1 inhibitor(s) and PDE2 inhibitor(s) may be administered in the same or different amounts.

(17) 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

(18) The PDE1 inhibitors of the invention and their pharmaceutically acceptable salts may be made using the methods as described and exemplified or similarly described or similarly exemplified in WO 2006/133261, WO 2007/143705, WO 2008/063505, WO 2008/070095, WO 2009/075784, WO 2009/073210, WO 2010/065153, WO 2010/065148, WO 2010/065151, WO 2010/065149, WO 2010/065147, WO 2010/065152, WO 2010/098839, WO 2010/132127, WO 2011/153129, WO 2011/153135, WO 2011/153136, WO 2011/153138, WO 2012/171016, the contents of each of which are incorporated by reference in their entirety. The PDE2 inhibitors of the invention may be made using the methods as described and exemplified or similarly described or similarly exemplified in WO 2010/054260, WO 2010/054253, WO 2012/104293, WO 2013/000924, WO 2013/034755, WO 2013/034758, WO 2013/034761, WO 2006/072615, WO 2006/024640, WO 2006/072612, WO 2012/114222, WO 2012/168817, WO 2005/041957, WO 2005/061497, WO 2011/011312, EP 1749824, EP 1548011, EP 1556055, U.S. Pat. No. 4,766,122, WO 2002/068423, WO 2002/050078, WO 2002/009713, WO 98/32755, U.S. Pat. No. 5,861,396, WO 2004/089953, U.S. Pat. No. 5,861,396, and U.S. Pat. No. 6,573,263, the contents of each of which are incorporated by reference in their entirety. 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.

(19) In particular, the synthetic methods for (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one and (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one, in free or salt form, may be prepared using the procedure set forth or similarly set forth below in Examples 1 and 2.

Example 1—(6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

(20) This compound may be prepared as described or similarly described in WO 2006/133261. Step (a) 7-(4-Methoxybenzyl)-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione: Phenyl isothiocyanate (3.9 mL, 32.7 mmol) is added to a suspension of 6-hydrazinyl-1-(4-methoxybenzyl)-3-methylpyrimidine-2,4(1H,3H)-dione (0.45 g, 1.6 mmol) in DMF (12 mL). The reaction mixture is heated at 120° C. for 40 hours, and then evaporated to remove solvent under reduced pressure. The residue is washed with hexanes, and then treated with MeOH (125 mL), and stored at −15° C. for 2 days to give a crystalline solid. The solid is recrystallized from CH.sub.3OH-EtOAc to afford 2.5 g product.

(21) Step (b) 5-Methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione: AlCl.sub.3 (0.733 g, 5.50 mmol) is added to a solution of 7-(4-methoxybenzyl)-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (0.692 g, 1.83 mmol) and anisole (40 μL, 0.367 mmol) in 1,2-dichloroethane (10 mL) under argon. The reaction mixture is stirred at room temperature for 30 min, and then quenched with water with cooling. The resulting suspension is filtered through a layer of celite and the celite is washed with MeOH (20 mL). The product is eluted from the celite with a large amount of THF. The THF eluent is evaporated to afford 0.47 g of product.

(22) Step (c) 6-Chloro-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one: 5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (450 mg, 1.75 mmol) is refluxed in POCl.sub.3 (20 mL) for 60 hours, and the mixture is evaporated to dryness. The residue is purified by silica gel flash chromatography to give 122 mg product as white solids and 207 mg starting material is recovered.

(23) Step (d) 6-((1R,2R)-2-hydroxycyclopentylamino)-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one: A solution of 6-chloro-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (75.8 mg, 0.275 mmol), (1R,2R)-2-amino-cyclopentanol (0.55 mmol) and DIPEA (144 μL, 0.825 mmol) in DMF (3 mL) is heated at 110° C. overnight. The reaction mixture is evaporated to remove DMF under reduced pressure. The residue is then purified by chromatography to give the desired product.

(24) Step (e) (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one: 2.0 M solution of thionyl chloride in CH.sub.2Cl.sub.2 (267 μL, 0.534 mmol) is added to a solution of 6-((1R,2R)-2-hydroxycyclopentylamino)-5-methyl-3-(phenylamino)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (30 mg, 0.088 mmol) in CH.sub.2Cl.sub.2 (1 mL) and THF (1.5 mL). The reaction mixture is stirred at r.t. overnight, and then quenched with 29 μL of 28% NH.sub.4OH. The resulting mixture is concentrated and purified by chromatography to give 26 mg product as white solids.

(25) Step (f) (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one: A mixture of (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (22 mg, 0.068 mmol), 2-(4-(bromomethyl)phenyl)pyridine (16.9 mg, 0.068 mmol), and K.sub.2CO.sub.3 (9.4 mg, 0.068 mmol) in DMF (2.5 mL) is stirred at room temperature overnight under argon. The reaction mixture is purified by a semi-preparative HPLC to give the final product.

Example 2—(6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

(26) ##STR00001##

(27) This compound may be prepared as described or similarly described in WO 2007/143705, WO 2013/192556, U.S. Prov. Appl. Nos. 61/838,105 and 61/919,424, the contents of which are incorporated by reference in their entirety.

Example 3—Measurement of PDE1 Inhibition In Vitro Using IMAP Phosphodiesterase Assay Kit

(28) 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.

(29) 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.

(30) 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.

(31) 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 CaCl.sub.2, 10 U/ml of calmodulin (Sigma P2277), 10 mM Tris-HCl pH 7.2, 10 mM MgCl.sub.2, 0.1% BSA, 0.05% NaN.sub.3) 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.

(32) 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).

(33) A decrease in GMP concentration, measured as decreased Δmp, is indicative of inhibition of PDE activity. IC.sub.50 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 IC.sub.50 values to be estimated using nonlinear regression software (XLFit; IDBS, Cambridge, Mass.).

(34) The PDE1 inhibitors may be selected and tested in an assay as described or similarly described herein for PDE1 inhibitory activity. Using the procedure described or similarly described in this example, the compound of Example 1 has an IC.sub.50 value of 105+/−10.9 pM.

Example 4—2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550)

(35) The compound of this example may be prepared as described or similarly described in U.S. Pat. No. 6,573,263, the contents of which are incorporated by reference in their entirety. The details are reproduced below:

Compound 4A: 2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-4-phenylbutyl]-5-methylimidazo-[5,14][1,2,4]triazin-4(3H)-one

(36) 110 mg (0.22 mmol) of 7-(1-acetyl-4-phenylbutyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one are dissolved in 5 ml of ethanol and treated in portions with 20 mg (0.53 mmol) of sodium borohydride to give 2-(3,4-dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one.

(37) Compound 4A is separated into the two diastereomeric compounds by chromatography under reversed phase conditions (Stability C30, 5 μm) using acetonitrile/water (1/1, v/v) as eluent. The corresponding enantiomerically pure compounds (Examples 4B, 4C, 4D and 4E below) can be obtained by chromatographic separation of the racemic diastereomers on a chiral stationary silica gel phase.

(38) Particularly suitable chiral stationary polyamide silica gel phases (CSP) for the separation of the racemates are those based on the monomers N-methacryloyl-L-leucine-d-menthylamide or N-methacryloyl-L-leucine-1-menthylamide (cf. EP-A-0 379 917) using, for example, ethyl acetate as eluent.

(39) The chromatographic resolution of the first-eluting diastereomers from Example 4A yields the two enantiomers Compound 4B 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550) and Compound 4C 2-(3,4-Dimethoxybenzyl)-7-{(1S)-1-[(1S)-1-hydroxyethyl]-4-phenylbutyl}-5-methyl-imidazo[5,1-f][1,2,4]triazin-4(3H)-one. Analogously to this, the two enantiomers Compound 4D 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1S)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one and Compound 4E 2-(3,4-Dimethoxybenzyl)-7-{(1S)-1-[(1R)-1-hydroxyethyl]4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one are obtained from the later-eluting diastereomers. The separation of the enantiomers Compound 4D and Compound 4E is carried out analogously to Compound 4B starting from the later eluting diastereomer.

(40) Moreover, the Compound 4B and Compound 4C can also preferably be obtained by diastereoselective reduction of 7-(1-acetyl-4-phenylbutyl)-2-(3,4-dimethoxybenzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one. For this, 190 mg (0.41 mmol) of 7-(1-acetyl-4-phenylbutyl)-2-(3,4-dimethoxy-benzyl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one are dissolved in 20 ml of dichloromethane/methanol 100/1 and, treated with 6.10 mg (0.45 mmol) of zinc chloride, stirred at room temperature for 30 min. After cooling to 0° C., 30 mg of sodium borohydride are added in portions and the mixture is then stirred for 2.5 h with ice-bath cooling. The batch is then neutralized using a few drops of 2 N hydrochloric acid, concentrated in vacuo and chromatographed using the eluent dichloromethane/methanol 80/1 and 40/1. The diastereomer mixture is then purified under reversed phase chromatographic conditions as mentioned in Example 4B and separated into the pure enantiomers by chromatography on the chiral stationary phase.

Example 5—Measurement of PDE2 Inhibition In Vitro

(41) Inhibition of PDE2 may be measured by using an assay similarly described in Example 3 except PDE2 enzyme (human recombinant) is used instead of PDE1 enzyme, fluorescent-cAMP is used as substrate, and cGMP (1 μM) is added to stimulate the enzyme.

(42) Alternatively, PDE2 inhibition may be measured as described or similarly described in Boess et al., Neuropharmacology, Volume 47, Issue 7, December 2004, Pages 1081-1092, the contents of which are incorporated by reference in their entirety. Using the procedure described or similarly described in Boess et al., the exemplified Compound 4B (Bay-60-7550) is reported to have an IC.sub.50 value of 2.0 nM (bovine) and 4.7 nM (human).

Example 6—Effect of PDE1 and PDE2 Inhibitor on Nitric Oxide Signaling in the Brain

(43) Animals:

(44) All handling and use of animals follow a protocol approved by Institutional Animal Care and Use Committee (IACUC) of Columbia University, in accordance with NIH guidelines. Male, C57BL/6 mice (7-8 weeks of age) are obtained from Jackson Laboratory. Up to five mice are housed per cage and are maintained under 12-hour light/dark cycles with standard Purina rodent chow and water ad libitum.

(45) Slice Preparation:

(46) Slice preparation may be carried out as described below or similarly described in Nishi et al., J. Neuroscience (2008) 28:10460-71, the contents of which are incorporated by reference. Male C57BL/6 mice at 6-8 weeks old are purchased from Japan SLC. All mice used are handled in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and the specific protocols are approved by the Institutional Animal Care and Use Committee of Kurume University School of Medicine. Male C57BL/6 mice are killed by decapitation. The brains are rapidly removed and placed in ice-cold, oxygenated Krebs-HCO.sub.3.sup.− buffer [(in mM) 124 NaCl, 4 KCl, 26 NaHCO.sub.3, 1.5 CaCl.sub.2, 1.25 KH.sub.2PO.sub.4, 1.5 MgSO4, and 10 D-glucose, pH 7.4]. Coronal slices (350 μm) are prepared using a vibrating blade microtome, VT1000S (Leica Microsystems), as described previously (Nishi et al., PNAS (2005) 102:1199-1204). Striata are dissected from the slices in ice-cold Krebs-HCO.sub.3.sup.− buffer.

(47) Slice Treatment:

(48) Each slice is placed in a polypropylene incubation tube with 2 ml of fresh Krebs-HCO.sub.3.sup.− buffer containing adenosine deaminase (10 μg/ml). The slices are pre-incubated at 30° C. under constant oxygenation with 95% O2/5% CO2 for 60 min. The buffer is replaced with fresh Krebs-HCO.sub.3.sup.− buffer after 30 min of pre-incubation. Slices are treated for 45 minutes with a PDE1 inhibitor alone, PDE2 inhibitor alone, PDE1 inhibitor in combination with PDE2 inhibitor or a control (DMSO vehicle with no PDE1 or PDE2 inhibitor). The PDE1 inhibitor compound (6aR,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-Pyridin-2yl)-benzyl)-cyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (Example 1) at 1 μM is used. PDE2 is blocked using 0.1 μM of 2-(3,4-Dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one (BAY 60-7550 or Example 4B). Following the pretreatment, the slices are stimulated with 5 μM of the NO donor, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) for 4 min, in Krebs as indicated, without adenosine deaminase. DEA/NO is used for its ability to elicit a dose-dependent change in cGMP levels. DEA/NO in a stock solution of 0.1N NaOH are prepared and diluted in Krebs buffer to either 0.5 μM or 5 μM immediately before application to each slice. Immediately following stimulation, the Krebs is removed and replaced with 5% trichloroacetic acid and sonicated. Samples are stored on ice before being centrifuged at 15,000×g for 20 minutes at 4° C. The supernatant is reserved for the cyclic nucleotide enzyme immunoassay and the precipitated protein pellet is re-suspended in 60 ul Laemmli sample buffer. Each is stored at −80° C. until use.

(49) Enzyme Immunoassay:

(50) The reserved supernatants are washed three times with 5× volume ethyl ether to remove the TCA. The samples are then dried under vacuum (Speedvac, Savant SPD111V) at room temperature. In preparation for either the cGMP or cAMP enzyme immunoassay (Cayman Chemical Co, Ann Arbor, Mich.), they are resuspended in 100 μL EIA buffer. The samples are acetylated and analyzed in comparison to provided standards according to kit instructions. The results from the colorometric assay are recorded by the SoftMax 4.8 software (Molecular Devices, Sunnyvale, Calif.). Each data point is converted to % B/B.sub.0 (100*[(sample or standard OD−average non-specific binding)/(average B.sub.0−average non-specific binding)]). The standards are plotted and fit to a 4-parameter logistic equation. The concentrations of the samples are interpolated from the standard curve using Microsoft Excel and GraphPad Prizm. The result of this experiment is illustrated in FIG. 1. This experiment shows that the combination of inhibition of PDE1 and PDE2 has a greater than additive effect on cGMP levels in the striatum under conditions of stimulation. While at low concentrations of DEA/NO (0.5 μM) the effect of PDE1 and PDE2 inhibitors is additive, at maximal DEA/NO (5.0 μM) there is an apparent more than additive effect, such that the level of cGMP attained by the combined inhibitors is greater than the sum of the effect of each inhibitor alone.