HK1243933B - Organic compounds - Google Patents

Description

organic compounds

Technical Field

The present invention relates to PDE2 inhibitory compounds of formula I as described below, their use as medicaments and pharmaceutical compositions comprising them. These compounds are useful, for example, in the treatment of PDE2-mediated disorders such as anxiety, depression, autism spectrum disorder (ASD), schizophrenia and cognitive impairment.

Background of the Invention

PDE2 is a 105-kDa homodimer expressed in various tissues and cell types, including the brain (including the hippocampus, striatum, and prefrontal cortex), heart, platelets, endothelial cells, adrenal glomerular cells, and macrophages. Although cGMP is the preferred substrate and effector molecule for this enzyme, PDE2 hydrolyzes both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and is thought to be involved in many physiological processes. In particular, inhibition of nitric oxide synthase (NOS), which reduces cGMP signaling, has been shown to attenuate the behavioral effects of the anxiolytic compound benzodiazepine chlordiazepoxide. In addition, commercially available PDE2 inhibitors such as Bay60-7550 have been shown to increase cyclic nucleotide levels in the brain and have significant anxiolytic and antidepressant effects in normal and stressed rodents (Xu et al., Eur. J. Pharmacol. (2005) 518: 40-46; Masood et al., J. Pharmacol. Exp. Ther. (2008) 326: 369-379; Masood et al., JPET (2009) 331: 690-699; Xu et al., Intl. J. Neuropsychopharmacol. (2013) 16: 835-847). Inhibition of PDE2 by Bay60-7550 has also been shown to increase cGMP and cAMP levels in stimulated primary neuronal cultures in a dose-responsive manner; enhance LTP in response to electrical stimulation in hippocampal slice preparations; enhance learning in a rat novel object recognition animal model and social cognition tasks; improve the acquisition and consolidation phases of novel object memory in aged, impaired rats; and improve behavior in object location and recognition tasks administered post-training. Gomez et al., Bioorg. Med. Chem. Lett. (2013) 23: 6522-6527. Bay 60-7550 has also been shown to improve cognitive and memory function in rats by enhancing nNOS activity in the brain (Domek-Lopacinska et al. (2008) Brain Res. 1216: 68-77). Therefore, PDE2 plays an important role in efficient behavior and cognitive function.

In addition to potent behavioral and cognitive functions, it has been observed that PDE2A mRNA and activity are highly induced in endothelial cells in response to tumor necrosis factor-α stimulation in vitro. Selective inhibition of PDE2 activity with 9-(6-phenyl-2-oxohexan-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one (PDP) significantly alters endothelial cell barrier function, suggesting that PDE2 may play an important role in regulating the fluid and protein integrity of the circulatory system under pathological conditions. Therefore, PDE2 may be a good pharmacological target in sepsis or more localized inflammatory responses.

In a recent study, PDE2 inhibition has also been shown to induce lung expansion, prevent pulmonary vascular remodeling, and reduce right ventricular hypertrophy characteristic of pulmonary arterial hypertension, suggesting the therapeutic potential of PDE2 inhibition in pulmonary arterial hypertension. Bubb et al., “Inhibition of Phosphodiesterase 2 Augments cGMP and cAMP Signaling to Ameliorate Pulmonary Hypertension,” Circulation, August 5, 2014, p. 496-507, DOI: 10.1161/CIRCULATIONAHA.114.009751.

Despite promising preclinical data and the identification of PDE2 as a promising drug target, no PDE2 inhibitors are currently known to be in clinical development, in part due to the poor metabolic stability and brain penetration of existing PDE2 compounds. Therefore, there is a need for compounds that selectively inhibit PDE2 activity while exhibiting excellent biophysical properties.

SUMMARY OF THE INVENTION

The present disclosure provides novel compounds having potent and selective PDE2 inhibitory properties with improved oral bioavailability and brain entry. Thus, in a first aspect, the present disclosure provides compounds of formula I:

in:

(i) _R1 and _R2 together with the nitrogen atom form a _heteroC3-7 cycloalkyl group (e.g., forming an azetidin-1-yl group);

(ii) R ₃ is H or C _1-4 alkyl (e.g., methyl);

(iii) R ₄ is heteroaryl or aryl (e.g. phenyl), which is optionally substituted by one or more groups selected from C _1-4 alkyl (e.g. ethyl), C _3-7 cycloalkyl (e.g. cyclopropyl), C _1-4 alkoxy (e.g. methoxy) and halo C _1-4 alkyl (e.g. trifluoromethyl);

(iv) R ₆ is H or C _1-4 alkyl (e.g., methyl);

The compound is in free or salt form.

The present disclosure also provides the following compounds of formula I:

1.1 Formula I, wherein R ₁ and R ₂ together with the nitrogen atom form a heteroC _3-7 cycloalkyl group (e.g., azetidin-1-yl);

1.2 Formula 1.1, wherein R ₁ and R ₂ together with the nitrogen atom form azetidin-1-yl;

1.3 Any of Formula I or 1.1-1.2, wherein R ₃ is H or C _1-4 alkyl (eg, methyl);

1.4 Any of Formula I or 1.1-1.2, wherein R ₃ is C _1-4 alkyl (eg, methyl);

1.5 Any of Formula I or 1.1-1.4, wherein R ₄ is heteroaryl or aryl (e.g., phenyl), which is optionally substituted with one or more groups selected from C _1-4 alkyl (e.g., ethyl), C _3-7 cycloalkyl (e.g., cyclopropyl), C _1-4 alkoxy (e.g., methoxy), and halo C _1-4 alkyl (e.g., trifluoromethyl);

1.6 Any of Formula I or 1.1-1.4, wherein R ₄ is aryl (e.g., phenyl) substituted with one or more groups selected from C _1-4 alkyl (e.g., ethyl), C _3-7 cycloalkyl (e.g., cyclopropyl), C _1-4 alkoxy (e.g., methoxy), and halogenated C _1-4 alkyl (e.g., trifluoromethyl);

1.7 Any of Formula I or 1.1-1.4, wherein R ₄ is aryl (eg, phenyl) substituted with C _1-4 alkyl (eg, ethyl);

1.8 Any of Formula I or 1.1-1.4, wherein R ₄ is aryl (eg, phenyl) substituted with C _3-7 cycloalkyl (eg, cyclopropyl);

1.9 Any of Formula I or 1.1-1.4, wherein R ₄ is aryl (eg, phenyl) substituted with C _1-4 alkoxy (eg, methoxy);

1.10 Any of Formula I or 1.1-1.4, wherein R ₄ is aryl (eg, phenyl) substituted with a halo-substituted C _1-4 alkyl (eg, trifluoromethyl);

1.11 Any of Formula I or 1.1-1.10, wherein R ₆ is H or C _1-4 alkyl (eg, methyl);

1.12 Any of Formula I or 1.1-1.10, wherein R ₆ is C _1-4 alkyl (eg, methyl);

1.13 Any of the above formulae, wherein the compound is selected from:

7-(azetidin-1-yl)-3-methyl-1-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidine;

7-(azetidin-1-yl)-1-(5-(4-methoxy-2-methylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine;

7-(azetidin-1-yl)-1-(5-(4-cyclopropylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine;

7-(azetidin-1-yl)-1-(5-(4-ethylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine;

1.14 Any of the above formulae, wherein the compound inhibits phosphodiesterase-mediated (e.g., PDE2-mediated) cGMP hydrolysis, e.g., wherein the _IC50 is less than 1 μM, more preferably less than or equal to 250 nM, more preferably less than or equal to 10 nM in an immobilized-metal affinity ion reagent PDE assay, e.g., as described in Example 5,

The compounds are in free or salt form.

In a second aspect, the present disclosure provides pharmaceutical compositions comprising a compound of the present disclosure, ie, a compound of Formula I or any one of Formulas 1.1-1.14, in free or pharmaceutically acceptable salt form, and a pharmaceutically acceptable diluent or carrier.

The present disclosure also provides methods of treating PDE2-mediated disorders, such as those listed below (particularly anxiety, depression, autism spectrum disorder (ASD), schizophrenia, cognitive impairment) using the compounds of the present disclosure. It is intended that this list is not exhaustive and may include other diseases and disorders listed below.

Thus, in a third aspect, the present disclosure provides a method of treating a PDE2-mediated disorder comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the present disclosure, i.e., a compound of Formula I or any one of Formulas 1.1-1.14 in free or pharmaceutically acceptable salt form, or a pharmaceutical composition disclosed herein.

In another embodiment of the third aspect, the present disclosure provides a method for treating the following disorders in an individual:

Neurological disorders (e.g., migraine; epilepsy; Alzheimer's disease; Parkinson's disease; brain injury; stroke; cerebrovascular disease (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, and cerebral hypoxia-ischemia); spinal muscular atrophy; lateral sclerosis; multiple sclerosis;

Cognitive impairment (including amnesia, senile dementia, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, medication-induced dementia, delirium, and mild cognitive impairment); cognitive impairment associated with Parkinson's disease and depression;

Mental disabilities (including Down syndrome and Fragile X syndrome);

Sleep disorders (including hypersomnia, circadian rhythm sleep disorders, insomnia, parasomnias, and sleep deprivation);

Psychiatric disorders (e.g., anxiety (including acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder (PTSD), obsessive-compulsive disorder, specific phobia, social phobia, chronic anxiety disorder, and obsessive-compulsive disorder);

factitious disorder (including acute hallucinatory mania);

Impulse control disorders (including pathological gambling, pathological pyromania, pathological stealing, and intermittent explosive disorder);

Mood disorders (including bipolar I disorder, bipolar II disorder, mania, mixed affective states, major depression, chronic depression, seasonal depression, psychotic depression, and postpartum depression);

Psychomotor disorders (extrapyramidal and movement disorders, such as Parkinson's disease, Lewy body disease, tremor, drug-induced tremor, drug-induced tardive dyskinesia, L-dopa-induced dyskinesia, and restless legs syndrome);

Psychosis (including schizophrenia (e.g., continuous or episodic, paranoid, juvenile, catatonic, poorly differentiated, and residual schizophrenia), schizoaffective disorder, schizophreniform psychosis, and delusional disorder;

Drug dependence (including narcotic dependence, alcoholism, amphetamine dependence, cocaine addiction, nicotine dependence and drug withdrawal syndrome);

Eating disorders (including anorexia, bulimia, bulimia, hyperphagia, and pagophagia);

Pediatric psychiatric disorders (including attention deficit disorder, attention-deficit/hyperactivity disorder, behavioral disorders (e.g., tic movement disorders, such as transient, chronic, motor, or vocal tics), autism, and autism spectrum disorder (ASD));

Mental and behavioral disorders caused by the use of psychoactive substances;

Cardiovascular disorders (such as pulmonary hypertension and pulmonary arterial hypertension); and

Pain (e.g., bone and joint pain (osteoarthritis), repetitive motion pain, dental pain, cancer pain, myofascial pain (muscle injury, fibromyalgia), perioperative pain (general surgery, gynecological), chronic pain, and neuropathic pain),

The subject is preferably a mammal, preferably a human, and the method comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure, i.e., a compound of Formula I or any one of Formulas 1.1-1.14 in free or pharmaceutically acceptable salt form, or a pharmaceutical composition disclosed herein.

In one embodiment, the disease or disorder is selected from anxiety, depression, autism spectrum disorder and schizophrenia, such as anxiety and/or depression in a patient with autism and/or schizophrenia. In another embodiment, the disease or disorder is cognitive impairment associated with schizophrenia or dementia.

In a fourth aspect, the present disclosure provides compounds disclosed herein, ie, compounds of Formula I or any one of Formulas 1.1-1.14 in free or pharmaceutically acceptable salt form (for the preparation of a medicament) for use in treating a PDE2-mediated disorder as disclosed herein.

In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising a compound disclosed herein, i.e., a compound of Formula I or a compound of any one of Formulas 1.1-1.14 in free or pharmaceutically acceptable salt form, and a pharmaceutically acceptable diluent or carrier, for use in treating a PDE2-mediated disorder as disclosed herein.

Detailed Description of the Invention

Unless otherwise specified or clear from the context, the following terms have the following meanings as used herein:

(a) As used herein, "alkyl" is a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, which may be straight or branched and may be optionally mono-, di- or tri-substituted, for example, by halogen (e.g., chlorine or fluorine), hydroxyl or carboxyl.

(b) "Aryl" as used herein is a monocyclic or bicyclic aromatic hydrocarbon, preferably phenyl, which is optionally substituted, for example, by alkyl (eg, methyl), halogen (eg, chlorine or fluorine), haloalkyl (eg, trifluoromethyl), or hydroxy.

(c) As used herein, "heteroaryl" is an aromatic moiety in which one or more of the atoms forming the aromatic ring is sulfur or nitrogen rather than carbon, such as pyridyl or thiadiazolyl, which may be optionally substituted, for example, with alkyl, halogen, haloalkyl, or hydroxy.

The compounds of the present disclosure, such as compounds of Formula I or any of Formulas 1.1-1.14, may exist in free or salt form, for example, as acid addition salts. In this specification, unless otherwise indicated, language such as "a compound of the present disclosure" should be understood to encompass the compound in any form, for example, in free or acid addition salt form, or where the compound contains an acidic substituent, in base addition salt form. The compounds of the present disclosure are intended for use as pharmaceuticals, and pharmaceutically acceptable salts are therefore preferred. Salts that are not suitable for pharmaceutical use may be useful, for example, for isolating or purifying a free compound of the present disclosure or a pharmaceutically acceptable salt thereof, and are therefore also encompassed.

Compounds of the present disclosure may also exist in prodrug form in some cases. Prodrug form is a compound that is converted into a compound of the present disclosure in vivo. For example, when a compound of the present disclosure includes a hydroxyl or carboxyl substituent, these substituents can form physiologically hydrolyzable and acceptable esters. As used herein, "physiologically hydrolyzable and acceptable esters" refer to esters of compounds of the present disclosure that can be hydrolyzed under physiological conditions to produce an acid (in the case of a compound of the present disclosure with a hydroxyl substituent) or an alcohol (in the case of a compound of the present disclosure with a carboxyl substituent), which itself is physiologically tolerable at the dose to be administered. Therefore, if a compound of the present disclosure includes a hydroxyl group, such as compound-OH, then on the one hand, the acyl ester prodrug of such a compound, i.e., compound-OC (O) -C _1-4 alkyl, can be hydrolyzed in vivo to form a physiologically hydrolyzable alcohol (compound-OH), and on the other hand, an acid (e.g., HOC (O) -C _1-4 alkyl). Alternatively, if a compound of the present disclosure contains a carboxylic acid, such as compound -C(O)OH, then the acid ester prodrug compound -C(O)OC _1-4 alkyl of such compound can be hydrolyzed to form compound -C(O)OH and HO-C _1-4 alkyl. As will be appreciated, the term thus encompasses conventional drug prodrug forms.

The compounds of the present disclosure include their enantiomers, diastereomers, and racemates, as well as polymorphs, hydrates, solvates, and complexes thereof. Some individual compounds within the scope of the present invention may contain double bonds. Reference to a double bond herein is intended to include both E and Z isomers of the double bond. In addition, some compounds within the scope of the present invention may contain one or more asymmetric centers. The present invention includes the use of any optically pure stereoisomer as well as any combination of stereoisomers.

It is also expected that the compounds of the present disclosure encompass stable and unstable isotopes thereof. A stable isotope is a non-radioactive isotope that contains an extra neutron compared to an abundant nuclide of the same kind (i.e., element). It is expected that the activity of compounds containing such isotopes will be retained, and such compounds may also have uses for measuring the pharmacokinetics of non-isotopic analogs. For example, deuterium (a non-radioactive stable isotope) can be used to replace a hydrogen atom at a certain position on a compound of the present disclosure. Examples of known stable isotopes include, but are not limited to, deuterium, ¹³ C, ¹⁵ N, ¹⁸ O. Alternatively, an unstable isotope of a radioactive isotope containing an extra neutron compared to an abundant nuclide of the same kind (i.e., element), such as ¹²³ I, ¹³¹ I, ¹²⁵ I, ¹¹ C, ¹⁸ F, can replace the corresponding abundant species of I, C, and F. Another example of a useful isotope of the compounds of the present invention is the ¹¹ C isotope. These radioactive isotopes can be used for radioimaging and/or pharmacokinetic studies of the compounds of the present invention. Isotopically labeled compounds of formula I can generally be prepared by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

The term "compounds of the disclosure" or "PDE 2 inhibitors of the disclosure" encompasses any and all compounds disclosed herein, eg, compounds of Formula I as described above or compounds of any one of Formulas 1.1-1.14, in free or salt form.

Thus, the term "treating" should be understood to encompass treating or ameliorating the symptoms of a disease as well as treating the cause of the disease. In one embodiment, the present invention provides a method of treating a disease or disorder disclosed herein. In another embodiment, the present invention provides a method of preventing a disease or disorder disclosed herein.

With respect to therapeutic methods, the phrase "effective amount" is intended to encompass an amount that is therapeutically effective to treat the particular disease or disorder.

The term "pulmonary hypertension" is intended to encompass pulmonary arterial hypertension.

The term "subject" includes humans and non-humans (i.e., animals). In specific embodiments, the present invention includes humans and non-humans. In another embodiment, the present invention includes non-humans. In another embodiment, the term includes humans.

The term "comprising" as used in this disclosure is intended to be open ended and does not exclude additional not shown elements or method steps.

The term "cognitive impairment" refers to any disorder comprising symptoms of cognitive deficits (i.e., subnormal or suboptimal function in one or more cognitive aspects such as memory, intelligence, learning, logic, attention, or executive function (working memory) relative to other individuals in the same general age group). Thus, cognitive impairment includes, but is not limited to, amnesia, senile dementia, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-related dementia, delirium, and mild cognitive impairment. Cognitive impairment may also be primarily, but not exclusively, a disorder associated with psychosis (schizophrenia), mood disorders, bipolar disorder, stroke, frontotemporal dementia, progressive supranuclear palsy, brain trauma and substance abuse, Asperger's syndrome, and age-related memory impairment.

The compounds of the present disclosure, such as compounds of Formula I or any of Formulas 1.1-1.14 described above, in free or pharmaceutically acceptable salt form, can be used as sole therapeutic agents, but can also be used in combination or co-administered with additional active agents.

The dosage for implementing the present invention will of course vary depending on, for example, the specific disease or condition to be treated, the specific compound of the present disclosure used, the mode of administration, and the desired therapy. The compounds of the present disclosure can be administered by any suitable route, including oral, parenteral, transdermal, or by inhalation, but are preferably administered orally. Typically, satisfactory results, such as for treating diseases as described above, are obtained by oral administration at a dosage of about 0.01 to 2.0 mg/kg. In larger mammals, such as humans, the specified daily dose for oral administration is thus within the range of about 0.75 to 150 mg, conveniently administered once daily, or administered in divided doses 2 to 4 times daily, or administered in a sustained-release form. Therefore, for example, a unit dosage form for oral administration can comprise about 0.2 to 75 or 150 mg, such as about 0.2 or 2.0 to 50, 75, or 100 mg of the compound of the present disclosure and its pharmaceutically acceptable diluent or carrier.

Conventional diluents or excipients and techniques known in the galenic field can be used to prepare pharmaceutical compositions comprising compounds of the present disclosure. Pharmaceutically acceptable carriers can include any conventional pharmaceutical carriers or excipients. Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents (e.g., hydrates and solvates). If desired, the pharmaceutical composition can include additional ingredients, such as flavorings, adhesives, excipients, etc. Therefore, for oral administration, tablets comprising various excipients, such as citric acid, can be used together with various disintegrants such as starch, alginic acid, and certain composite silicates, as well as adhesives such as sucrose, gelatin, and gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc can generally be used for tableting purposes. Similar types of solid compositions can also be used in soft and hard-filled gelatin capsules. Therefore, non-limiting examples of materials include lactose or milk sugar and high molecular weight polyethylene glycols. When an aqueous suspension or elixir is desired for oral administration, the active compound therein may be combined with various sweeteners or flavorings, colorants or dyes, and, if desired, with an emulsifier or suspending agent, a diluent such as water, ethanol, propylene glycol, glycerol, or a combination thereof. For example, the pharmaceutical composition may be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained-release formulation, solution or suspension, in a form suitable for parenteral injection as a sterile solution, suspension or emulsion, in a form suitable for topical administration as an ointment or cream, or in a form suitable for rectal administration as a suppository.

The compounds of the present disclosure and their pharmaceutically acceptable salts can be prepared using the methods described and exemplified herein and by methods analogous thereto and by methods known in the chemical art. Such methods include, but are not limited to, those described below. If not commercially available, the raw materials used in these methods can be prepared by methods selected from the chemical art using techniques similar or analogous to the synthesis of known compounds. All references cited herein are incorporated herein by reference in their entirety.

Example 1

7-(azetidin-1-yl)-3-methyl-1-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidine

(a) 5-Chloro-1-methyl-4-nitro-1H-pyrazole

Lithium bis(trimethylsilyl)amide (1.0 M, 65 mL, 65 mmol) in THF was added dropwise to a solution of 1-methyl-4-nitro-1H-pyrazole (5.50 g, 43.3 mmol) and hexachloroethane (10.54 g, 44.5 mmol) in dichloromethane (120 mL) at 25°C. The reaction mixture was stirred at 25°C for 60 min and then quenched with water (1 mL). The mixture was evaporated to dryness. The residue was washed sequentially with water (50 mL), saturated NaHCO₃ _x2 (2 x 30 mL), and brine (30 mL), then dried under vacuum to give 6.50 g of product (93% yield). MS (ESI) m/z 162.0 [M+H] ^⁺ . ^1H NMR (500 MHz, _CDCl₃ ) δ 8.15 (s, 1H), 3.92 (s, 3H).

(b) 5-Chloro-1-methyl-1H-pyrazol-4-amine hydrochloride

To a suspension of 5-chloro-1-methyl-4-nitro-1H-pyrazole (6.50 g, 40.2 mmol) in 12N HCl (15 mL) and ethanol (15 mL) was added tin(II) chloride (35.5 g, 160.9 mmol). The reaction mixture was stirred at 90°C until the reaction was complete. The reaction mixture was evaporated to dryness. The residue was treated with 12N HCl (25 mL) and then cooled at 5°C for 2 h. After filtration, the filter cake was washed with 6N HCl (2 x 25 mL) and then dried under vacuum to give 6.08 g of product (yield: 90%). MS (ESI) 132.0 [M+H] ⁺ . ¹ H NMR (500 MHz, CD ₃ OD) δ 7.66 (s, 1H), 3.89 (s, 3H).

(c) 5-Chloro-4-hydrazino-1-methyl-1H-pyrazole hydrochloride

To a stirred solution of 5-chloro-1-methyl-1H-pyrazol-4-amine hydrochloride (6.08 g, 36.2 mmol) in HCl (12 N, 35 mL) at 0°C was added aqueous _NaNO₂ (5.50 g, 80.0 mmol). The reaction mixture was stirred at 0°C for 45 min, then tin(II) chloride (22.8 g, 120 mmol) was added. After the addition was complete, the reaction mixture was stirred at 0°C for 30 min, then stirred at room temperature overnight. The resulting mixture was cooled in an ice bath for 2 h and then filtered. The filter cake was washed with HCl (12 N, 20 mL) and then dried under vacuum to give 7.77 g of crude product (yield: 98%), which was used in the next reaction without further purification. MS (ESI) m/z 147.0 [M+H] ^⁺ .

(d) 5-Bromo-4-chloro-6-(1-ethoxyvinyl)pyrimidine

A suspension of 5-bromo-4,6-dichloropyrimidine (5.00 g, 21.9 mmol) and tributyl(1-ethoxyvinyl)stannane (7.92 g, 21.9 mmol) in DMF (20 mL) was degassed with argon, followed by the addition of tetrakis(triphenylphosphine)palladium(0) (1.27 mg, 1.10 mmol). The suspension was further degassed and then heated at 110° C. under argon for 8 h. After removal of the solvent under reduced pressure, the residue was purified by silica gel column chromatography using a gradient of 0-40% ethyl acetate in hexanes over 30 min to afford 2.72 g of the product (yield: 47%). MS (ESI) m/z 263.0 [M+H] ⁺ . ¹ H NMR (500MHz, CDCl ₃ ) δ 8.85 (s, 1H), 4.71 (d, J=3.1Hz, 1H), 4.60 (d, J=3.1Hz, 1H), 3.98 (q, J=7.0Hz, 2H), 2.67 (s, 0H), 1.42 (t, J=7.0Hz, 3H).

(e) 5-Bromo-4-chloro-6-(1-(2-(5-chloro-1-methyl-1H-pyrazol-4-yl)hydrazonoyl)ethyl)pyrimidine

A suspension of 5-bromo-4-chloro-6-(1-ethoxyvinyl)pyrimidine (1.60 g, 6.07 mmol) and 5-chloro-4-hydrazino-1-methyl-1H-pyrazole hydrochloride (3.11 g, 12.1 mmol) in acetic acid (32 mL) was stirred at 60°C for 6 h. After removing the solvent under reduced pressure, the residue was treated with saturated _NaHCO₃ (40 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic phases were washed with brine (70 mL) and evaporated to dryness. The residue was dried under vacuum to give 1.0 g of crude product (yield: 45%), which was used in the next step without further purification. MS (ESI) m/z 362.9 [M+H] ^⁺ . ¹ H NMR (500MHz, CDCl ₃ ) δ 8.79 (s, 1H), 7.66 (s, 1H), 7.05 (s, 1H), 3.83 (s, 3H), 2.32 (s, 3H).

(f) 7-Chloro-1-(5-chloro-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine

A mixture of 5-bromo-4-chloro-6-(1-(2-(5-chloro-1-methyl-1H-pyrazol-4-yl)hydrazono)ethyl)pyrimidine (334 mg, 0.92 mmol), 1,10-phenanthroline (497 _mg , 2.76 mmol), and _K₂CO₃ (127 mg, 0.92 mmol) in toluene (4 mL) was heated at 100° C. for 1.5 h in a sealed tube. After cooling to room temperature, the reaction mixture was diluted with toluene (3 mL) and filtered. The solid was washed twice with toluene (2×3 mL). The combined filtrates were washed three times with saturated _FeSO₄ · _7H₂O (3×4 mL) and evaporated to dryness. The resulting residue was dried in vacuo to give 147 mg of crude product (yield: 57%), which was used in the next step without further purification. MS (ESI) m/z 283.0 [M+H] ^⁺ .

(g) 7-(azetidin-1-yl)-1-(5-chloro-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine

A mixture of 7-chloro-1-(5-chloro-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine (147 mg, 0.52 mmol), azetidine hydrochloride (64 mg, 0.68 mmol), and _Et₃N (105 mg, 1.04 mmol) in toluene (1.5 mL) was heated at room temperature in a sealed tube until the reaction was complete. The mixture was poured into 2N NaOH (18 mL) and extracted three times with _CH₂Cl₂ (3×25 mL). _The combined organic phases were washed with brine (20 mL) and evaporated to dryness. The resulting residue was dried under vacuum to give 181 g of crude product, which was used in the next step without further purification. MS (ESI) 304.1 [M+H] ^⁺ . ¹ H NMR (500MHz, CDCl ₃ ) δ 8.51 (s, 1H), 7.66 (s, 1H), 3.95 (s, 3H), 3.87 (t, J = 7.8Hz, 4H), 2.61 (s, 3H), 2.33-2.25 (m, 2H).

(h) 7-(azetidin-1-yl)-3-methyl-1-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidine

A suspension of 7-(azetidin-1-yl)-1-(5-chloro-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine (58 mg, 0.19 mmol), 4-(trifluoromethyl)-phenylboronic acid (51 mg, 0.27 mmol) and tripotassium phosphate (92 mg, 0.43 mmol) in ethanol (0.70 mL) and water (0.080 mL) was heated at 70° C. under argon for 10 min, followed by the addition of tetrakis(triphenylphosphine)palladium(0) (19 mg, 0.017 mmol). The suspension was degassed again and then heated in a microwave at 130° C. for 2 h. Tetrakis(triphenylphosphine)palladium(0) (9 mg, 0.0079 mmol) was further added. The mixture was heated in a microwave at 140° C. for 7 h. After removal of the solvent, the residue was purified by semi-preparative HPLC using a gradient of 0-33% acetonitrile in water containing 0.1% formic acid over 16 min to afford 12 mg of the final product as an off-white solid (HPLC purity: 98%; yield: 15%). MS (ESI) m/z 414.2 [M+H] ⁺ . ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.43 (s, 1H), 7.71 (s, 1H), 7.63 (d, J=8.1 Hz, 2H), 7.50 (d, J=8.1 Hz, 2H), 3.96 (s, 3H), 3.81 (t, J=7.8 Hz, 4H), 2.59 (s, 3H), 2.32-2.21 (m, 2H).

Example 2

7-(azetidin-1-yl)-1-(5-(4-methoxy-2-methylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine

_The title compound was prepared in a manner similar to that described in Example 1, except that 4-(trifluoromethyl)phenylboronic acid and _K₂CO₃ were added in the final step instead of 4-(trifluoromethyl)phenylboronic acid and _K₃PO₄ , respectively. The final product was obtained as an off-white solid (HPLC purity: ₉₉ %; yield: 32%). MS (ESI) m/z 390.2 [M+H] ^⁺ . ¹ H NMR (500MHz, CDCl ₃ )δ8.37 (s, 1H), 7.77 (s, 1H), 7.06 (d, J = 8.4Hz, 1H), 6.68 (d, J = 2.5Hz, 1H), 6.64 (dd, J = 8.5, 2.6Hz , 1H), 4.04-3.82(m, 4H), 3.74(s, 3H), 3.71(s, 3H), 2.55(s, 3H), 2.35-2.26(m, 2H), 2.08(s, 3H).

Example 3

7-(azetidin-1-yl)-1-(5-(4-cyclopropylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine

_The title compound was prepared in a manner analogous to the method described in the synthesis of Example 1, wherein cyclopropylphenyl-boronic acid and _Na2CO3 were added in the final step instead _of 4-(trifluoromethyl)phenylboronic acid and _K3PO4 , respectively. MS (ESI) m/z 386.2 [M+H] ⁺ .

Example 4

7-(azetidin-1-yl)-1-(5-(4-ethylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine

The title compound was prepared in a manner analogous to the method described in the synthesis of Example 1, wherein 4-ethylphenyl-boronic acid and _{Cs2CO3 were} added in the final step instead of 4-(trifluoromethyl)phenylboronic acid and _K3PO4 , _{respectively.MS} (ESI)m/z374.2[M+H] ⁺ .

Example 5

In vitro PDE2 inhibition assay

A mammalian expression cloning vector containing a recombinant cDNA copy of r-hPDE2A (accession number NM_002599, human phosphodiesterase 2A, cGMP-stimulated, transcript variant 1) was purchased from Origene. The protein was expressed by transient transfection of HEK293 cells. Cells were harvested 48 hours after transfection, washed once with TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), and then lysed by sonication in cold homogenization buffer (50 mM Tris-HCl, pH 7.5, _{5 mM} MgCl₂, 1X protease inhibitor cocktail). The homogenate was centrifuged at 15,000 g for 30 min at 4°C to obtain the soluble cytosolic fraction. Cytosolic protein concentration was determined using a BCA protein assay kit (Pierce) using bovine serum albumin as a standard.

Assay: PDE2A is assayed using FL-cAMP as a substrate. An enzyme titration is first performed to determine the working concentration of PDE. The concentration of enzyme that gives an activity of 100 ΔmP in the absence of inhibitor is considered an appropriate working concentration for PDE.

Based on the titration curve, the PDE enzyme was diluted in standard reaction buffer (10 mM Tris-HCl pH 7.2, _{10 mM} MgCl₂, 0.1% BSA, 0.05% _NaN₃ ). For the PDE2 assay, the reaction buffer was supplemented with 1 μM cGMP to fully activate the enzyme. 99 μl of the diluted enzyme solution was added to each well of a flat-bottomed 96-well polystyrene plate, followed by the addition of 1 μl of the test compound dissolved in 100% DMSO. The compound was mixed and pre-incubated with the enzyme for 10 minutes at room temperature.

The FL-cNMP conversion reaction was initiated by the addition of substrate (final concentration 45 nM). The enzyme and inhibitor mixture (16 μl) and substrate solution (4 μl, 0.225 μM) were combined in a 384-well microtiter plate. The reaction was incubated in the dark at room temperature for 15 min. The reaction was stopped by adding 60 μl of binding reagent (IMAP beads diluted 1:400 in binding buffer supplemented with a 1:1800 dilution of defoamer) to each well of the 384-well plate. The plate was incubated at room temperature for 1 hour to allow IMAP binding to proceed to completion and then placed in an Envision multimode microplate reader (PerkinElmer, Shelton, CT) to measure fluorescence polarization (Δmp).

The reduction of cAMP concentration, measured as reduced Δmp, indicates inhibition of PDE activity. Enzyme activity was measured in the presence of 8 to 16 compound concentrations ranging from 0.00037 nM to 80,000 nM, and then a schematic diagram of the relationship between drug concentration and ΔmP was drawn to determine the IC ₅₀ value. The test well values were normalized to control the reaction run on the same plate (converted to the value of % control). IC ₅₀ values were estimated using nonlinear regression software, fitting a four-parameter single-point dose response model (XLFit; IDBS, Cambridge, MA). The lower portion of the curve was fixed to 0% of the control.

Quality Control: To determine the _IC50 of an inhibitor, select an enzyme concentration that gives an optimal signal range of 100-200 millipolarization units. Measure the total fluorescence intensity of each sample well to calculate the mean and standard deviation. If the total fluorescence intensity of any sample well is not within the range of the mean ± 3SD, the mP value for that particular well is discarded.

Using the IMAP method described above or similar to that described above, we screened a proprietary PDE-focused compound library to identify novel compounds with nanomolar PDE2 inhibitory activity. Exemplary compounds of the present disclosure (e.g., compounds of Examples 1-4) were tested and demonstrated to be active at nanomolar concentrations, for example, as follows: Example 1: IC ₅₀ 23.1 nM; Example 2: IC ₅₀ 92 nM; Example 3: IC ₅₀ 9.5 nM; Example 4, IC ₅₀ 18.5 nM. Furthermore, the compounds were selective for PDE2; the compound of Example 4 was tested and demonstrated to be over 20-fold selective for PDE2 over PDE1, PDE3, PDE4D, PDE5, PDE6, PDE7B, PDE8A, PDE9A, PDE10A, and PDE11A.

Example 6

Pharmacokinetic studies in mice

Mice were given a single oral dose of the test compound (10 mg/kg, PO), and plasma and brain availability (0.25-4 h) were measured using HPLC and LC-MS, using methods similar to those described in Zhao et al., J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. (2005) 819(1): 73-80 and Appels, N.M. et al., Rapid Commun. Mass Spec. 2005. 19(15): p. 2187-92. This experiment demonstrated that the compound of Example 1 had good brain entry, as shown in Figure 1.

Example 7

Effects of treatment with PDE2 inhibitors on memory performance in Wistar rats during a targeted cognitive task

The memory-enhancing effect of the compound of Example 1 was tested in an animal model of cognition, namely the target recognition task. See Ennaceur, A., Delacour, J., 1988. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav. Brain Res. 31, 47-59. When tested one hour later, the rats remembered the target they had explored in the previous trial. However, when a 24-hour inter-trial interval was used, they could not remember which target was presented to them in the first trial. It was tested whether the compound of Example 1, when administered 2 hours before the first trial, could attenuate this time-induced target memory deficit. The compound was administered to 2-3 month old male Wistar rats.

Two hours before learning, the compound of Example 1 was orally injected (2 ml/kg) at doses of 0, 0.3, 1, 3, and 10 mg/kg. None of the doses tested had an effect on exploratory behavior. This study demonstrates that the compound of Example 1 can completely prevent time-dependent forgetting at a dose of 1.0 mg/kg. Moderate memory improvements were observed at 0.3 and 3.0 mg/kg.

Example 8

Effects of a novel PDE2 inhibitor on cGMP signaling in hippocampal cells and brain

We propose that HT-22 cells (immortalized mouse hippocampus-neuronal precursor cells subcloned from their parental HT-4 cells) are valuable models for understanding the cellular and molecular processes associated with hippocampal-dependent mood changes. We anticipate that in cell-based assays, target 1 will produce 10-20 novel PDE2 inhibitors for functional evaluation. Compounds that induce a significant increase in cGMP accumulation only in cell-based HT-22 cell assays will enter behavioral assessments. Cell-based assay data will be the minimum requirement for any PDE2 inhibitor with potential for CNS indications, and will provide guidance for the dosage selection of behavioral tests.

Claims (14)

1. Compounds of Formula I in: (i) _R1 and _R2 together with nitrogen atoms form a nitrogen-1-butane-1-yl heterocyclic ring; (ii) _R3 is an H or _C1-4 alkyl group; (iii) _R4 is a heteroaryl or aryl group, which is optionally substituted by one or more groups selected from _C1-4 alkyl, _C3-7 cycloalkyl, _C1-4 alkoxy and halo- _C1-4 alkyl groups; (iv) _R6 is an H or _C1-4 alkyl group; The compound exists in free or salt form. 2. The compound of claim 1, wherein _R4 is an aryl group, optionally substituted by one or more groups selected from _C1-4 alkyl, _C3-7 cycloalkyl, _C1-4 alkoxy, and halo- _C1-4 alkyl groups, the compound being in free or salt form. 3. The compound according to claim 1 or 2, wherein R ₆ is a _C1-4 alkyl group, and the compound is in free or salt form. 4. The compound in its free or salt form according to claim 1, 2 or 3, wherein the compound is selected from: 7-(azacyclobutan-1-yl)-3-methyl-1-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidine; 7-(azacyclobutane-1-yl)-1-(5-(4-methoxy-2-methylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine; 7-(azacyclobutane-1-yl)-1-(5-(4-cyclopropylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine; 7-(azacyclobutane-1-yl)-1-(5-(4-ethylphenyl)-1-methyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[4,3-d]pyrimidine. 5. A pharmaceutical composition comprising a compound according to any one of claims 1-4, in combination or in combination with a pharmaceutically acceptable diluent or carrier. 6. Use of the compound of any one of claims 1-4 or the pharmaceutical composition of claim 5 for the preparation of a medicament for treating PDE2-mediated disorders selected from neurological disorders, cognitive impairments, cognitive impairments associated with Parkinson's disease and depression, mental disorders, mood disorders, pediatric psychosis and cardiovascular disorders. 7. The use of claim 6, wherein the disorder is selected from the following: anxiety, depression, amnesia, Alzheimer's disease, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-related dementia, cognitive impairment, migraine, epilepsy, Alzheimer's disease, Parkinson's disease, brain injury, and stroke. 8. The use of claim 6, wherein the disorder is selected from the following: amnesia, Alzheimer's disease, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-related dementia, delirium, and mild cognitive impairment. 9. The use of claim 6, wherein the disorder is selected from acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder, specific phobias, social phobia, chronic anxiety disorder, attention deficit disorder, and attention deficit hyperactivity disorder. 10. The use of claim 6, wherein the disorder is selected from type I bipolar disorder, type II bipolar disorder, mania, mixed affective state, major depression, chronic depression, seasonal depression, psychotic depression, and postpartum depression. 11. The use of claim 6, wherein the barrier is selected from pulmonary hypertension and pulmonary arterial hypertension. 12. A compound in free or pharmaceutically acceptable salt form according to any one of claims 1-4, or a pharmaceutical composition according to claim 5, for the preparation of a medicament for the treatment or preventive treatment of a disease, disorder, or ailment according to any one of claims 6-8. 13. A pharmaceutical composition comprising a compound according to any one of claims 1-4 in free or pharmaceutically acceptable salt form, the pharmaceutical composition being used to prepare a pharmaceutical treatment or preventative treatment for a disease, disorder, or ailment according to any one of claims 6-8. 14. A pharmaceutical composition comprising a compound according to any one of claims 1-4 in free or pharmaceutically acceptable salt form, the pharmaceutical composition being used as a medicine.