Organic compound

Abstract

The present invention relates to novel compounds with PDE1 inhibitory capacity that can be used as tracers for use in diagnostic techniques, biomarkers for phosphodiesterase 1 (PDE1) in vivo, methods for the treatment and / or development of new therapies for PDE1-related conditions, and methods of detection and treatment.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority and benefit of U.S. Provisional Application No. 62 / 833,481, filed on April 12, 2019, the content of which is hereby incorporated by reference in its entirety as part of this specification.

[0002] Disclosed technical field The present invention relates to tracers for use in diagnostic techniques, particularly radiolabeled tracers for SPECT and positron emitter - labeled compositions for PET, detection of phosphodiesterase 1 (PDE1) activation in vivo, methods for the treatment and / or development of new therapies for PDE1 - related conditions such as cancer or tumors, such as glioblastoma multiforme, and methods of detection and treatment. Particularly interesting compositions are radiolabeled compositions that selectively bind to PDE1 associated with a condition of interest in various tissues and organs. The positron emitter - labeled compositions targeting PDE1 form the basis for new therapies for cancer, central nervous system, and cardiovascular disorders.

Background Art

[0003] Disclosed background Gamma - ray - based imaging techniques use tracer compounds that are introduced into the body to be imaged. The tracer compounds contain radionuclides from which photons are emitted either directly or indirectly, and the location of photon generation within the body is calculated from intercepted data collected by gamma - ray detectors. Two commonly used gamma - ray - based imaging techniques are positron emission tomography (referred to as PET) and single - photon emission computed tomography (referred to as SPECT). In PET, the radionuclide indirectly emits a pair of photons in opposite directions. PET radionuclides emit positrons, which, when they come into contact with an electron in their immediate vicinity, cause the antimatter annihilation of both particles, resulting in the emission of a pair of photons. In SPECT, the radionuclide is a direct gamma emitter. Examples of isotopes useful for gamma - ray - based imaging include carbon - 11 ( 11carbon-11 (also known as C or C11), fluorine-18 ( 18 also known as 18F or F18), technetium-99m ( 99 also known as 99mTc or Tc99m), indium-111 ( 111 also known as In or In111), iodine-123 ( 123 also known as I or I123), and tritium (T or 3 also known as 3H).

[0004] In addition to the radionuclide, the tracer compound includes a ligand that provides affinity of the tracer for a selected target related to one or more tissues, organs or conditions of interest.

[0005] Type 1 cyclic nucleotide phosphodiesterase (PDE1) comprises an enzyme family consisting of isoforms A, B and C, all of which are regulated by calcium-calmodulin (Ca / CaM) and hydrolyze cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). This enzyme acts as an important regulator of these intracellular signaling messengers, particularly in excitable cells where calcium levels increase during excitation and contraction. The enzymology of PDE1 has been well studied in vitro and the tissue distribution of the three isoforms is well characterized. For other clinically interesting phosphodiesterases, these parameters have been useful in the design of specific positron emission probes that are helpful in quantifying the enzyme occupancy in humans of candidate small molecule phosphodiesterase inhibitors.

[0006] Recently, there has been considerable interest in the development of PDE1 inhibitors as therapeutic agents for human diseases, including cognitive impairment and degenerative diseases such as heart failure and Parkinson's disease. The enzymology of the PDE1 family has been widely studied and reviewed. The focus of recent research in this field is the calcium-dependence of enzyme activity due to regulation by calcium and the protein calmodulin. Since this enzyme becomes active only when intracellular calcium levels rise to micromolar concentrations, the contribution of PDE1 to the control of cyclic nucleotide levels in most systems is transient and almost certainly varies depending on the intracellular microdomain / environment. Proteolytic fragments of PDE1 lacking the N-terminal regulatory domain and calmodulin-binding domain are not subject to the calcium-calmodulin requirement. This suggests that, as in the case of other PDE families, the N-terminal regulatory domain of PDE1 occludes the cyclic nucleotide binding site, and in the case of the PDE1 enzyme, binding of calcium-calmodulin to this regulatory domain releases this occlusion, allowing substrate binding and enzyme hydrolytic activity.

[0007] Glioblastoma is the most common malignant brain tumor in adults, occurring at a frequency of 2-3 per 100,000 in developed countries in Europe and the United States, accounting for more than half of all primary brain tumors. The prognosis is almost invariably poor, with a median survival of 6-9 months and a 5-year survival rate of less than 3%. Brodbelt A, et al.; (UK) National Cancer Information Network Brain Tumour Group. Glioblastoma in England: 2007-2011. Eur. J. Cancer. 2015 Mar; 51(4):533-42.

[0008] Glioblastoma is an aggressive tumour characterized by rapid growth and invasion of normal brain tissue. The effectiveness of current treatment methods is hampered by the need to maintain normal brain function during surgery, tumour-specific resistance to radiotherapy, and the inability of many drugs to cross the blood-brain barrier. In the subsequent Phase III clinical trials of targeted agents, no improvement in survival was seen (Touat M, et al., Glioblastoma targeted therapy: updated approaches from recent biological insights. Ann. Oncol. 2017 Jul 1;28(7):1457-1472), and little improvement has been seen in adult glioblastoma patients over the past 30 years, as temozolomide (an alkylating compound that can cross the blood-brain barrier) remains one of the only effective chemotherapeutic agents available for the treatment of glioblastoma. Even with maximal treatment with debulking surgery, radiotherapy, and temozolomide, the median survival is only 14.6 months. Stupp R, et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma; N Engl J Med. 2005 Mar 10;352(10):987-96.

[0009] In particular, with regard to PDE1C, recent evidence has shown that PDE1C is a proliferation-related gene as it is exclusively expressed in proliferating vascular smooth muscle cells. Rybalkin SD, et al., Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest 1997;100:2611-2621. In addition, PDE1C expression in experimental tumor models such as melanoma (Watanabe Y, et al., Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells. Exp Ther Med 2012;4:205-210), neuroblastoma (Jang IS, Juhnn YS. Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active G protein alpha, Q227L Gsalpha. Exp Mol Med 2001;33:37-45), and osteosarcoma (Ahlstroem M, et al., Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation. Cell Mol Biol Lett 2005;10:305-319) has been sporadically reported along with other PDE subtypes.

[0010] Previous studies have shown that inhibition of PDE1 (especially isoform PDE1B) induces apoptosis in human leukemia cells. Jiang X, Paskind M, Weltzien R, Epstein PM. Expression and regulation of mRNA for distinct isoforms of mitogen-stimulated and leukemic human lymphocytes. Cell Biochem Biophys 1998; 28:135-60. Studies have also shown that 5 out of 6 human glioblastoma cell lines from the National Cancer Institute show high PDE1 expression (mainly PDE1C) and only minor PDE4 expression. Marko D, Pahlke G, Merz KH, Eisenbrand G. Cyclic 3’,5’-nucleotide phosphodiesterases: potential targets for antitumor therapy. Chem Res Toxicol 2000;13:944-8. Similarly, PDE1C mRNA is overexpressed in human malignant melanoma-associated antigen (MAA) cells and proliferation is inhibited by vinpocetine, a non-selective PDE1 inhibitor. Zhao AZ, et al., Recent advances in the study of Ca2+ / CaM-activated phosphodiesterases: expression and physiological functions. Adv Second Messenger Phosphoprotein Res 1997;31:237-51. More recent studies have shown that PDE1C is significantly overexpressed in 20% of glioblastomas compared to normal human brain and siRNA-mediated silencing of PDE1C inhibits proliferation (45 - 50%) and invasion (40 - 60%) in glioblastoma patient-derived cell cultures.Rowther FB, et al., Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro. Mol Carcinog. 2016 Mar; 55(3):268-79。

[0011] Currently, there are largely unmet needs for effective methods of treating central nervous system tumors such as glioblastoma multiforme. There is an urgent need for improved therapeutic compositions as well as methods for treatment and diagnosis in such conditions.

Summary of the Invention

[0012] Summary of the Disclosure We have previously shown that inhibition of PDE1 activity with the disclosed compounds can safely maintain or restore cAMP function in a wide range of pathological conditions including models of neurodegeneration and neuroinflammation, heart failure, pulmonary hypertension and peripheral inflammation, as well as in humans with certain diseases. More recently, we have shown that PDE1 inhibitors decrease cell migration of microglia and monocytes. Recent evidence indicates that PDE1, particularly the PDE1C isoform, is overexpressed in experimental tumor models such as melanoma, neuroblastoma and osteosarcoma. In addition, focal genomic overrepresentation of PDE1C in glioblastoma multiforme (GBM) cells has been demonstrated. The genomic gain of PDE1C is associated with increased expression in GBM-derived cell cultures and is essential for promoting cell proliferation, migration and invasion in cancer cells.

[0013] Many types of cancer cells overexpress PDE1 activity, which is identified through various biomarkers such as increased RNA expression, DNA copy number, PDE1 binding (PET or radioisotope retention of PDE1 inhibitor molecules), or enzyme activity. These cancer cells also exhibit low levels of cAMP, which allows for more rapid cancer cell invasion and proliferation but can be increased by PDE1 inhibitors. Such properties can be treated with PDE-1 inhibitors alone or in combination with chemotherapeutic agents, gene therapy agents, and / or immunological approaches. Additionally, by inhibiting PDE1, apoptotic cell death is induced, migration is prevented, metastasis is limited, and inflammation is reduced. Thus, PDE1 inhibitors have a synergistic effect with chemotherapeutic agents and immunological approaches.

[0014] Also, in many types of cells and in certain cancers, such as glioblastoma tumors, it is known that intracellular calcium levels are elevated. The elevated calcium levels activate calcium-activated proteases such as calpain. Calpain is known to cleave the N-terminal regulatory domain of PDE1, providing an active catalytic domain that is no longer calcium / calmodulin sensitive. Activation of PDE1 can decrease intracellular cyclic nucleotide concentrations and enhance pathways involved in cancer development.

[0015] Since many types of cancer show an increase in the amount of PDE1, radiolabeled PDE1 inhibitors are thought to be useful for the diagnosis and subsequent treatment of such cancers or other conditions that result in an increase in the amount of PDE1. However, efforts to create radiolabeled compounds for selectively detecting PDE1 have not been relatively successful. The inventors have created novel compounds and radioligands that are effective and selective PDE1 radiotracers.

[0016] Accordingly, in various embodiments, the present disclosure provides novel compounds of formula Ia and / or formula II, which may be substituted with T (tritium, 3 H) in place of H at one or more positions.

[0017] In one aspect, the present application provides a method for mapping functional PDE1 activity in a tissue and / or organ of interest using positron emission tomography, the method comprising administering to the tissue and / or organ an effective amount of a PDE1 radiolabeled tracer compound according to Formula Ia et seq. or II et seq.; allowing sufficient time for the PDE1 radiolabeled tracer to effectively associate with PDE1 in the tissue and organ of interest; and analyzing the tissue and organ of interest using positron emission tomography.

[0018] In a further aspect, the present disclosure provides a method for treating a PDE1-mediated disease, disorder or condition, the method comprising administering to a subject in need thereof an effective amount of a compound according to Formula Ia et seq. and / or II et seq.; imaging the subject with a positron emission tomography device; administering to the subject a PDE1 inhibitor free of a radionuclide at a predetermined dose; imaging the subject with a positron emission tomography device; comparing the data thus obtained; and evaluating the effective delivery of the PDE1 inhibitor to a tissue of interest in a PDE1-mediated condition.

[0019] In another aspect, the present disclosure provides a method for diagnosing a PDE1-mediated disease, disorder or condition characterized by upregulation of PDE1 expression in a subject, the method comprising obtaining a first tissue sample from a patient suspected of having a PDE1-mediated disease, disorder or condition or at risk of a PDE1-mediated disease, disorder or condition; contacting the first tissue sample with an effective amount of a compound according to Formula Ia et seq. or II et seq.; imaging the first tissue sample with a positron emission tomography device; and comparing the results of the previous step with a second tissue sample in which PDE1 expression is not upregulated [Method 3]. In some embodiments, the method further comprises quantifying PDE1 expression ex vivo via a biopsy taken from a patient suffering from glioblastoma.

[0020] In various embodiments, the present disclosure also provides pharmaceutical compositions comprising a compound of the present disclosure, prepared using conventional diluents or excipients and techniques known in the art. Accordingly, oral dosage forms can include tablets, capsules, solutions, suspensions, and the like.

[0021] In various embodiments, the present disclosure also provides PDE1 inhibitors of formula Ia and / or II, in free form or salt form, for use in the treatment of a condition selected from cancer or tumor, inhibition of the proliferation, migration and / or invasion of tumor cells, or the treatment of glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

[0023]

Figure 2

[0024]

Figure 3

[0025]

Figure 4

[0026] **DETAILED DESCRIPTION OF THE DISCLOSURE**

[0027] **COMPOUNDS FOR USE IN THE DISCLOSED METHODS** In one embodiment, the PDE1 inhibitor for use in the therapeutic, diagnostic, detection, and prevention methods described herein is a selective PDE1 inhibitor.

[0028] **PDE1 INHIBITOR** In one embodiment, the present invention provides a PDE1 inhibitor for use particularly in the therapeutic, diagnostic, detection and prevention methods described herein, in free form, pharmaceutically acceptable salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), of formula Ia: [Chemical formula] [wherein, (i) R2 and R5 are independently H or hydroxy, and R3 and R4 together form a trimethylene bridge or a tetramethylene bridge [preferably, the carbons carrying R3 and R4 each have the R configuration and the S configuration]; or, R2 and R3 are each methyl, and R4 and R5 are each H; or, R2, R4 and R5 are H, and R3 is isopropyl [preferably, the carbon carrying R3 has the R configuration]; (ii) R6 is phenylamino (which may be halo-substituted), benzylamino (which may be halo-substituted), C 1-4 alkyl, or C 1-4 alkylsulfide; for example, phenylamino or 4-fluorophenylamino; (iii) R 10 is C 1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, phenyl (which may be halo-substituted or hydroxy-substituted), pyridyl (which may be halo-substituted or hydroxy-substituted) (for example, 6-fluoropyrid-2-yl), thiadiazolyl (for example, 1,2,3-thiadiazol-4-yl), or pyrrolidyl optionally substituted with C 1-4 alkyl (for example, 1-methylpyrrolidin-2-yl); (iv) X and Y are independently C or N] a compound, wherein at least one position on the compound is substituted with tritium (T) or deuterium (D) instead of H.

[0029] In a further embodiment, the present invention provides a compound of formula I as set forth in the following formula:

[0030] 1.1 A compound of formula Ia containing at least one T substituent instead of 1.1 H.

[0031] 1.2 A compound of formula Ia or 1.1, wherein R2 and R5 are each independently H, T or hydroxy, and R3 and R4 together form a trimethylene bridge or a tetramethylene bridge [preferably, the carbons bearing R3 and R4 each have an R configuration and an S configuration].

[0032] 1.3 A compound of formula Ia or 1.1 - 1.2, wherein R2 and R3 are each methyl, and R4 and R5 are each H or T.

[0033] 1.4 A compound of formula Ia or 1.1 - 1.3, wherein R2, R4 and R5 are H or T, and R3 is isopropyl [preferably, the carbon bearing R3 has an R configuration].

[0034] 1.5 A compound of formula Ia or 1.1 - 1.4, wherein R6 is phenylamino.

[0035] 1.6 A compound of formula Ia or 1.1 - 1.5, wherein R6 is phenylamino which may be halogen - substituted.

[0036] 1.7 A compound of formula Ia or 1.1 - 1.6, wherein R6 is halogen - substituted phenylamino.

[0037] 1.8 A compound of formula Ia or 1.1 - 1.7, wherein R6 is phenylamino which may be fluoro - substituted.

[0038] 1.9 A compound of formula Ia or 1.1 - 1.8, wherein R6 is 4 - fluorophenylamino.

[0039] 1.10 R 10 is C 1-4Alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, phenyl which may be halo-substituted or hydroxy-substituted, pyridyl which may be halo-substituted (e.g., 6-fluoropyridin-2-yl), or C 1-4 A compound of formula Ia or 1.1 to 1.9, which is pyrrolidyl which may be alkyl-substituted (e.g., 1-methylpyrrolidin-2-yl).

[0040] 1.11 R 10 is C 1-4 An alkyl, a compound of formula Ia or 1.1 to 1.10.

[0041] 1.12 R 10 is methylcarbonyl, a compound of formula Ia or 1.1 to 1.10.

[0042] 1.13 R 10 is pyridyl which may be halo-substituted (e.g., 6-fluoropyridin-2-yl), a compound of formula Ia or 1.1 to 1.10.

[0043] 1.14 R 10 is 6-fluoropyridin-2-yl, a compound of formula Ia or 1.1 to 1.10.

[0044] 1.15 R 10 is C 1-4 An alkyl-substituted pyrrolidyl which may be alkyl-substituted (e.g., 1-methylpyrrolidin-2-yl), a compound of formula Ia or 1.1 to 1.10.

[0045] 1.16 R 10 is methyl-substituted pyrrolidyl (e.g., 1-methylpyrrolidin-2-yl), a compound of formula Ia or 1.1 to 1.10.

[0046] 1.17 R 10 is 1-methylpyrrolidin-2-yl, a compound of formula Ia or 1.1 to 1.10.

[0047] 1.18 R 10A compound of formula Ia or 1.1 to 1.10, wherein at least one position is substituted with tritium (T) instead of H, and is 1-methylpyrrolidin-2-yl.

[0048] 1.19 R 10 A compound of formula Ia or 1.1 to 1.10, wherein R is pyrrolidyl substituted with tritiated methyl.

[0049] 1.20 R 10 A compound of formula Ia or 1.1 to 1.10, wherein R is 1-methylpyrrolidin-2-yl in which the methyl substituent is fully or partially tritiated.

[0050] 1.21 R 10 A compound of formula Ia or 1.1 to 1.10, wherein R is 1-methylpyrrolidin-2-yl in which the methyl substituent is fully tritiated.

[0051] 1.22 A compound of formula Ia or 1.1 to 1.21, wherein X and Y are C.

[0052] 1.23 R2 and R3 are each methyl, R4 and R5 are each H or T; R6 is phenylamino which may be halo-substituted; R 10 is 1-4 pyrrolidyl which may be alkyl-substituted (e.g., 1-methylpyrrolidin-2-yl), and X and Y are C, a compound of formula Ia or 1.1 to 1.22.

[0053] 1.24 The compound is in free form, pharmaceutically acceptable salt form or prodrug form,

Chemical Structure

[0054] 1.25 The compound is in free form, pharmaceutically acceptable salt form or prodrug form,

Chemical Structure

[0055] In another embodiment, the present invention provides a PDE1 inhibitor for use in the therapeutic and prophylactic methods described herein, in free form, pharmaceutically acceptable salt form or prodrug form (including its enantiomers, diastereoisomers and racemates), of formula II: [Chemical formula] [wherein, R1, R2 and R5 are independently C which may be substituted with T 1-4 alkyl, H or T (tritium, 3 H); R3, R4, R 11 , R 12 , R 14 and R 15 are independently selected from H or T; R6, R7, R8, R9 and R 10 are independently selected from halogen (e.g., F), H or T; R 13 is C 1-4 alkyl, methylcarbonyl, hydroxyethyl, carboxylic acid, sulfonamide, phenyl which may be substituted with halo or hydroxy, pyridyl which may be substituted with halo or hydroxy (e.g., 6-fluoropyridin-2-yl), thiadiazolyl (e.g., 1,2,3-thiadiazol-4-yl), or pyrrolidyl which may be substituted with C 1-4 alkyl (e.g., 1-methylpyrrolidin-2-yl), and each of these may be substituted with tritium (T) in place of H at one or more positions] The compound of, wherein at least one of R1 to R 15 is T, or the compound is substituted with tritium (T) in place of H at at least one position.

[0056] In a further embodiment, the present invention provides a compound of formula I as described in the following formula:

[0057] 2.1 Compounds of formula II wherein R1, R2 and R5 are each independently optionally T-substituted C 1-4 alkyl.

[0058] 2.2 Compounds of formula II or 2.1 wherein R1, R2 and R5 are T-substituted C 1-4 alkyl.

[0059] 2.3 Compounds of formula II or any of 2.1 - 2.2 wherein R1, R2 and R5 are each optionally T-substituted methyl.

[0060] 2.4 Compounds of formula II or any of 2.1 - 2.3 wherein R3, R4, R 11 , R 12 , R 14 and R 15 are H.

[0061] 2.5 Compounds of formula II or any of 2.1 - 2.4 wherein R 13 is C 1-4 alkyl-substituted pyrrolidyl (e.g., 1-methylpyrrolidin-2-yl).

[0062] 2.6 Compounds of formula II or any of 2.1 - 2.5 wherein R 13 is [Chemical formula] selected from.

[0063] 2.7 Compounds of formula II or 2.6 wherein any of R 16 -R 23 is independently optionally T-substituted C 1-4 alkyl, H or T.

[0064] 2.8 Compounds of formula II or 2.6 wherein R 13 is [Chemical formula] [wherein, R 16 、R 18 and R 19 are each independently selected from H or T; R 17 is T-substituted C 1-4 alkyl] is a compound of formula II or any of 2.1 to 2.7.

[0065] 2.9 R 13 is

Chemical formula

[0066] 2.10 R 17 is T3C, the compound of 2.9.

[0067] 2.11 R 13 is

Chemical formula

[0068] 2.12 R 13 is

Chemical formula

[0069] 2.13 R 20 is T3C, the compound of 2.12.

[0070] 2.14 The compound is in free form, pharmaceutically acceptable salt form or prodrug form,

Chem.

[0071] 2.15 The compound is in free form, pharmaceutically acceptable salt form or prodrug form,

Chem.

[0072] In some embodiments, the present invention provides a PDE1 inhibitor for potentially using in the treatment, diagnosis, detection and prevention methods described herein, wherein the inhibitor is in free form or pharmaceutically acceptable salt form of the following:

Chem.

[0073] In some embodiments, the present invention provides a PDE1 inhibitor for potentially using in the treatment, diagnosis, detection and prevention methods described herein, wherein the inhibitor is in free form or pharmaceutically acceptable salt form of the following:

Chem.

[0074] In some embodiments, the invention is a PDE1 inhibitor for potential use in the therapeutic, diagnostic, detection and prevention methods described herein, wherein the inhibitor is in free form or in a pharmaceutically acceptable salt form of the following:

Chemical formula

[0075] In some embodiments, the invention is a PDE1 inhibitor for potential use in the therapeutic, diagnostic, detection and prevention methods described herein, wherein the inhibitor is in free form or in a pharmaceutically acceptable salt form of the following:

Chemical formula

[0076] In some embodiments, the invention is a PDE1 inhibitor for potential use in the therapeutic, diagnostic, detection and prevention methods described herein, wherein the inhibitor is in free form or in a pharmaceutically acceptable salt form of the following:

Chemical formula

[0077] In some embodiments, the present invention is a PDE1 inhibitor for potentially use in the treatment, diagnosis, detection and prevention methods described herein, wherein the inhibitor is in free form or a pharmaceutically acceptable salt form of the following:

Chemical formula

[0078] In one embodiment, any of the selective PDE1 inhibitors of the above formula (e.g., formula Ia or II) is a compound that inhibits phosphodiesterase-mediated (e.g., PDE1-mediated, particularly PDE1B-mediated) hydrolysis of cGMP and, by inference, cAMP. For example, preferred compounds, in free or salt form, have an IC 50 less than 1 μM, preferably less than 500 nM, preferably less than 50 nM, preferably less than 5 nM in an immobilized metal affinity particle reagent PDE assay.

[0079] In some embodiments, the present invention is a PDE1 inhibitor for potentially use in the treatment, diagnosis, detection and prevention methods described herein, wherein the inhibitor is in free form or a pharmaceutically acceptable salt form of the following:

Chemical formula

[0080] Further examples of PDE1 inhibitors that can be proton or radiolabels for use in the methods and treatments described herein can be found in International Publication No. WO 2006 / 133261 A2; U.S. Patent No. 8,273,750; U.S. Patent No. 9,000,001; U.S. Patent No. 9,624,230; International Publication No. WO 2009 / 075784 A1; U.S. Patent No. 8,273,751; U.S. Patent No. 8,829,008; U.S. Patent No. 9,403,836; International Publication No. WO 2014 / 151409 A1, U.S. Patent No. 9,073,936; U.S. Patent No. 9,598,426; U.S. Patent No. 9,556,186; U.S. Patent Application Publication No. 2017 / 0231994 A1, International Publication No. WO 2016 / 022893 A1, and U.S. Patent Application Publication No. 2017 / 0226117 A1 (each of which is hereby incorporated by reference in its entirety).

[0081] Further examples of PDE1 inhibitors that are suitable for use in the methods and treatments described herein and that can be radiolabeled (e.g., tritiated) can be found in International Publication No. WO 2018 / 007249 A1; U.S. Patent Application Publication No. 2018 / 0000786; International Publication No. WO 2015 / 118097 A1; U.S. Patent No. 9,718,832; International Publication No. WO 2015 / 091805 A1; U.S. Patent No. 9,701,665; U.S. Patent Application Publication No. 2015 / 0175584 A1; U.S. Patent Application Publication No. 2017 / 0267664 A1; International Publication No. WO 2016 / 055618 A1; U.S. Patent Application Publication No. 2017 / 0298072 A1; International Publication No. WO 2016 / 170064 A1; U.S. Patent Application Publication No. 2016 / 0311831 A1; International Publication No. WO 2015 / 150254 A1; U.S. Patent Application Publication No. 2017 / 0022186 A1; International Publication No. WO 2016 / 174188 A1; U.S. Patent Application Publication No. 2016 / 0318939 A1; U.S. Patent Application Publication No. 2017 / 0291903 A1; International Publication No. WO 2018 / 073251 A1; International Publication No. WO 2017 / 178350 A1; and U.S. Patent Application Publication No. 2017 / 0291901 A1 (each of which is hereby incorporated by reference in its entirety as part of this specification). In the event of a conflict or lack of compatibility between the description of a document incorporated by reference as part of this specification and the description in this disclosure, the description in this disclosure shall govern.

[0082] Further examples of PDE1 inhibitors and compounds suitable for use as PDE1 radiotracers are disclosed in International Publication No. WO 2011 / 043816 A1 and U.S. Patent No. 8,858,911 (the contents of which are hereby incorporated by reference as part of this specification).

[0083] Unless otherwise specified or apparent from the context, the following terms as used herein have the following meanings:

[0084] (a) As used herein, the term "selective PDE1 inhibitor" refers to a PDE1 inhibitor having at least 100-fold selectivity over the inhibition of any other PDE isoform family that PDE1 inhibition covers, namely PDE2 - PDE11.

[0085] (b) As used herein, the term "alkyl" refers to a saturated or unsaturated hydrocarbon moiety, preferably saturated, preferably having 1 - 6 carbon atoms, which may be straight-chain or branched-chain, and which may be mono-substituted, di-substituted or tri-substituted, for example, by halogen (e.g., chloro or fluoro), hydroxy or carboxy.

[0086] (c) As used herein, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3 - 9 carbon atoms, at least some of which form a non-aromatic monocyclic or bicyclic or bridged cyclic structure, and which may be substituted, for example, by halogen (e.g., chloro or fluoro), hydroxy or carboxy. When the cycloalkyl contains one or more atoms selected from N and O and / or S, the cycloalkyl may be a heterocycloalkyl.

[0087] (d) Unless otherwise specified, the term "heterocycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon moiety, preferably saturated, preferably containing 3 - 9 carbon atoms, at least some of which form a non-aromatic monocyclic or bicyclic or bridged cyclic structure, with at least one carbon atom replaced by N, O or S, and the heterocycloalkyl may be substituted, for example, by halogen (e.g., chloro or fluoro), hydroxy or carboxy.

[0088] (e) As used herein, "aryl" is a monocyclic or bicyclic aromatic hydrocarbon, preferably phenyl, which may be substituted, for example, with alkyl (e.g., methyl), halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoromethyl), hydroxy, carboxy, or further aryl or heteroaryl (e.g., biphenyl or pyridylphenyl).

[0089] (f) As used herein, "heteroaryl" is an aromatic moiety in which one or more of the atoms constituting the aromatic ring are not carbon but sulfur or nitrogen, such as pyridyl or thiadiazolyl, which may be substituted, for example, with alkyl, halogen, haloalkyl, hydroxy or carboxy.

[0090] (g) "Substituted with T (tritium, 3 H) or D (deuterium, 2 H) instead of H at one or more positions" or similar expressions mean that the frequency of a particular hydrogen isotope (tritium or deuterium) at that position is higher than the frequency of H (protium) at that position.

[0091] The disclosed compounds, for example, PDE1 inhibitors as described herein, may exist in free form or in salt form, for example, as acid addition salts. As used herein, unless otherwise specified, terms such as "the disclosed compounds" should be understood to include the compounds in any form, for example, the free form or the acid addition salt form, or, when the compound contains an acidic substituent, the base addition salt form. Since the disclosed compounds are intended for use as pharmaceuticals, pharmaceutically acceptable salts are preferred. Salts not suitable for pharmaceutical use may be useful, for example, for the isolation or purification of the free disclosed compounds or their pharmaceutically acceptable salts, and thus such salts are also included.

[0092] The compounds of the present disclosure may, in some cases, also exist in prodrug form. A prodrug form is a compound that is converted in the body to the compound of the present disclosure. For example, when the compound of the present disclosure contains a hydroxy substituent or a carboxy substituent, these substituents may form physiologically hydrolysable and acceptable esters. As used herein, "physiologically hydrolysable and acceptable ester" means an ester of the compound of the present disclosure that is hydrolysable under physiological conditions and yields an acid (in the case of a compound of the present disclosure having a hydroxy substituent) or an alcohol (in the case of a compound of the present disclosure having a carboxy substituent) that is physiologically acceptable at the dosage to be administered. Thus, when the compound of the present disclosure contains a hydroxy group, for example, the compound -OH, the acyl ester prodrug of the compound, i.e., the compound -O-C(O)-C1-4 alkyl, can hydrolyse in the body to form, on the one hand, a physiologically hydrolysable alcohol (compound -OH) and, on the other hand, an acid (e.g., HOC(O)-C1-4 alkyl). Alternatively, when the compound of the present disclosure contains a carboxylic acid, for example, the compound -C(O)OH, the acid ester prodrug of the compound, the compound -C(O)O-C1-4 alkyl, can hydrolyse to form the compound -C(O)OH and HO-C1-4 alkyl. Thus, as will be understood, this term encompasses conventional pharmaceutical prodrug forms.

[0093] In another embodiment, the present disclosure further provides a pharmaceutical composition comprising a PDE1 inhibitor in combination with an anti-tumor agent, each in free form or in pharmaceutically acceptable salt form, mixed with a pharmaceutically acceptable carrier. The term "in combination" as used herein encompasses simultaneous, sequential or contemporaneous administration of the PDE1 inhibitor and the anti-tumor agent. In another embodiment, the present disclosure provides a pharmaceutical composition containing such compounds. In some embodiments, the combination of the PDE1 inhibitor and the anti-tumor agent allows the anti-tumor agent to be administered in an amount lower than the amount that would be effective when administered as a single monotherapy.

[0094] Methods of Using the Compounds of the Disclosure In another embodiment, the present application is a method for mapping or detecting the level of activated PDE1 activity in a tissue and / or organ of interest using positron emission tomography, comprising: (a) administering an effective amount of a PDE1 tracer compound according to Formula Ia et seq. or II et seq. to the tissue and / or organ (which may be by administering the PDE1 tracer compound to a subject in vivo or by administering the PDE1 tracer compound to a tissue sample ex vivo); (b) allowing sufficient time for the PDE1 tracer to effectively associate with PDE1 in the tissue and / or organ of interest; and (c) detecting, using positron emission tomography, the binding of the PDE1 tracer compound to activated PDE1 in the tissue and / or organ of interest wherein the method comprises: the PDE1 tracer compound is a compound that binds to PDE1 and contains a radioisotope at at least one position, for example, substituted with T (tritium, 3 3H) instead of H at one or more positions, to provide a method [Method 1].

[0095] In a further embodiment, the present invention provides Method 1 as described in the following method:

[0096] 1.1 Method 1, wherein at least one position on the compound according to Formula Ia and / or II is substituted with tritium (T) instead of H.

[0097] 1.2 The compound is in free form or pharmaceutically acceptable salt form,

Chemical formula

[0098] 1.3 Sufficient time for the tracer to effectively associate with PDE1 in the tissue and / or organ of interest is about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes, any of the above methods.

[0099] 1.4 Sufficient time for the tracer to effectively associate with PDE1 in the tissue and / or organ of interest is about 60 minutes, any of the above methods.

[0100] 1.5 The mapping is selective and reversible, any of the above methods.

[0101] 1.6 Binding of the PDE1 tracer compound in the tissue or organ to activated PDE1 is detected in vitro, any of the above methods.

[0102] 1.7 Detectable binding of the PDE1 tracer compound in the tissue or organ to activated PDE1 is detected in vivo, any of the above methods.

[0103] 1.8 A compound according to formula Ia or II is administered intravenously, any of the above methods.

[0104] 1.9 Positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography, any of the above methods.

[0105] 1.10 The tissue is from a patient suffering from a PDE1-mediated disease, disorder or condition, any of the above methods.

[0106] 1.11 Method 1.10, wherein the PDE1-mediated disease, disorder or condition is a cardiovascular-related disorder, a neurodegenerative disease, cancer or a tumor.

[0107] 1.12 The PDE1-mediated disease, disorder or condition is a. A tumor selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma (e.g., brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumor, anaplastic glioma, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, adenoma (e.g., basophilic adenoma, eosinophilic adenoma, chromophobic adenoma, parathyroid adenoma, pancreatic islet adenoma, fibroadenoma), fibromatosis (fibrous histiocytoma), fibroma, hemangioma, lipoma (e.g., angiolipoma, medullary lipoma, fibrolipoma, spindle cell lipoma, brown fat tumor, atypical lipoma), myxoma, osteoma, preleukemia, rhadomyoma, papilloma, seborrheic keratosis, skin appendage tumor, hepatic adenoma, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, cystic teratoma, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C-cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoid, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (e.g., pulmonary metaplasia), leukoplakia, hemangioma, lymphangioma, carcinoma (e.g., squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, embryonal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, oat cell carcinoma, pancreatic islet cell carcinoma, malignant carcinoid), sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), blastoma (e.g., medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, retinal tumor of the eye, osteoblastoma, bone tumor, neuroblastoma), germ cell tumor, mesothelioma, malignant skin appendage tumor, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloides, or Wilms tumor; b. cancer selected from leukemia (i.e., lymphocytic leukemia or myelogenous leukemia); c. a cardiovascular disorder, inflammatory disease or disorder, selected from angina pectoris, stroke, renal insufficiency, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, diabetes-related hypertension, atherosclerosis-related hypertension, renovascular hypertension, congestive heart failure, myocardial infarction, angina pectoris, stroke and renal insufficiency, hypertension, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy, and connective tissue diseases or disorders (e.g., Marfan syndrome); d. a neurodegenerative disease selected from Parkinson's disease, restless legs syndrome, tremors, dyskinesia, Huntington's disease, Alzheimer's disease and drug-induced movement disorders; mental disorders including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar disorder, anxiety, sleep disorders (e.g., narcolepsy), agnosia, dementia, Tourette syndrome, autism, fragile X syndrome, psychostimulant withdrawal and drug dependence; e. a respiratory and inflammatory disorder, and an autoimmune and inflammatory disease, selected from asthma, chronic obstructive pulmonary disease and allergic rhinitis; f. a disease or condition characterized by low levels of cAMP and / or cGMP in cells expressing PDE1 (or, inhibition of the cAMP and / or cGMP signaling pathway); and / or g. a disease or condition characterized by a decrease in dopamine D1 receptor signaling activity is Method 1.10.

[0108] 1.13 Method 1.10, wherein the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

[0109] 1.14 Method 1.10, wherein the PDE1-mediated disease, disorder or condition is glioma (e.g., ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma or mixed glioma, e.g., oligoastrocytoma).

[0110] Method 1.10, wherein the glioma is a gliocytoma (e.g., glioblastoma multiforme).

[0111] Method 1.10 to 1.15, wherein the condition is glioblastoma multiforme.

[0112] 1.17 Further, in free form or pharmaceutically acceptable salt form, [Chemical formula] Any of the above methods 1.10 to 1.16, comprising treating a PDE1-mediated disease, disorder or condition by administering a PDE1 inhibitor selected from

[0113] In another embodiment, the present invention provides a PET scan image generated by any of the methods after Method 1.

[0114] In another embodiment, the present disclosure is a method for treating a PDE1-mediated disease, disorder or condition in a subject in need thereof, a) administering to the subject an effective amount of a compound according to Formula Ia et seq. and / or II et seq., wherein the compound according to Formula Ia et seq. and / or II et seq. is radioactively labeled, for example, at one or more positions H is replaced by T (tritium, 3 H); b) imaging the subject with a positron emission tomography device; c) administering to the subject a PDE1 inhibitor, wherein the PDE1 inhibitor is not replaced by T (tritium, 3 H) at one or more positions; d) imaging the subject with a positron emission tomography device; e) comparing the data thus obtained, and f) evaluating the delivery of the PDE1 inhibitor to the tissue of interest in the PDE1-mediated condition by detecting the substitution of the compound according to Formula Ia et seq. and / or II et seq. Provided is a method [Method 2] that includes.

[0115] In a further embodiment, the present invention provides Method 2 as described in the following method:

[0116] 2.1 Method 2, wherein at least one position on the compound according to Formula Ia and / or II and later in step a) is substituted with tritium (T) instead of H.

[0117] 2.2 The compound in step a) is in free form or in a pharmaceutically acceptable salt form,

Chemical formula

[0118] 2.3 Positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography, any of the above methods.

[0119] 2.4 The tissue is from a patient suffering from a PDE1-mediated disease, disorder or condition, any of the above methods.

[0120] 2.5 The PDE1-mediated disease, disorder or condition is a cardiovascular-related disorder, neurodegenerative disease, cancer or tumor, any of the above methods.

[0121] 2.6 The PDE1-mediated disease, disorder or condition is a. A tumor selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma (e.g., brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, adenoma (e.g., basophilic adenoma, eosinophilic adenoma, chromophobe adenoma, parathyroid adenoma, pancreatic islet adenoma, fibroadenoma), fibroid (fibrous histiocytoma), fibroma, hemangioma, lipoma (e.g., angiolipoma, medullary lipoma, fibrolipoma, spindle cell lipoma, brown fat tumor, atypical lipoma), myxoma, osteoma, preleukemia, rhadomyoma, papilloma, seborrheic keratosis, skin appendage tumor, hepatic adenoma, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, cystic teratoma, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoid, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (e.g., lung metaplasia), leukoplakia, hemangioma, lymphangioma, carcinoma (e.g., squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, embryonal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, oat cell carcinoma, pancreatic islet cell carcinoma, malignant carcinoid), sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), blastoma (e.g., medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, retinal tumor of the eye, osteoblastoma, bone tumor, neuroblastoma), germ cell tumor, mesothelioma, malignant skin appendage tumor, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloides, or Wilms tumor; b. cancer selected from leukemia (i.e., lymphocytic leukemia or myelogenous leukemia); c. a cardiovascular disorder, inflammatory disease or disorder selected from angina pectoris, stroke, renal insufficiency, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, diabetes-related hypertension, atherosclerosis-related hypertension, renovascular hypertension, congestive heart failure, myocardial infarction, angina pectoris, stroke and renal insufficiency, hypertension; Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy; and connective tissue diseases or disorders (e.g., Marfan syndrome); d. a neurodegenerative disease selected from Parkinson's disease, restless legs syndrome, tremor, dyskinesia, Huntington's disease, Alzheimer's disease and drug-induced movement disorders; mental disorders including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar disorder, anxiety, sleep disorders (e.g., narcolepsy), agnosia, dementia, Tourette syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and drug dependence; e. a respiratory and inflammatory disorder, and an autoimmune and inflammatory disease selected from asthma, chronic obstructive pulmonary disease and allergic rhinitis; f. a disease or condition characterized by low levels of cAMP and / or cGMP in cells expressing PDE1 (or inhibition of the cAMP and / or cGMP signaling pathway); and / or g. a disease or condition characterized by a decrease in dopamine D1 receptor signaling activity which is any of the above methods.

[0122] 2.7 which is any of the above methods, wherein the condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

[0123] 2.8 which is any of the above methods, wherein the PDE1-mediated disease, disorder or condition is glioma (e.g., ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, or mixed glioma, e.g., oligoastrocytoma).

[0124] 2.9 Any of the above methods, wherein the glioma is an astrocytoma (e.g., glioblastoma multiforme).

[0125] 2.10 Any of the above methods, wherein the condition is glioblastoma multiforme.

[0126] 2.11 The PDE1 inhibitor administered in step c) is one of the following in free form or in pharmaceutically acceptable salt form: [Chemical formula] Any of the above methods, selected from:

[0127] 2.12 The PDE1 inhibitor administered in step c) is one of the following in free form or in pharmaceutically acceptable salt form: [Chemical formula] Any of the above methods, which is:

[0128] In yet another embodiment, the present disclosure is a method for diagnosing a PDE1-mediated disease, disorder or condition characterized by upregulation of PDE1 expression in a subject, comprising: a) obtaining a first tissue sample from a patient suspected of having a PDE1-mediated disease, disorder or condition or at risk of a PDE1-mediated disease, disorder or condition; b) contacting the first tissue sample with an effective amount of a compound according to formula Ia et seq. or II et seq. (wherein the compound according to formula Ia et seq. or II et seq. is radiolabeled, e.g., substituted with T (tritium, 3 H) at one or more positions instead of H); c) imaging the first tissue sample with a positron emission tomography device; d) optionally comparing the result of step c) with a second tissue sample in which PDE1 expression is not upregulated to provide a method [Method 3].

[0129] In a further embodiment, the present invention provides Method 1 as described in the following method:

[0130] 3.1 Further, Method 3 including the step of imaging a second tissue sample with a positron emission tomography device.

[0131] 3.2 Any of the above methods, wherein the second tissue sample is obtained from a subject not suffering from a PDE1-mediated disease, disorder or condition.

[0132] 3.3 Any of the above methods, wherein both the first tissue sample and the second tissue sample are of the same type of tissue (e.g., human brain tissue).

[0133] 3.4 Any of the above methods, wherein the first tissue sample is human brain tissue taken from a subject suspected of suffering from glioblastoma multiforme, and the second tissue sample is non-cancerous human brain tissue.

[0134] 3.5 Any of the above methods, wherein if it is shown by comparison in step d) that the PDE1 expression in the first tissue sample is greater than that in the second sample, the patient is suffering from a PDE1-mediated disease, disorder or condition.

[0135] 3.6 Any of the above methods, wherein at least one position on the compound according to formula Ia and / or II and later in step a) is substituted with tritium (T) instead of H.

[0136] 3.7 The compound in step a) is in free form or pharmaceutically acceptable salt form,

Chemical formula

[0137] 3.8 Any of the above methods, wherein the positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography.

[0138] 3.9 Any of the above methods, wherein the PDE1-mediated disease, disorder or condition is a cardiovascular-related disorder, a neurodegenerative disease, cancer or a tumor.

[0139] 3.10 The PDE1-mediated disease, disorder or condition is a. A tumor selected from one or more of acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma (e.g., brainstem glioma, ependymoma, mixed glioma, optic nerve glioma), subependymoma, medulloblastoma, meningioma, metastatic brain tumor, anaplastic glioma, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, adenoma (e.g., basophilic adenoma, eosinophilic adenoma, chromophobic adenoma, parathyroid adenoma, pancreatic islet adenoma, fibroadenoma), fibroid (fibrous histiocytoma), fibroma, hemangioma, lipoma (e.g., angiolipoma, medullary lipoma, fibrolipoma, spindle cell lipoma, brown lipoma, atypical lipoma), myxoma, osteoma, preleukemia, rhadomyoma, papilloma, seborrheic keratosis, skin appendage tumor, hepatic adenoma, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, cystic teratoma, ganglioneuroma, meningoma, neurilemmoma, neurofibroma, C-cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoid, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia (e.g., pulmonary metaplasia), leukoplakia, hemangioma, lymphangioma, carcinoma (e.g., squamous cell carcinoma, epidermoid carcinoma, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, cholangiocarcinoma, transitional cell carcinoma, embryonal cell carcinoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, oat cell carcinoma, pancreatic islet cell carcinoma, malignant carcinoid), sarcoma (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, malignant fibrous histiocytoma, hemangiosarcoma, angiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, neurofibrosarcoma), blastoma (e.g., medulloblastoma and glioblastoma, types of brain tumor, retinoblastoma, retinal tumor of the eye, osteoblastoma, bone tumor, neuroblastoma), germ cell tumor, mesothelioma, malignant skin appendage tumor, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloides, or Wilms tumor; b. cancer selected from leukemia (i.e., lymphocytic leukemia or myeloid leukemia); c. a cardiovascular disorder, inflammatory disease or disorder selected from angina, stroke, renal failure, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, diabetes-related hypertension, atherosclerosis-related hypertension, renovascular hypertension, congestive heart failure, myocardial infarction, angina, stroke and renal failure, hypertension; inflammatory diseases or disorders, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy, and connective tissue diseases or disorders (e.g., Marfan syndrome); d. neurodegenerative diseases selected from Parkinson's disease, restless legs syndrome, tremors, dyskinesia, Huntington's disease, Alzheimer's disease and drug-induced movement disorders; mental disorders including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar disorder, anxiety, sleep disorders (e.g., narcolepsy), agnosia, dementia, Tourette syndrome, autism, fragile X syndrome, psychostimulant withdrawal and drug dependence; e. respiratory and inflammatory disorders, and autoimmune and inflammatory diseases selected from asthma, chronic obstructive pulmonary disease and allergic rhinitis; f. a disease or condition characterized by low levels of cAMP and / or cGMP in cells expressing PDE1 (or inhibition of the cAMP and / or cGMP signaling pathway); and / or g. a disease or condition characterized by a decrease in dopamine D1 receptor signaling activity any of the above methods.

[0140] 3.11 Any of the above methods, wherein the PDE1-mediated disease, disorder or condition is glioma, osteosarcoma, melanoma, leukemia or neuroblastoma.

[0141] 3.12 Any of the above methods, wherein the PDE1-mediated disease, disorder or condition is glioma (e.g., ependymoma, astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma or mixed glioma, e.g., oligoastrocytoma).

[0142] 3.13 Any of the above methods, wherein the glioma is an astrocytoma (e.g., glioblastoma multiforme).

[0143] 3.14 Any of the above methods, wherein the condition is glioblastoma multiforme.

[0144] 3.15 Any of the above methods, further comprising treating the subject for a PDE1-mediated disease, disorder or condition by administering a pharmaceutically acceptable amount of a PDE1 inhibitor.

[0145] 3.16 The PDE1 inhibitor administered for the treatment of a PDE1-mediated disease, disorder or condition is one of the following in free form or in pharmaceutically acceptable salt form:

Chemical formula

[0146] 3.17 The PDE1 inhibitor administered in step c) is one of the following in free form or in pharmaceutically acceptable salt form:

Chemical formula

[0147] The present invention further provides the use of a PDE1 inhibitor, such as any of the compounds of formula Ia or later or formula II or later, in the manufacture of a medicament for use in any of methods 1 or later, methods 2 or later or methods 3 or later.

[0148] The present invention further provides any of the compounds of a PDE1 inhibitor, such as any of the compounds of formula Ia or later or formula II or later, for use in any of methods 1 or later, methods 2 or later or methods 3 or later.

[0149] The present invention further provides a pharmaceutical composition for use in any of Method 1 onwards, Method 2 onwards or Method 3 onwards, comprising a PDE1 inhibitor, for example, any of the compounds of Formula Ia onwards or Formula II onwards.

[0150] Method for producing the compounds of the present disclosure

[0151] The PDE1 inhibitors of the present disclosure and their pharmaceutically acceptable salts can be prepared using the methods as described and exemplified in U.S. Patent No. 8,273,750, U.S. Patent Application Publication No. 2006 / 0173878, U.S. Patent No. 8,273,751, U.S. Patent Application Publication No. 2010 / 0273753, U.S. Patent No. 8,697,710, U.S. Patent No. 8,664,207, U.S. Patent No. 8,633,180, U.S. Patent No. 8,536,159, U.S. Patent Application Publication No. 2012 / 0136013, U.S. Patent Application Publication No. 2011 / 0281832, U.S. Patent Application Publication No. 2013 / 0085123, U.S. Patent Application Publication No. 2013 / 0324565, U.S. Patent Application Publication No. 2013 / 0338124, U.S. Patent Application Publication No. 2013 / 0331363, International Publication No. 2012 / 171016 and International Publication No. 2013 / 192556, as well as by methods similar thereto and by methods known in the chemical art. Such methods include, but are not limited to, the methods described below. The starting materials for these processes can be prepared by procedures selected from chemical techniques using the same or similar techniques as the methods for synthesizing known compounds if they are not commercially available.

[0152] Various PDE1 inhibitors and their starting materials can be produced using the methods described in U.S. Patent Application Publication No. 2008-0188492 (A1), U.S. Patent Application Publication No. 2010-0173878 (A1), U.S. Patent Application Publication No. 2010-0273754 (A1), U.S. Patent Application Publication No. 2010-0273753 (A1), International Publication No. 2010 / 065153, International Publication No. 2010 / 065151, International Publication No. 2010 / 065151, International Publication No. 2010 / 065149, International Publication No. 2010 / 065147, International Publication No. 2010 / 065152, International Publication No. 2011 / 153129, International Publication No. 2011 / 133224, International Publication No. 2011 / 153135, International Publication No. 2011 / 153136, International Publication No. 2011 / 153138. All references cited herein are hereby incorporated by reference in their entirety as part of this specification.

[0153] The disclosed compounds include their enantiomers, diastereomers and racemates, as well as their polymorphs, hydrates, solvates and complexes. Some individual compounds within the scope of the present disclosure may contain double bonds. The representation of double bonds in the present disclosure means that both the E isomer and the Z isomer of the double bond are included. Further, some compounds within the scope of the present disclosure may contain one or more asymmetric centers. The present disclosure includes the use of any optically pure stereoisomers, and combinations of stereoisomers.

[0154] It is also intended that the disclosed compounds include their stable and unstable isotopes. Stable isotopes are non-radioactive isotopes that contain one additional neutron compared to the abundant nuclides of the same species (i.e., element). The activity of compounds containing such isotopes is retained, and such compounds are also considered to have utility for measuring the pharmacokinetics of non-isotope analogs. For example, a hydrogen atom at a certain position of the disclosed compound can be replaced with deuterium (a non-radioactive stable isotope). Examples of known stable isotopes include deuterium, 13 C, 15 N, 18Examples include, but are not limited to, O. Alternatively, unstable isotopes that are radioactive isotopes containing a plurality of additional neutrons compared to abundant nuclides of the same species (i.e., elements), such as 123 I, 131 I, 125 I, 11 C, 18 F may be replaced by the corresponding abundant species of I, C, and F. Another example of a useful isotope of the compounds of the present disclosure is 11 the C isotope. Of particular interest in this case is tritium, which is a radioactive isotope of hydrogen (protium). These radioactive isotopes are useful for radiation imaging and / or pharmacokinetic studies of the compounds of the present disclosure.

[0155] As used herein, a compound referred to as a "radioactive label" contains at least one radioactive isotope in place of a non-radioactive isotope at a specific position, such as containing tritium in place of hydrogen, or has a substituent containing a radioactive isotope.

[0156] Melting points are uncorrected and (dec) indicates decomposition. Temperatures are given in degrees Celsius (°C); unless otherwise noted, operations are carried out at room temperature or ambient temperature, i.e., in the range of 18 - 25 °C. Chromatography means silica gel flash chromatography; thin layer chromatography (TLC) is carried out on silica gel plates. NMR data are reported as delta values of the major diagnostic protons in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Conventional abbreviations for signal shapes are used. Coupling constants (J) are expressed in Hz. For mass spectra (MS), for molecules where multiple mass spectral peaks occur due to isotope splitting, the major ion of the minimum mass is reported. The composition of solvent mixtures is indicated as volume percent or volume ratio. When NMR spectra are complex, only diagnostic signals are reported.

[0157] The terms "treatment" and "treating" should thus be understood to include the treatment or alleviation of the symptoms of a disease, as well as the treatment of the cause of the disease.

[0158] For a method of treatment, the term "effective amount" is intended to encompass a therapeutically effective amount for treating a particular disease or disorder.

[0159] The term "patient" includes human or non-human (i.e., animal) patients. In certain embodiments, the disclosure encompasses both humans and non-humans. In other embodiments, the disclosure encompasses non-humans. In yet other embodiments, the term encompasses humans.

[0160] As used in this disclosure, the term "comprising" is intended to be open-ended and does not exclude additional elements or method steps not recited.

[0161] The dosages used in practicing the present disclosure will, of course, vary depending, for example, on the particular disease or condition being treated, the particular disclosed compound being used, the mode of administration, and the desired therapy. The disclosed compounds can be administered by suitable routes including oral, parenteral, transdermal, or inhalation, but are preferably administered orally. In general, satisfactory results for the treatment of diseases such as those noted above have been shown to be obtainable by oral administration at dosages on the order of about 0.01 - 2.0 mg / kg. In large mammals, such as humans, the indicated daily dose for oral administration of both PDE1 inhibitors is, accordingly, in the range of about 0.50 - 300 mg, conveniently administered once daily, or in divided doses of 2 - 4 times daily, or in sustained release dosage forms. Thus, unit dosage forms for oral administration can contain, for example, about 0.2 - 150 or 300 mg of the disclosed compound, such as about 0.2 or 2.0 - 10, 25, 50, 75, 100, 150 or 200 mg, together with a pharmaceutically acceptable diluent or carrier.

[0162] The compounds of the present disclosure can be administered by satisfactory routes including oral, parenteral (intravenous, intramuscular or subcutaneous) or transdermal routes, but are preferably administered orally. In certain embodiments, for example in depot formulations, the compounds of the present disclosure are preferably administered parenterally, for example by injection.

[0163] The compounds of the present disclosure and the pharmaceutical compositions of the present disclosure can be combined with one or more additional therapeutic agents and used at lower doses, particularly when the individual agents are used as monotherapies, than when used alone, to enhance the therapeutic activity of the combined agents without causing the undesirable side effects that generally occur with conventional monotherapies. Accordingly, the compounds of the present disclosure can be administered simultaneously, separately, sequentially or contemporaneously with other agents useful for treating the disease. In another example, side effects can be reduced or minimized by administering the compounds of the present disclosure in combination with one or more additional therapeutic agents in free form or in salt form, where (i) the dose of the second therapeutic agent or (ii) both the dose of the compound of the present disclosure and the second therapeutic agent is lower than when the agent / compound is administered as a monotherapy. Non-limiting examples of such additional therapeutic agents can include ACE inhibitors, angiotensin II receptor antagonists, calcium channel blockers, and the like.

[0164] The term "simultaneously" when referring to therapeutic use means administering two or more active ingredients by the same route of administration simultaneously or almost simultaneously.

[0165] The term "separately" when referring to therapeutic use means administering two or more active ingredients by different routes of administration simultaneously or almost simultaneously.

[0166] The pharmaceutical compositions containing the compounds of the present disclosure can be prepared using conventional diluents or excipients and techniques known in the field of herbal medicine technology. Accordingly, oral dosage forms can include tablets, capsules, solutions, suspensions, and the like.

Example

[0167] Example 1: Synthesis of a novel PDE1 inhibitor Synthesis of 3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(4-(1-methylpyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 1):

Chem.

[0168] (a) tert-Butyl 2-(4-((5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)phenyl)pyrrolidine-1-carboxylate A suspension of 5,7,7-trimethyl-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (300 mg, 1.37 mmol), tert-butyl 2-(4-(bromomethyl)phenyl)pyrrolidine-1-carboxylate (650 mg, 1.78 mmol) and K2CO3 (189 mg, 1.37 mmol) in DMF (3 mL) is stirred at room temperature overnight. The solvent is removed under reduced pressure. The obtained residue is dissolved in ethyl acetate (300 mL), sonicated and washed with water (3×80 ml). Ethyl acetate is removed and the residue is dried under high vacuum to obtain 1.07 g of a crude product (containing salts), which is used in the next step without further purification. MS (ESI) m / z 479.2 [M+H]+.

[0169] (b) 3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(4-(pyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one A solution of tert-butyl 2-(4-((5,7,7-trimethyl-4-oxo-4,5,7,8-tetrahydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-2-yl)methyl)phenyl)pyrrolidine-1-carboxylate (1 g, 1.37 mmol) and hexachloroethane (494 mg, 2.01 mmol) in a mixture of toluene (15 mL) and THF (15 mL) is added dropwise with LiHMDS (1.0 M in hexane, 3.13 mL, 3.13 mmol). The reaction mixture is stirred at room temperature for 30 minutes, 4-fluoroaniline (0.30 mL, 3.13 mmol) is added, and then LiHMDS (1.0 M in hexane, 3.13 mL, 3.13 mmol) is added. The mixture is stirred at room temperature for an additional 10 minutes, and 4-(pyrrolidin-1-yl)pyridine (31 mg, 0.21 mmol) is added. The resulting solution is heated to 80 °C and stirred at this temperature for 30 minutes. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate (200 mL). The ethyl acetate solution is washed with water (3 × 50 mL) and evaporated to dryness under reduced pressure. Dichloromethane (5 mL) is added to the resulting residue, and then TFA (5 mL) is added at room temperature. The mixture is stirred at room temperature overnight. After removing the solvent, the crude product is loaded onto a basic alumina column to obtain the crude product. This product is further purified by preparative HPLC equipped with a reverse-phase C18 column (gradient eluent: 0 - 26% acetonitrile in water containing 0.1% formic acid over 16 minutes). The title product is obtained as a pale yellow solid (200 mg; overall yield of three steps: 30%). MS (ESI) m / z 488.4 [M+H]+. 1 H NMR (500 MHz, Chloroform-d) δ 8.19 (s, 2H), 7.34 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 6.95 - 6.92 (m, 2H), 6.88 - 6.85 (m, 2H), 4.91 (s, 2H), 3.84 (s, 2H), 3.42 - 3.24 (m, 3H), 3.20 (s, 3H), 2.40 - 2.29 (m, 1H), 2.25 - 2.12 (m, 3H), 1.48 (s, 6H).

[0170] (c) 3-((4-Fluorophenyl)amino)-5,7,7-trimethyl-2-(4-(1-methylpyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one To a solution of 3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(4-(pyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (40 mg, 0.075 mmol) in methanol (0.4 mL) is added 37% formaldehyde (6 μL, 0.075 mmol). The mixture is stirred at room temperature for 10 minutes, and sodium cyanoborohydride (5 mg, 0.075 mmol) is added. The resulting mixture is stirred at room temperature for 30 minutes and then filtered. The filtrate is concentrated and purified by a preparative HPLC system (gradient eluent: 0 - 17% acetonitrile in water containing 0.1% formic acid over 16 minutes). The title compound is obtained as a white solid (27 mg, 71% yield). MS (ESI) m / z 502.3. 1 H NMR (500 MHz, Chloroform-d) δ 8.32 (s, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.05 - 6.98 (m, 3H), 6.95 (dd, J = 9.0, 8.0 Hz, 2H), 6.89 (dd, J = 9.0, 4.6 Hz, 2H), 4.89 (s, 2H), 3.85 (s, 2H), 3.77 (s, 2H), 3.35 (s, 3H), 2.91 (s, 1H), 2.51 (s, 3H), 2.40 - 2.30 (m, 3H), 2.13 - 2.07 (m, 1H), 1.46 (s, 6H).

[0171] Synthesis of 3-((4-fluorophenyl)amino)-5-[3H]methyl-7,7-dimethyl-2-(4-(1-methylpyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one (Compound 2) [Chemistry] The labeled compound was synthesized by reacting 3-((4-fluorophenyl)amino)-5,7,7-trimethyl-2-(4-(pyrrolidin-2-yl)benzyl)-7,8-dihydro-2H-imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one with [3H]methyl iodide. The crude product obtained was purified by HPLC to give the final compound with radiochemical purity >99% and specific activity 58 Ci / mol. The product identity was confirmed by HPLC co-elution with the authentic standard.

[0172] The inhibition constant (Ki) values of Compound 1 were determined against the entire panel of phosphodiesterase family members. Compound 1 showed a potent inhibitory effect against the PDE1 enzyme and more than 1000-fold selectivity against all other PDE families (Table 1). In contrast, the potencies of Compound 1 against PDE1 A, B, and C were almost equivalent.

[0173] [Table 1]

[0174] Example 2: Saturation Binding of a Novel Radioligand Homogenates of mouse striatum, mouse prefrontal cortex, or dog heart were incubated in duplicate at room temperature in binding buffer containing nanomolar concentrations of Compound 2. Specific high-affinity binding of Compound 2 to the brain and heart was saturated. The B max value in mouse striatum was 1252 fmol / mg protein, 269 fmol / mg protein in mouse cortex, and 124 fmol / mg protein in dog heart. The B max value of binding to the striatum was significantly higher than that in the cortex and heart, consistent with the high expression of PDE1B in this region. The apparent K d values for the binding of Compound 2 were 2.77, 1.32, and 0.41 (nM) in mouse striatum, mouse cortex, and dog heart, respectively.

[0175] Example 3: Competitive Binding of Compound 2 Various concentrations of a very potent PDE1 inhibitor were competed with Compound 2 at a fixed concentration (1.5 nM) that is lower than the K d value. The IC 50 values of the competing PDE1 inhibitor were determined from a competition assay using Compound 2 binding to homogenates of mouse striatum and cortex, and were 3.45 nM and 0.97 nM, respectively. In a competition assay using Compound 2 binding to dog heart homogenate, a similar IC 50 value (1.11 nM) was determined for the competing PDE1 inhibitor. The Hill values for displacement of the radioligand were -1.20 in the striatum, -1.27 in the cortex, and -1.03 in dog left ventricular tissue.

[0176] Furthermore, follow-up tests were conducted using four more potent and specific PDE1 inhibitors. These were tested for their efficacy in inhibiting Compound 2 binding to homogenates of mouse striatum or cortex. The K i values determined by the radioligand competition assay were consistent with the inhibition constant values from in vitro enzyme assays. The K i values of these inhibitors in the binding assay were approximately three times the activity measured in the in vitro PDE1 enzyme activity assay.

[0177] Example 4: Calcium Dependence of PDE1 Cellular Activity To further investigate the regulation of PDE1 by intracellular calcium concentration, striatal tissue slice specimens were used. Striatal slice specimens maintain a part of the intact striatal circuit, including interneurons, cortical and substantia nigra nerve inputs to nerve terminals, and medium spiny neurons that make up the majority (95%) of neurons. Depolarization of the cell membrane by increasing the external potassium (K+) concentration is an established method of stimulating striatal slices, especially to cause the release of neurotransmitters such as glutamate from nerve terminals. Under this condition, both voltage-gated calcium channels and NMDA receptors are expected to open, allowing calcium influx into the cell. An increase in intracellular calcium can activate PDE1 in medium spiny neurons and also activate nitric oxide synthase (NOS) in interneurons via calcium-calmodulin binding. Nitric oxide released from interneurons activates soluble guanylyl cyclase in medium spiny neurons, causing an increase in cGMP levels. The global nature of K+ depolarization causes simultaneous stimulation of medium spiny neurons by neurotransmitter release and depolarization.

[0178] When striatal slices were stimulated with short-term K+ depolarization, a significant increase in cGMP levels was revealed by pre-incubation of the slices with 1 μM of two potent PDE1 inhibitors (Figure 1).

[0179] Ionomycin, a calcium ionophore, was used to control intracellular calcium concentration in a depolarization-free state. Ionomycin (10 μM, in Krebs buffer) increased intracellular calcium, as confirmed by the increased calcium-sensitive phosphorylation of Ca2+ / calmodulin-dependent protein kinase II (CaMKII), as shown in Fig. 2A. No increase in cGMP levels occurred with ionomycin-induced calcium influx alone (Fig. 2B). However, inhibition of PDE1 activity in the presence of ionomycin significantly increased cGMP levels (p < 0.01, n = 8). Removal of extracellular calcium by replacing 3 mM calcium chloride in the Krebs solution with 3 mM EGTA blocked the inhibition of PDE1 and the increase in cGMP seen with ionomycin.

[0180] In another study, 1321N1 human astrocytoma cells were pretreated with 10 μM ionomycin for 15 min in the absence of extracellular Ca2+ to deplete Ca2+. Subsequently, addition of Ca2+ to the extracellular medium in the presence of the calcium ionophore ionomycin caused a concentration-dependent increase in intracellular Ca2+ concentration, as previously reported (8). This increase in Ca2+ levels activated calcium-calmodulin-dependent PDE1, resulting in a decrease in cAMP signaling. Fig. 1 shows that addition of 3 mM Ca2+ to the extracellular medium caused substantial inhibition of isoproterenol-induced cAMP accumulation. This effect was reversed by potent PDE1 inhibition at concentrations in the 0.1 - 1 nanomolar range (Fig. 3).

[0181] In a further study, the binding of compound 2 (1.5 nM) to mouse cortex was titrated with different concentrations of the calcium chelator EGTA to determine the calcium-dependence of ligand binding. EGTA decreased the specific binding of compound 2 in a concentration-dependent manner, with an IC 50The value was 7.7 μM (Figure 4). The level of binding activity remaining in the presence of excess (200 μM) EGTA was approximately 5% compared to the binding level in the absence of EGTA. These results using binding are reasonably consistent with the tests of enzyme activity and establish the absolute calcium dependence of PDE1 inhibitor binding, i.e., compound 2 binding, to the PDE1 enzyme. This application also encompasses the following aspects. [Aspect 1] The compound of formula Ia in free form, pharmaceutically acceptable salt form or prodrug form (including its enantiomers, diastereoisomers and racemates):

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Claims

1. The compound of formula Ia in free form or in a pharmaceutically acceptable salt form (including its enantiomers, diastereoisomers and racemates): 【Chemical 1】 Wherein: (i) R 2 and R 3 are each methyl, and R 4 and R 5 are each H; (ii) R 6 is phenylamino (which may be halo-substituted), benzylamino (which may be halo-substituted), C 1-4 alkyl, or C 1-4 alkyl sulfide; (iii) R 10 is 1-methylpyrrolidin-2-yl in which at least one position is substituted with tritium (T) instead of H; (iv) X and Y are C The compound.

2. The compound according to claim 1, wherein the compound is in free form or in a pharmaceutically acceptable salt form of 【Chemical 2】

3. A method for mapping PDE1 activity in a tissue and / or organ of interest using positron emission tomography, comprising administering an effective amount of a PDE1 tracer compound which is the compound according to claim 1 or 2 (wherein at least one position on the compound is substituted with tritium (T) instead of H), and imaging the tissue and organ of interest using positron emission tomography in a subject after allowing sufficient time for the tracer to effectively associate with PDE1 in the tissue and / or organ of interest.

4. The method according to claim 3, wherein the sufficient time for the tracer to effectively associate with PDE1 in the tissue and / or organ of interest is 60 minutes.

5. The method according to claim 3 or 4, wherein the mapping is selective and reversible.

6. The method according to any one of claims 3 to 5, wherein the tissue and / or organ is imaged in vitro.

7. The method according to any one of claims 3 to 5, wherein the tissue and / or organ is imaged in vivo.

8. The method according to any one of claims 3 to 5, wherein the subject is a subject administered with the PDE1 tracer compound intravenously.

9. The method according to any one of claims 3 to 8, wherein the positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography.

10. The method according to any one of claims 3 to 9, wherein the tissue is from a patient suffering from a PDE1-mediated disease, disorder or condition.

11. A medicament for treating a PDE1-mediated disease, disorder or condition in a subject in need thereof, comprising the radiolabeled compound according to claim 1 or 2, wherein the treatment comprises: a) administering the medicament to the subject; ​ b) imaging the subject with a positron emission tomography device; c) administering to the subject a PDE1 inhibitor that does not contain a radionuclide at a predetermined dose; d) imaging the subject with a positron emission tomography device; e) comparing the data thus obtained, and f) evaluating the effective delivery of the PDE1 inhibitor to the tissue of interest in a PDE1-related condition A medicament comprising the same.

12. The medicament according to claim 11, wherein the positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography.

13. The medicament according to claim 11 or 12, wherein the PDE1-mediated disease, disorder or condition is a cardiovascular-related disorder, a neurodegenerative disease, cancer or a tumor.

14. The PDE1-mediated disease, disorder or condition is a. one or more selected from acoustic neuroma, astrocytoma, chordoma, CNS lymphoma, craniopharyngioma, glioma, subependymal tumor, medulloblastoma, meningioma, metastatic brain tumor, anaplastic glioma, pituitary tumor, primitive neuroectodermal tumor (PNET), schwannoma, adenoma, fibroma, fibroma, hemangioma, lipoma, myxoma, osteoma, preleukemia, rhadomyoma, papilloma, seborrheic keratosis, skin appendage tumor, hepatic adenoma, renal tubular adenoma, bile duct adenoma, transitional cell papilloma, cystic teratoma, ganglioneuroma, meningoma, schwannoma, neurofibroma, C-cell hyperplasia, pheochromocytoma, insulinoma, gastrinoma, carcinoid, chemodectoma, paraganglioma, nevus, actinic keratosis, cervical dysplasia, metaplasia, leukoplakia, hemangioma, lymphangioma, carcinoma, sarcoma, blastoma, germ cell tumor, mesothelioma, malignant skin appendage tumor, hypernephroma, seminoma, glioma, malignant meningioma, malignant schwannoma, malignant pheochromocytoma, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloides, or Wilms tumor; a tumor; b. a cancer selected from leukemia; c. A cardiovascular disorder, inflammatory disease or disorder, selected from angina pectoris, stroke, renal insufficiency, essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, diabetes-related hypertension, atherosclerosis-related hypertension, renovascular hypertension, congestive heart failure, myocardial infarction, angina pectoris, stroke and renal insufficiency, hypertension; Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, and Emery-Dreifuss muscular dystrophy; and connective tissue disease or disorder; d. A neurodegenerative disease selected from Parkinson's disease, restless legs syndrome, tremor, dyskinesia, Huntington's disease, Alzheimer's disease and drug-induced movement disorder; a mental disorder including depression, attention deficit disorder, attention deficit hyperactivity disorder, bipolar disorder, anxiety, sleep disorder, agnosia, dementia, Tourette syndrome, autism, fragile X syndrome, psychostimulant withdrawal, and drug dependence; e. A respiratory and inflammatory disorder, and an autoimmune and inflammatory disease selected from asthma, chronic obstructive pulmonary disease and allergic rhinitis; f. A disease or condition characterized by low levels of cAMP and / or cGMP in cells expressing PDE1 (or inhibition of the cAMP and / or cGMP signaling pathway); and / or g. A disease or condition characterized by a decrease in dopamine D1 receptor signaling activity The medicament according to claim 11 or 12, which is

15. The medicament according to claim 11 or 12, wherein the condition is glioma, osteosarcoma, melanoma, leukemia, or neuroblastoma.

16. The medicament according to claim 11 or 12, wherein the PDE1-mediated disease, disorder or condition is glioma.

17. The medicament according to claim 16, wherein the glioma is astrocytoma.

18. The medicament according to claim 11 or 12, wherein the condition is glioblastoma multiforme.

19. The PDE1 inhibitor administered in step c) is one of the following in free form or pharmaceutically acceptable salt form: [Chemical Formula 3] The medicament according to any one of claims 11 to 18, selected from

20. A method for assisting in the diagnosis of a PDE1-mediated disease, disorder or condition characterized by upregulation of PDE1 expression in a subject, comprising a) contacting a first tissue sample obtained from a patient suspected of having or at risk of a PDE1-mediated disease, disorder or condition with an effective amount of the compound according to claim 1 or 2; b) imaging the first tissue sample with a positron emission tomography device; c) comparing the result of step b) with a second tissue sample in which PDE1 expression is not up-regulated A method comprising.

21. The method according to claim 20, further comprising the step of imaging the second tissue sample with a positron emission tomography device.

22. The method according to claim 20 or 21, wherein the second tissue sample is obtained from a subject not suffering from a PDE1-mediated disease, disorder or condition.

23. The method according to any one of claims 20 to 22, wherein both the first tissue sample and the second tissue sample are of the same type of tissue.

24. The method according to any one of claims 20 to 23, wherein the first tissue sample is human brain tissue taken from a subject suspected of having glioblastoma multiforme, and the second tissue sample is non-cancerous human brain tissue.

25. If the comparison in step c) shows that PDE1 expression in the first tissue sample is greater than that in the second sample, the patient is suffering from a PDE1-mediated disease, disorder or condition. The method according to any one of claims 20 to 24.

26. The method according to any one of claims 20 to 25, wherein at least one position on the compound in step a) is substituted with tritium (T) instead of H.

27. The compound of step a) is in free form or pharmaceutically acceptable salt form, [Chemical Formula 4] The method according to any one of claims 20 to 26.

28. The method according to any one of claims 20 to 27, wherein the positron emission tomography is positron emission tomography / computed tomography or single photon emission computed tomography / computed tomography.

Images